Hematopoiesis. The mystery of blood Cell and vascular Formation

Hematopoiesis. The mystery of blood Cell and vascular Formation

https://reasonandscience.catsboard.com/t2295-hematopoiesis-the-mystery-of-blood-cell-and-vascular-formation

Little attention has been given to the evolutionary origin of blood. Very little scientific literature exists on the subject. And remarkably: Understanding how the blood system is formed is an ongoing fundamental research challenge, even in 2015. Hematopoiesis is the description of how blood cellular components form.   Blood and the developing blood cells and their precursors are produced  in the bone marrow through pluripotential Hematopoietic stem cells (HSCs)  which give rise to all  ten different type of blood cells through the process of haematopoiesis  These blood cells are: macrophages , neutrophils, basophils, eosinophils, erythrocytes, dendritic cells, platelets, T cells, B cells, and natural killer cells.

Question: At least six of the different cell types in blood are life essential. If one is missing, life ceases to exist. Had these blood types not to emerge together, to make the life of a multicellular organism possible? 
Most blood types require intermediate steps in their development until becoming final cell types used in blood. These intermediate cell types have no function in the organism. Why would evolution produce them? 

Bruce Alberts and his colleagues noted: “Blood contains many types of cells with very different functions, ranging from the transport of oxygen to the production of antibodies. Some of these cells function entirely within the vascular system, while others use the vascular system only as a means of transport and perform their function elsewhere (1994, p. 1161).

The process of developing the diverse blood cell repertoire from stem and progenitor cells termed hematopoiesis has been subject to considerable investigation. However, key steps in the complex process of hematopoiesis, including hematopoietic stem cell generation during embryogenesis, hematopoietic stem, and progenitor cell expansion remain incompletely understood. 

Furthermore: Red blood cells, most white blood cells, and platelets are produced in the bone marrow, the soft fatty tissue inside bone cavities. 
And bone formation is an irreducible complex, extremely complex process:


http://reasonandscience.heavenforum.org/t2296-origin-and-development-of-bones-osteogenesis



Natural killer cells are a type of immune cell that has granules (small particles) with enzymes that can kill tumor cells or cells infected with a virus. A natural killer cell is a type of white blood cell. Also called NK cell and NK-LGL. They  are part of the innate immune defense against infection and cancer, and are especially useful in combating certain viral pathogens9
T cells are essential for human immunity. 8
B cells  are a type of white blood cell that makes antibodies. B lymphocytes are part of the immune system and develop from stem cells in the bone marrow. Primary B-cell immunodeficiencies (B-PID) constitute a heterogeneous group of immunodeficiencies characterized by defective production of antigen-specific antibodies and predisposition to recurrent and severe infections 9
Dendritic cells are needed to control B and T lymphocytesm, and capture and process antigens, express lymphocyte co-stimulatory molecules, migrate to lymphoid organs and secrete cytokines to initiate immune responses.3
Macrophages are essential components of the innate immune system. They are a type of white blood cell that engulfs and digests cellular debris, foreign substances, microbes, cancer cells, and anything else that does not have the types of proteins specific of healthy body cells on its surface in a process called phagocytosis.2
Neutrophils  are a required type of immune cell that is one of the first cell types to travel to the site of an infection. Neutrophils help fight infection by ingesting microorganisms and releasing enzymes that kill the microorganisms. A neutrophil is a type of white blood cell,
Basophils contain anticoagulant heparin, which prevents blood from clotting too quickly. They also contain the vasodilator histamine, which promotes blood flow to tissues.
Eosinophils effector functions include production of: cationic granule proteins and their release by degranulation, the production of reactive oxygen species such as hypobromite, superoxide, and peroxide, production of lipid mediators like the eicosanoids, enzymes, such as elastase. growth factors such as TGF beta, VEGF, and PDGF. etc....
Red blood cells ( erythrocytes ) are the most common type of blood cell and essential for the vertebrate organism's  delivering oxygen (O2) to the body tissues—via blood flow through the circulatory system 6
Platelets  are a essential component of blood whose function (along with the coagulation factors) is to stop bleeding by clumping and clotting blood vessel injuries 7




1) https://en.wikipedia.org/wiki/Hematopoietic_stem_cell
2) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2956013/
3) http://www.ncbi.nlm.nih.gov/pubmed/9521319
4) http://www.ncbi.nlm.nih.gov/pubmedhealth/PMHT0022058/
5) https://en.wikipedia.org/wiki/Basophil
6) https://en.wikipedia.org/wiki/Red_blood_cell
7) https://en.wikipedia.org/wiki/Platelet
8  http://www.tcells.org/beginners/tcells/
9) http://www.ncbi.nlm.nih.gov/pubmedhealth/PMHT0022043/
10) https://en.wikipedia.org/wiki/Blood
11) http://www.evolutionnews.org/2015/09/controlling_blo099611.html
12) http://creation.com/bone-building-perfect-protein-osteocalcin#r1
13) http://reasonandscience.heavenforum.org/t2296-origin-and-development-of-bones-osteogenesis?highlight=bones


Red Blood Cells: Centerpiece in the Evolution of the Vertebrate Circulatory System
http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.521.8022&rep=rep1&type=pdf

Blood is a bodily fluid in animals that delivers necessary substances such as nutrients and oxygen to the cells and transports metabolic waste products away from those same cells. In vertebrates, it is composed of blood cells suspended in blood plasma. Plasma, which constitutes 55% of blood fluid, is mostly water, and contains dissipated proteins, glucose, mineral ions, hormones, carbon dioxide (plasma being the main medium for excretory product transportation), and blood cells themselves. Albumin is the main protein in plasma, and it functions to regulate the colloidal osmotic pressure of blood. According to H.Glicksman, the regulation of blood pressure requires a irreducible complex system .
 The blood cells are mainly red blood cells, white blood cells  and platelets. The most abundant cells in vertebrate blood are red blood cells. These contain hemoglobin, an iron-containing protein, which facilitates oxygen transport by reversibly binding to this respiratory gas and greatly increasing its solubility in blood. In contrast, carbon dioxide is almost entirely transported extracellularly dissolved in plasma as bicarbonate ion. 

The bone marrow, which is the flexible tissue in the interior of bones, forms a suitable environment for stem cell survival, growth and development. It is composed of stromal cells and a microvascular network. Bones are an amazing example of design, present in all vertebrates. They have a huge advantage over man-made girders, in that they are constantly rebuilding and redesigning themselves to cope with changing stress directions.12 This involves a fine balance of the activity of bone-depositing cells (osteoblasts) and bone resorbing cells (osteoclasts).  It’s been recently shown that thyroid-stimulating hormone (TSH), best known for what its name says—stimulating the production of hormones in the thyroid gland—has an important role. It oversees both types of cells—without it, bones have osteoporosis in some parts (too little bone, so very weak), and are too dense in other patches. So both are essential. For bone formation, over 24 different proteins, cells, Vitamins, transcription factors etc. are required. So, we can confidently say, the production of blood requires a hudge number of different body parts, which are essential. Blood by its own has no function. The vascular system without blood has no function. Bones require a vascular system, which by their own are required to make blood. The vertebrate body is a hudge irreducible complex sytem, that could not have emerged gradually. Each multicellular organism had to emerge fully, in its interdependent form. 

Hematopoiesis. The mystery of blood Cell and vascular formation

http://reasonandscience.heavenforum.org/t2295-hematopoiesis-the-mystery-of-blood-cell-and-vascular-formation

OVERVIEW OF BLOOD

Little attention has been given to the evolutionary basis of blood. Here, i will employ a description of what blood consists of, hematopoiesis ( how blood cellular components form ), function and essentiality of each blood component in health and pathological consequences through desease or a component inexistence, and elucidating if a gradual evolutionary step by step pathway is  hypothetically and theoretically possible, or if the origin of blood is better explained through intelligent design and sudden creation.  


Blood is a fluid connective tissue that circulates through the cardiovascular system. Like the other connective tissues, blood consists of cells and an extracellular component. Total blood volume in the average adult is about 6 L or 7% to 8% of total body weight. The heart’s pumping action propels blood through the cardiovascular system to the body tissues. 

Blood’s many functions include:
• delivery of nutrients and oxygen directly or indirectly to
cells,
• transport of wastes and carbon dioxide away from cells,
• delivery of hormones and other regulatory substances to
and from cells and tissues,
• maintenance of homeostasis by acting as a buffer and
participating in coagulation and thermoregulation, and
• transport of humoral agents and cells of the immune
system that protect the body from pathogenic agents,
foreign proteins, and transformed cells (i.e., cancer cells).

Blood consists of cells and their derivatives and a protein-rich fluid called plasma. Blood cells and their derivatives include:

• erythrocytes, also called red blood cells (RBCs);
• leukocytes, also known as white blood cells (WBCs); and
• thrombocytes, also termed platelets.

Plasma is the liquid extracellular material that imparts fluid properties to blood. The relative volume of cells and plasma in whole blood is approximately 45% and 55%, respectively. The volume of packed erythrocytes in a sample of blood is called the hematocrit (HCT) or packed cell volume (PCV). The hematocrit is measured by centrifuging a blood sample to which anticoagulants have been added, and then calculating the percentage of the centrifuge tube volume occupied by the erythrocytes compared with that of the whole blood (Fig. 10.1).



A normal hematocrit reading is about 39% to 50% in men and 35% to 45% in women; thus, 39% to 50% and 35% to 45% of the blood volume for men and women, respectively, consists of erythrocytes. Low hematocrit values often reflect reduced numbers of circulating erythrocytes (a condition called anemia) and may indicate significant blood loss caused by internal or external bleeding. Leukocytes and platelets constitute only 1% of the blood volume. In a blood sample that has been centrifuged, the cell fraction (the part of the sample that contains the cells) consists mainly of packed erythrocytes (99%). The leukocytes and platelets are contained in a narrow, light-colored layer between the erythrocytes and plasma called the buffy coat (see Fig. 10.1).



As Table 10.1 indicates, there are nearly 1,000 times more erythrocytes (5  10^12 cells/L of blood) than leukocytes (7 10^9/L of blood).

PLASMA

Although the blood cells are the major objects of interest in histology, a brief examination of plasma is also useful. The composition of plasma is summarized in Table 10.2.



More than 90% of plasma by weight is water, which serves as the solvent for a variety of solutes, including proteins, dissolved gases, electrolytes, nutrients, regulatory substances, and waste materials. The solutes in the plasma help maintain homeostasis, a steady state that provides optimal pH and osmolarity for cellular metabolism. Plasma proteins consist primarily of albumin, globulins, and fibrinogen. Albumin is the main protein constituent of the plasma, accounting for approximately half of the total plasma proteins. It is the smallest plasma protein (about 70 kDa) and is made in the liver. Albumin is responsible for exerting the concentration gradient between blood and extracellular tissue fluid. This major osmotic pressure on the blood vessel wall, called the colloid osmotic pressure, maintains the correct proportion of blood to tissue fluid volume. If a significant amount of albumin leaks out of the blood vessels into the loose connective tissue or is lost from the blood to urine in the kidneys, then the colloid osmotic pressure of the blood decreases, and fluid accumulates in the tissues. (This increase in tissue fluid is most readily noted by swelling of the ankles at the end of a day.) Albumin also acts as a carrier protein; it binds and transports hormones (thyroxine), metabolites ( bilirubin), and drugs (barbiturates). Globulins include the immunoglobulins ( γ-globulins), the largest component of the globulin fraction, and nonimmune globulins (α-globulin and β-globulin). The immunoglobulins are antibodies, a class of functional immune-system molecules secreted by plasma cells. Nonimmune globulins are secreted by the liver. They help maintain the osmotic pressure within the vascular system and also serve as carrier proteins for various substances such as copper (by ceruloplasmin), iron (by transferrin), and the protein hemoglobin (by haptoglobin). Nonimmune globulins also include fibronectin, lipoproteins, coagulation factors, and other molecules that may exchange between the blood and the extravascular connective tissue. Fibrinogen, the largest plasma protein (340 kilodaltons), is made in the liver. In a series of cascade reactions with other coagulation factors, soluble fibrinogen is transformed into the insoluble protein fibrin (323 kilodaltons).

During conversion of fibrinogen to fibrin, fibrinogen chains are broken to produce fibrin monomers that rapidly polymerize to form long fibers. These fibers become cross-linked to form an impermeable net at the site of damaged blood vessels, thereby preventing further blood loss. With the exception of these large plasma proteins and regulatory substances, which are small proteins or polypeptides, most plasma constituents are small enough to pass through the blood vessel wall into the extracellular spaces of the adjacent connective tissue. In general, plasma proteins react with common fixatives; they are often retained within the blood vessels in tissue sections. Plasma proteins do not possess a structural form above the molecular level; thus, when they are retained in blood vessels in the tissue block, they appear as a homogeneous substance that stains evenly with eosin in hematoxylin and eosin (H&E)–stained sections.

Serum is the same as blood plasma except that clotting factors have been removed.


For laboratory purposes, samples of blood are often drawn from a vein (the procedure called venipuncture).When blood is removed from the circulation, it immediately clots. A blood clot consists mostly of erythrocytes entangled in a network of fine fibers composed of fibrin. To prevent clotting of a blood sample, an anticoagulant such as citrate or heparin is added to the blood specimen as it is obtained. Citrate binds calcium ions, which are essential for triggering the cascade of coagulation reactions; heparin deactivates the clotting factors in the plasma. Plasma that lacks coagulation factors is called serum. 

ERYTHROCYTES

Erythrocytes are anucleate, biconcave discs.Erythrocytes or red blood cells (RBCs), are anucleatecells devoid of typical organelles. They function only within the bloodstream to bind oxygen for delivery to the tissues and, in exchange, bind carbon dioxide for removal from the tissues. Their shape is that of a biconcave discs with a diameter of 7.8 μm, an edge thickness of 2.6 μm, and a central thickness of 0.8 μm. This shape maximizes the cell’s surface area (140 μm2), an important attribute in gas exchange.


The life span of erythrocytes is approximately 120 days, after which most (90%) of them are phagocytosed by macrophages in the spleen, bone marrow, and liver. The remaining aged erythrocytes (10%) break down intravascularly, releasing insignificant amounts of hemoglobin into the blood. In H&E–stained sections, erythrocytes are usually 7 to 8 μm in diameter. Because their size is relatively consistent in fixed tissue, they can be used to estimate the size of other cells and structures in tissue sections; in this role, erythrocytes are appropriately referred to as the “histologic ruler.” Because both living and preserved erythrocytes usually appear as biconcave discs, they can give the impression that their form is rigid and inelastic (Fig. 10.2).



They are, in fact, extremely deformable. They pass easily through the narrowest capillaries by folding over on themselves. They stain uniformly with eosin. In thin sections viewed with the transmission electron microscope (TEM), the contents of an erythrocyte appear as a dense, finely granular material. 

The shape of the erythrocyte is maintained by membrane proteins.

The cell membrane of an erythrocyte is composed of a typical lipid bilayer and contains two functionally significant groups of proteins.
• Integral membrane proteins represent most of the proteins in the lipid bilayer. They consist of two major families: glycophorins and band 3 proteins. The extracellular domains of these integral membrane proteins are glycosylated and express specific blood group antigens. Glycophorin C, a member of the glycophorin family of transmembrane proteins, plays an important role in attaching the underlying cytoskeletal protein network to the cell membrane. Band 3 protein binds hemoglobin and acts as an additional anchoring site for the cytoskeletal proteins (Fig. 10.3).



• Peripheral membrane proteins reside on the inner surface of the cell membrane. They are organized into a two-dimensional hexagonal lattice network that laminates the inner layer of the membrane. The lattice itself, which is positioned parallel to the membrane, is composed mainly of cytoskeletal proteins including spectrin tetramers, actin, band 4.1 protein, adducin, band 4.9 protein, and tropomyosin (see Fig. 10.3) that form a network or mesh. The lattice is anchored to the lipid bilayer by the globular protein ankyrin, which interacts with band 4.2 protein as well as with band 3 integral membrane protein.

This unique cytoskeletal arrangement contributes to the shape of the erythrocyte and imparts elastic properties and stability to the membrane. The cytoskeleton is not static; it undergoes continuous rearrangement in response to various physical factors and chemical stimuli as the cell moves through the vascular network.
  Any defect in the expression of genes that encode these cytoskeleton proteins can result in abnormally shaped and fragile erythrocytes. For instance, hereditary spherocytosis is caused by a primary defect in spectrin gene expression that results in spherical erythrocytes. Hereditary elliptocytosis is caused by a deficiency in band 4.1 proteins that results in elliptical erythrocytes. In
both conditions, erythrocytes are unable to adapt to changes in their environment (e.g., osmotic pressure and mechanical deformations), which results in premature destruction of the cells, or hemolysis.

Erythrocytes contain hemoglobin, a protein specialized for the transport of oxygen and carbon dioxide.

Erythrocytes transport oxygen and carbon dioxide bound to the protein hemoglobin (68 kilodaltons). A monomer of hemoglobin is similar in composition and structure to myoglobin, the oxygen-binding protein found in striated muscle. A high concentration of hemoglobin is present within erythrocytes and is responsible for their uniform staining with eosin and the cytoplasmic granularity seen with the TEM. The disc shape of the erythrocyte facilitates gas exchange because more hemoglobin molecules are closer to the plasma membrane than they would be in a spherical cell. Thus, gases have less distance to diffuse within the cell to reach a binding site on the hemoglobin. Hemoglobin consists of four polypeptide chains of globin, each complexed to an iron-containing heme alpha, beta, gamma, and sigma group (Fig. 10.4). The structure of the polypeptide chains varies.


LEUKOCYTES

Leukocytes are subclassified into two general groups. The basis for this division is the presence or absence of prominent specific granules in the cytoplasm. Cells containing specific granules are classified as granulocytes (neutrophils, eosinophils, and basophils), and cells that lack specific granules are classified as agranulocytes (lymphocytes and monocytes). However, both agranulocytes and granulocytes possess small number of nonspecific
azurophilic granules, which are lysosomes. The relative number of the various leukocytes is given in Table 10.1.

The function of blood vessels is to carry blood. Blood contains many types of cells, with functions that range from the transport of oxygen to the production of antibodies. Some of these cells stay within the vascular system, while others use the vascular system only as a means of transport and perform their function elsewhere. All blood cells, however, have certain similarities in their life history. They all have limited life-spans and are produced throughout the life of the animal. Most remarkably, they are all generated ultimately from a common stem cell, located (in adult humans) in the bone marrow. This hematopoietic (blood-making) stem cell is thus multipotent, giving rise to all the types of terminally differentiated blood cells as well as some other types of cells, such as the osteoclasts in bone. The hematopoietic system is the most complex of the stem-cell systems in the mammalian body. 

Neutrophils are the most numerous WBCs as well as the most common granulocytes


Neutrophils measure 10 to 12 μm in diameter in blood smears and are obviously larger than erythrocytes. Although named for their lack of characteristic cytoplasmic staining, they are also readily identified by their multilobal nucleus; thus, they are also called polymorphonuclear neutrophils or polymorphs. Mature neutrophils possess two to four lobes of nuclear material joined by thinner nuclear strands . The arrangement is not static; rather, in living neutrophils the lobes and connecting strands change their shape, position, and even number. The chromatin of the neutrophil has a characteristic arrangement. Wide regions of heterochromatin are located chiefly at
the periphery of the nucleus in contact with the nuclear envelope. Regions of euchromatin are located primarily at the center of the nucleus with relatively smaller regions contacting the nuclear envelope (Fig. 10.5). In women, the Barr body (the condensed, single, inactive X chromosome) forms a drumstickshaped appendage on one of the nuclear lobes.


Neutrophils contain three types of granules.

The cytoplasm of a neutrophil contains three kinds of granules. The different types of granules reflect the various phagocytotic functions of the cell.
• Specific granules (secondary granules) are the smallest granules and are at least twice as numerous as azurophilic granules. They are barely visible in the light microscope; in electron micrographs, they are ellipsoidal (see Fig. 10.5). Specific granules contain various enzymes (i.e., type IV collagenase, phospholipase) as well as complement activators and other antimicrobial peptides (i.e., lysozymes, lactoferrins).
• Azurophilic granules (primary granules) are larger and less numerous than specific granules. They arise early in granulopoiesis and occur in all granulocytes, as well as in monocytes and lymphocytes. The azurophilic granules are the lysosomes of the neutrophil and contain myeloperoxidase (MPO) (a peroxidase enzyme), which appears as a finely stippled material with the TEM. Myeloperoxidase helps to generate highly reactive bactericidal hypochlorite and chloramines. In addition to containing a variety of the typical acid hydrolases, azurophilic granules also contain cationic proteins called defensins, which function analogously to antibodies and the antimicrobial peptide cathelicidin to kill pathogens.
• Tertiary granules in neutrophils are of two types. One  type contains phosphatases (enzymes that remove a phosphate group from a substrate) and is sometimes called a phosphasome. The other type contains metalloproteinases, such as gelatinases and collagenases, which are  thought to facilitate the migration of the neutrophil through the connective tissue. Aside from these granules, membrane-bounded organelles
are sparse. A small Golgi apparatus is evident in the center of the cell, and mitochondria are relatively few in number (see Fig. 10.5).

Neutrophils are motile cells; they leave the circulation and migrate to their site of action in the connective tissue.An important property of neutrophils and other leukocytes is their motility. Neutrophils are the most numerous of the first wave of cells to enter an area of tissue damage. Their migration is controlled by the expression of adhesion molecules on the neutrophil surface that interact with corresponding ligands on endothelial cells (Fig. 10.6) and are often involved in cell binding.



The initial phase of neutrophil migration occurs in the postcapillary venules and is regulated by a mechanism involving neutrophil–endothelial cell recognition. Selectins (a type of cell adhesion molecule) on the surface of the circulating neutrophil (CD62L) interact with receptors (GlyCAM-1) on the surface of the endothelial cells. The neutrophil becomes partially tethered to the endothelial cell as a result of this interaction, which slows the neutrophil and causes it to roll on the surface of the endothelium. In the second phase, another group of adhesion molecules on the neutrophil surface, called integrins (VLA-5), are activated by chemokine signals from the endothelial cells. In the third phase, integrins and other adhesion molecules from the immunoglobulin superfamily (e.g., intercellular adhesion molecule-1 [ICAM-1], vascular cell adhesion molecule-1 [VCAM-1]) expressed on the neutrophil surface engage with their specific receptors on the endothelial cells, attaching the neutrophil to the endothelial cell. The neutrophil then extends a pseudopod to an intercellular junction. Histamine and heparin released at the injury site by perivascular mast cells open the intercellular junction, allowing the neutrophil to migrate into the connective tissue. With the TEM, the cytoplasmic contents of a neutrophil pseudopod appear as an expanse of finely granular cytoplasmic matrix with no membranous organelles (see Fig. 10.5). The finely granular appearance is attributable to the presence of actin filaments, some microtubules, and glycogen, which are involved in the extension of the cytoplasm to form the pseudopod and the subsequent contraction that pulls the cell forward. Once the neutrophil enters the connective tissue, further migration to the injury site is directed by a process known as chemotaxis, the binding of chemoattractant molecules and extracellular matrix proteins to specific receptors on the surface of the neutrophil.

Neutrophils are active phagocytes that utilize a variety of surface receptors to recognize bacteria and other infectious agents at the site of inflammation

Once at the site of tissue injury, the neutrophil must first recognize any foreign substances before phagocytosis can occur. Like most phagocytic cells, neutrophils have a variety of receptors on their cell membrane that can recognize and bind to bacteria, foreign organisms, and other infectious agents (Fig 10.7).



Some of these organisms and agents bind directly to neutrophils (no modifications of their surfaces are required), whereas others must be opsonized (coated with antibodies or complement) to make them more attractive to the neutrophil. The most common receptors used by neutrophils during phagocytosis include the following.
• Fc receptors on the neutrophil surface bind to the exposed Fc region of IgG antibodies that coat bacterial surfaces (see Fig 10.7). Binding of IgG-coated bacteria activates the neutropil’s phagocitic activity and causes a rapid
surge in intracellular metabolism.
• Complement receptors (CRs) facilitate binding and uptake of immune complexes that are opsonized by active C3 complement protein, namely, C3b. Binding of bacteria or other C3b-coated antigens to CRs triggers phagocytosis, resulting in activation of a neutrophil’s lytic pathways and respiratory burst reactions.
• Scavenger receptors (SRs) are a structurally diverse group of transmembrane glycoproteins that bind to modified (acetylated or oxidized) forms of low-density lipoproteins (LDLs), polyanionic molecules that are often on the surface of both Gram-positive and Gram-negative bacteria and apoptotic bodies. Binding of these receptors increases the phagocitic activity of neutrophils.
• Toll-like receptors, also known as pattern recognition receptors (PRRs), are neutrophil receptors that recognize pathogenic molecules such as endotoxins, lipopolysaccharides, peptidoglycanes, and lipoteichoic acids that are arranged in predictable pathogen-associated molecular patterns (PAMPs) and are commonly expressed on bacterial surfaces and other infectious agents. Like other phagocytic cells, neutrophils possess a variety of tolllike receptors that recognize PAMPs.

Phagocytosed bacteria are killed within phagolysosomes by the toxic reactive oxygen intermediates produced during respiratory burst.

Phagocytosis begins when the neutrophil recognizes and attaches to the antigen. Extended pseudopods of the neutrophil engulf the antigen and internalize it to form a phagosome (see Fig. 10.7). Specific and azurophilic granules fuse with the phagosome membrane, and the lysosomal hydrolases of the azurophilic granules digest the foreign material. During phagocytosis, the neutrophil’s glucose and oxygen utilization increases noticeably and is referred to as the respiratory burst. It results in synthesis of several oxygen- containing compounds called reactive oxygen intermediates (ROIs). They include free radicals such as oxygen and hydroxyl radicals that are used in immobilizing and killing live bacteria within the phagolysosomes. By definition, free radicals possess an unpaired electron within their chemical structure, which makes them highly reactive and therefore capable of damaging intracellular molecules, including lipids, proteins, and nucleic acids. The process by which microorganisms are killed within neutrophils is termed oxygen-dependent intracellular killing. In general,
two biochemical pathways are involved in this process: the first is the phagocyte oxidase (phox) system that utilizes the phagocyte’s nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex in the phagolysosome membrane; the second is associated with the lysosomal enzyme myeloperoxidase (MPO) found in the azurophilic granules of neutrophils (Fig 10.. Within the phagocyte oxidase pathway, or phox system, phagocytosis proceeds by signaling the cell to produce sufficient amounts of NADPH needed to generate superoxide anions. Increased glucose uptake and shunting of NADPH metabolism is achieved via the pentose phosphate pathway (also known as pentose shunt). The cytosolic NADPH becomes an electron donor: The NADPH oxidase enzyme complex transports electrons across the membrane to molecular O2 inside the phagolysosome to generate the free radical superoxide anions (O–2). These superoxide anions are converted into ROIs. The superoxide dismutase converts superoxide anions to singlet oxygen (1O2) and hydrogen peroxide (H2O2), which further reacts with superoxide anions to produce bactericidal hydroxyl radicals (OH–) (the neutral form of the hydroxyl ion) and more singlet oxygen molecules (see Fig 10..




Oxygen-dependent killing with MPO involvement occurs when azurophilic granules containing MPO fuse with phagosomes containing phagocytosed bacteria. During the neutrophil’s respiratory burst, MPO, using heme as a cofaccofactor, catalyzes a reaction that produces hypochlorous acid (HOCl) from hydrogen peroxide (H2O2) and a chloride anion (Cl–). Hypochlorous acid, which is about 1,000 times more effective in bacterial killing than hydrogen peroxide, is further metabolized to a highly toxic hypochlorite OCl– (bleach) and chlorine (Cl2). Some of the hypochloride may spontaneously break down to yield toxic singlet oxygen (1O2) and chloride ions (Cl–) (see Fig. 10.8 ). In addition, nitric oxide (NO) and other reactive nitrogen intermediates (RNIs) have also been implicated in the intracellular microbial killing mechanisms. NO has been found in neutrophils; however, it is believed that RNI-mediated killing mechanisms do not appear to have a critical role in humans. The main role of neutrophil-derived NO is to induce vasodilatation, which in turn facilitates the migration of neutrophils from blood vessels to surrounding connective tissue.

Phagocytosed bacteria can also be killed by a diverse arsenal of oxygen-independent killing mechanisms utilizing bacteriolytic enzymes and antimicrobial peptides.

In addition to the oxygen-dependent respiratory burst reactions, microorganisms can be killed by bacteriolytic enzymes and cationic antimicrobial peptides that are stored within the granules of the neutrophil’s cytoplasm. These oxygenindependent killing mechanisms are directed toward the bacterial cell membrane, causing its breakdown and leakage.
Neutrophils contain particularly large amounts of cationic antimicrobial proteins such as defensins and antimicrobial peptides called cathelicidins. Similar to lysozymes and
cathepsins stored in the specific granules, these cationic antimicrobial proteins break down the bacterial wall. In addition, lysosomal hydrolytic enzymes that digest bacterial
proteins and lactoferrins that chelate iron from nutritional bacterial pathways contribute to the destruction of the invading bacteria.

These mechanisms are not as efficient as oxygen- dependent killing pathways. Neutrophils from patients with defects in oxygen-dependent pathways, such as those with chronic granulomatous disease (see Folder 10.4), are still able to destroy phagocytosed bacteria to some degree. However, because of the low efficiency of these processes, individuals with these defects are more susceptible to serious infections. After intracellular digestion by the neutrophil, the remnants of degraded material are stored in residual bodies or
exocytosed. Most neutrophils die in this process; the accumulation of dead bacteria and dead neutrophils constitutes the thick exudate called pus. The yellow–green color of the
pus and of mucus secretions (e.g., from infected lungs) comes from the heme pigment of MPO enzyme in azurophilic granules of neutrophils.

Inflammation and wound healing also involve monocytes, lymphocytes, eosinophils, basophils, and fibroblasts

Monocytes also enter the connective tissue as a secondary response to tissue injury. At the site of tissue injury, they transform into macrophages that phagocytose cell and tissue
debris, fibrin, remaining bacteria, and dead neutrophils. Normal wound healing depends on the participation of macrophages in the inflammatory response; they become the
major cell type in the inflammatory site after the neutrophils are spent. At the same time that the macrophages become active at the site of inflammation, fibroblasts near the site and undifferentiated mesenchymal cells in the adventitia of small vessels at the site begin to divide and differentiate into fibroblasts and myofibroblasts that then secrete the fibers and ground substance of the healing wound. Like neutrophils, monocytes are attracted to the inflammatory site by chemotaxis.

Eosinophils

Eosinophils are about the same size as neutrophils, and their nuclei are typically bilobed (Fig. 10.9). As in neutrophils, the compact heterochromatin of eosinophils is chiefly adjacent to the nuclear envelope, whereas the euchromatin is located in the center of the nucleus.



Eosinophils are named for the large, eosinophilic, refractile granules in their cytoplasm

The cytoplasm of eosinophils contains two types of granules: numerous, large, elongated specific granules and azurophilic granules (otherwise, the eosinophil contains only a sparse representation of membranous organelles).. Specific granules of eosinophils contain a crystalloid body that is readily seen with the TEM, surrounded by a less electron-dense matrix. These crystalloid bodies are responsible for the refractivity of the granules in the light microscope. They contain four major proteins: an arginine-rich protein called major basic protein (MBP), which accounts for the intense acidophilia of the granule; eosinophil cationic protein (ECP); eosinophil peroxidase (EPO); and eosinophil-derived neurotoxin (EDN). MBP is localized in the crystalloid body; the other three proteins are found in the granule matrix. MBP, ECP, and EPO have a strong cytotoxic effect on protozoans and helminthic parasites; EDN causes nervous system dysfunction in parasitic organisms; histaminase neutralizes the activity of histamine; and arylsulfatase neutralizes leukotrienes secreted by basophils and mast cells.Specific granules also contain histaminase, arylsulfatase, collagenase, and cathepsins.
• Azurophilic granules are lysosomes. They contain a variety of the usual lysosomal acid hydrolases and other hydrolytic enzymes that function in destruction of parasites and hydrolysis of antigen–antibody complexes internalized by the eosinophil.

Eosinophils are associated with allergic reactions, parasitic infections, and chronic inflammation.

Eosinophils develop and mature in the bone marrow. Once released from the bone marrow, they circulate in peripheral blood and then migrate to the connective tissue. Eosinophils are activated by interactions with IgG, IgA, or secretory IgA antibodies.

The release of arylsulfatase and histaminase by eosinophils at sites of allergic reaction moderates the potentially deleterious effects of inflammatory vasoactive mediators. The eosinophil also participates in other immunologic responses and phagocytoses antigen–antibody complexes. Thus, the count of eosinophils in blood samples of individuals with allergies and parasitic infections is usually high. Eosinophils play a major role in host defense against helminthic parasites. They are also found in large numbers in the lamina propria of the intestinal tract and at other sites of potential chronic inflammation (i.e.,lung tissues in patients with asthma).


Basophils

Basophils are about the same size as neutrophils and are so named because the numerous large granules in their cytoplasm stain with basic dyes.

Basophils are the least numerous of the WBCs, accounting for less than 0.5% of total leukocytes.

Often, several hundred WBCs must be examined in a blood smear before one basophil is found. The lobed basophil nucleus is usually obscured by the granules in stained blood
smears, but its characteristics are evident in electron micrographs (Fig. 10.10). Heterochromatin is chiefly in a peripheral location, and euchromatin is chiefly centrally located;
typical cytoplasmic organelles are sparse. The basophil plasma membrane possesses numerous high-affinity Fc receptors for IgE antibodies. In addition, a specific 39-kilodalton protein called CD40L is expressed on the basophil’s surface. CD40L interacts with a complementary receptor (CD40) on B lymphocytes, which results in increased synthesis of IgE.



The basophil cytoplasm contains two types of granules: specific granules, which are larger than the specific granules of the neutrophil, and nonspecific azurophilic granules.

• Specific granules exhibit a grainy texture and myelin figures when viewed with the TEM. These granules contain a variety of substances, namely, heparin, histamine, heparan sulfate, leukotrienes, IL-4, and IL-13. Heparin, a sulfated glycosaminoglycan, is an anticoagulant. Histamine and heparan sulfate are vasoactive agents that among other actions cause dilation of small blood vessels. Leukotrienes are modified lipids that trigger prolonged constriction of smooth muscles in the pulmonary airways. Interleukin-4 (IL-4) and interleukin-13 (IL-13) promote synthesis of IgE antibodies. The intense basophilia of these specific granules correlates with the high concentration of sulfates within the glycosaminoglycan molecules of heparin and heparan sulfate.
• Azurophilic granules are the lysosomes of basophils and contain a variety of the usual lysosomal acid hydrolases that are similar to those in other leukocytes.

The function of basophils is closely related to that of mast cells.

Basophils are functionally related to, but not identical with, mast cells of the connective tissue. Both mast cells and basophils bind an antibody secreted by plasma cells, IgE, through high-affinity Fc receptors expressed on their cell surface. The subsequent exposure to, and reaction with, the antigen (allergen) specific for IgE triggers the activation of basophils and mast cells and the release of vasoactive agents from cell granules. These substances are responsible for the severe vascular disturbances associated with hypersensitivity reactions and anaphylaxis. Furthermore, both basophils and mast cells are derived from the same basophil–mast cell progenitor (BMCP) cell. If a specific BMCP expresses the granulocyte-related transcription factor CCAAT/enhancer-binding protein  (C/EBP), the cell becomes committed to differentiate into a basophil progenitor (BaP) cell. Basophils develop and differentiate in the bone marrow and are released to the peripheral blood as mature cells. In the absence of C/EBP transcription factor, a BMCP cell migrates to the spleen and after further differentiation travels as a mast cell precursor (MPC) to the intestine, where it becomes a mature mast cell.

Lymphocytes

Lymphocytes are the main functional cells of the lymphatic or immune system

Lymphocytes are the most common agranulocytes and account for about 30% of the total blood leukocytes.

Lymphocytes are essential for immunologic defense of the organism.

 In understanding the function of the lymphocytes, one must realize that most lymphocytes found in blood or lymph represent recirculating immunocompetent cells (i.e., cells that have developed the capacity to recognize and respond to antigens and are in transit from one lymphatic tissue to another). In the tissues associated with the immune system, three groups of lymphocytes can be identified according to size: small, medium, and large lymphocytes, ranging in diameter from 6 to 30 μm. The large lymphocytes are either activated lymphocytes, which possess surface receptors that interact with a specific antigen, or natural killer (NK) lymphocytes. In the bloodstream, most lymphocytes are small or medium sized, 6 to 15  μm in diameter. The majority—more than 90%—are small lymphocytes. In blood smears, the mature lymphocyte approximates the size of an erythrocyte. When observed in the light microscope in a blood smear, small lymphocytes have an intensely staining, slightly indented, spherical nucleus. The cytoplasm appears as a very thin, pale blue rim surrounding the nucleus. In general, there are no recognizable cytoplasmic organelles other than an occasional fine azurophilic granule. The TEM reveals that the cytoplasm primarily contains free ribosomes and a few mitochondria. Other organelles are so sparse that they are usually not seen in a thin section. Small, dense lysosomes that correspond to the azurophilic granules seen in the light microscope are occasionally observed; a pair of centrioles and a small Golgi apparatus are located in the cell center, the area of the indentation of the nucleus. In the medium lymphocyte, the cytoplasm is more abundant, the nucleus is larger and less heterochromatic, and the Golgi apparatus is somewhat more developed (Fig. 10.11). Greater numbers of mitochondria and polysomes and small profiles of rough endoplasmic reticulum are also seen in these medium-sized cells. The ribosomes are the basis for the slight basophilia displayed by lymphocytes in stained blood smears.



Three functionally distinct types of lymphocytes are present in the body: T lymphocytes, B lymphocytes, and NK cells.

The characterization of lymphocyte types is based on their function, not on their size or morphology. T lymphocytes (T cells) are so named because they undergo differentiation in the thymus. B lymphocytes (B cells) are so named because they were first recognized as a separate population in the bursa of Fabricius in birds or bursa-equivalent organs (e.g., bone marrow) in mammals. Natural killer (NK) cells develop from the same precursor cell as B and T cells and are so named because they are programmed to kill certain types of transformed cells.
• T cells have a long life span and are involved in cellmediated immunity. T cells are characterized by the presence of cell-surface recognition proteins called T-cell receptors (TCRs), which in a majority of T cells comprise two glycoprotein chains called - and -TCR chains. They express CD2, CD3, CD5, and CD7 marker proteins on their surface; however, they are subclassified on the basis of the presence or absence of CD4 and CD8 proteins. CD4 T lymphocytes possess the CD4 marker and recognize antigens bound to major histocompatability complex II (MHC II) molecules. CD8 T lymphocytes possess the CD8 marker and recognize antigen bound to MHC I molecules.
• B cells have variable life spans and are involved in the production of circulating antibodies. Mature B cells in blood express IgM and IgD and MHC II molecules on their surface. Their specific markers are CD9, CD19,
CD20, and CD24.
• NK cells are programmed during their development to kill certain virus-infected cells and some types of tumor cells. They also secrete an antiviral agent, interferon  (IFN-). NK cells are larger than B and T cells (15  μm
in diameter) and have a kidney-shaped nucleus. Because NK cells have several large cytoplasmic granules easily seen by light microscopy, they are also called large granular lymphocytes (LGLs). Their specific markers include CD16, CD56, and CD94. T and B cells are indistinguishable in blood smears and tissue sections; immunocytochemical staining for different types of markers and receptors on their cell surface must be used to identify them. NK lymphocytes can be identified in the light microscope by size, nuclear shape, and presence of cytoplasmic granules; however, immunocytochemical staining for their specific markers is used to confirm microscopic identification.

T and B lymphocytes express different surface molecules.

Although the T and B cells cannot be distinguished on the basis of their morphology, their distinctive surface proteins (CD proteins) can be used to identify the cells with immunolabeling techniques. In addition, immunoglobulins are expressed on the surface of B cells that function as antigen receptors. In contrast, T cells do not have antibodies but express TCRs. These recognition proteins appear during discrete stages in the maturation of the cells within the thymus. In general, the surface molecules mediate or augment specific T-cell functions and are required for the recognition or binding of T cells to antigens displayed on the surface of target cells. In human blood, 60% to 80% of lymphocytes are mature T cells, and 20% to 30% are mature B cells. Approximately 5% to 10% of the cells do not demonstrate the surface markers associated with either T or B cells. These are NK cells and the rare circulating hemopoietic stem cells (see below). The size differences described above may have functional significance; some of the large lymphocytes may be cells that have been stimulated to divide whereas others may be plasma cell precursors that are undergoing differentiation in response to the presence of antigen.

Several different types of T lymphocytes have been identified: cytotoxic, helper, suppressor, and gamma/delta

The activities of cytotoxic, helper, suppressor, and gamma/delta T lymphocytes are mediated by molecules located on their surface. Immunolabeling techniques have made it possible to identify specific types of T cells and study their function.
• Cytotoxic CD8 T cells serve as the primary effector cells in cell-mediated immunity. CD8 cells are specifically sensitized T lymphocytes that recognize antigens through the TCRs on viral or neoplastic host cells. Cytotoxic CD8 T lymphocytes only recognize antigens bound to MHC I molecules. After the TCR binds the antigen–MHC I complex, the cytotoxic CD8T cells secrete lymphokines and perforins that produce ion channels in the membrane of the infected or neoplastic cell, leading to its lysis (see Chapter 14, Lymphatic System). Cytotoxic CD8 T lymphocytes play a significant role in rejection of allografts and in tumor immunology.
• Helper CD4 T cells are critical for induction of an immune response to a foreign antigen. Antigen bound to MHC II molecules is presented by antigen-presenting cells such as macrophages to a helper CD4 T lymphocyte. Binding of the TCR to the antigen–MHC II complex activatesthe helper CD4 T cells. The activated helper CD4 T lymphocytes then produce interleukins (mainly IL-2), which act in an autocrine mode to simulate the proliferation and differentiation of more helper CD4 T lymphocytes. Newly differentiated cells synthesize and secrete lymphokines that affect function as well as differentiation of B cells, T cells, and NK cells. B cells differentiate into plasma cells and synthesize antibody.
• Regulatory (suppressor) T-cells represent a phenotypically diverse population of T lymphocytes that can functionally suppress an immune response to foreign and self-antigen by influencing the activity of other cells in the immune system. The CD4CD25FOXP3 regulator T cells represent a classical example of cells that can downregulate the ability of T lymphocytes to initiate immune responses. The FOXP3 marker indicates an expression of forkhead family transcription factors that are characteristic of many T cells. Furthermore, tumor-associated CD8CD45RO T suppressor cells secrete IL-10 and
also suppress T-cell activation. The suppressor T cells may also function in suppressing B-cell differentiation and in regulating erythroid cell maturation in the bone marrow.
• Gamma/delta ( ) T cells represent a small population of T cells that possess a distinct TCR on their surface. Most T cells have a TCR receptor composed of two glycoprotein chains called - and -
TCR chains. In contrast, T cells possess TCR receptors made up of one -chain and one -chain.

These cells develop in the thymus and migrate into various epithelial tissues (e.g., skin, oral mucosa, intestine, and vagina). Once they colonize an epithelial tissue, they do not recirculate between blood and lymphatic organs. They are also known as intraepithelial lymphocytes. Their location within the skin and mucosa of internal organs allows them to function in the first line of defense against invading organisms.


Monocytes

Monocytes are the precursors of the cells of the mononuclear phagocytotic system.


Monocytes are the largest of the WBCs in a blood smear (average diameter, 18 μm). They travel from the bone marrow to the body tissues, where they differentiate into the various phagocytes of the mononuclear phagocytotic system—  that is, connective tissue macrophages, osteoclasts, alveolar macrophages, perisinusoidal macrophages in the liver (Kupffer cells), and macrophages of lymph nodes, spleen, and bone marrow among others. Monocytes remain in the blood for only about 3 days. The nucleus of the monocyte is typically more indented than that of the lymphocyte (Fig. 10.12 ).



The indentation is the site of the cell center where the welldeveloped Golgi apparatus and centrioles are located. Monocytes also contain smooth endoplasmic reticulum, rough endoplasmic reticulum, and small mitochondria. Although these cells are classified as agranular, they contain small, dense, azurophilic granules. These granules contain typical lysosomal enzymes similar to those found in the azurophilic granules of neutrophils.

Monocytes transform into macrophages, which function as antigen-presenting cells in the immune system

During inflammation, the monocyte leaves the blood vessel at the site of inflammation, transforms into a tissue macrophage, and phagocytoses bacteria, other cells, and tissue debris

The monocyte–macrophage is an antigen-presenting cell and plays an important role in immune responses by partially degrading antigens and presenting their fragments on the MHC II molecules located on the macrophage surface of helper CD4 T lymphocytes for recognition.

THROMBOCYTES


Thrombocytes are small, membrane-bounded, anucleate cytoplasmic fragments derived from megakaryocytes.

Thrombocytes (platelets) are derived from large polyploid cells (cells whose nuclei contain multiple sets of chromosomes) in the bone marrow called megakaryocytes (Fig. 10.13).



In platelet formation, small bits of cytoplasm are separated from the peripheral regions of the megakaryocyte by extensive platelet demarcation channels. The membrane that lines these channels arises by invagination of the plasma membrane; therefore, the channels are in continuity with the extracellular space. The continued development and fusion of the platelet demarcation membranes result in the complete partitioning of cytoplasmic fragments to form individual platelets. After entry into the vascular system from the bone marrow, the platelets circulate as discoid structures about 2 to 3 μm in diameter. Their life span is about 10 days. Structurally, platelets may be divided into four zones based on organization and function. The TEM reveals a structural organization of the thrombocyte cytoplasm that can be categorized into the following four zones (Fig. 10.14).



•The peripheral zone consists of the cell membrane covered by a thick surface coat of glycocalyx. The glycocalyx consists of glycoproteins, glycosaminoglycans, and several coagulation factors adsorbed from the plasma.
The integral membrane glycoproteins function as receptors in platelet function.
• The structural zone comprises microtubules, actin filaments, myosin, and actin-binding proteins that form a network supporting the plasma membrane. From 8 to 24 microtubules reside as a bundle immediately below the actin filament network. They are circumferentially arranged and are responsible for maintaining the platelet’s disc shape.
• The organelle zone occupies the center of the platelet. It consists of mitochondria, peroxisomes, glycogen particles, and at least three types of granules dispersed within the
cytoplasm.

The most numerous granules are  granules (300 to 500 nm in diameter) that contain mainly fibrinogen, coagulation factors, plasminogen, plasminogen activator inhibitor, and platelet-derived growth factor. The contents of these granules play an important role in the initial phase of vessel repair, blood coagulation, and platelet aggregation. The smaller, denser, and less numerous granules mainly contain adenosine diphosphate (ADP), adenosine triphosphate (ATP), serotonin, and histamine, which facilitate platelet adhesion and vasoconstriction in the area of the injured vessel. The  granules are similar to lysosomes found in other cells and contain several hydrolytic enzymes. The contents of granules function in clot resorption during the later stages of vessel repair.


• The membrane zone consists of two types of membrane channels. The open canalicular system (OCS) is the first type of membrane channel. The OCS is a developmental remnant of the platelet demarcation channels and
is simply a membrane that did not participate in subdividing the megakaryocyte cytoplasm. In effect, open canaliculi are invaginations into the cytoplasm from the plasma membrane. The dense tubular system (DTS) is the second type of membrane channel. The DTS contains an electron-dense material originating from the rough endoplasmic reticulum of the megakaryocyte, which serves as a storage site for calcium ions. DTS channels do not connect with the surface of the platelet; however, both the OCS and DTS fuse in various areas of the platelet to form membrane complexes that are important in regulation of the intraplatelet calcium concentration.


Platelets function in continuous surveillance of blood vessels, blood clot formation, and repair of injured tissue.

Platelets are involved in several aspects of hemostasis (control of bleeding). They continuously survey the endothelial lining of blood vessels for gaps and breaks. When a blood vessel wall is injured or broken, the exposed connective tissue at the damaged site promotes platelet adhesion. Adhesion of the platelets at the damaged site triggers their degranulation and release of serotonin, ADP, and thromboxane A2.

Serotonin is a potent vasoconstrictor that causes the vascular smooth muscle cells to contract, thereby reducing local blood flow at the site of injury. Adenosine diphosphate (ADP), a nucleotide, and the signaling molecule thromboxane A2, are responsible for further aggregation of platelets into a primary hemostatic plug. The mass of aggregated platelets stop extravasation of blood. At the same time, the activated platelets release their  and granules, which contain among other substances coagulation factors such as platelet thromboplastic factor (PF3) and additional serotonin.

The glycocalyx of the platelets provides a reaction surface for the conversion of soluble fibrinogen into fibrin. Fibrin then forms a loose mesh over the initial plug and is further stabilized by covalent cross-links that produce a dense aggregation of fibers (Fig 10.15). Platelets and red blood cells become trapped in this mesh. The initial platelet plug is transformed into a definitive clot known as a secondary hemostatic plug by additional tissue factors secreted by the damaged blood vessel.



After the definitive clot is formed, platelets cause clot retraction, probably as a function of the actin and myosin found in the structural zone of the platelet. Contraction of the clot permits the return of normal blood flow through the vessel.

Finally, after the clot has served its function, it is lysed by plasmin, a fibrinolytic enzyme that circulates in the plasma in an inactive form known as plasminogen. The hydrolytic enzymes released from the  granules assist in this process. The activator for plasminogen conversion, tissue plasminogen activator (TPA), is derived principally from endothelial cells. A synthetic form of TPA is currently used as an emergency treatment to minimize the damage caused by clots that lead to strokes.

An additional role of platelets is to help repair the injured tissues beyond the vessel itself. Platelet-derived growth factor released from the  granules stimulates smooth muscle cells and fibroblasts to divide and allow tissue repair.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3410208/

Red Blood Cells Are All Alike; White Blood Cells Can Be Grouped in Three Main Classes

Blood cells can be classified as red or white. The red blood cells, or erythrocytes, remain within the blood vessels and transport O2 and CO2 bound to hemoglobin. The white blood cells, or leukocytes, combat infection and in some cases phagocytose and digest debris. Leukocytes, unlike erythrocytes, must make their way across the walls of small blood vessels and migrate into tissues to perform their tasks. In addition, the blood contains large numbers of platelets, which are not entire cells but small, detached cell fragments or “minicells” derived from the cortical cytoplasm of large cells called megakaryocytes. Platelets adhere specifically to the endothelial cell lining of damaged blood vessels, where they help to repair breaches and aid in blood clotting. All red blood cells belong in a single class, following the same developmental trajectory as they mature, and the same is true of platelets; but there are many distinct types of white blood cells. White blood cells are traditionally grouped into three major categories—granulocytes, monocytes, and lymphocytes—based on their appearance in the light microscope. Granulocytes contain numerous lysosomes and secretory vesicles (or granules) and are subdivided into three classes according to the morphology and staining properties of these organelles (Figure 22–27).




The differences in staining reflect major differences of chemistry and function. Neutrophils (also called polymorphonuclear leukocytes because of their multilobed nucleus) are the most common type of granulocyte; they phagocytose and destroy microorganisms, especially bacteria, and thus have a key role in innate immunity to bacterial infection. Basophils secrete histamine (and, in some species, serotonin) to help mediate inflammatory reactions; they are closely related to mast cells, which reside in connective tissues but are also generated from the hematopoietic stem cells. Eosinophils help to destroy parasites and modulate allergic inflammatory responses. Once they leave the bloodstream, monocytes (see Figure 22–27D) mature into macrophages, which, together with neutrophils, are the main “professional phagocytes” in the body. Both types of phagocytic cells contain specialized lysosomes that fuse with newly formed phagocytic vesicles (phagosomes), exposing phagocytosed microorganisms to a barrage of enzymatically produced, highly reactive molecules of superoxide (O2 –) and hypochlorite (ClO–, the active ingredient in bleach), as well as to attack by a concentrated mixture of lysosomal hydrolase enzymes that become activated in the phagosome. Macrophages, however, are much larger and longer-lived than neutrophils. They recognize and remove senescent, dead, and damaged cells in many tissues, and they are unique in being able to ingest large microorganisms such as protozoa. Monocytes also give rise to dendritic cells. Like macrophages, dendritic cells are migratory cells that can ingest foreign substances and organisms, but they do not have as active an appetite for phagocytosis and instead have a crucial role as presenters of foreign antigens to lymphocytes to trigger an immune response. Dendritic cells in the epidermis (called Langerhans cells), for example, ingest foreign antigens and carry these trophies back from the skin to present to lymphocytes in lymph nodes. There are two main classes of lymphocytes, both involved in immune responses: B lymphocytes make antibodies, while T lymphocytes kill virusinfected cells and regulate the activities of other white blood cells. In addition, there are lymphocyte-like cells called natural killer (NK) cells, which kill some types of tumor cells and virus-infected cells. . Here we concentrate mainly on the development of the other blood cells, often referred to collectively as myeloid cells. Table 22–1 summarizes the various types of blood cells and their functions.



The Production of Each Type of Blood Cell in the Bone Marrow Is Individually Controlled

Most white blood cells function in tissues other than the blood; blood simply transports them to where they are needed. A local infection or injury in any tissue rapidly attracts white blood cells into the affected region as part of the inflammatory response, which helps fight the infection or heal the wound. The inflammatory response is complex and is governed by many different signal molecules produced locally by mast cells, nerve endings, platelets, and white blood cells, as well as by the activation of complement. Some of these signal molecules act on the endothelial lining of nearby capillaries, helping white blood cells to first stick and then make an exit from the bloodstream into the tissue. Damaged or inflamed tissues and local endothelial cells secrete other molecules called chemokines, which act as chemoattractants for specific types of white blood cells, causing them to become polarized and crawl toward the source of the attractant. As a result, large numbers of white blood cells enter the affected tissue (Figure 22–28).



Other signal molecules produced during an inflammatory response escape into the blood and stimulate the bone marrow to produce more leukocytes and release them into the bloodstream. The regulation tends to be cell-type specific: some bacterial infections, for example, cause a selective increase in neutrophils, while infections with some protozoa and other parasites cause a selective increase in eosinophils. (For this reason, physicians routinely use differential white blood cell counts to aid in the diagnosis of infectious and other inflammatory diseases.) In other circumstances, erythrocyte production is selectively increased—for example, in response to anemia (lack of hemoglobin) due to blood loss, and in the process of acclimatization when one goes to live at high altitude, where oxygen is scarce. Thus, blood cell formation, or hematopoiesis, necessarily involves complex
controls, which regulate the production of each type of blood cell individually to meet changing needs.

Bone Marrow Contains Multipotent Hematopoietic Stem Cells, Able to Give Rise to All Classes of Blood Cells

In the bone marrow, the developing blood cells and their precursors, including the stem cells, are intermingled with one another, as well as with fat cells and other stromal cells (connective-tissue cells), which produce a delicate supporting meshwork of collagen fibers and other extracellular matrix components. In addition, the whole tissue is richly supplied with thin-walled blood vessels, called blood sinuses, into which the new blood cells are discharged. Megakaryocytes are also present; these, unlike other blood cells, remain in the bone marrow when mature and are one of its most striking features, being extraordinarily large (diameter up to 60 μm) with a highly polyploid nucleus. They normally lie close beside blood sinuses, and they extend processes through holes in the endothelial lining of these vessels; platelets pinch off from the processes and are swept away into the blood (Figure 22–29 ).



Because of the complex arrangement of the cells in bone marrow, it is difficult to identify in ordinary tissue sections any but the immediate precursors of the mature blood cells. There is no obvious visible characteristic by which we can recognize the ultimate stem cells. In the case of hematopoiesis, the stem cells were first identified by a functional assay that exploited the wandering lifestyle of blood cells and their precursors. When an animal is exposed to a large dose of x-rays, most of the hematopoietic cells are destroyed and the animal dies within a few days as a result of its inability to manufacture new blood cells. The animal can be saved, however, by a transfusion of cells taken from the bone marrow of a healthy, immunologically compatible donor. Among these cells there are some that can colonize the irradiated host and permanently reequip it with hematopoietic tissue. Such experiments prove that the marrow contains hematopoietic stem cells. They also show how we can assay for the presence of hematopoietic stem cells and hence discover the molecular features that distinguish them from other cells. For this purpose, cells taken from bone marrow are sorted (using a fluorescence- activated cell sorter) according to the surface antigens that they display, and the different fractions are transfused back into irradiated mice. If a fraction rescues an irradiated host mouse, it must contain hematopoietic stem cells. In this way, it has been possible to show that the hematopoietic stem cells are characterized by a specific combination of cell-surface proteins, and by appropriate sorting we can obtain virtually pure stem-cell preparations. The stem cells turn out to be a tiny fraction of the bone marrow population—about 1 cell in 50,000–100,000; but this is enough. A single such cell injected into a host mouse with defective hematopoiesis is sufficient to reconstitute its entire hematopoietic system, generating acomplete set of blood cell types, as well as fresh stem cells. This and other experiments (using artificial lineage markers) show that the individual hematopoietic stem cell is multipotent and can give rise to the complete range of blood cell types, both myeloid and lymphoid, as well as to new stem cells like itself (Figure 22–31).


The sequence of cell fate restrictions shown  in Figure 22–31  conveys the impression of a program executed with computer-like logic and precision. 


Commitment Is a Stepwise Process

Hematopoietic stem cells do not jump directly from a multipotent state into a commitment to just one pathway of differentiation; instead, they go through a series of progressive restrictions. The first step, usually, is commitment to either a myeloid or a lymphoid fate. This is thought to give rise to two kinds of progenitor cells, one capable of generating large numbers of all the different types of myeloid cells, and the other giving rise to large numbers of all the different types of lymphoid cells. Further steps give rise to progenitors committed to the production of just one cell type. The steps of commitment correlate with changes in the expression of specific transcription regulators, needed for the production of different subsets of blood cells.

Divisions of Committed Progenitor Cells Amplify the Number of Specialized Blood Cells

Hematopoietic progenitor cells generally become committed to a particular pathway of differentiation long before they cease proliferating and terminally differentiate. The committed progenitors go through many rounds of cell division to amplify the ultimate number of cells of the given specialized type. In this way, a single stem-cell division can lead to the production of thousands of differentiated progeny, which explains why the number of stem cells is such a small fraction of the total population of hematopoietic cells. For the same reason, a high rate of blood cell production can be maintained even though the stem-cell division rate is low. The smaller the number of division cycles that the stem cells themselves have to undergo in the course of a lifetime, the lower the risk of generating stem-cell mutations, which would give rise to persistent mutant clones of cells in the body—a particular danger in the hematopoietic system where a relatively small accumulation of mutations can be sufficient to cause cancer. A low rate of stem-cell division also slows the process of replicative cell senescence. The stepwise nature of commitment means that the hematopoietic system can be viewed as a hierarchical family tree of cells. Multipotent stem cells give rise to committed progenitor cells, which are specified to give rise to only one or a few blood cell types. The committed progenitors divide rapidly, but only a limited number of times, before they terminally differentiate into cells that divide no further and die after several days or weeks. Figure 22–31 depicts the hematopoietic family tree. It should be noted, however, that variations are thought to occur: not all stem cells generate the identical patterns of progeny via precisely the same sequence of steps.

Stem Cells Depend on Contact Signals From Stromal Cells

Like the stem cells of other tissues, hematopoietic stem cells depend on signals from their niche, in this case created by the specialized connective tissue of the bone marrow. (This is the site in adult humans; during development, and in nonhuman mammals such as the mouse, hematopoietic stem cells can also make their home in other tissues—notably liver and spleen.) When they lose contact with their niche, the hematopoietic stem cells tend to lose their stem-cell potential (Figure 22–32).



Evidently the loss of potency is not absolute or instantaneous, however, since the stem cells can still survive journeys via the bloodstream to colonize other sites in the body.

Factors That Regulate Hematopoiesis Can Be Analyzed in Culture

While the stem cells depend on contact with bone marrow stromal cells for longterm maintenance, their committed progeny do not, or at least not to the same degree. These cells can thus be dispersed and cultured in a semisolid matrix of dilute agar or methylcellulose, and factors derived from other cells can be added artificially to the medium. The semisolid matrix inhibits migration, so that the progeny of each isolated precursor cell remain together as an easily distinguishable colony. A single committed neutrophil progenitor, for example, may give rise to a clone of thousands of neutrophils. Such culture systems have provided a way to assay for the factors that support hematopoiesis and hence to purify them and explore their actions. These substances are glycoproteins and are usually called colony-stimulating factors (CSFs). Some of these factors circulate in the blood and act as hormones, while others act in the bone marrow as secreted local mediators; still others take the form of membrane-bound signals that act through cell–cell contact. An important example of the latter is a protein called Steel or Stem Cell Factor (SCF ). This is expressed both in the bone marrow stroma (where it helps to define the stem-cell niche) and along pathways of migration, and it occurs both in a membrane-bound and a soluble form. It binds to a receptor tyrosine kinase called Kit, and it is required during development for guidance and survival not only of hematopoietic cells but also of other migratory cell types—specifically,
germ cells and pigment cells.

Erythropoiesis Depends on the Hormone Erythropoietin

The best understood of the CSFs that act as hormones is the glycoprotein erythropoietin, which is produced in the kidneys and regulates erythropoiesis, the formation of red blood cells, to which we now turn. The erythrocyte is by far the most common type of cell in the blood (see Table 22–1). When mature, it is packed full of hemoglobin and contains hardly any of the usual cell organelles. In an erythrocyte of an adult mammal, even the nucleus,
endoplasmic reticulum, mitochondria, and ribosomes are absent, having been extruded from the cell in the course of its development (Figure 22–33).



The erythrocyte therefore cannot grow or divide, and it has a limited life-span—about 120 days in humans or 55 days in mice. Worn-out erythrocytes are phagocytosed and digested by macrophages in the liver and spleen, which remove more than 10^11 senescent erythrocytes in each of us each day. Young erythrocytes actively protect themselves from this fate: they have a protein on their surface that binds to an inhibitory receptor on macrophages and thereby prevents their phagocytosis. A lack of oxygen or a shortage of erythrocytes stimulates specialized cells in the kidney to synthesize and secrete increased amounts of erythropoietin into the bloodstream. The erythropoietin, in turn, boosts the production of erythrocytes. The effect is rapid: the rate of release of new erythrocytes into the bloodstream rises steeply 1–2 days after an increase in erythropoietin levels in the bloodstream. Clearly, the hormone must act on cells that are close precursors of the mature erythrocytes. The cells that respond to erythropoietin can be identified by culturing bone marrow cells in a semisolid matrix in the presence of erythropoietin. In a few days, colonies of about 60 erythrocytes appear, each founded by a single committed erythroid progenitor cell. This progenitor depends on erythropoietin for its survival as well as its proliferation. It does not yet contain hemoglobin, and it is derived from an earlier type of committed erythroid progenitor whose survival and proliferation are governed by other factors.

Multiple CSFs Influence Neutrophil and Macrophage Production

CSFs are defined as factors that promote the production of colonies of differentiated blood cells. But precisely what effect does a CSF have on an individual hematopoietic
cell? The factor might control the rate of cell division or the number of division cycles that the progenitor cell undergoes before differentiating; it might act late in the hematopoietic lineage to facilitate differentiation; it might act early to influence commitment; or it might simply increase the probability of cell survival (Figure 22–34).



By monitoring the fate of isolated individual hematopoietic cells in culture, it has been possible to show that a single CSF, such as granulocyte/macrophage CSF, can exert all these effects, although it is still not clear which are most important in vivo. Studies in vitro indicate, moreover, that there is a large element of chance in the way a hematopoietic cell behaves—a reflection, presumably, of “noise” in the genetic control system. If two sister cells are taken immediately after a cell division and cultured apart under identical conditions, they frequently give rise to colonies that contain different types of blood cells or the same types of blood cells in different numbers. Thus, both the programming
of cell division and the process of commitment to a particular path of differentiation seem to involve random events at the level of the individual cell, even though the behavior of the multicellular system as a whole is regulated in a reliable way. The sequence of cell fate restrictions shown earlier, in Figure 22–31, conveys the impression of a program executed with computer-like logic and precision. Individual cells may be more varied, quirky, and erratic, and may sometimes progress by other decision pathways from the stem-cell state toward terminal differentiation.


Regulation of Cell Survival Is as Important as Regulation of Cell Proliferation

The default behavior of hematopoietic cells in the absence of CSFs is death by apoptosis , and the control of cell survival plays a central part in regulating the numbers of blood cells. The amount of apoptosis in the vertebrate hematopoietic system is enormous: billions of neutrophils die in this way each day in an adult human, for example. In fact, most neutrophils produced in the bone marrow die there without ever functioning. This futile cycle of production and destruction presumably serves to maintain a reserve supply of cells that can be promptly mobilized to fight infection whenever it flares up, or phagocytosed and digested for recycling when all is quiet. Compared with the life of the organism, the lives of cells are cheap. Too little cell death can be as dangerous to the health of a multicellular organism as too much proliferation. Mutations that inhibit cell death by causing excessive production of the intracellular apoptosis inhibitor Bcl2 promote the development of cancer in B lymphocytes. Indeed, the capacity for unlimited self-renewal is a dangerous property for any cell to possess. Many cases of leukemia arise through mutations that confer this capacity on committed hematopoietic precursor cells that would normally be fated to differentiate and die after a limited number of division cycles.

Summary

The many types of blood cells, including erythrocytes, lymphocytes, granulocytes, and macrophages, all derive from a common multipotent stem cell. In the adult, hematopoietic stem cells are found mainly in bone marrow, and they depend on signals from the marrow stromal (connective-tissue) cells to maintain their stem-cell character. The stem cells are few and far between, and they normally divide infrequently to produce more stem cells (self-renewal) and various committed progenitor cells (transit amplifying cells), each able to give rise to only one or a few types of blood cells. The committed progenitor cells divide extensively under the influence of various protein signal molecules (colony-stimulating factors, or CSFs) and then terminally differentiate into mature blood cells, which usually die after several days or weeks. Studies of hematopoiesis have been greatly aided by in vitro assays in which stem cells or committed progenitor cells form clonal colonies when cultured in a semisolid matrix. The progeny of stem cells seem to make their choices between alternative developmental pathways in a partly random manner. Cell death by apoptosis, controlled by the availability of CSFs, also plays a central part in regulating the numbers of mature differentiated blood cell

related issues: Unicellular and multicellular Organisms are best explained through design
http://reasonandscience.heavenforum.org/t2010-unicellular-and-multicellular-organisms-are-best-explained-through-design?highlight=unicellular

Embryonic Hematopoiesis

Hemangiogenic precursor cells first arise in the posterolateral mesoderm during gastrulation and from there migrate to the earliest blood-forming organs (Fig. 17.1). Under the influence of Runx-1, some of their progeny follow the hematopoietic lineage, whereas others, responding to Hoxa3 enter the endothelial lineage. Still other progeny will enter a third lineage and eventually form vascular smooth muscle cells. Although blood cell formation (hematopoiesis) begins in the yolk sac, the yolk sac–derived cells are soon replaced by blood cells that are independently derived from other sites of hematopoiesis (Fig. 17.2).



The upper graph highlights the relative importance of the various sites of hematopoiesis. The lower graph shows the percentages of the various hemoglobin polypeptide chains present in the blood at any given time. The α chain is treated separately from the others. AGM, aorta/genital ridge/mesonephros region.
(Based on Carlson B: Patten’s foundations of embryology, ed 6, New York, 1996, McGraw-Hill).

The blood islands contain pluripotential hematopoietic stem cells, which can give rise to most types of cells found in the embryonic blood. The erythrocytes produced in the yolk sac are large nucleated cells that enter the bloodstream just before the heart tube begins to beat at about 22 days’ gestation. For the first 6 weeks, the circulating erythrocytes are largely yolk sac derived, but during that time, preparations for the next stages of hematopoiesis are taking place.
Analysis of human embryos has shown that, starting at 28 days, definitive intraembryonic hematopoiesis begins in small clusters of cells (para-aortic clusters) in the splanchnopleuric mesoderm associated with the ventral wall of the dorsal aorta and shortly thereafter in the aorta/genital ridge/mesonephros (AGM) region. Precursor cells from the AGM region make their way via the blood to blood-forming sites in the liver, the yolk sac, and the placenta. Hematopoietic stem cells formed in the AGM, the yolk sac, and the placenta become transported to the liver via the circulation to the liver (see Fig. 17.1). By 5 to 6 weeks of gestation, sites of hematopoiesis become prominent in the liver. In both the yolk sac and the early sites of embryonic hematopoiesis, the endothelial cells themselves briefly retain the capacity for producing blood-forming cells. There is now evidence that in the AGM region, nitric oxide gas signaling, resulting from shear stress caused by blood flow on the endothelial cells, can induce their transformation into hematopoietic stem cells.
The erythrocytes produced by the liver are quite different from the erythrocytes derived from the yolk sac. Although still considerably larger than normal adult red blood cells, liver-derived erythrocytes are non-nucleated and contain different types of hemoglobin. By 6 to 8 weeks of gestation in humans, the liver replaces the yolk sac as the main source of blood cells. Although the liver continues to produce red blood cells until the early neonatal period, its contribution begins to decline in the sixth month of pregnancy. At this time, the formation of blood cells shifts to the bone marrow, the definitive site of adult hematopoiesis. This shift is controlled by cortisol secreted by the fetal adrenal cortex. In the absence of cortisol, hematopoiesis remains confined to the liver. Before hematopoiesis becomes well established in the bone marrow, small amounts of blood formation may also occur in the omentum and possibly the spleen.

Cellular Aspects of Hematopoiesis

The first hematopoietic stem cells that arise in the embryo are truly pluripotential in that they can give rise to all the cell types found in the blood (Fig. 17.3). These pluripotent stem cells, sometimes called hemocytoblasts, have great proliferative ability. They produce vast numbers of progeny, most of which are cells at the next stage of differentiation, but they also produce small numbers of their original stem cell type, which act as a reserve capable of replenishing individual lines of cells should the need arise. Very early in development, the line of active blood-forming cells subdivides into two separate lineages. Lymphoid stem cells ultimately form the two lines of lymphocytes: B lymphocytes (which are responsible for antibody production) and T lymphocytes (which are responsible for cellular immune reactions). Myeloid stem cells are precursors to the other lines of blood cells: erythrocytes, the granulocytes (neutrophils, eosinophils, and basophils), monocytes, and platelets. The second-generation stem cells (lymphoid and myeloid) are still pluripotent, although their developmental potency is restricted because neither lymphoid cells nor myeloid cells can form the progeny of the other type.



Mature blood cells are shown on the right. CFU, colony-forming unit; GM, granulocyte and monocyte; L, lymphocyte; ML, myeloid and lymphoid; S, spleen.
Stemming from their behavior in certain experimental situations, the hematopoietic stem cells are often called colony-forming units (CFUs). The first-generation stem cell is called the CFU-ML because it can give rise to myeloid and lymphoid lines of cells. Stem cells of the second generation are called CFU-L (lymphocytes) and CFU-S (spleen) (determined from experiments in which stem cell differentiation was studied in irradiated spleens). In some cases, the progeny of CFU-ML and CFU-S are committed stem cells, which are capable of forming only one type of mature blood cell. For each lineage, the forming cell types must pass through several stages of differentiation before they attain their mature phenotype.
What controls the diversification of stem cells into specific cell lines? Experiments begun in the 1970s provided evidence for the existence of specific colony-stimulating factors (CSFs) for each line of blood cell. CSFs are diffusible proteins that stimulate the proliferation of hematopoietic stem cells. Some CSFs act on several types of stem cells; others stimulate only one type. Although much remains to be learned about the sites of origin and modes of action of CSFs, many CSFs seem to be produced locally in stromal cells of the bone marrow, and some may be stored on the local extracellular matrix. CSFs are bound by small numbers of surface receptors on their target stem cells. Functionally, CSFs represent mechanisms for stimulating the expansion of specific types of blood cells when the need arises. Recognition of the existence of CSFs has prompted considerable interest in their clinical application to conditions characterized by a deficiency of white blood cells (leukopenia).
Certain Hox genes, especially those of the Hoxa and Hoxb families, play an important role in some aspects of hematopoiesis. Exposure of bone marrow to antisense oligonucleotides against specific Hox genes results in the suppression of specific lines of differentiation of blood cells. Conversely, engineered overexpression of genes, such as Hoxb8, Hoxa9, and Hoxa10, causes leukemia in mice. Evidence is increasing for the involvement of Hox genes in the pathogenesis of human leukemias. One important function of the Hox genes in hematopoiesis is the regulation of proliferation. Several growth factors, especially bone morphogenetic protein-4 (BMP-4), Indian hedgehog, and Wnt proteins, are important in stimulating and maintaining hematopoietic stem cell activity.


Erythropoiesis


Red blood cell formation (erythropoiesis) occurs in three waves during the embryonic period. The first wave begins with precursors within the yolk sac, which produce primitive nucleated erythrocytes that mature within the bloodstream. The second wave also begins in the yolk sac, but the precursor cells then colonize the embryonic liver and produce the first of a generation of definitive fetal erythrocytes that are dominant during the prenatal period. The third wave consists of precursor cells that enter the liver from the AGM mesoderm and the placenta. Some of these definitive erythroid progenitor cells send progeny directly from the liver into the bloodstream as definitive fetal erythrocytes. Others seed the bone marrow and produce adult-type erythrocytes later in the fetal period.
The erythrocyte lineage represents one line of descent from the CFU-S cells. Although the erythroid progenitor cells are restricted to forming only red blood cells, there are many generations of precursor cells (Fig. 17.4). The earliest stages of erythropoiesis are recognized by the behavior of the precursor cells in culture, rather than by morphological or biochemical differences. These are called erythroid burst-forming units (BFU-E) and erythroid CFUs (CFU-E). Each responds to different stimulatory factors. The pluripotent CFU-S precursors (see Fig. 17.3) respond to interleukin-3, a product of macrophages in adult bone marrow. A hormone designated as burst-promoting activity stimulates mitosis of the BFU-E precursors (see Fig. 17.4). A CFU-E cell, which has a lesser proliferative capacity than a BFU-E cell, requires the presence of erythropoietin as a stimulatory factor.


Erythropoietin is a glycoprotein that stimulates the synthesis of the mRNA for globin and is first produced in the fetal liver. Later in development, synthesis shifts to the kidney, which remains the site of erythropoietin production in adults. Under conditions of hypoxia (e.g., from blood loss or high altitudes), the production of erythropoietin by the kidneys increases, thereby stimulating the production of more red blood cells to compensate for the increased need. In adult erythropoiesis, the CFU-E stage seems to be the one most responsive to environmental influences. The placenta is apparently impervious to erythropoietin, and this property insulates the embryo from changes in erythropoietin levels of the mother and eliminates the influence of fetal erythropoietin on the blood-forming apparatus of the mother.
One or two generations after the CFU-E stage, successive generations of erythrocyte precursor cells can be recognized by their morphology. The first recognizable stage is the proerythroblast (Fig. 17.5), a large, highly basophilic cell that has not yet produced sufficient hemoglobin to be detected by cytochemical analysis. Such a cell has a large nucleolus, much uncondensed nuclear chromatin, numerous ribosomes, and a high concentration of globin mRNAs. These are classic cytological characteristics of an undifferentiated cell.



Succeeding stages of erythroid differentiation (basophilic, polychromatophilic, and orthochromatic erythroblasts) are characterized by a progressive change in the balance between the accumulation of newly synthesized hemoglobin and the decline of first the RNA-producing machinery and later the protein-synthesizing apparatus. The overall size of the cell decreases, and the nucleus becomes increasingly pyknotic (smaller with more condensed chromatin) until it is finally extruded at the stage of the orthochromatic erythrocyte. After the loss of the nucleus and most cytoplasmic organelles, the immature red blood cell, which still contains a small number of polysomes, is a reticulocyte. Reticulocytes are released into the bloodstream, where they continue to produce small amounts of hemoglobin for 1 or 2 days. The final stage of hematopoiesis is the mature erythrocyte, which is a terminally differentiated cell because of the loss of its nucleus and most of its cytoplasmic organelles. Erythrocytes in embryos are larger than their adult counterparts and have a shorter life span (50 to 70 days in the fetus versus 120 days in adults).

The Blood  1

In order to perform the functions of (1) transportation, (2) regulation, and (3) protection, the circulatory system relies on several key components. All of these components can be found in the fluid that travels within these living tubules—blood. Commenting on the remarkable properties of this fluid, Bruce Alberts and his colleagues noted: “Blood contains many types of cells with very different functions, ranging from the transport of oxygen to the production of antibodies. Some of these cells function entirely within the vascular system, while others use the vascular system only as a means of transport and perform their function elsewhere (1994, p. 1161).
The total volume of blood represents only 8% of the total weight of a human (Van de Graaff and Fox, p. 655). Ironically, it is classified as a tissue, but it is one of the few substances in the human body that is not “fixed” in place. Tissues such as nerves, muscles, and organs have a specific function and are limited in movement. Blood, however, is not limited to any one part of the body. This ability to move allows blood to provide these “fixed” tissues with nourishment and then carry off waste products. Blood itself is composed of a cellular portion referred to as formed elements, and a fluid portion designated as plasma. The formed elements constitute approximately 45% of the total volume of blood (Van de Graaff and Fox, p. 655) and are comprised of erythrocytes (red blood cells), leukocytes (white blood cells), and platelets. Plasma is a straw-colored liquid that consists primarily of water and dissolved solutes. Approximately 89% of plasma is water, 9% is protein material (e.g., albumins), 0.9% is salts, and 0.9% is sugar, urea, etc.
In considering whether the circulatory system could be the product of evolution, one must consider that the acidity of blood is critical to survival. Key salts provide basic ions, such as sodium, potassium, phosphate, and magnesium that help maintain a steady pH value for the blood. These bicarbonate ions remove carbon dioxide from the tissues and help maintain a slightly alkaline pH of 7.4. During traumatic injuries or surgeries, a great deal of attention is given to the pH of the blood since a significant decrease or loss of this alkalinity can cause rapid and violent breathing, with death likely to occur at a pH of 7.0 or below. Conversely, if the pH of the blood is allowed to go beyond 7.6, it also can prove fatal. The Lange medical book on “Fluid and Electrolytes” records: “The control of blood pH is critically important since modest swings—e.g., 0.10-0.20 pH unit in either direction—can cause symptoms referable to impaired cardiopulmonary performance and neurologic function. More extreme pH changes can be fatal” (see Co­gan, 1991, p. 175). This narrow, “unforgiving” pH range would not be expected if blood were the product of millions of years of evolution. If blood were the product of evolution and millions of years of random change, one would expect nature to have selected for a fluid that was not dependent on such a critical pH level—especially since slight pH variations can prove fatal!
The salinity in our blood stream has made some evolutionists speculate that this is evidence we evolved out of the sea. For instance, Robert Lehrman noted:

One human characteristic, a chemical one, harks back to our ancestry in the ocean.... The percentage of sodium, potassium, calcium, magnesium, iodine, chlorine, and other minerals in human blood salt coincide with those of sea water. Our ocean-living ancestors developed cells adapted to the chemical environment of sea water. When they left the ocean, they took a part of the environment with them in the form of a fluid that bathes the cells; later it was incorporated into the blood stream (as quoted in Batten, 1997, p. 24).

However, on average, the concentration of sodium chloride (salt) in sea water is 2.7% higher than we find in the human blood stream (additionally, the sea contains 0.8% other salts, some of which are not present in blood and would not benefit the cardiovascular system). Creationist Don Batten carried out an extensive comparison demonstrating the actual percentages of salts and minerals found in human blood and seawater. He noted that there is little similarity between human blood and seawater and reported that “even the blood of sea creatures such as crabs is quite different from seawater” (1997, p. 24). If evolutionists took the time to do the math, they would find that the Baltic Sea—one of the “fresher” large bodies of water—still is much too salty to have played any physiological part in the production of blood.
Erythrocytes (also known as red blood cells) are the most common of the formed elements. Adult humans have approximately 2-3 x 1013 red blood cells at any given time (see Wikipedia, n.d.). These cells provide oxygen to tissues, and assist in the disposal of carbon dioxide. In humans, red blood cells are devoid of nuclei (i.e., they are anucleated) and intracellular organelles, while birds, amphibians, and other animals have red blood cells that are nucleated. This key difference should not be overlooked in light of our alleged evolutionary origins. All cells require a nucleus for replication and maturation. Even red blood cells have a nucleus during their very early stages of development. However, in humans, the production of red blood cells occurs in the bone marrow, and thus we do not normally see these nucleated cells in the circulation (although they occasionally are found in newborns). An obvious question is: How did humans “evolve” cells that would mature without a nucleus? And furthermore, why would nature select for this? By losing their nuclei, these cells are unable to replicate like other cells within the body. The body is dependent on pluripotent stem cells within bone marrow for future erythrocyte production. With a lifespan of only 120 days and no nuclei, they must be constantly produced in order to carry oxygen throughout the body. Each second about 2.5 million new erythrocytes are produced or about 200 billion each day (see “Cardiovascular System,” 2004)! Some animals produce these cells intravascularly (i.e., in the blood stream), whereas humans and other animals produce them extra­vas­cu­larly (in the bone marrow or other hema­topoietic tissue). Additionally, this loss of cellular organelles means that these cells are unable to produce energy, and thus, they must get energy from anaerobic respiration. Anaerobic respiration in red blood cells is a complex cascade of events that puts even more impossible explanatory demands on evolutionists.
Red blood cells are formed by a process known as erythropiesis. It takes approximately seven days for these cells to develop, and then they are released into the blood stream. Old red blood cells are “engulfed by phagocytes, destroyed, and their materials are released in the blood. The main sites of destruction are the liver and spleen” (see Wikipedia, n.d.). During their lifetime these specialized cells travel

over 100 miles, are buffeted at high velocities during their passage through the heart, and have to negotiate tiny capillaries.... As they age, subtle structural changes occur which render them identifiable to scavenger cells in the spleen and elsewhere, and they end their days being devoured and digested by these predators (Blakemore and Jennett, 2001, p. 85).

However, this process must be orchestrated or else the individual will suffer from having too many or insufficient numbers of red blood cells in the blood stream. Consider the fate of an individual unable to breakdown aged red blood cells, or someone who is unable to produce replacements. How did this feedback mechanism arise? Blakemore and Jennett observed:

Their rate of production is beautifully adapted to this function. It is regulated by a hormone called erythropoietin, produced in the kidney in the adult and in the liver in the fetus. Close to the gene that regulates erythropoietin production are regions of DNA that sense oxygen tension; when this falls, erythropoietin synthesis is stimulated, and more red cells are produced in the bone marrow. When adequate oxygenation of tissues is achieved, erythropoietin production is reduced. By this biological feedback loop the body is able to respond to varying oxygen demands by modifying the rate of red cell production (2001, p. 85, emp. added).

This system is also irreducibly complex. All of the parts are necessary in order for the feedback mechanism to work properly. So how was this “beautifully adapted” feedback loop able to evolve in a series of evolutionary steps? The truth is that it could not!
As the red blood cell matures and is ready to leave the bone marrow, it expels its nucleus. The reason for anucleated red blood cells in humans is directly related to function—the unique shape and loss of nucleus provides added surface area through which gas can diffuse (Van de Graaff and Fox, 1989, p. 656; Blakemore and Jennett, p. 85). The anucleated biconcave shape increases surface area and allows the cell to remain flexible enough to squeeze through small capillaries. Even an anucleated red blood cell is larger (8µm) than capillaries (2-3µm). However, without the nucleus present, the red blood cell is flexible and able to fold over on itself. For how many “millions of years” was development limited as red blood cells slowly “evolved” the ability to shed their nucleus, develop anaerobic respiration for energy needs, and finally become flexible and able to fold into capillaries? The functional design of the anucleated red blood cell’s shape (a biconcave disc) can only be explained by the ultimate Designer.
In addition, red blood cells contain hemoglobin, which is responsible for carrying oxygen to every cell in the body. Hemoglobin is a complex protein that has two pairs of chains (referred to as alpha and beta) which bind to the red-pigmented molecule known as heme. As Blakemore and Jennett described: “In most mammals, adult hemoglobin (hemoglobin A) comprises two unlike pairs of chains of amino acids, or globin chains, called α and β, each of which is folded round one iron-containing heme molecule, to which oxygen can bind” (p. 85). This configuration allows hemoglobin to transport four molecules of oxygen. Given the added surface area from the anucleated biconcave disc, each cell would contain “about 280,000,000 molecules of hemoglobin” (see “Cardiovascular System,” 2004). What are the odds that this engineering accomplishment happened by random chance? Consider that an evolutionary origin of hemoglobin would require a minimum of 120 mutations to convert an alpha chain to a beta. At least 34 of those changes require changeovers in 2 or 3 nucleotides. Yet, if a single nucleotide change occurred through mutation, the result would ruin the blood and kill the organism. Simply put, evolution cannot explain the existence of hemoglobin molecules in the circulatory system of humans.

Another component of formed elements are leukocytes (white blood cells). Unlike red blood cells, leukocytes contain nuclei and mitochondria and can purposefully move in an amoeboid fashion (Van de Graaff and Fox, p. 657). Leukocytes serve as the primary line of defense in the vascular system. There are five different leukocytes present in the blood: neutrophils, eosinophils, basophils, lymphocytes, and monocytes. These cells will be covered in greater detail with the immune system, but the point should not be missed that these specialized cells traverse the circulatory system and are critical to the survival of individuals (e.g., consider immune deficient individuals). Yet, evolutionary theory is unable to explain adequately their origin.

The final formed element found within the blood is platelets. Platelets are much smaller than red blood cells, and serve to stop blood loss from wounds (hemo­stasis). Consider for just a moment the conundrum posed by blood clotting. It is critical that the circulatory system have a means to prevent blood loss when it is damaged, but this process must only occur when needed, and humans cannot afford to have this clot spread to healthy vessels. Not only must a clotting network be available, but also there must be an effective means of terminating the clotting cascade once the vessels have been repaired. In his book Darwin’sBlack Box, biochemist Michael Behe observed:

When a pressurized blood circulation system is punctured, a clot must form quickly or the animal will bleed to death. If blood congeals at the wrong time or place, though, then the clot may block circulation as it does in heart attacks and strokes. Furthermore, a clot has to stop bleeding all along the length of the cut, sealing it completely. Yet blood clotting must be confined to the cut or the entire blood system of the animal might solidify, killing it. Consequently, the clotting of blood must be tightly controlled so that the clot forms only when and where it is required (2003, pp. 78-79).

Behe then goes into painstaking detail to record the biochemical cascade that must transpire in order for blood clotting to occur. For over fifteen pages he records all of the events that are required in order for this process to occur (see pages 81-97). Figure 4-3 of his book shows a flowchart of the proteins involved in the blood coagulation cascade. Aside from there being dozens of proteins required, many are produced in response to complex feedback loops within the cascade itself. The statistical odds of evolving those specific proteins in just the right manner, in just the right location, and having them available when that first land animal was getting scratched up as it slowly crawled over rocks and shells onto dry land are incalculable! Only God could create such a complex system that can heal itself so precisely.

1) http://www.apologeticspress.org/ApPubPage.aspx?pub=1&issue=571&article=450

An Evolving Paradigm for Stem Cell Biology

Abstract

Establishment and maintenance of the blood system relies on self-renewing hematopoietic stem cells (HSCs) that normally reside in small numbers in the bone marrow niche of adult mammals. This Review describes the developmental origins of HSCs and the molecular mechanisms that regulate lineage-specific differentiation. Studies of hematopoiesis provide critical insights of general relevance to other areas of stem cell biology including the role of cellular interactions in development and tissue homeostasis, lineage programming and reprogramming by transcription factors, and stage- and age-specific differences in cellular phenotypes.

Introduction

The blood system serves as a paradigm for understanding tissue stem cells, their biology, and involvement in aging, disease, and oncogenesis. Because mature blood cells are predominantly short lived, stem cells are required throughout life to replenish multilineage progenitors and the precursors committed to individual hematopoietic lineages. Hematopoietic stem cells (HSCs) reside as rare cells in the bone marrow in adult mammals and sit atop a hierarchy of progenitors that become progressively restricted to several or single lineages (Orkin, 2000). These progenitors yield blood precursors devoted to unilineage differentiation and production of mature blood cells, including red blood cells, megakaryocytes, myeloid cells (monocyte/macrophage and neutrophil), and lymphocytes. As with all other stem cells, HSCs are capable of self-renewal—the production of additional HSCs—and differentiation, specifically to all blood cell lineages.
HSCs are defined operationally by their capacity to reconstitute the entire blood system of a recipient. In general, preparation of patients for transplantation with donor bone marrow containing HSCs entails destruction of host bone marrow by irradiation or by treatment with high-dose cytotoxic drugs, in part to provide ‘‘space’’ for donor HSCs within the marrow microenvironment (the niche) of the recipient. HSCs can be prospectively identified by monoclonal antibodies directed to surface markers, by dye efflux, or on the basis of their metabolic properties; HSCs can be separated from more-committed progenitors and other marrow cells by fluorescence-activated cell sorting (FACS). With contemporary methods, HSCs may be highly purified such that as few as one cell may provide long-term (>4 months) hematopoietic reconstitution in a recipient. Technical considerations regarding the assays for quantitation of HSCs and evaluation of their function have recently been reviewed (Purton and Scadden, 2007). Because no ex vivo assays can replace in vivo transplantation for measuring biological activity of HSCs, characterizing cell populations based on the expression of cell-surface markers cannot be considered synonymous with determining their function. During stress or other manipulations (such as in mutant animals), the surface marker profile of HSCs and their progenitors may be distorted.
Here, we discuss the developmental origins of the hematopoietic system and the molecular control of self-renewal and lineage determination. The process of hematopoiesis is generally conserved throughout vertebrate evolution. Manipulation of animal models, such as the mouse and zebrafish, has complemented and greatly extended studies of human hematopoiesis. Although not an entirely ideal experimental system, partial reconstitution of the blood system of immunodeficient mice (such as NOD/SCID strains) has been commonly employed to study human hematopoiesis. The remarkable regenerative properties of human HSCs are best illustrated by the success of marrow transplantation in human patients, a current mainstay of therapy for a variety of genetic disorders, acquired states of bone marrow failure, and cancers.

Emergence of HSCs

In vertebrates, the production of blood stem cells is accomplished by the allocation and specification of distinct embryonic cells in a variety of sites that change during development (Galloway and Zon, 2003) (Figure 1 and Figure 2). In mammals, the sequential sites of hematopoiesis include the yolk sac, an area surrounding the dorsal aorta termed the aorta-gonad mesonephros (AGM) region, the fetal liver, and finally the bone marrow (Figure 1). Recently, the placenta has been recognized as an additional site that participates during the AGM to fetal liver period. The properties of HSCs in each site differ, presumably reflecting diverse niches that support HSC expansion and/or differentiation and intrinsic characteristics of HSCs at each stage. For instance, HSCs present in the fetal liver are in cycle, whereas adult bone marrow HSCs are largely quiescent.


Developmental Regulation of Hematopoiesis in the Mouse


Developmental Regulation of Hematopoiesis in the Mouse
(A) Hematopoiesis occurs first in the yolk sac (YS) blood islands and later at the aorta-gonad mesonephros (AGM) region, placenta, and fetal liver (FL). YS blood islands are visualized by LacZ staining of transgenic embryo expression GATA-1- driven LacZ. AGM and FL are stained by LacZ in Runx1-LacZ knockin mice. (Photos courtesy of Y. Fujiwara and T. North.).
(B) Hematopoiesis in each location favors the production of specific blood lineages. Abbreviations: ECs, endothelial cells; RBCs, red blood cells; LTHSC, long-term hematopoietic stem cell; ST-HSC, short-term hematopoietic stem cell; CMP, common myeloid progenitor; CLP, common lymphoid progenitor; MEP, megakaryocyte/erythroid progenitor; GMP, granulocyte/macrophage progenitor.

(C) Developmental timewindows for shifting sites of hematopoiesis.

Hematopoietic Development in the Zebrafish


Hematopoietic Development in the Zebrafish
(A) Hematopoiesis occurs first in the intermediate cell mass (ICM) and subsequently in the aorta-go-nad mesonephros (AGM) region and caudal hematopoietic tissue (CHT). Later hematopoietic cells are found in the kidney as well as in the thymus. In situ hybridization for GATA-1 at 30 hr (ICM), for c-myb at 36 hr (AGM), for SCL/tal1 at days 4 and 6.5 (CHT), and for c-mybat day 6 (top view) to demonstrate expression in the kidney marrow and thymus. (Photos courtesy of X. Bai and T. Bowman.)
(B) Developmental time windows for hematopoietic sites in the zebrafish.

Although there is little dispute regarding where HSCs are found during development, few topics have polarized investigators as much as the origin of HSCs. HSCs are derived from ventral mesoderm (see Review by C.E. Murry and G. Keller, page 661 of this issue). The contribution of each hematopoietic site (such as the yolk sac and fetal liver) to circulating blood in the fetus or adult was seemingly answered more than 25 years ago. Recent studies in mice and zebrafish, however, challenge the field with divergent views.

Multiple Waves of Hematopoiesis during Development

The initial wave of blood production in the mammalian yolk sac is termed ‘‘primitive.’’ The primary function for primitive hematopoiesis is production of red blood cells that facilitate tissue oxygenation as the embryo undergoes rapid growth. The hallmark of primitive erythroid cells is expression of embryonic globin proteins. The primitive hematopoietic system is transient and rapidly replaced by adult-type hematopoiesis that is termed “definitive.”
In mammals, the next site of hematopoietic potential is the AGM region. Hematopoietic cells were first detected in the aorta of the developing pig more than 80 years ago. Subsequently, studies of chick-quail chimeras and diploid-triploid Xenopus embryos demonstrated analogous AGM-like regions. Morphological examination revealed that a sheet of lateral mesoderm migrates medially, touches endoderm, and then forms a single aorta tube. Clusters of hematopoietic cells subsequently appear in the ventral wall. Similarly, an intraembryonic source of adult HSCs in mice capable of long-term reconstitution of irradiated hosts resides in the AGM region (Muller et al., 1994). At embryonic day 10.5, little HSC activity is detectable, whereas by day 11 engrafting activity is present.

Additional hematopoietic activity in the mouse embryo was detected subsequently in other sites, including the umbilical arteries and the allantois in which hematopoietic and endothelial cells are colocalized (Inman and Downs, 2007). Umbilical veins lack hematopoietic potential, suggesting that a hierarchy exists during definitive hematopoiesis in which HSCs arise predominantly during artery specification. In addition, significant numbers of HSCs are found in the mouse placenta (Gekas et al., 2005; Ottersbach and Dzierzak, 2005), nearly coincident with the appearance of HSCs in the AGM region and for several days thereafter. Placental HSCs could arise through de novo generation or colonization upon circulation, or both. The relative contribution of each of the above sites to the final pool of adult HSCs remains largely unknown.
Subsequent definitive hematopoiesis involves the colonization of the fetal liver, thymus, spleen, and ultimately the bone marrow. It is believed that none of these sites is accompanied by de novo HSC generation. Rather, their niches support expansion of populations of HSCs that migrate to these new sites. However, until very recently (as discussed below), there has been no evidence by fate mapping or direct visualization that HSCs from one site colonize subsequent sites.

Hemangioblasts and Hemogenic Endothelium

A common origin for blood and vascular cells, the “hemangio-blast,” was hypothesized a century ago, based largely on the intimate association of these lineages in the blood islands of the developing yolk sac. Sharing of markers between blood and blood vessel cells, and the impairment of both tissues in mutants, such as the mouse flk1 knockout (Shalaby et al., 1997) and zebrafish cloche (Stainier et al., 1995), are consistent with a common origin. Clonal studies using in vitro differentiating mouse embryonic stem (ES) cells provide the strongest evidence in favor of the existence of hemangioblasts (Choi et al., 1998). Furthermore, hemangioblast activity has been detected at the mid-streak stage of gastrulation and during the neural plate stage but is extremely transient in vivo (Huber et al., 2004). Despite these findings, formal proof of the hemangioblast hypothesis requires direct demonstration that a single cell divides asymmetrically to form blood and vascular derivatives in vivo.
Clonal analysis in mouse chimeras, however, presents contradictory evidence regarding the existence of the hemangioblast (Ueno and Weissman, 2006). Three different, stably marked ES cells were mixed and coinjected into host blastocysts. According to the hemangioblast hypothesis, each blood island of the yolk sac should be clonally derived. However, in these experiments more than a single ES cell often contributed to each blood island of the chimeric mice. The existence of the hemangioblast has also been addressed in zebrafish. A primitive wave of hematopoiesis occurs in a region called the intermediate cell mass that contains erythroid cells surrounded by venous endothelial cells (see Figure 2). Hematopoietic and endothelial markers segregate between the 3- to 10-somite period of development. By this time, there are few, if any, cells that might be considered hemangioblasts based on overlapping blood and blood vessel gene expression. Alternatively, hemangioblasts could appear before the 3-somite stage and also exhibit wider developmental potential than solely blood and blood vessels. Ventral mesodermal cells are dedicated specifically to hematopoietic and endothelial fates. Fate-mapping studies have been performed in which a caged fluorescent dye is injected into the zebrafish embryo at the one-cell stage, and then at a later time the fluorescent dye is uncaged in single cells using a laser. Individual cells appear dedicated to hematopoietic and endothelial lineages at the 0- to 3-somite stage (Vogeli et al., 2006). However, other cell fates may also be present at this early time. Similarly, smooth muscle cells can be derived from populations of in vitro differentiated mouse ES cells exhibiting blood and blood vessel fates (Ema et al., 2003; Ema and Rossant, 2003). These studies support the existence of hemangioblasts, although it may be necessary to redefine the potential of these cells to include additional lineages (such as smooth muscle).
Principally based on morphology it has been proposed that as the AGM forms, “hemogenic endothelial” cells in the ventral wall of the aorta, rather than hemangioblasts, bud off HSCs. The program of hemogenic endothelial cell development may be regulated differently from that of presumptive hemangioblasts, given that the transcription factor requirements differ. For example, the transcription factor Runx1 is necessary for blood formation from hemogenic endothelium but not from yolk sac hemangioblasts (North et al., 1999, 2002). The potential to generate hematopoietic, endothelial, and smooth muscle cells has been attributed to another cell type, termed the mesoangioblast, present in the aorta (Cossu and Bianco, 2003). Perhaps, the presumptive mesoangioblast might be a precursor of the hemogenic endothelial cell.
Other work has indicated that mesenchymal cell populations in the subaortic region poke through the aorta and bud off HSCs (Bertrand et al., 2005). As this occurs, mesenchymal cells express endothelial-specific genes and ultimately express HSC-associated markers. These observations suggest an alternative model in which subaortic mesenchymal cells, which may also have smooth muscle potential, rather than hemogenic endothelial cells, are the source of future definitive HSCs.

Developmental Relationships between the Yolk Sac and the AGM

As with mesodermal derivatives, all blood cells in embryonic, fetal, and adult animals might arise from a small set of cells during development. Evidence for and against this notion is present in the literature. Fate mapping in the pre-gastrula Xenopus embryo with fluorescent dye injected into individual blastomeres of the 32-cell embryo demonstrated that different blastomeres contribute to primitive hematopoiesis and definitive HSC production (Ciau-Uitz et al., 2000). This finding contradicts the conclusion derived from diploid-triploid chimeric frogs that ventral mesoderm is the common origin of both primitive and definitive populations (Turpen et al., 1997). Technical aspects of fate mapping of the 32-cell embryo have been challenged (Lane and Sheets, 2002).
In situ hybridization and chimera studies in amphibians and birds suggest that the yolk sac and the AGM are derived independently and arise at different times in development (Turpen et al., 1997). With short-term culture and subsequent transplantation, mouse AGM tissue (isolated one day prior to the appearance of HSCs in vivo) generates cells with the capacity for long-term engraftment, whereas mouse yolk sac tissue does not (Cumano et al., 1996; Medvinsky and Dzierzak, 1996). The origin of HSCs in the AGM can be traced by Runx1 expression in the embryonic day 8.5 (E8.5) mouse embryo, just before the onset of circulation. Because functional activity of stem cells as determined by transplantation into irradiated adults occurs much later (at day 11), it is possible that cells of the yolk sac colonize the AGM through the circulation. In fact, HSC-like activity of yolk sac cells (as defined by a neonatal transplantation assay) (Palis et al., 2001) is detected as early as day 9, although circulation has started by that time. Conclusive resolution of the developmental relationship between cells of the yolk sac and AGM requires direct visualization of the migration event. Furthermore, the specific assay used to determine stem cell activity for one population of cells (such as immune reconstitution following irradiation of adult animals) may not be appropriate for a different stem cell population. Distinct host requirements, such as the use of neonatal recipients for cells of the yolk sac, may be necessary. Some of the intrinsic differences between cell populations, such as developmental stage, ease of access, the local niche, and whether they are dividing, may preclude a host transplant assay from detecting engraftment and multilineage reconstitution. Such questions will plague studies of other tissue stem cells, as these stem cells are defined by functional and biological readouts.

Does the Yolk Sac Contain HSCs?

Based on cell fate mapping and transplantation experiments in avian and amphibian species, the AGM has been widely viewed as the principal site for HSC production during vertebrate development. Accordingly, the yolk sac has often been relegated to a subservient position, despite older experiments suggesting that the yolk sac might be the source of adult hematopoiesis. Metcalf and Moore cultured E7.5 mouse embryos from which the yolk sac had been removed (Moore and Metcalf, 1970). Given that no hematopoietic cells appeared in the fetal liver following several days in culture, they concluded that the yolk sac was the major site of adult blood formation for the embryo. Although hematopoiesis in the yolk sac is largely primitive in character, progenitors within the yolk sac do give rise to definitive type cells in hematopoietic colony assays, an observation consistent with a yolk sac origin for definitive cells. This view was supported by other experiments in which specific donor-derived T cell populations appeared following transplantation of cells of the yolk sac into fetuses (Weissman et al., 1978).
In more recent work, Nishikawa and colleagues have also challenged the dogma that the yolk sac lacks definitive hematopoietic stem cells (Samokhvalov et al., 2007). The fate of early embryonic tissues was traced in transgenic mice in whichRunx1 regulatory elements drive expression of hormonally activated Cre recombinase. Administration of tamoxifen to pregnant female mice at a particular developmental window permits the fate of cells expressing Runx1(visualized by activation of a Flox-LacZ allele) to be assessed. Treatment of embryos at E7.5 led to prominent marking of fetal liver cells and adult hematopoietic cells. As the yolk sac is the only hematopoietic site at E7.5 and the only tissue known to express Runx1 at E7.5, these findings suggest that the yolk sac contains definitive HSCs (or cells that may give rise to HSCs). These experiments were interpreted to support the yolk sac as a site of HSC formation prior to the AGM, although consensus in the field is far from unanimous (DeWitt, 2007). Diploid-triploid transplants in frogs reveal that ~20% of adult blood in some animals is derived from the ventral blood island (the equivalent to the yolk sac), providing independent evidence that adult hematopoiesis may arise from the yolk sac region. Despite this finding, it is also clear that the analogous AGM region in Xenopus is the predominant contributor to adult hematopoiesis. The precise origin of HSCs in the adult remains a topic for further debate and study.

In Vivo Fate Mapping of Migrating Cells

Presumptive HSCs in the zebrafish express the transcription factors c-myb andRunx1 (see Figure 2). Caged fluorescein dye fate mapping of AGM cells has revealed a new hematopoietic region, the caudal hematopoietic tissue. Laser uncaging is targeted to a region of cells in which transgenic expression of green fluorescent protein (GFP) driven by an HSC-specific promoter marks HSCs in the AGM (Ferkowicz et al., 2003). This approach ensures that laser uncaging occurs specifically within HSCs. Multiple cells are uncaged and their fate is followed (Jin et al., 2007; Murayama et al., 2006). Uncaged cells of the AGM region that express CD41 (a surface marker of early HSCs) or c-myb appear later as fluorescent cell populations in the caudal hematopoietic tissue. The larval and adult site of hematopoiesis in the zebrafish is the kidney. Later on in the fate map experiments, the larval kidney becomes fluorescent, demonstrating that cells of the caudal hematopoietic tissue colonize the kidney. In addition, fluorescence is detected in the thymus. Recent evidence suggests direct population of thymic primordia through tissue planes, a finding consistent with earlier experiments in birds showing migration of progenitors to the thymus along the thoracic duct. Thus, population of the thymus may occur through circulation and direct migration through tissues. Alternatively, the caudal hematopoietic tissue may represent a site similar to the placenta or fetal liver prior to the onset of definitive hematopoiesis in the kidney.
It is generally stated that HSCs of the fetal liver circulate to the adult bone marrow and, hence, are the source of adult hematopoiesis in birds and mammals (see Review by D.J. Laird et al., page 612 of this issue). In contrast, developmental studies reveal that the fetal liver and marrow are seeded at similar times during development (Delassus and Cumano, 1996). Direct tracking of cellular migration is required to distinguish these possibilities.

Pathways Involved in the Emergence of HSCs

The AGM has been characterized largely by morphology and functional assays, but the pathways involved in HSC generation remain incompletely defined. Studies of chick embryos demonstrate that endoderm has a prominent role and secretes inducing factors. Somitic mesoderm also contributes to the dorsal aspect of the aorta, and the addition of factors—such as VEGF, TGF-β, and FGF—to the somitic mesoderm leads to induction of hematopoietic tissue. In contrast, TGF-α and EGF suppressed formation of hematopoietic cells (Pardanaud and Dieterlen-Lievre, 1999).
Signaling pathways that regulate the induction of the AGM have recently been uncovered in mouse and zebrafish. Notch 1 is required for artery identity and aortic HSC production (Kumano et al., 2003). In the zebrafish mutant mindbomb that lacks Notch signaling, Runx1 overexpression rescues HSC production (Burns et al., 2005). Similarly, a Notch1 mutant is rescued by Runx1 overexpression, suggesting that Runx1 lies downstream or parallel to Notch signaling. Other pathways participate in the process including CoupTF-II (Pereira et al., 1999), as well as the CDX-HOX pathway (Davidson et al., 2003).
The Wnt/β -catenin and Notch-Delta signaling pathways influence the function of adult HSCs. Treatment of purified HSCs with Wnt3a protein leads to a modest increase in engrafting cells (Reya et al., 2003). Whereas a pulse of Wnt signaling appears to induce HSCs, constitutive Wnt activation by stabilized β-catenin leads to anemia, possibly by stem cell exhaustion as a consequence of prolonged Wnt signaling (Kirstetter et al., 2006; Scheller et al., 2006). Wnt signaling may be dispensable for adult HSC homeostasis, given that conditional knockout of β- or γ-catenin in hematopoietic cells fails to affect HSC number or engraftment potential (Cobas et al., 2004; Scheller et al., 2006). Stimulation of the Notch pathway also increases HSC activity and appears to be required for the increased self-renewal upon Wnt activation (Duncan et al., 2005). In addition to the Wnt and Notch pathways, new growth factors such as angio-poietin-like proteins appear capable of supporting ex vivo expansion of HSCs (Zhang et al., 2006).
A chemical genetic screen has recently revealed a role for the prostaglandin pathway in the production of HSCs in the zebra-fish. Treatment of embryos with prostaglandin E2 (PGE2) augments stem cell production (North et al., 2007), most likely through the EP4 receptor, a G-coupled receptor specifically expressed in the aorta region and activated by PGE2 (Villablanca et al., 2007). Prostaglandins also affect the homeostasis of definitive adult hematopoiesis, as shown by irradiation recovery assays, 5-fluorouracil stimulation assays, and long-term hematopoietic reconstitution. Thus, the emergence of HSCs in the aorta involves the prostaglandin pathway and the Notch-Runx pathways, which appear to be independent based on genetic relationships.c   n  
The hematopoietic system of the Drosophila embryo generates myeloid-like cells critical for tissue remodeling and engulfment and phagocytosis of dead cells. The emergence of sites of hematopoiesis during embryogenesis is remarkably similar to that of vertebrates. Drosophila progenitors are also formed adjacent to the circulatory system. Hematopoietic progenitors bud off from head mesoderm. These myeloid cells are transient and ultimately replaced by cells that bud off near the heart region and the great vessel. Vascular endothelial growth factor (VEGF) ligands are required for derivation of adult hematopoietic cells, as well as for attracting myeloid cells at specific sites (Cho et al., 2002). Genetic analysis demonstrates that specific signaling pathways, such as Notch, are required for the derivation of the lymph gland and a hemangioblast-like cell population (Mandal et al., 2004). Recent studies demonstrate that the lymph gland of the third instar larva of the fruit fly is patterned and contains a signaling center that expresses Hedgehog ligand (Krzemien et al., 2007; Mandal et al., 2007). Hedgehog cooperates with Notch ligands expressed in these regions to form a stem cell niche and regulates the cycling of hematopoietic progenitors. The search is underway for a similar signaling center in vertebrates. Hedgehog is also required for AGM hematopoiesis in the zebrafish (Gering and Patient, 2005). More recently, studies of human embryonic stem cells have indicated that factors such as hedgehog and bone morphogenetic protein (BMP) promote blood production during in vitro differentiation.

Niches

Stem cells depend on their microenvironment, the niche, for regulation of self-renewal and differentiation. Studies of Drosophila testes and ovarian stem cells have led to formulation of concepts that may be applicable to the niche in other tissues (Decotto and Spradling, 2005; see also the Review by S.J. Morrison and A.C. Spradling, page 598 of this issue). For instance, in the ovary, a hub cell directly binds to a stem cell and regulates its self-renewal and differentiation, in part though BMP signaling (see Minireview by R.M. Cinalli et al., page 559 of this issue). In the testis, an apical hub cell expresses the ligand Upd, an activator of the JAK-STAT signaling pathway in adjacent germ cells to control their self-renewal. By analogy to the Drosophila reproductive organs, investigators have sought an equivalent of the hub cell for the HSC.
As the site of hematopoiesis changes during vertebrate development, the nature of the stem cell niche must also change. The adult bone marrow niche (depicted in Figure 3) has received most attention. Mutant mice in which the BMP pathway is disrupted have increased numbers of osteoblasts and HSCs (Calvi et al., 2003; Zhang et al., 2003). These findings suggest that osteoblasts may represent a critical component of the bone marrow niche for HSCs. As assessed by intravital microscopy, HSCs appear to reside in the periosteal region of calverium marrow (Sipkins et al., 2005). Transplanted GFP-marked or LacZ-marked HSCs appear to lodge adjacent to osteoblasts. Many factors, including ligands for Notch receptors and N-cadherin, are liberated by osteoblasts, although the contribution of these to adult hematopoiesis remains to be established. The role of N-cadherin as a mediator of interactions with osteoblasts (Zhang et al., 2003), as well as the prominence of osteoblasts for HSC adherence, have been challenged (Kiel et al., 2007). Recent findings suggest that HSCs are maintained in a quiescent state through interaction with thrombopoietin-producing osteoblasts (Yoshihara et al., 2007). The association of HSCs with osteo-blasts is countered by other studies that place HSCs adjacent to vascular cells. The chemokine CXCL12 regulates the migration of HSCs to the vascular cells (now called the vascular niche) (Kiel and Morrison, 2006). Taken together, these findings suggest that HSCs reside in various sites within the marrow and that their function might depend on their precise localization. Much of the existing debate may be semantic, however, if the osteoblastic and vascular niches are intertwined and not physically separate. Alternatively, HSCs may truly reside in distinct subregions, which may endow them with different activities. Cellular dynamics within the niche are relevant to clinical marrow transplantation. For example, recent findings suggest that antibody-mediated clearance of host HSCs facilitates occupancy of the niche and transplantation by exogenous HSCs (Czechowicz et al., 2007).




Stem Cell Niche in the Adult Bone Marrow
HSCs are found adjacent to osteoblasts that are under the regulation of bone morphogenetic protein (BMP) (the osteobast niche). HSCs are also found adjacent to blood vessels (the vascular niche). The chemokine CXCL12 regulates the migration of HSCs from the circulation to the bone marrow. The osteoblast and vascular niches in vivo lie in close proximity or may be interdigitated. The marrow space also contains stromal cells that support hematopoiesis including the production of cytokines, such as c-Kit ligand, that stimulate stem cells and progenitors. Cytokines, including interleukins, thrombopoietin (Tpo), and erythropoietin (Epo), also influence progenitor function and survival.


How niches modulate self-renewal is a challenge for future studies. The generation of premalignant myeloproliferative syndromes in mice with abnormal niches underscores the need for precise control in vivo (Perry and Li, 2007). Remarkably, the site of hematopoiesis is not conserved in vertebrate evolution. For instance, the site of adult hematopoiesis is the kidney in fish. The frog forms adult blood in the liver, and birds and mammals form blood in the marrow. In the frog Rana temporaria, the site of hematopoiesis switches between the liver and bone marrow depending on the season (Maslova and Tavrovskaia, 1993).
Little is known regarding the nature of the niche for embryonic hematopoietic sites. Are diverse properties attributed to embryonic, fetal, and adult HSCs due to differences in their respective niches? To what extent are factors shared among different developmental or anatomical niches?

Transcription Factors in Hematopoietic Development

As intrinsic determinants of cellular phenotype, transcription factors provide an entry point for unraveling how HSCs develop during embryogenesis and how lineage-restricted differentiation is programmed (Orkin, 2000). Here we focus on principles and concepts that have emerged and consider how these may inform other organ/tissue systems. Recent reviews provide additional discussion of transcription factors in different hematopoietic lineages (Nutt and Kee, 2007; Iwasaki and Akashi, 2007; Kim and Bresnick, 2007;Rothenberg, 2007). Insights into the functions of the critical transcription factors have rested predominantly on findings from either conventional or conditional gene knockouts in mice and from forced expression experiments, all complemented by developmental studies in other model organisms (e.g., zebrafish, chicken, Drosophila, Xenopus). The transcription factors that are critical for hematopoiesis encompass virtually all classes of DNA-binding proteins, rather than favoring a specific family. A remarkable feature of transcription factors in the hematopoietic system is that the majority are involved in chromosomal translocations or with somatic mutations in human hematopoietic malignancies. Furthermore, experimental manipulation of the genes for such factors in mice often promotes malignancy. Hematopoietic cell fate is intertwined with the origins of leukemias. Requirements for several transcription factors, as established through conventional gene targeting, are summarized in Figure 4 (see also the SnapShot by S.H. Orkin and L.I. Zon, page 631 of this issue).


Requirements of Transcription Factors in Hematopoiesis
The stages at which hematopoietic development is blocked in the absence of a given transcription factor, as determined through conventional gene knockouts, are indicated by red bars. The factors depicted in black have been associated with oncogenesis. Those factors in light font have not yet been found translocated or mutated in human/mouse hematologic malignancies. Abbreviations: LT-HSC, long-term hematopoietic stem cell; ST-HSC, short-term hematopoietic stem cell; CMP, common myeloid progenitor; CLP, common lymphoid progenitor; MEP, megakaryocyte/erythroid progenitor; GMP, granulocyte/macrophage progenitor; RBCs, red blood cells.

For discussion purposes, one may distinguish between factors required for HSC formation or function and those employed in lineage-specific differentiation. Among the ‘‘HSC transcription factors’’ are MLL (for mixed lineage-leukemia gene), Runx1, TEL/ ETV6, SCL/tal1, and LMO2, whose genes account in toto for the majority of known leukemia-associated translocations in patients. In these instances, the translocations either deregulate expression of the locus, as in the case of SCL/tal1 and LMO2 in T cell acute leukemias, or generate chimeric fusion proteins, as in myeloid and lymphoid leukemias associated with MLL, Runx1, andTEL/ETV6. The above distinction is arbitrary in that all of these HSC transcription factors also serve roles later within differentiation of individual blood lineages, and conversely, factors that appear to have more lineage-restricted roles (such as PU.1, Gfi-1, C/EBPα) act within HSCs. The redeployment of transcription factors at different stages of blood cell development is reflected in part in the dynamic patterns of expression of key regulators and complicates analysis of in vivo requirements. Both temporal- and lineage-restricted conditional inactivation are often needed to reveal a meaningful phenotype. These circumstances reflect parsimony with regard to the repertoire of factors required to achieve complex gene regulation and differentiation.

Factors Essential for Formation of HSCs

The transcription factors required to program mesoderm toward a hematopoietic fate are of special interest. The basic-helix-loop- helix (bHLH) factor SCL/tal-1 and its associated protein partner, the Lim-domain containing LMO2, are individually essential for development of both the primitive and definitive (or adult) hematopoietic systems (Kim and Bresnick, 2007). In their absence, no blood cells are generated. Within the primitive system at the yolk sac stage, these factors are thought to function within the hemangioblast to specify a blood rather than a vascular fate. The genes encoding the SET-domain containing histone methyltransferase MLL and runt-domain Runx1 proteins are essential for generation of HSCs within the AGM (and possibly at other sites) (Orkin, 2000). In the absence of Runx1, no hematopoietic clusters (representing presumptive HSCs) form in the dorsal aorta in mice. As noted above, in zebrafish Runx1 lies downstream of Notch signaling, which is required for the induction of hematopoiesis. In addition, BMP signaling restricts hemato-vascular development of lateral mesoderm, possibly acting through a pathway involving LMO2 and presumably GATA-2 (Burns et al., 2005). MLL, like its Drosophila counterpart trithorax, functions in maintenance of, but not initiation of, HOX gene expression. MLL also appears to lie upstream of HOXB4 (and presumably other HOX genes) in HSC specification. Leukemias in which MLL function is perturbed due to chromosomal translocations may arise in part as a secondary consequence of changes in HOX gene expression. Recently, it has been demonstrated that expression of an MLL fusion gene (MLL-AF9) in granulocyte/ macrophage progenitors (GMPs) induces a “HSC stem cell-like” signature that includes various HOX genes (Krivtsov et al., 2006). The acquisition of a stem cell signature by leukemic GMPs may contribute to self-renewal of leukemia stem cells.
Study of a zebrafish mutant defective for blood formation identified the caudal-related factor Cdx4 as an inducer of blood specification (Davidson et al., 2003). Indeed, Cdx4-deficient embryos are rescued by expression of various homeobox genes (e.g., HoxA9) but not by SCL/tal1 (Yan et al., 2006). Activation ofCdx4 expression in mouse ES cells alters the pattern of HOX gene expression, augments in vitro blood formation, and cooperates with HOXB4 in the generation of long-term reconstituting hematopoietic progenitors (Wang et al., 2005). Recent evidence indicates that the pathway to Cdx4 in the zebrafish is initiated by the TATA-box binding protein-related factor 3 (Trf3) (Hart et al., 2007).

Temporal and Stage-Specific Requirements for Hematopoietic Regulators

Within the definitive hematopoietic system, fetal liver and bone marrow HSCs differ in many properties, including cell-surface markers, developmental potential, and cell-cycle status. Transplantation experiments in mice suggest that some characteristics that distinguish fetal liver and adult HSCs are intrinsically regulated (Bowie et al., 2007). Furthermore, HSCs in older age mice exhibit different self-renewal and gene expression patterns than those from younger animals (see Review by D. Rossi et al., page 681 of this issue). How the distinctive properties of HSCs at different developmental stages are programmed is of particular interest in correlating biological read-outs with molecular determinants. Recently, the HMG-box containing factor Sox17, which is also critical to endoderm specification, has been identified as critical for generation of fetal, but not adult, HSCs (Kim et al., 2007). “Geriatric” HSCs are less efficient at homing to and engrafting in the bone marrow, possibly linked to their increased cycling frequency. In addition, the differentiation potential of older HSCs is biased toward myeloid versus lymphoid lineages (Sudo et al., 2000). Several changes in gene expression of old versus young HSCs have been described, including increased expression of leukemia-associated genes and decreased expression of genes contributing to DNA damage repair, genomic integrity, and chromatin remodeling (Rossi et al., 2005; Nijnik et al., 2007). Some of these properties are posited to predispose older HSCs to myeloid leukemias (see Review by D. Rossi et al.).
Moreover, the transcription factors required for specification and formation of HSCs may not be required continuously for the subsequent survival or self-renewal of HSCs. Although SCL/tal1 is an obligate factor for hematopoietic fate specification during development, conditional inactivation in adult HSCs has surprisingly little consequence on maintenance or self-renewal of HSCs and multipotent progenitors (Mikkola et al., 2003). Under these circumstances, the role of this factor in maturation of erythroid and megakaryocytic cells is revealed. Similarly, inactivation of Runx1 in adult HSCs does not ablate HSC properties but instead perturbs differentiation of specific lineages (megakaryocytes, lymphocytes) (Ichikawa et al., 2004). Such observations point to differences in the transcription factor composition of emerging HSCs and adult HSCs and suggest that the phenotype of HSCs is quite stable.

Multilineage Gene Expression in HSCs

Generally, the expression of the lineage-affiliated transcription factors can be readily reconciled with the simple hierarchy diagrams of hematopoiesis (see Figure 1 and Figure 4). For instance, GATA-1 is highly expressed in megakaryocytic/erythroid progenitors (called MEPs) that give rise to megakaryocyte and red blood cell precursors, whereas a “myeloid factor,” such as C/EBPα, is present in GMPs. Indeed, in committed progenitors and precursors one can conveniently match cell-surface phenotypes (defined by monoclonal antibodies) and the subset of hematopoietic transcription factors expressed in these cells. However, this relationship breaks down at earlier stages in the hierarchy. A simple one-to-one correspondence of lineage-restricted transcription factors and progenitors is challenged by findings that earlier multipotential progenitors and HSCs express markers of disparate lineages even within single cells, albeit generally at low levels (Orkin, 2003). This phenomenon, termed lineage priming, suggests that the fate of these immature cells is not sealed and that lineage selection is largely a process in which alternative possibilities are extinguished rather than one in which new programs are imposed on an otherwise blank slate.
Lineage priming may be an efficient means by which chromatin invested in important hematopoietic programs is maintained in an available or open configuration in HSCs. Transient repression of alternative fates, followed by more permanent silencing, maintains the inherent plasticity of multipotential progenitors. Moreover, the coexistence of transcription factors representing different lineages within a common cell (the HSC or immature progenitor) offers the potential for immediate “crosstalk” between different fates at the molecular level (see below).
Recently, by FACS sorting of cells initiating expression GATA- 1 or PU.1, it has been demonstrated that short-term repopulating HSCs may be further subdivided into those committed to myeloerythroid and myelolymphoid lineages (Arinobu et al., 2007). As these findings illustrate, continued fractionation of HSC or progenitor populations reveals increasing diversity in the choice of lineage. Thus, the schematic lineage diagrams that are generally presented cannot be taken literally but rather as guides to the options available to progenitors. The extent to which HSCs exhibit developmental potential beyond hematopoiesis remains controversial (Graf, 2002). Experiments purportedly demonstrating “plasticity” through transplantation of marrow cells to recipient mice are plagued by possible cell fusion of differentiated hematopoietic cells with host cells and by inadequate characterization of input populations.

Mechanisms of Action for Principal Hematopoietic Regulators

The requirements and functions of the principal transcriptional regulators are context dependent (Orkin, 2000). The key lineage- restricted factors are endowed with the complementary tasks of promoting their own lineage differentiation while simultaneously acting against factors favoring other choices (Figure 5). Combining positive and antagonistic roles in the major regulators provides an efficient means for resolving and reinforcing lineage choices. Numerous examples of this principle of lineage programming have been described. GATA-1 and PU.1 promote erythroid/ megakaryocytic/eosinophil and myeloid differentiation, respectively. The proteins physically interact and antagonize each other’s actions. In vivo confirmation of the complementary roles of GATA-1 and PU.1 has been shown in zebrafish. Inhibition of GATA-1expression by morpholinos shifts hematopoietic progenitors to a myeloid fate, whereas the converse occurs upon inhibition of PU.1 expres​sion(Galloway et al., 2005; Rhodes et al., 2005). Other examples of direct antagonism by hematopoietic transcription factors include the relative relationships of C/ EBP and FOG1with respect to eosinophil and multipotential cell fates (Querfurth et al., 2000), EKLF and Fli-1 for erythroid and megakaryocytic choice (Starck et al., 2003), GATA-3 and T-bet for TH1 and TH2 cells (Usui et al., 2006), and Gfi1 and PU.1 for neutrophil versus monocyte outcomes (Dahl et al., 2007). In the absence of Gfi1 in mice, neutrophil precurors fail to mature and also incompletely silence monocyte/macrophage gene expres​sion(Hock et al., 2003).


Transcription Factor Antagonism in Lineage Determination
Examples of antagonism are depicted in red. The transcription factors present in the mature precursors following choice of specific lineage are shown at the bottom in black. Abbreviations: CMP, common myeloid progenitor; MEP, megakaryocyte/erythroid progenitor; GMP, granulocyte/macrophage progenitor; RBCs, red blood cells.


http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2628169/

Red Blood Cells Are All Alike; White Blood Cells Can Be Grouped in Three Main Classes

Blood cells can be classified as red or white. The red blood cells, or erythrocytes, remain within the blood vessels and transport O2 and CO2 bound to hemoglobin. The white blood cells, or leukocytes, combat infection and in some cases phagocytose and digest debris. Leukocytes, unlike erythrocytes, must make their way across the walls of small blood vessels and migrate into tissues to perform their tasks. In addition, the blood contains large numbers of platelets, which are not entire cells but small, detached cell fragments or “minicells” derived from the cortical cytoplasm of large cells called megakaryocytes. Platelets adhere specifically to the endothelial cell lining of damaged blood vessels, where they help to repair breaches and aid in blood clotting. All red blood cells belong in a single class, following the same developmental trajectory as they mature, and the same is true of platelets; but there are many distinct types of white blood cells. White blood cells are traditionally grouped into three major categories—granulocytes, monocytes, and lymphocytes—based on their appearance in the light microscope. Granulocytes contain numerous lysosomes and secretory vesicles (or granules) and are subdivided into three classes according to the morphology and staining properties of these organelles (Figure 22–27).




The differences in staining reflect major differences of chemistry and function. Neutrophils (also called polymorphonuclear leukocytes because of their multilobed nucleus) are the most common type of granulocyte; they phagocytose and destroy microorganisms, especially bacteria, and thus have a key role in innate immunity to bacterial infection. Basophils secrete histamine (and, in some species, serotonin) to help mediate inflammatory reactions; they are closely related to mast cells, which reside in connective tissues but are also generated from the hematopoietic stem cells. Eosinophils help to destroy parasites and modulate allergic inflammatory responses. Once they leave the bloodstream, monocytes (see Figure 22–27D) mature into macrophages, which, together with neutrophils, are the main “professional phagocytes” in the body. Both types of phagocytic cells contain specialized lysosomes that fuse with newly formed phagocytic vesicles (phagosomes), exposing phagocytosed microorganisms to a barrage of enzymatically produced, highly reactive molecules of superoxide (O2 –) and hypochlorite (ClO–, the active ingredient in bleach), as well as to attack by a concentrated mixture of lysosomal hydrolase enzymes that become activated in the phagosome. Macrophages, however, are much larger and longer-lived than neutrophils. They recognize and remove senescent, dead, and damaged cells in many tissues, and they are unique in being able to ingest large microorganisms such as protozoa. Monocytes also give rise to dendritic cells. Like macrophages, dendritic cells are migratory cells that can ingest foreign substances and organisms, but they do not have as active an appetite for phagocytosis and instead have a crucial role as presenters of foreign antigens to lymphocytes to trigger an immune response. Dendritic cells in the epidermis (called Langerhans cells), for example, ingest foreign antigens and carry these trophies back from the skin to present to lymphocytes in lymph nodes. There are two main classes of lymphocytes, both involved in immune responses: B lymphocytes make antibodies, while T lymphocytes kill virusinfected cells and regulate the activities of other white blood cells. In addition, there are lymphocyte-like cells called natural killer (NK) cells, which kill some types of tumor cells and virus-infected cells. . Here we concentrate mainly on the development of the other blood cells, often referred to collectively as myeloid cells. Table 22–1 summarizes the various types of blood cells and their functions.



The Production of Each Type of Blood Cell in the Bone Marrow Is Individually Controlled

Most white blood cells function in tissues other than the blood; blood simply transports them to where they are needed. A local infection or injury in any tissue rapidly attracts white blood cells into the affected region as part of the inflammatory response, which helps fight the infection or heal the wound. The inflammatory response is complex and is governed by many different signal molecules produced locally by mast cells, nerve endings, platelets, and white blood cells, as well as by the activation of complement. Some of these signal molecules act on the endothelial lining of nearby capillaries, helping white blood cells to first stick and then make an exit from the bloodstream into the tissue. Damaged or inflamed tissues and local endothelial cells secrete other molecules called chemokines, which act as chemoattractants for specific types of white blood cells, causing them to become polarized and crawl toward the source of the attractant. As a result, large numbers of white blood cells enter the affected tissue (Figure 22–28).



Other signal molecules produced during an inflammatory response escape into the blood and stimulate the bone marrow to produce more leukocytes and release them into the bloodstream. The regulation tends to be cell-type specific: some bacterial infections, for example, cause a selective increase in neutrophils, while infections with some protozoa and other parasites cause a selective increase in eosinophils. (For this reason, physicians routinely use differential white blood cell counts to aid in the diagnosis of infectious and other inflammatory diseases.) In other circumstances, erythrocyte production is selectively increased—for example, in response to anemia (lack of hemoglobin) due to blood loss, and in the process of acclimatization when one goes to live at high altitude, where oxygen is scarce. Thus, blood cell formation, or hematopoiesis, necessarily involves complex
controls, which regulate the production of each type of blood cell individually to meet changing needs.

Bone Marrow Contains Multipotent Hematopoietic Stem Cells, Able to Give Rise to All Classes of Blood Cells

In the bone marrow, the developing blood cells and their precursors, including the stem cells, are intermingled with one another, as well as with fat cells and other stromal cells (connective-tissue cells), which produce a delicate supporting meshwork of collagen fibers and other extracellular matrix components. In addition, the whole tissue is richly supplied with thin-walled blood vessels, called blood sinuses, into which the new blood cells are discharged. Megakaryocytes are also present; these, unlike other blood cells, remain in the bone marrow when mature and are one of its most striking features, being extraordinarily large (diameter up to 60 μm) with a highly polyploid nucleus. They normally lie close beside blood sinuses, and they extend processes through holes in the endothelial lining of these vessels; platelets pinch off from the processes and are swept away into the blood (Figure 22–29 ).



Because of the complex arrangement of the cells in bone marrow, it is difficult to identify in ordinary tissue sections any but the immediate precursors of the mature blood cells. There is no obvious visible characteristic by which we can recognize the ultimate stem cells. In the case of hematopoiesis, the stem cells were first identified by a functional assay that exploited the wandering lifestyle of blood cells and their precursors. When an animal is exposed to a large dose of x-rays, most of the hematopoietic cells are destroyed and the animal dies within a few days as a result of its inability to manufacture new blood cells. The animal can be saved, however, by a transfusion of cells taken from the bone marrow of a healthy, immunologically compatible donor. Among these cells there are some that can colonize the irradiated host and permanently reequip it with hematopoietic tissue. Such experiments prove that the marrow contains hematopoietic stem cells. They also show how we can assay for the presence of hematopoietic stem cells and hence discover the molecular features that distinguish them from other cells. For this purpose, cells taken from bone marrow are sorted (using a fluorescence- activated cell sorter) according to the surface antigens that they display, and the different fractions are transfused back into irradiated mice. If a fraction rescues an irradiated host mouse, it must contain hematopoietic stem cells. In this way, it has been possible to show that the hematopoietic stem cells are characterized by a specific combination of cell-surface proteins, and by appropriate sorting we can obtain virtually pure stem-cell preparations. The stem cells turn out to be a tiny fraction of the bone marrow population—about 1 cell in 50,000–100,000; but this is enough. A single such cell injected into a host mouse with defective hematopoiesis is sufficient to reconstitute its entire hematopoietic system, generating acomplete set of blood cell types, as well as fresh stem cells. This and other experiments (using artificial lineage markers) show that the individual hematopoietic stem cell is multipotent and can give rise to the complete range of blood cell types, both myeloid and lymphoid, as well as to new stem cells like itself (Figure 22–31).


The sequence of cell fate restrictions shown  in Figure 22–31  conveys the impression of a program executed with computer-like logic and precision. 


Commitment Is a Stepwise Process

Hematopoietic stem cells do not jump directly from a multipotent state into a commitment to just one pathway of differentiation; instead, they go through a series of progressive restrictions. The first step, usually, is commitment to either a myeloid or a lymphoid fate. This is thought to give rise to two kinds of progenitor cells, one capable of generating large numbers of all the different types of myeloid cells, and the other giving rise to large numbers of all the different types of lymphoid cells. Further steps give rise to progenitors committed to the production of just one cell type. The steps of commitment correlate with changes in the expression of specific transcription regulators, needed for the production of different subsets of blood cells.

Divisions of Committed Progenitor Cells Amplify the Number of Specialized Blood Cells

Hematopoietic progenitor cells generally become committed to a particular pathway of differentiation long before they cease proliferating and terminally differentiate. The committed progenitors go through many rounds of cell division to amplify the ultimate number of cells of the given specialized type. In this way, a single stem-cell division can lead to the production of thousands of differentiated progeny, which explains why the number of stem cells is such a small fraction of the total population of hematopoietic cells. For the same reason, a high rate of blood cell production can be maintained even though the stem-cell division rate is low. The smaller the number of division cycles that the stem cells themselves have to undergo in the course of a lifetime, the lower the risk of generating stem-cell mutations, which would give rise to persistent mutant clones of cells in the body—a particular danger in the hematopoietic system where a relatively small accumulation of mutations can be sufficient to cause cancer. A low rate of stem-cell division also slows the process of replicative cell senescence. The stepwise nature of commitment means that the hematopoietic system can be viewed as a hierarchical family tree of cells. Multipotent stem cells give rise to committed progenitor cells, which are specified to give rise to only one or a few blood cell types. The committed progenitors divide rapidly, but only a limited number of times, before they terminally differentiate into cells that divide no further and die after several days or weeks. Figure 22–31 depicts the hematopoietic family tree. It should be noted, however, that variations are thought to occur: not all stem cells generate the identical patterns of progeny via precisely the same sequence of steps.

Stem Cells Depend on Contact Signals From Stromal Cells

Like the stem cells of other tissues, hematopoietic stem cells depend on signals from their niche, in this case created by the specialized connective tissue of the bone marrow. (This is the site in adult humans; during development, and in nonhuman mammals such as the mouse, hematopoietic stem cells can also make their home in other tissues—notably liver and spleen.) When they lose contact with their niche, the hematopoietic stem cells tend to lose their stem-cell potential (Figure 22–32).



Evidently the loss of potency is not absolute or instantaneous, however, since the stem cells can still survive journeys via the bloodstream to colonize other sites in the body.

Factors That Regulate Hematopoiesis Can Be Analyzed in Culture

While the stem cells depend on contact with bone marrow stromal cells for longterm maintenance, their committed progeny do not, or at least not to the same degree. These cells can thus be dispersed and cultured in a semisolid matrix of dilute agar or methylcellulose, and factors derived from other cells can be added artificially to the medium. The semisolid matrix inhibits migration, so that the progeny of each isolated precursor cell remain together as an easily distinguishable colony. A single committed neutrophil progenitor, for example, may give rise to a clone of thousands of neutrophils. Such culture systems have provided a way to assay for the factors that support hematopoiesis and hence to purify them and explore their actions. These substances are glycoproteins and are usually called colony-stimulating factors (CSFs). Some of these factors circulate in the blood and act as hormones, while others act in the bone marrow as secreted local mediators; still others take the form of membrane-bound signals that act through cell–cell contact. An important example of the latter is a protein called Steel or Stem Cell Factor (SCF ). This is expressed both in the bone marrow stroma (where it helps to define the stem-cell niche) and along pathways of migration, and it occurs both in a membrane-bound and a soluble form. It binds to a receptor tyrosine kinase called Kit, and it is required during development for guidance and survival not only of hematopoietic cells but also of other migratory cell types—specifically,
germ cells and pigment cells.

Erythropoiesis Depends on the Hormone Erythropoietin

The best understood of the CSFs that act as hormones is the glycoprotein erythropoietin, which is produced in the kidneys and regulates erythropoiesis, the formation of red blood cells, to which we now turn. The erythrocyte is by far the most common type of cell in the blood (see Table 22–1). When mature, it is packed full of hemoglobin and contains hardly any of the usual cell organelles. In an erythrocyte of an adult mammal, even the nucleus,
endoplasmic reticulum, mitochondria, and ribosomes are absent, having been extruded from the cell in the course of its development (Figure 22–33).



The erythrocyte therefore cannot grow or divide, and it has a limited life-span—about 120 days in humans or 55 days in mice. Worn-out erythrocytes are phagocytosed and digested by macrophages in the liver and spleen, which remove more than 10^11 senescent erythrocytes in each of us each day. Young erythrocytes actively protect themselves from this fate: they have a protein on their surface that binds to an inhibitory receptor on macrophages and thereby prevents their phagocytosis. A lack of oxygen or a shortage of erythrocytes stimulates specialized cells in the kidney to synthesize and secrete increased amounts of erythropoietin into the bloodstream. The erythropoietin, in turn, boosts the production of erythrocytes. The effect is rapid: the rate of release of new erythrocytes into the bloodstream rises steeply 1–2 days after an increase in erythropoietin levels in the bloodstream. Clearly, the hormone must act on cells that are close precursors of the mature erythrocytes. The cells that respond to erythropoietin can be identified by culturing bone marrow cells in a semisolid matrix in the presence of erythropoietin. In a few days, colonies of about 60 erythrocytes appear, each founded by a single committed erythroid progenitor cell. This progenitor depends on erythropoietin for its survival as well as its proliferation. It does not yet contain hemoglobin, and it is derived from an earlier type of committed erythroid progenitor whose survival and proliferation are governed by other factors.

Multiple CSFs Influence Neutrophil and Macrophage Production

CSFs are defined as factors that promote the production of colonies of differentiated blood cells. But precisely what effect does a CSF have on an individual hematopoietic
cell? The factor might control the rate of cell division or the number of division cycles that the progenitor cell undergoes before differentiating; it might act late in the hematopoietic lineage to facilitate differentiation; it might act early to influence commitment; or it might simply increase the probability of cell survival (Figure 22–34).



By monitoring the fate of isolated individual hematopoietic cells in culture, it has been possible to show that a single CSF, such as granulocyte/macrophage CSF, can exert all these effects, although it is still not clear which are most important in vivo. Studies in vitro indicate, moreover, that there is a large element of chance in the way a hematopoietic cell behaves—a reflection, presumably, of “noise” in the genetic control system. If two sister cells are taken immediately after a cell division and cultured apart under identical conditions, they frequently give rise to colonies that contain different types of blood cells or the same types of blood cells in different numbers. Thus, both the programming
of cell division and the process of commitment to a particular path of differentiation seem to involve random events at the level of the individual cell, even though the behavior of the multicellular system as a whole is regulated in a reliable way. The sequence of cell fate restrictions shown earlier, in Figure 22–31, conveys the impression of a program executed with computer-like logic and precision. Individual cells may be more varied, quirky, and erratic, and may sometimes progress by other decision pathways from the stem-cell state toward terminal differentiation.


Regulation of Cell Survival Is as Important as Regulation of Cell Proliferation

The default behavior of hematopoietic cells in the absence of CSFs is death by apoptosis , and the control of cell survival plays a central part in regulating the numbers of blood cells. The amount of apoptosis in the vertebrate hematopoietic system is enormous: billions of neutrophils die in this way each day in an adult human, for example. In fact, most neutrophils produced in the bone marrow die there without ever functioning. This futile cycle of production and destruction presumably serves to maintain a reserve supply of cells that can be promptly mobilized to fight infection whenever it flares up, or phagocytosed and digested for recycling when all is quiet. Compared with the life of the organism, the lives of cells are cheap. Too little cell death can be as dangerous to the health of a multicellular organism as too much proliferation. Mutations that inhibit cell death by causing excessive production of the intracellular apoptosis inhibitor Bcl2 promote the development of cancer in B lymphocytes. Indeed, the capacity for unlimited self-renewal is a dangerous property for any cell to possess. Many cases of leukemia arise through mutations that confer this capacity on committed hematopoietic precursor cells that would normally be fated to differentiate and die after a limited number of division cycles.

Summary

The many types of blood cells, including erythrocytes, lymphocytes, granulocytes, and macrophages, all derive from a common multipotent stem cell. In the adult, hematopoietic stem cells are found mainly in bone marrow, and they depend on signals from the marrow stromal (connective-tissue) cells to maintain their stem-cell character. The stem cells are few and far between, and they normally divide infrequently to produce more stem cells (self-renewal) and various committed progenitor cells (transit amplifying cells), each able to give rise to only one or a few types of blood cells. The committed progenitor cells divide extensively under the influence of various protein signal molecules (colony-stimulating factors, or CSFs) and then terminally differentiate into mature blood cells, which usually die after several days or weeks. Studies of hematopoiesis have been greatly aided by in vitro assays in which stem cells or committed progenitor cells form clonal colonies when cultured in a semisolid matrix. The progeny of stem cells seem to make their choices between alternative developmental pathways in a partly random manner. Cell death by apoptosis, controlled by the availability of CSFs, also plays a central part in regulating the numbers of mature differentiated blood cell

related issues: Unicellular and multicellular Organisms are best explained through design
http://reasonandscience.heavenforum.org/t2010-unicellular-and-multicellular-organisms-are-best-explained-through-design?highlight=unicellular

Embryonic Hematopoiesis

Hemangiogenic precursor cells first arise in the posterolateral mesoderm during gastrulation and from there migrate to the earliest blood-forming organs (Fig. 17.1). Under the influence of Runx-1, some of their progeny follow the hematopoietic lineage, whereas others, responding to Hoxa3 enter the endothelial lineage. Still other progeny will enter a third lineage and eventually form vascular smooth muscle cells. Although blood cell formation (hematopoiesis) begins in the yolk sac, the yolk sac–derived cells are soon replaced by blood cells that are independently derived from other sites of hematopoiesis (Fig. 17.2).



The upper graph highlights the relative importance of the various sites of hematopoiesis. The lower graph shows the percentages of the various hemoglobin polypeptide chains present in the blood at any given time. The α chain is treated separately from the others. AGM, aorta/genital ridge/mesonephros region.
(Based on Carlson B: Patten’s foundations of embryology, ed 6, New York, 1996, McGraw-Hill).

The blood islands contain pluripotential hematopoietic stem cells, which can give rise to most types of cells found in the embryonic blood. The erythrocytes produced in the yolk sac are large nucleated cells that enter the bloodstream just before the heart tube begins to beat at about 22 days’ gestation. For the first 6 weeks, the circulating erythrocytes are largely yolk sac derived, but during that time, preparations for the next stages of hematopoiesis are taking place.
Analysis of human embryos has shown that, starting at 28 days, definitive intraembryonic hematopoiesis begins in small clusters of cells (para-aortic clusters) in the splanchnopleuric mesoderm associated with the ventral wall of the dorsal aorta and shortly thereafter in the aorta/genital ridge/mesonephros (AGM) region. Precursor cells from the AGM region make their way via the blood to blood-forming sites in the liver, the yolk sac, and the placenta. Hematopoietic stem cells formed in the AGM, the yolk sac, and the placenta become transported to the liver via the circulation to the liver (see Fig. 17.1). By 5 to 6 weeks of gestation, sites of hematopoiesis become prominent in the liver. In both the yolk sac and the early sites of embryonic hematopoiesis, the endothelial cells themselves briefly retain the capacity for producing blood-forming cells. There is now evidence that in the AGM region, nitric oxide gas signaling, resulting from shear stress caused by blood flow on the endothelial cells, can induce their transformation into hematopoietic stem cells.
The erythrocytes produced by the liver are quite different from the erythrocytes derived from the yolk sac. Although still considerably larger than normal adult red blood cells, liver-derived erythrocytes are non-nucleated and contain different types of hemoglobin. By 6 to 8 weeks of gestation in humans, the liver replaces the yolk sac as the main source of blood cells. Although the liver continues to produce red blood cells until the early neonatal period, its contribution begins to decline in the sixth month of pregnancy. At this time, the formation of blood cells shifts to the bone marrow, the definitive site of adult hematopoiesis. This shift is controlled by cortisol secreted by the fetal adrenal cortex. In the absence of cortisol, hematopoiesis remains confined to the liver. Before hematopoiesis becomes well established in the bone marrow, small amounts of blood formation may also occur in the omentum and possibly the spleen.

Cellular Aspects of Hematopoiesis

The first hematopoietic stem cells that arise in the embryo are truly pluripotential in that they can give rise to all the cell types found in the blood (Fig. 17.3). These pluripotent stem cells, sometimes called hemocytoblasts, have great proliferative ability. They produce vast numbers of progeny, most of which are cells at the next stage of differentiation, but they also produce small numbers of their original stem cell type, which act as a reserve capable of replenishing individual lines of cells should the need arise. Very early in development, the line of active blood-forming cells subdivides into two separate lineages. Lymphoid stem cells ultimately form the two lines of lymphocytes: B lymphocytes (which are responsible for antibody production) and T lymphocytes (which are responsible for cellular immune reactions). Myeloid stem cells are precursors to the other lines of blood cells: erythrocytes, the granulocytes (neutrophils, eosinophils, and basophils), monocytes, and platelets. The second-generation stem cells (lymphoid and myeloid) are still pluripotent, although their developmental potency is restricted because neither lymphoid cells nor myeloid cells can form the progeny of the other type.



Mature blood cells are shown on the right. CFU, colony-forming unit; GM, granulocyte and monocyte; L, lymphocyte; ML, myeloid and lymphoid; S, spleen.
Stemming from their behavior in certain experimental situations, the hematopoietic stem cells are often called colony-forming units (CFUs). The first-generation stem cell is called the CFU-ML because it can give rise to myeloid and lymphoid lines of cells. Stem cells of the second generation are called CFU-L (lymphocytes) and CFU-S (spleen) (determined from experiments in which stem cell differentiation was studied in irradiated spleens). In some cases, the progeny of CFU-ML and CFU-S are committed stem cells, which are capable of forming only one type of mature blood cell. For each lineage, the forming cell types must pass through several stages of differentiation before they attain their mature phenotype.
What controls the diversification of stem cells into specific cell lines? Experiments begun in the 1970s provided evidence for the existence of specific colony-stimulating factors (CSFs) for each line of blood cell. CSFs are diffusible proteins that stimulate the proliferation of hematopoietic stem cells. Some CSFs act on several types of stem cells; others stimulate only one type. Although much remains to be learned about the sites of origin and modes of action of CSFs, many CSFs seem to be produced locally in stromal cells of the bone marrow, and some may be stored on the local extracellular matrix. CSFs are bound by small numbers of surface receptors on their target stem cells. Functionally, CSFs represent mechanisms for stimulating the expansion of specific types of blood cells when the need arises. Recognition of the existence of CSFs has prompted considerable interest in their clinical application to conditions characterized by a deficiency of white blood cells (leukopenia).
Certain Hox genes, especially those of the Hoxa and Hoxb families, play an important role in some aspects of hematopoiesis. Exposure of bone marrow to antisense oligonucleotides against specific Hox genes results in the suppression of specific lines of differentiation of blood cells. Conversely, engineered overexpression of genes, such as Hoxb8, Hoxa9, and Hoxa10, causes leukemia in mice. Evidence is increasing for the involvement of Hox genes in the pathogenesis of human leukemias. One important function of the Hox genes in hematopoiesis is the regulation of proliferation. Several growth factors, especially bone morphogenetic protein-4 (BMP-4), Indian hedgehog, and Wnt proteins, are important in stimulating and maintaining hematopoietic stem cell activity.


Erythropoiesis


Red blood cell formation (erythropoiesis) occurs in three waves during the embryonic period. The first wave begins with precursors within the yolk sac, which produce primitive nucleated erythrocytes that mature within the bloodstream. The second wave also begins in the yolk sac, but the precursor cells then colonize the embryonic liver and produce the first of a generation of definitive fetal erythrocytes that are dominant during the prenatal period. The third wave consists of precursor cells that enter the liver from the AGM mesoderm and the placenta. Some of these definitive erythroid progenitor cells send progeny directly from the liver into the bloodstream as definitive fetal erythrocytes. Others seed the bone marrow and produce adult-type erythrocytes later in the fetal period.
The erythrocyte lineage represents one line of descent from the CFU-S cells. Although the erythroid progenitor cells are restricted to forming only red blood cells, there are many generations of precursor cells (Fig. 17.4). The earliest stages of erythropoiesis are recognized by the behavior of the precursor cells in culture, rather than by morphological or biochemical differences. These are called erythroid burst-forming units (BFU-E) and erythroid CFUs (CFU-E). Each responds to different stimulatory factors. The pluripotent CFU-S precursors (see Fig. 17.3) respond to interleukin-3, a product of macrophages in adult bone marrow. A hormone designated as burst-promoting activity stimulates mitosis of the BFU-E precursors (see Fig. 17.4). A CFU-E cell, which has a lesser proliferative capacity than a BFU-E cell, requires the presence of erythropoietin as a stimulatory factor.


Erythropoietin is a glycoprotein that stimulates the synthesis of the mRNA for globin and is first produced in the fetal liver. Later in development, synthesis shifts to the kidney, which remains the site of erythropoietin production in adults. Under conditions of hypoxia (e.g., from blood loss or high altitudes), the production of erythropoietin by the kidneys increases, thereby stimulating the production of more red blood cells to compensate for the increased need. In adult erythropoiesis, the CFU-E stage seems to be the one most responsive to environmental influences. The placenta is apparently impervious to erythropoietin, and this property insulates the embryo from changes in erythropoietin levels of the mother and eliminates the influence of fetal erythropoietin on the blood-forming apparatus of the mother.
One or two generations after the CFU-E stage, successive generations of erythrocyte precursor cells can be recognized by their morphology. The first recognizable stage is the proerythroblast (Fig. 17.5), a large, highly basophilic cell that has not yet produced sufficient hemoglobin to be detected by cytochemical analysis. Such a cell has a large nucleolus, much uncondensed nuclear chromatin, numerous ribosomes, and a high concentration of globin mRNAs. These are classic cytological characteristics of an undifferentiated cell.



Succeeding stages of erythroid differentiation (basophilic, polychromatophilic, and orthochromatic erythroblasts) are characterized by a progressive change in the balance between the accumulation of newly synthesized hemoglobin and the decline of first the RNA-producing machinery and later the protein-synthesizing apparatus. The overall size of the cell decreases, and the nucleus becomes increasingly pyknotic (smaller with more condensed chromatin) until it is finally extruded at the stage of the orthochromatic erythrocyte. After the loss of the nucleus and most cytoplasmic organelles, the immature red blood cell, which still contains a small number of polysomes, is a reticulocyte. Reticulocytes are released into the bloodstream, where they continue to produce small amounts of hemoglobin for 1 or 2 days. The final stage of hematopoiesis is the mature erythrocyte, which is a terminally differentiated cell because of the loss of its nucleus and most of its cytoplasmic organelles. Erythrocytes in embryos are larger than their adult counterparts and have a shorter life span (50 to 70 days in the fetus versus 120 days in adults).

The Blood  1

In order to perform the functions of (1) transportation, (2) regulation, and (3) protection, the circulatory system relies on several key components. All of these components can be found in the fluid that travels within these living tubules—blood. Commenting on the remarkable properties of this fluid, Bruce Alberts and his colleagues noted: “Blood contains many types of cells with very different functions, ranging from the transport of oxygen to the production of antibodies. Some of these cells function entirely within the vascular system, while others use the vascular system only as a means of transport and perform their function elsewhere (1994, p. 1161).
The total volume of blood represents only 8% of the total weight of a human (Van de Graaff and Fox, p. 655). Ironically, it is classified as a tissue, but it is one of the few substances in the human body that is not “fixed” in place. Tissues such as nerves, muscles, and organs have a specific function and are limited in movement. Blood, however, is not limited to any one part of the body. This ability to move allows blood to provide these “fixed” tissues with nourishment and then carry off waste products. Blood itself is composed of a cellular portion referred to as formed elements, and a fluid portion designated as plasma. The formed elements constitute approximately 45% of the total volume of blood (Van de Graaff and Fox, p. 655) and are comprised of erythrocytes (red blood cells), leukocytes (white blood cells), and platelets. Plasma is a straw-colored liquid that consists primarily of water and dissolved solutes. Approximately 89% of plasma is water, 9% is protein material (e.g., albumins), 0.9% is salts, and 0.9% is sugar, urea, etc.
In considering whether the circulatory system could be the product of evolution, one must consider that the acidity of blood is critical to survival. Key salts provide basic ions, such as sodium, potassium, phosphate, and magnesium that help maintain a steady pH value for the blood. These bicarbonate ions remove carbon dioxide from the tissues and help maintain a slightly alkaline pH of 7.4. During traumatic injuries or surgeries, a great deal of attention is given to the pH of the blood since a significant decrease or loss of this alkalinity can cause rapid and violent breathing, with death likely to occur at a pH of 7.0 or below. Conversely, if the pH of the blood is allowed to go beyond 7.6, it also can prove fatal. The Lange medical book on “Fluid and Electrolytes” records: “The control of blood pH is critically important since modest swings—e.g., 0.10-0.20 pH unit in either direction—can cause symptoms referable to impaired cardiopulmonary performance and neurologic function. More extreme pH changes can be fatal” (see Co­gan, 1991, p. 175). This narrow, “unforgiving” pH range would not be expected if blood were the product of millions of years of evolution. If blood were the product of evolution and millions of years of random change, one would expect nature to have selected for a fluid that was not dependent on such a critical pH level—especially since slight pH variations can prove fatal!
The salinity in our blood stream has made some evolutionists speculate that this is evidence we evolved out of the sea. For instance, Robert Lehrman noted:

One human characteristic, a chemical one, harks back to our ancestry in the ocean.... The percentage of sodium, potassium, calcium, magnesium, iodine, chlorine, and other minerals in human blood salt coincide with those of sea water. Our ocean-living ancestors developed cells adapted to the chemical environment of sea water. When they left the ocean, they took a part of the environment with them in the form of a fluid that bathes the cells; later it was incorporated into the blood stream (as quoted in Batten, 1997, p. 24).

However, on average, the concentration of sodium chloride (salt) in sea water is 2.7% higher than we find in the human blood stream (additionally, the sea contains 0.8% other salts, some of which are not present in blood and would not benefit the cardiovascular system). Creationist Don Batten carried out an extensive comparison demonstrating the actual percentages of salts and minerals found in human blood and seawater. He noted that there is little similarity between human blood and seawater and reported that “even the blood of sea creatures such as crabs is quite different from seawater” (1997, p. 24). If evolutionists took the time to do the math, they would find that the Baltic Sea—one of the “fresher” large bodies of water—still is much too salty to have played any physiological part in the production of blood.
Erythrocytes (also known as red blood cells) are the most common of the formed elements. Adult humans have approximately 2-3 x 1013 red blood cells at any given time (see Wikipedia, n.d.). These cells provide oxygen to tissues, and assist in the disposal of carbon dioxide. In humans, red blood cells are devoid of nuclei (i.e., they are anucleated) and intracellular organelles, while birds, amphibians, and other animals have red blood cells that are nucleated. This key difference should not be overlooked in light of our alleged evolutionary origins. All cells require a nucleus for replication and maturation. Even red blood cells have a nucleus during their very early stages of development. However, in humans, the production of red blood cells occurs in the bone marrow, and thus we do not normally see these nucleated cells in the circulation (although they occasionally are found in newborns). An obvious question is: How did humans “evolve” cells that would mature without a nucleus? And furthermore, why would nature select for this? By losing their nuclei, these cells are unable to replicate like other cells within the body. The body is dependent on pluripotent stem cells within bone marrow for future erythrocyte production. With a lifespan of only 120 days and no nuclei, they must be constantly produced in order to carry oxygen throughout the body. Each second about 2.5 million new erythrocytes are produced or about 200 billion each day (see “Cardiovascular System,” 2004)! Some animals produce these cells intravascularly (i.e., in the blood stream), whereas humans and other animals produce them extra­vas­cu­larly (in the bone marrow or other hema­topoietic tissue). Additionally, this loss of cellular organelles means that these cells are unable to produce energy, and thus, they must get energy from anaerobic respiration. Anaerobic respiration in red blood cells is a complex cascade of events that puts even more impossible explanatory demands on evolutionists.
Red blood cells are formed by a process known as erythropiesis. It takes approximately seven days for these cells to develop, and then they are released into the blood stream. Old red blood cells are “engulfed by phagocytes, destroyed, and their materials are released in the blood. The main sites of destruction are the liver and spleen” (see Wikipedia, n.d.). During their lifetime these specialized cells travel

over 100 miles, are buffeted at high velocities during their passage through the heart, and have to negotiate tiny capillaries.... As they age, subtle structural changes occur which render them identifiable to scavenger cells in the spleen and elsewhere, and they end their days being devoured and digested by these predators (Blakemore and Jennett, 2001, p. 85).

However, this process must be orchestrated or else the individual will suffer from having too many or insufficient numbers of red blood cells in the blood stream. Consider the fate of an individual unable to breakdown aged red blood cells, or someone who is unable to produce replacements. How did this feedback mechanism arise? Blakemore and Jennett observed:

Their rate of production is beautifully adapted to this function. It is regulated by a hormone called erythropoietin, produced in the kidney in the adult and in the liver in the fetus. Close to the gene that regulates erythropoietin production are regions of DNA that sense oxygen tension; when this falls, erythropoietin synthesis is stimulated, and more red cells are produced in the bone marrow. When adequate oxygenation of tissues is achieved, erythropoietin production is reduced. By this biological feedback loop the body is able to respond to varying oxygen demands by modifying the rate of red cell production (2001, p. 85, emp. added).

This system is also irreducibly complex. All of the parts are necessary in order for the feedback mechanism to work properly. So how was this “beautifully adapted” feedback loop able to evolve in a series of evolutionary steps? The truth is that it could not!
As the red blood cell matures and is ready to leave the bone marrow, it expels its nucleus. The reason for anucleated red blood cells in humans is directly related to function—the unique shape and loss of nucleus provides added surface area through which gas can diffuse (Van de Graaff and Fox, 1989, p. 656; Blakemore and Jennett, p. 85). The anucleated biconcave shape increases surface area and allows the cell to remain flexible enough to squeeze through small capillaries. Even an anucleated red blood cell is larger (8µm) than capillaries (2-3µm). However, without the nucleus present, the red blood cell is flexible and able to fold over on itself. For how many “millions of years” was development limited as red blood cells slowly “evolved” the ability to shed their nucleus, develop anaerobic respiration for energy needs, and finally become flexible and able to fold into capillaries? The functional design of the anucleated red blood cell’s shape (a biconcave disc) can only be explained by the ultimate Designer.
In addition, red blood cells contain hemoglobin, which is responsible for carrying oxygen to every cell in the body. Hemoglobin is a complex protein that has two pairs of chains (referred to as alpha and beta) which bind to the red-pigmented molecule known as heme. As Blakemore and Jennett described: “In most mammals, adult hemoglobin (hemoglobin A) comprises two unlike pairs of chains of amino acids, or globin chains, called α and β, each of which is folded round one iron-containing heme molecule, to which oxygen can bind” (p. 85). This configuration allows hemoglobin to transport four molecules of oxygen. Given the added surface area from the anucleated biconcave disc, each cell would contain “about 280,000,000 molecules of hemoglobin” (see “Cardiovascular System,” 2004). What are the odds that this engineering accomplishment happened by random chance? Consider that an evolutionary origin of hemoglobin would require a minimum of 120 mutations to convert an alpha chain to a beta. At least 34 of those changes require changeovers in 2 or 3 nucleotides. Yet, if a single nucleotide change occurred through mutation, the result would ruin the blood and kill the organism. Simply put, evolution cannot explain the existence of hemoglobin molecules in the circulatory system of humans.

Another component of formed elements are leukocytes (white blood cells). Unlike red blood cells, leukocytes contain nuclei and mitochondria and can purposefully move in an amoeboid fashion (Van de Graaff and Fox, p. 657). Leukocytes serve as the primary line of defense in the vascular system. There are five different leukocytes present in the blood: neutrophils, eosinophils, basophils, lymphocytes, and monocytes. These cells will be covered in greater detail with the immune system, but the point should not be missed that these specialized cells traverse the circulatory system and are critical to the survival of individuals (e.g., consider immune deficient individuals). Yet, evolutionary theory is unable to explain adequately their origin.

The final formed element found within the blood is platelets. Platelets are much smaller than red blood cells, and serve to stop blood loss from wounds (hemo­stasis). Consider for just a moment the conundrum posed by blood clotting. It is critical that the circulatory system have a means to prevent blood loss when it is damaged, but this process must only occur when needed, and humans cannot afford to have this clot spread to healthy vessels. Not only must a clotting network be available, but also there must be an effective means of terminating the clotting cascade once the vessels have been repaired. In his book Darwin’sBlack Box, biochemist Michael Behe observed:

When a pressurized blood circulation system is punctured, a clot must form quickly or the animal will bleed to death. If blood congeals at the wrong time or place, though, then the clot may block circulation as it does in heart attacks and strokes. Furthermore, a clot has to stop bleeding all along the length of the cut, sealing it completely. Yet blood clotting must be confined to the cut or the entire blood system of the animal might solidify, killing it. Consequently, the clotting of blood must be tightly controlled so that the clot forms only when and where it is required (2003, pp. 78-79).

Behe then goes into painstaking detail to record the biochemical cascade that must transpire in order for blood clotting to occur. For over fifteen pages he records all of the events that are required in order for this process to occur (see pages 81-97). Figure 4-3 of his book shows a flowchart of the proteins involved in the blood coagulation cascade. Aside from there being dozens of proteins required, many are produced in response to complex feedback loops within the cascade itself. The statistical odds of evolving those specific proteins in just the right manner, in just the right location, and having them available when that first land animal was getting scratched up as it slowly crawled over rocks and shells onto dry land are incalculable! Only God could create such a complex system that can heal itself so precisely.

1) http://www.apologeticspress.org/ApPubPage.aspx?pub=1&issue=571&article=450

The critical contribution of repression to lineage selection is illustrated by loss-of-function studies of Pax5. Proper B cell development requires Pax5 (Nutt and Kee, 2007). In its absence, proB cells assume a multipotential phenotype and differentiate (under appropriate growth factor conditions) to T-, NK-, or dendritic cells, macrophages, neutrophils, or erythroid precursors. Repression of critical growth factor receptors, for example macrophage-colony-stimulating factor (M-CSF) receptor, restricts lineage choice during normal development. In effect, Pax5 commits progenitors to a B cell fate, while other B cell transcription factors, such as E2A and EBP, specify lineage-appropriate gene activation.
In T-lymphoid development, Notch signaling serves a similar but more focused role as it functions as a commitment factor, principally by repressing factors, such as PU.1, associated with other outcomes. Although GATA-3 has been viewed as a T cell-specific transcription factor, recent work indicates that it functions only in the context of Notch signaling to promote T cell development (Rothenberg, 2007). As recently stated, lineage programming in the T cell lineage is more the consequence of “negotiation” rather than instruction.

Mechanisms of Lineage Programming

The context-dependent action and direct antagonism between key transcriptional regulators are best accommodated by models in which factors interact directly within protein complexes (Orkin, 2000). In this regard, the GATA factors are illustrative (Kim and Bresnick, 2007). A protein complex in erythroid cells comprised of GATA-1 (or its close relative, GATA-2), LMO2 and its partner Ldb1, and SCL/tal1 and its heterodimeric partner E2A recognizes a consensus GATA-E-box DNA motif. Knockout of either LMO2 orSCL/tal1 leads to the absence of any hematopoietic progenitors in the early embryo. The identical phenotypes are consistent with the action of these proteins within a single complex that is required prior to any commitment to erythroid differentiation. Indeed, forced expression of GATA-1/2, SCL, and LMO2 converts Xenopus mesoderm efficiently to a hematopoietic fate. Remarkably, although the GATA/SCL/ LMO2/Lbd1 complex recognizes a composite DNA-binding site, later studies demonstrated that the DNA-binding activity of SCL (in a heterodimer with E2A) is dispensable for hematopoietic specification but required for full erythroid and megakaryocytic cell maturation (Porcher et al., 1999). The recruitment of DNA-binding-defective SCL to the protein complex accounts for in vivo function despite its inability to bind to DNA.
GATA factors form alternative protein complexes with a specific cofactor known as FOG (for friend of GATA) (Kim and Bresnick, 2007). Targeted mutation in mice has demonstrated the essential role of the GATA/FOG interaction for erythroid and megakaryocytic development. The GATA-1 (or GATA-2)/FOG1 complex in both erythroid and megakaryocytic lineages is physically associated with the NuRD chromatin remodeling complex via an NuRD binding motif at the N terminus of FOG1. The interaction of FOG1 with GATA factors mediates transcription repression and also may facilitate accessibility of the GATA factor to its DNA-binding motif in chromatin. GATA/SCL/LMO2/Ldb1 and GATA/FOG1 complexes appear to be mutually exclusive.
Other connections between hematopoietic factors and chromatin-associated proteins amplify this theme. Ikaros proteins also associate with the NuRD complex and participate in repression and the formation of heterochromatin (Kim et al., 1999). The erythroid factor EKLF interacts with Brg1, a critical component of the Swi/Snf ATP-dependent chromatin remodeling complex (Brown et al., 2002). Consistent with the relevance of this relationship to biological function, a Brg1 mutant protein, which retains ATPase activity and assembles into the Swi/Snf complex, fails to generate DNase I hypersensitivity and leads to embryonic lethality due to failure to activate normal β-globin expres​sion(Bultman et al., 2005). Furthermore, another erythroid factor NF-E2 associates with the MLL2 complex, which is responsible for H3K4 histone methylation (Demers et al., 2007). In addition, the zinc-finger repressor Gfi protein recruits a CoREST/lysine demethylase (LSD1) complex to target genes in erythroid, megakaryocytic, and myeloid cells to control maturation of these lineages (Saleque et al., 2007). Further elucidation of the ways in which hematopoietic transcription factors interact and function with chromatin-associated factors in HSCs and individual lineages is an on-going challenge for the field.
The involvement of multiprotein complexes in the action of he-matopoietic transcription factors predicts that relative concentrations of particular factors should influence the choice of lineage. Protein complexes constitute a convenient platform for direct competition, as well as for cooperative action. Considerable data argue for concentration-dependent effects in lineage choice and differentiation. Experiments in zebrafish cited above in which the pattern of erythroid versus myeloid development is affected by inhibition of expression of GATA-1 or PU.1 (Galloway et al., 2005; Rhodes et al., 2005) are consistent with this model. The relative action of GATA-1 and PU.1 fits a simple quantitative model that predicts a metastable undifferentiated progenitor state when both proteins are present at low levels and differentiation to one of two alternatives when one of the proteins is present at higher levels (Roeder and Glauche, 2006). Taking advantage of PU.1-null fetal liver progenitors, Singh and colleagues have proposed that high-level PU.1 expression directs macrophage differentiation, whereas low-level expression promotes B cell formation (DeKoter and Singh, 2000). Subsequently, they proceeded to show that low-level PU.1-expressing bi-phenotypic (macrophage/neutrophil) cells are relatively stable, and increased PU.1 levels promote macrophage differentiation by influencing a circuit of counterantagonistic repressors, Egr-1/2/Nab-2 and Gfi1 (Laslo et al., 2006). A mathematical model, similar to that described above for GATA-1 and PU.1, has been derived to account for these observed effects in the two myeloid lineages.
MicroRNAs (miRNAs) provide an additional level of control beyond the transcription factors (Shivdasani, 2006) (see Minireview by B. Stadler and H. Ruohola-Baker, page 563 of this issue). Ongoing studies of the involvement of miRNAs in hematopoiesis will reveal their roles in lineage decisions, stem cell to progenitor transitions, niche control, and cell function. The transcription of blood cell-specific miRNAs is likely to be driven by the complexes discussed above, providing a complex regulatory network. Several miRNAs are highly expressed in specific hematopoietic lineages and manipulation of their levels has been correlated with changes in cellular properties or differentiation (Chen et al., 2004). For example, miR-150 impinges on B cell differentiation by targeting c-myb mRNA (Xiao et al., 2007), whereas miR-155 is required for T helper cell generation and germinal center activity (Rodriguez et al., 2007; Thai et al., 2007). Moreover, conditional inactivation of the gene encoding Dicer, an essential component in the processing of pre-miRNAs to miRNAs, leads to multiple defects in T-lineage lymphopoiesis (Muljo et al., 2005; Cobb et al., 2006). Unpublished experiments indicate that Dicer is also required in the setting of bone marrow transplantation for radioprotection of lethally irradiated recipient mice (cited in Martinez and Busslinger, 2007).

Lineage Reprogramming

Cellular differentiation was once considered unidirectional, that is, once progenitors have committed to a particular linear pathway their fate is sealed. Accumulating evidence in the hematopoietic system and in other systems (see Review by R. Jaenisch and R. Young, page 567 of this issue) dispels this notion and provides a strong foundation for cellular reprogramming. Indeed, cells of one hematopoietic lineage can be converted to another through the forced expression of carefully chosen transcription factors (Figure 6). Knowledge of the rules governing how transcription factors that direct cellular lineages act informs directed attempts to reprogram one lineage to another. In an early example based on such logic, Querfurth et al. (2000) queried the significance of the lack of FOG-1 in eosinophils, despite their dependence on GATA-1 for differentiation. Forced expression of FOG-1 in avian eosinophils downregulated expression and function of C/EBP β, an essential eosinophil factor, leading to acquisition of a multipotent phenotype. Conversely, downregulation of FOG-1 by C/EBP β is a critical step in eosinophil lineage commitment. In other experiments, forced expression of GATA-1 was shown to drive early myeloid avian progenitors to erythroid, eosinophilic, or thromboblastic (megakaryocytic) precursors (Kulessa et al., 1995). Moreover, introduction of GATA-1 into GMPs and common lymphoid progenitors redirects their commitment to megakaryocytic/erythroid progenitors or to erythroid cells/mast cells/basophils (Iwasaki and Akashi, 2007). Committed B- and T-lymphoid cells can be reprogrammed to functional macrophages through expression of C/EBPα (Laiosa et al., 2006; Xie et al., 2004). Furthermore, preT cells can be reprogrammed to myeloid dendritic cells through PU.1 expression. Cells that are reprogrammed transit through an intermediate state in which markers of both myeloid and lymphoid lineages are expressed, indicative of the stepwise nature of the process. Resolution of the intermediate state leads to stable unilineage differentiation. Notch and GATA-3 appear to counteract reprogramming of preT cells to macrophages or dendritic cells. Recently, GATA- 3, traditionally viewed as a T cell-restricted factor, has been shown to direct mast cell reprogramming from proT cells (Taghon et al., 2007). In addition, mid-stage thymocytes (at the DN2 stage) can be converted to mast cells through growth in culture medium containing interleukin (IL)-3 and stem cell factor (Kit-ligand) in the absence of Notch.



Reprogramming of Hematopoietic Lineages

The orange arrows depict lineage reprogramming upon expression of the transcription factors GATA-1, C/EBP, or GATA-3. Abbreviations: HSC, hematopoietic stem cell; CMP, common myeloid progenitor; CLP, common lymphoid progenitor; MEP, megakaryocyte/erythroid progenitor; GMP, granulocyte/ macrophage progenitor.

Cancer: A Perturbation of the Hematopoietic Transcriptional Network

Of the more than two dozen regulators designated “hematopoietic transcription factors,” nearly all are intimately associated with hematopoietic malignancy (Figure 4). Indeed, the majority of genes encoding these factors were discovered either through analysis of chromosomal translocations found in human leukemias or study of cooperating leukemia genes during insertional mutagenesis in the mouse. Disturbance of the homeostatic balance of the critical transcriptional regulators is a defining feature of leukemias. In the setting of chromosomal translocations that lead to misexpression of a factor, such asSCL/tal1 or LMO2, target genes may be inappropriately activated or repressed in early lymphoid progenitors. Chimeric transcription factors generated through chromosomal translocations exert multiple downstream effects, including improper target gene activation or repression, inhibition of function of other critical factors, and recruitment of alternative chromatin-modifying enzymes to target loci (Rosenbauer and Tenen, 2007). Leukemia is not the consequence of nonspecific transcriptional effects but rather the end result of attacks at vulnerable points in a network. This observation is best exemplified by somatic mutations in GATA-1 in Down Syndrome- associated megakaryocytic leukemia (Wechsler et al., 2002),PU.1 and C/EBPα in myeloid leukemias (Mueller et al., 2002; Pabst et al., 2001), and Pax5 and other B cell factors (for example, E2A and EBF) in B-lymphoid leukemias (Mullighan et al., 2007). In addition to somatic mutation of the principal hematopoietic transcription factors, lesions in more broadly utilized signaling pathways that control specific lineage differentiation may underlie hematopoietic malignancy. This circumstance is illustrated by consistent somatic mutation of Notch in T cell leukemias (Weng et al., 2004). As an essential component of the regulatory network for T cell commitment and development, Notch is a preferred target for somatic mutation in T cell leukemia.

Messages for the Wider Stem Cell Field

As arguably the most “mature” organ system under study, the hematopoietic system constitutes a model for other subfields of stem cell biology. The hematopoietic system continues to evolve as a model, however, as numerous critical issues remain to be addressed. It is likely that many of the concepts derived from work in the blood field will be revisited in other organ systems. Nonetheless, it is also apparent that nature has explored alternative pathways to tissue organization and development. Hence, differences should be anticipated, particularly with respect to the extent to which true stem cells are required for the maintenance of different tissues or cell populations within an organ. Adult stem cell populations are generated during embryogenesis. For other organs, distinct stage-specific programs regulate stem cell homeostasis and tissue differentiation. As illustrated by umbilical cord blood stem cells, transient populations may have therapeutic value. Reflection on the history and recent advances in the hematopoietic field leads to several “lessons” for the stem cell field:

• Precise characterization of the cells within the hematopoietic hierarchy has been instrumental in providing an adequate framework for biological and molecular studies. The prospective isolation of subsets of cells, coupled with in vitro colony-forming assays and quantitative in vivo transplantation methods, has greatly facilitated molecular studies of both normal and malignant hematopoiesis. HSCs differ in their properties depending on their location (fetal liver, bone marrow, placenta) and on the age of the organism. Hence, thorough cell biological studies are fundamental to approaching mechanisms that regulate stem cell function.
  
• The “classical” hierarchy diagram depicting progenitors arising in an orderly fashion from a prototypical HSC provides a seductive, but overly simplified view. HSCs may be described more accurately as groups of cells with varying developmental potentials based on intrinsic networks driven by transcription factors and inputs from the cellular niches in which they reside. Processes, such as lineage priming coupled with plastic decisions driven by transcription factor competition, confer great flexibility on the options of HSCs. The molecular events of self-renewal must be coordinated with these steps. Current views of HSCs must account for a spectrum of cells with varying engraftment potential and distinct biases toward subsequent myeloid or lymphoid lineage choices. These complex features of HSC biology are likely to be revisited in studies of other tissue-dedicated stem cells.
  
• Parallel investigation in diverse species has accelerated an understanding of blood cell development. Although an important goal is application of fundamental knowledge of hematopoietic stem cell biology to the treatment of human disease, studies in mice, fish, and flies are mutually reinforcing. Although the anatomical features of blood formation in the latter organisms differ from that of mammals, critical growth factor signaling and transcriptional pathways are shared. Parallel investigation of different species takes advantage of the strengths of each and complements work with human hematopoietic cells.
  
• Elucidation of the transcriptional network that controls lineage choice and differentiation sets the stage for directed reprogramming of cellular lineages. Knowing the principal factors that govern the cellular lineages identifies candidate regulators for functional testing. Further study of the sequence of molecular events accompanying lineage conversion is likely to provide important mechanistic clues regarding how complex cellular reprogramming occurs (Rossant, 2007).
  
• The remarkable link between hematopoietic transcription factors and malignancy underscores how disturbance of a transcriptional network lies at the crux of oncogenesis. As most hematopoietic transcription factors were discovered through study of chromosomal translocations in leukemia, investigators should pursue the normal developmental functions of genes disrupted or brought into fusion products in various solid tumors, both in sarcomas and epithelial cancers, given the recent appreciation for the importance of translocations in these settings. More generally, the transcriptional regulators participating in cell-specific gene expression provide entry points into the transcriptional network controlling self-renewal and differentiation, the hallmarks of stem cells.
  

Serial cell differentiation: intricate system of design 1

Single celled organisms replicate as fully functional cells, and they maintain cellular integrity through a system of direct epigenetic inheritance,1 or ‘cell memory’. Some tissues in multicellular organisms proliferate in the same way. However, the majority of tissues in adult multicellular organisms don’t.



Figure 1. The process of hematopoiesis (the generation of blood cells) is an example of the serial cell differentiation process.

Most tissues in mature multicellular organisms replicate via a method called serial differentiation.2 Cells go through a series of differentiation stages as they duplicate, ending in a fully differentiated cell, which eventually dies and passes out of the system, or is recycled by apoptosis (programmed cell death). There are three different types of cells in this system: stem cells, a class called ‘transient amplifying cells’ (TACs) and fully differentiated cells.

Serial differentiation

Stem cells

The undifferentiated cells are the only ones in this differentiation process that are self-renewing, i.e. they produce daughter cells that are exactly like the mother cell. These cells have the capacity to divide and change into many different types of cells. They are also very important during embryonic development, where new cell types are constantly needed.3 These stem cells are kept relatively few in number, and the cell lines proliferate through the differentiation process.

Transient amplifying cells

The daughters of stem cells do more than just self-renew; they differentiate into different kinds of cells. However, they don’t change into fully differentiated cells immediately; they change into a class called ‘transient amplifying cells’ (TACs). While TACs divide; unlike stem cells, TACs do not self-renew. Rather, the daughter cells of TACs are one stage further along the differentiation process than the ‘mother’ cell. These cells amplify the number of cells that will eventually become fully differentiated from the original stem cell that they started from.

Fully differentiated cells

A particular stem cell goes through a number of cell division events and the differentiation process of the TAC stage to produce fully differentiated cells. These are the mature cells that carry out the different jobs of the tissues, such as blood cells (figure 1), reproductive cells and epithelial cells. These cells no longer divide or differentiate, and once they have served their purpose, they are ‘deleted’ from the system and their components are recycled.4

Designed for maintenance

This is a rather elaborate system to conjure up if you just want to maintain tissues! It is also metabolically expensive because not only do the mature cells require nutrients, but so do the stem cells and TACs. Therefore, you’re feeding cells that don’t actually do anything in the body except replicate. So why bother using so much energy?

As Pepper et al. point out, the aim of this process is to separate the self-renewing and active proliferating properties of cells into different groups.2 This severely limits the number of duplications that any one cell line will undergo, which limits the possibility of mutational damage taking hold in a particular tissue.
This system actively works against natural selection of individual cells in favour of tissue integrity to suppress somatic evolution, which is the change that the body is subjected to due to mutation and selection within the body’s cell population. Pepper et al. comment:

“We hypothesize that this is achieved in animals by compartmentalizing self-renewing tissues such that one cell population (stem cells) undergoes self-renewal, while another (TACs) undergoes active proliferation. If no cell population combines both these necessary elements of somatic evolution, somatic evolution is thereby suppressed.”

The stem cells are maintained as a small and quiescent population through slow self-renewal. The maintenance of the self-renewing population at low levels militates against selection of highly proliferative strains of stem cells.
The later stages of the differentiation process are focussed on proliferation, but they don’t self-renew. Each duplication event moves the daughter cells along the next stage of differentiation, until the cells are shed after they have become fully differentiated.
While it would cost less energy to just have self-renewing mature cells, it would result in the quick death of the organism if something went wrong in comparison to serial differentiation.
Less energy would be used up because the body would not have to support stem cells and TACs, but only fully differentiated cells. However, there is a much higher chance a mutation that increases the reproductive success of a particular cell would gain a hold in such a setup when compared to serial differentiation. Therefore, the benefit of longevity far outweighs the energy cost incurred for maintaining the system.

Evolution of multicellularity and serial differentiation

Pepper et al. also comment on the prospects of serial cellular differentiation aiding the transition from unicellular to multicellular life:

“It is believed that multicellular organisms could not have arisen or been evolutionarily stable without possessing mechanisms to suppress somatic selection among cells within organisms, which would otherwise disrupt organismal integrity. Here, we propose that one such mechanism is a specific pattern of ongoing cell differentiation commonly found in metazoans with cell turnover, which we call ‘serial differentiation’.”5

They believe that this transition from unicellularity to multicellularity is controlled by epigenetic alterations:

“Thus, our results support the suggestion … that epigenetic inheritance played a central role in the transition from unicellular to multicellular life by helping to control selection among the cells of the newly emergent multicellular individual.”5

However, both serial differentiation and the multicellular organism have to be assumed for this to work. At best it suggests how multicellularity persisted, but it does not suggest its origin.There is a fundamental evolutionary conflict in a multicellular organism: cellular selection vs bodily integrity. Generally, natural selection at the cellular level will favour those cells that are better at reproductive competition and survival. However, if those cells are allowed to proliferate in an uncontrolled manner in a multicellular organism, it will inevitably disrupt the organism’s bodily integrity, and harm or kill the organism.6 This inevitably kills these ‘fitter’ cells too because they cause the host to die. Cancer is a prime example. A cancer is essentially a mess of excessively proliferating cells within a multicellular organism. In an environment with limited resources (the organism), such cells will naturally out-compete normal cells because normal cells generally don’t proliferate indefinitely. The cancer cells outstrip the normal cells for resources and take over the system. However, this leads to malfunction in the organism, and if left untreated, will inevitably kill the organism. At the organismal level, selection will favour those traits that preserve bodily integrity, which seeks to control proliferation of cells beyond what is necessary. Pepper et al. confer:

“Multicellular organisms could not emerge as functional entities before organism-level selection had led to the evolution of mechanisms to suppress cell-level selection.”7

However, this leads to a conundrum for the evolutionist: how do multicellular creatures evolve from single celled organisms when cellular selection is diametrically opposed to organism-level selection? A single cell seeks to proliferate more than its competitors; the multicellular organism seeks to control such proliferation to what is needed at a higher level of organisation. This can be seen in the process of apoptosis as well:

“Even today, apoptosis serves an essential role in terms of ‘cellular altruism’. It helps to ensure that an organism’s genetic integrity is not compromised, by removing some somatic cells that have sustained irreparable, genetic mutations. Crucially, apoptosis also helps to maintain a species’ genetic integrity, by eliminating aberrant germ cells that would otherwise carry intact but faulty genes into the next generation.”8

The system of serial differentiation is designed to enhance bodily integrity, not reduce it. The system has to be in place before it can be selected for, yet organism-level selection cannot take over without measures such as serial differentiation in place. The very existence of this system argues against the evolution of multicellularity.

Conclusion

Serial differentiation is an essential system for the maintenance of mature multicelled organisms. It serves to separate the self-renewal and proliferative stages of cell division, which limits the effect mutations have on tissues. Evolution cannot explain the origin of the system, and neither can it explain the origin of multicellularity. These features of life clearly speak of purposeful, intelligent creation consistent with the Bible’s account of creation.

Life is in the blood 3

So says [url=http://biblia.com/bible/esv/Leviticus 17.11]Leviticus 17:11[/url]. Everyone knows that we must have enough blood flowing around our body or else our bodily functions deteriorate and we die. Yet for a long time the exact function of blood was little understood. In what ways has modern science shown [url=http://biblia.com/bible/esv/Leviticus 17.11]Leviticus 17:11[/url] to be true?
Blood is fundamental to the function of every cell of every component in our bodies. Cells need food to survive, grow, repair themselves and to fulfill their specific functions, and, to reproduce. Cellular food is transported in blood to provide energy for all the cells’ needs. As humans are multicellular organisms, having separate specialized organs with highly sophisticated functions, transport and communication between these structures is essential.

Coordination

Do the cells of the body tell the blood how it should work? No. Does the blood carry around everything possible just in case? No. The cells and the blood work together to provide optimum conditions for correct functioning of all the cells—with their different requirements—in all the tissues and organs of the whole body, including the cells of the blood itself. Blood provides this coordinated environment by regulating acidity/alkalinity (pH), providing oxygen (and removing carbon dioxide and other waste products), and carrying essential vitamins and minerals. Also, blood has to be in the right places at the right times, at the right temperature and pressure, and it carries regulatory messages between organs via blood ‘messengers’ called hormones. All this is organized within very specific limits—straying outside these (through injury, disease, toxins, etc.) rapidly reduces functionality.

Hormonal feedback

Hormones, those important chemical messengers in the blood, are involved in self-regulating feedback systems. These systems stimulate hormone production in times of lack, and suppress it in times of plenty. For example, when we eat, the sugars in the intestine are digested and absorbed into the local bloodstream. This blood then passes through the pancreas and its higher sugar level stimulates production of the hormone insulin. As insulin is distributed in the bloodstream, it reduces the blood sugar to normal levels again by increasing the amount of sugar that all cells take in. In fact the brain relies almost entirely on sugar (specifically glucose) for its energy supply; hence this feedback system is absolutely critical for proper brain activity. If the blood glucose ever drops too much, we lose consciousness.
The body’s systems tend to be wisely over-engineered, so that one might predict that there is also a system to cope with low sugar levels, for example when we exercise and use sugar up. This system uses the hormone glucagon (also from the pancreas) and it works by releasing glucose into the blood from stores located mostly in the liver.
There are about fifteen organs classed as hormone-producing (endocrine) glands,1 and their products, carried by the blood, affect either every cell in general or specifically target certain cells. Widely known examples are the male and female hormones testosterone and estrogen, adrenaline (epinephrine in the US), the thyroid hormone thyroxine, and many more.

Why is blood red?

The red colour of blood reflects the colour of the hemoglobin inside the red blood cells. This is because the hemoglobin contains iron. The ‘heme’ of the hemoglobin molecule in vertebrates (creatures with a backbone) is a porphyrin ring which surrounds ferrous iron atoms. It is the spatial relationship between heme, iron and globin which makes it possible to bind oxygen molecules reversibly—one to each iron—and which makes the system so efficient.
For example, thyroxine regulates the speed of metabolism in every cell, and having the correct amount (within narrow limits) allows normal cellular activity. Too much and we become ‘hyper’, too little and we are slow and lethargic.
Another example is gastrin. The target organ for gastrin is that part of the inner lining of the stomach which produces hydrochloric acid for digestion. Food in the last part of the stomach stimulates the production of gastrin, which is carried back by the blood to stimulate acid production. This is a positive feedback mechanism in which blood is the essential communicating link.

Anticipation

Blood also has a major role in body protection in that it is an integral part of the immune or infection-fighting system, involving antibodies and white blood cells. It also possesses a highly complex mechanism to prevent its own loss from the body (clotting) and to prevent clotting inside the body (thrombosis). The capacity to quickly initiate clotting outside and to limit—even reverse—clotting on the inside is provided by ‘cascades’—cumulative processes in which each step of the process is dependent on the one before it (see box). The cascades are of such complexity that new factors, cofactors and regulators are being constantly added to our body of knowledge. It is now known that there are more than a hundred factors or steps that make up the clotting cascade.2 Such details add to our appreciation of how finely balanced, effective and versatile the system is. But a greater marvel is that such a system, which is there in anticipation of blood loss, internal injury or disease, should be there at all.

Unique red blood cells

Red blood cells (RBCs or erythrocytes) form the majority of the cells in the blood—and a quarter of all cells in the human body. They are unique among all others—in mammals, they have no nucleus and none of the usual energy-producing structures in the cell outside the nucleus. This is a design feature of mammals (creatures which, like us, suckle their young). Normally, a cellular nucleus carries the DNA which instructs the cell on how to perform its functions, including repair and reproduction, at the appropriate times. RBCs cannot do this because instead they are especially designed to carry oxygen, and in humans, having a nucleus would hinder this essential function. So the nucleus is lost after formation, leaving them with their characteristic biconcave shape.

Blood bytes

There are about 4–6 million red blood cells (RBCs) in every cubic millimetre of blood; 20–30 trillion of them in each person.
Every day about 1% of these are changed. New RBCs take about 7 days to form in the bone marrow, and are produced at the staggering rate of about 2 to 3 million every second.
Each RBC lasts about 120 days before its components are recycled to form new RBCs.
During its 4-month lifetime, each red cell travels some 500 km (300 miles) around the body, passing through the heart about 14,000 times per day.
Most of our blood vessels are the microscopic capillaries. If the blood vessels in one person were laid end to end, they would be about 150,000 km (100,000 miles) in length—enough to circle the earth at the equator about four times!
*All figures are for a healthy adult
Two reasons have been suggested for this. First, the relative size of RBCs (6–8 µm diameter and just 2 µm thick)3 and capillaries (tiny blood vessels) is such that red blood cells often have to deform in order to squeeze through. A nucleus (about 6 µm on average4) could prevent passage of the cell and make it get stuck, blocking the circulation.
Second, the shape and deformability of the red blood cell is optimized for the carrying and delivery of oxygen, and it maximizes the amount of hemoglobin that can be packed into the cell. Nevertheless birds, which have a very high oxygen requirement, do fine with nucleated RBCs, so there are other design features in birds that compensate for this.5
The system of the red blood cells giving oxygen to the cells of the tissues is reversed when the red blood cell reaches the lungs, where it gives up its carbon dioxide (though this is mostly carried by plasma6) and takes on a new load of oxygen. At rest, all the blood (5 litres in an adult) completes a circuit within a minute (spending 1 to 3 seconds in the capillaries). With exercise, circulation is as quick as every 10 seconds.7 Having a molecule such as hemoglobin which can handle oxygen so quickly and reversibly, when required, is amazing.

Conclusion

So is the life of the flesh in the blood? Although not confirmed by science until modern times, this statement from [url=http://biblia.com/bible/esv/Leviticus 17.11]Leviticus 17:11[/url] has always been true. Blood actively maintains life by providing a vital function for all cells, tissues and organs, and thus the life of the whole body. The more we find out about the astounding functional design and complexity of blood, the more marvellous it becomes to us, and the more honour and praise is due its Creator.

Irreducibly complex: The clotting cascade

The function of the blood clotting system is to prevent the escape of blood from a damaged vessel. To do this, the blood has a special and very complex repair procedure in place. Once initiated by a cut, the first component in the process is activated, which in turn activates the next component, and so on, in a series of cumulative, mutually-dependent steps. This physiological chain of production, or cascade, results in the formation of a solid obstruction (a clot) in order to seal over the damage.
Some of the main components of the clotting cascade are the proteins fibrinogen, prothrombin, Stuart (anti-hemophilic) factor and proaccelerin. None of these are used for any other purpose in the blood. The system is very finely tuned to result in a repair process that achieves just the repair needed at just the right place and time to stop bleeding and begin the process of healing. Importantly, the process is also self-limiting to ensure that coagulation (clotting) of the entire blood supply does not occur.
The Intelligent Design advocate Michael Behe, in his book Darwin’s Black Box, has noted that the clotting cascade is an example of irreducible complexity. The removal or degradation of just one, any one, of the components or steps would cause the cascade to fail. Obviously this would have dire consequences for the organism. It is exceedingly difficult to see how the clotting cascade could have evolved, as any postulated simplified or ‘primitive’ version of the process would result in failure.1

See also Behe, M., In defense of the irreducibility of the blood clotting cascade: Response to R. Doolittle, K. Miller and K. Robison, www.trueorigin.org/behe03.asp, 2000.

References and notes

Guyton, Arthur C., Textbook of Medical Physiology, Eds Arthur C. Guyton, John E. Hall, p. 838, W.B. Saunders Co., Philadelphia PA 19106, 10th Edition 2000.Return to text.

E.g. there is an international scientific journal dedicated solely to Thrombosis and Haemostasis (Schattauer, ISSN 0340-6245, 12 issues/yr).Return to text.

1 µm (micrometre) is a millionth of a metre, or 1/25,400 inch. Return to text.

‘Cell Nucleus’, Encyclopaedia Brittannica: Ultimate Reference Suite 2005.Return to text.

See e.g. Blown away by design; creation.com/bird-lung. Return to text.

Most of the oxygen in the bloodstream (98%) is carried on the hemoglobin in the RBCs; a little is dissolved in the plasma. Most of the carbon dioxide returning to the lungs is carried dissolved in the plasma, with a small amount in the RBCs. Return to text.

Guyton, Ref.5, p. 3. Return to text.


Understanding Cardiovascular Function: How Water Stays Within the Circulation 2



Moreover, we saw that it is the sodium-potassium pumps within the plasma membrane of the cells that allow the body to take control to maintain the 2/3:1/3 ratio in order to follow the rules of diffusion and osmosis. Now we need to find out what innovation maintains the 80:20 relationship between the interstitial fluid and the plasma. To begin to solve this problem we must first look at how the cardiovascular system works and how some of the laws of nature affect its function. The cardiovascular system consists of the heart and the blood vessels, and is responsible for circulating the blood throughout the body. The blood travels from the left side of the heart, through the arteries to the capillaries where the exchange of chemicals takes place between the circulation and the interstitial fluid. The interstitial fluid then exchanges chemicals with the cells, acting as a bridge between the cells and the circulation. On leaving the capillaries, the blood travels in the veins, picking up water, salt, glucose, and other nutrients from the gastrointestinal system, on its way to the right side of the heart. The blood then travels from the right side of the heart to the lungs where it picks up oxygen and drops off carbon dioxide. It then returns to the left side of the heart and the circuit is repeated. Since blood consists of matter, and thus has mass, it is subject to the law of inertia, which is the tendency of an object at rest to remain at rest. That means the blood requires a source of energy to power it to where it needs to go. The left side of the heart uses energy to pump the blood into the arteries. This generates a hydrostatic pressure, which is the force that the blood applies against the walls of the blood vessel as it flows through it.

Think of it like the air pressure in a tire or the pressure you feel against the walls of a pipe as water flows through. The blood flows through the arteries and arterioles, using up energy. By the time it travels from the heart to the capillaries, the hydrostatic pressure has dropped from about 100 units (at the heart) to about 35 units (at the capillary). As the blood then leaves the capillary, the pressure drops to about 15 units. The walls of the capillaries contain tiny pores and as the blood moves through them the hydrostatic pressure pushes water out of the blood into the interstitial fluid. It's like squeezing boiled potatoes through a ricer. In fact, as blood continues to flow into the capillaries, this pressure can force an amount of water equal to the total plasma volume out of the circulation within just a few minutes. It's obvious that if the body didn't have a way to force most of this squeezed out water immediately back into the circulation, human life would not exist. The force that opposes filtering water out of the circulation by the hydrostatic pressure is osmosis. Plasma mainly consists of water (90 percent) and chemicals in solution such as glucose and sodium. But it also contains plasma proteins that perform various functions. The main plasma protein that provides this osmotic effect is albumin. Remember, a solute exerts an osmotic pull on water across a membrane based on its inability to leave that solution. The total protein content of blood is much higher than in the interstitial fluid. And since the protein can't pass through the capillary wall, this means that, by the power of osmosis, water naturally tends to move from the interstitial fluid back into the circulation. This osmotic pressure exerted by albumin can be measured and is equal throughout the capillary. In fact, a normal plasma level of albumin provides an osmotic pull (to bring water back into the circulation) of about 25 units. Recall that the hydrostatic pressure squeezing water out of the circulation at the start of the capillary is about 35 units, while at the end it's about 15 units.

This means that when the blood enters the capillary, the net flow of water is +10 units (35 - 25), meaning that it's moving from the circulation into the interstitial fluid. When it leaves the capillary, the net flow of water is -10 units (15 - 25), meaning that it's moving from the interstitial fluid back into the circulation. Thus albumin helps the body bring back most of the water that is squeezed out of the capillaries by the hydrostatic pressure that moves blood through the cardiovascular system. Of course, the filtering of water through the capillary is dependent on many other factors as well. However, this provides a basis for understanding why having the right amount of albumin in the blood is vital for maintaining the circulation. So, in following the rules, the body must take control with its innovation of albumin to help preserve the 80:20 relationship between the plasma and the interstitial fluid. This is needed so the body can have enough blood volume and the circulation can adequately feed the cells. As we've noted before, when it comes to life and death, real numbers have real consequences. The normal range of albumin is 3.5-5.0 units and nobody really knows how the body controls its production nor how the liver knows how much to make to keep the body alive. Clinical experience shows that when severe malnutrition or certain liver, kidney, or gastrointestinal disorders cause the serum albumin to drop below 2.0 units, the osmotic pull of water back into the circulation is so diminished that it allows a lot of water to stay inside the interstitial fluid, forming what is called edema. Edema of the tissues results in organ malfunction. When it affects the lungs it can result in respiratory failure and death.

Because water tends to move out of the blood into the interstitial fluid, the blood volume drops as well, resulting in severe weakness, fatigue, and dizziness on standing. In fact, a plasma albumin level that is less than 1.0 unit is thought to be incompatible with life. It's mainly the sodium-potassium pumps in the plasma membrane of your cells and the albumin in your blood that together make sure the water in your body is properly distributed. Despite the fact that medical science really doesn't understand how the body controls the production of albumin or knows how much it should make, evolutionary biologists reassure us that all of this came about by chance and the laws of nature alone. But, what in the body controls its total water content? After all, even if water is properly distributed in our body, if we don't have enough water, we die. That is a subject we'll begin to tackle next time.


FORMATION OF BLOOD CELLS (HEMOPOIESIS)

Sites of Hemopoiesis
Hemopoiesis occurs in diff erent organs of the body, depending on the stage of development of the individual. In a developing embryo, hemopoiesis initially occurs in the yolk sac and later in the development in liver, spleen, lymph nodes, and bone marrow. After birth, hemopoiesis continues almost exclusively in the red marrow of different bones. In the newborn, all bone marrow is red and functions in hemopoiesis. The red bone marrow is a highly cellular structure and consists of hemopoietic stem cells and the precursors of   different blood cells. Red marrow also contains a loose arrangement of fine reticular fibers that form an intricate network. As the individual ages and becomes an adult, the
red marrow is found primarily in the flat bones of the skull, sternum and ribs, vertebrae, and pelvic bones. The remaining bones, primarily the long bones in the limbs of the body, gradually accumulate fat, and their marrow becomes yellow. Consequently, they lose the hemopoietic functions.

In this process, all blood cells originate from a common stem cell in the red bone marrow that is self-renewing. Because this stem cell type can produce all blood cell types, it is called the pluripotential hemopoietic stem cell. Pluripotential stem cells, in turn, produce two major cell lineages that form the pluripotential myeloid stem cells and pluripotential lymphoid stem cells. Before maturation and release into the bloodstream, the stem cells from each lineage undergo numerous divisions and intermediate stages of differentiation before maturation (Overview Figure 6.1).



Myeloid stem cells develop in the red bone marrow and eventually give rise to erythrocytes, eosinophils, neutrophils, basophils, monocytes, and megakaryocytes. Lymphoid stem
cells also develop in the red bone marrow. 

Bone marrow is the flexible tissue in the interior of bones. In humans, red blood cells are produced by cores of bone marrow in the heads of long bones in a process known as hematopoiesis. On average, bone marrow constitutes 4% of the total body mass of humans; in an adult having 65 kilograms of mass (143 lbs), bone marrow typically accounts for approximately 2.6 kilograms (5.7 lb). The hematopoietic component of bone marrow produces approximately 500 billion blood cells per day, which use the bone marrow vasculature as a conduit to the body's systemic circulation.Bone marrow is also a key component of the lymphatic system, producing the lymphocytes that support the body's immune system.

Some lymphoid cells remain in the bone marrow, proliferate, mature, and become B lymphocytes. 

A lymphocyte is one of the subtypes of white blood cell in a vertebrate's immune system. They include natural killer cells (NK cells) (which function in cell mediated, cytotoxic innate immunity), T cells (for cell-mediated, cytotoxic adaptive immunity), and B cells (for humoral, antibody-driven adaptive immunity). They are the main type of cell found in lymph, which prompted the name lymphocyte.

Others leave the bone marrow and migrate via the bloodstream to lymph nodes and the spleen, where they proliferate and differentiate into B lymphocytes, after which they colonize peripheral lympoid tissues (connective tissues, lymphoid tissues, and lymphoid organs). Other undifferentiated lymphoid cells migrate to the thymus gland, where they proliferate and differentiate into immunocompetent T lymphocytes. Afterward, T lymphocytes enter the bloodstream and migrate to reside in the connective tissues and specific regions of peripheral lymphoid organs of the body. Both B and T lymphocytes reside in numerous peripheral lymphoid tissues, lymph nodes, and spleen. Here, they initiate immune responses when exposed to antigens. Both the B and T lymphocytes are morphologically indistinguishable. Only the different protein markers on their cell surfaces allow these cells to be distinguished by immunohistochemical means. Because all blood cells have a limited life span, the pluripotential hemopoietic stem cells continually divide and differentiate to produce new progeny of cells. 

Hod did this mechanism of continuous division and differentiation evolve ? 

When the blood cells become worn out and die, they are destroyed by macrophages in different lymphoid organs such as the spleen.

Formed Elements: Major Blood Cell Types

Microscopic examination of a stained blood smear reveals the major blood cell types. Erythrocytes, or RBCs, are nonnucleated cells and are the most numerous blood cells. During their maturation process, the erythrocytes extrude their nuclei, and the mature blood cells enter the bloodstream, without their nuclei. Erythrocytes remain in the blood and perform their major functions within the blood vessels. In contrast, leukocytes, or WBCs, are nucleated and subdivided into granulocytes and agranulocytes, depending on the presence or absence of granules in their cytoplasm. Granulocytes are the neutrophils, eosinophils, and basophils. Agranulocytes are the monocytes and lymphocytes. Leukocytes perform their major functions outside the blood vessels. They migrate out of the blood vessels through capillary walls and enter the connective tissue, lymphatic tissue, and bone marrow. The primary function of leukocytes is to defend the body against bacterial invasion or the presence of foreign material. Consequently, most leukocytes are concentrated in the connective tissue of different organs.



Hemopoiesis (hematopoiesis) includes both erythropoiesis and leukopoiesis (development of red and white blood cells, respectively), as well as thrombopoiesis (development of platelets; Fig. 10.16).





Blood cells have a limited life span; they are continuously produced and destroyed. The ultimate objective of hemopoiesis is to maintain a constant level of the different cell types found in the peripheral blood. Both the human erythrocyte (life span of 120 days) and the platelet (life span of 10 days) spend their entire life in the circulating blood. Leukocytes, however, migrate out of the circulation shortly after entering it from the bone marrow and spend most of their variable life spans (and perform all of their functions) in the tissues. In the adult, erythrocytes, granulocytes, monocytes, and platelets are formed in the red bone marrow; lymphocytes are also formed in the red bone marrow and in the lymphatic tissues.

Hemopoiesis is initiated in early embryonic development.

During fetal life, both erythrocytes and leukocytes are formed in several organs before the differentiation of the bone marrow. The first or yolk-sac phase of hemopoiesis begins in the third week of gestation and is characterized by the formation of “blood islands” in the wall of the yolk sac of the embryo. In the second, or hepatic phase, early in fetal development, hemopoietic centers appear in the liver (Fig. 10.17). Blood cell formation in these sites is largely limited to erythroid cells although some leukopoiesis occurs in the liver. The liver is the major blood-forming organ in the fetus during the second trimester. The third or bone marrow phase of fetal hemopoiesis and leukopoiesis involves the bone marrow (and other lymphatic tissues) and begins during the second trimester of pregnancy. After birth, hemopoiesis takes place only in the red bone marrow and lymphatic tissues, as in the adult (Fig. 10.18). The precursors of both the blood cells and germ cells arise in the yolk sac.



Monophyletic Theory of Hemopoiesis

According to the monophyletic theory of hemopoiesis, blood cells are derived from a common hemopoietic stem cell.

Considerable circumstantial evidence has for many years supported the monophyletic theory of hemopoiesis in which all blood cells arise from a common stem cell. Decisive evidence for the validity of the monophyletic theory has come with the isolation and demonstration of the hemopoietic stem cell (HSC). The hemopoietic stem cell, also known as pluripotential stem cell (PPSC), is capable not only of differentiating into all the blood cell lineages but also of self-renewal ( i.e., the pool of stem cells is self-sustaining). Recent studies indicate that HSCs also have the potential to differentiate into multiple non–blood cell lineages and contribute to the cellular regeneration of various tissues and multiple organs. During embryonic development, HSCs are present in the circulation and undergo tissue-specific differentiation in different organs. Human HSCs have been isolated from umbilical cord blood, fetal liver, and fetal and adult bone marrow. In the adult, HSCs have the potential to repair tissues under pathologic conditions (e.g., ischemic injury, organ failure). Human HSCs express specific molecular marker proteins such as CD34 and CD90 and at the same time do not express lineage-specific markers (Lin–) that are found on lymphocytes, granulocytes, monocytes, megakaryocytes, and erythroid cells. It is now believed that human HSC can be identified by the Lin, CD34, CD90, and CD38 cell- surface markers. HSCs are not identifiable in the routine preparation; however, they can be identified and isolated using immunocytochemical methods.

A hemopoietic stem cell (HSC) in the bone marrow gives rise to multiple colonies of progenitor stem cells.

In the bone marrow, descendants of the HSC differentiate into two major colonies of multipotential progenitor cells: The common myeloid progenitor (CMP) cells and the common lymphoid progenitor (CLP) cells. Ultimately, common myeloid progenitor (CMP) cells which were previously called colony-forming units–granulocyte, erythrocyte, monocyte, megakaryocyte (CFU-GEMM), differentiate into specific lineage-restricted progenitors (Table 10.3). These include the following.



• Megakaryocyte/erythrocyte progenitor (MEP) cells: These bipotential stem cells give rise to monopotent megakaryocyte-committed progenitor cells (MKP or CFU-Meg) and other monopotent erythrocyte-committed progenitor cells (ErP or CFU-E) that give rise to the erythrocyte lineage.
• Granulocyte/monocyte progenitor (GMP or CFUGM) cells: Development of the GMP (CFU-GM) cells requires high-level expression of PU.1 transcription factor. These cells then give rise to the neutrophil progenitors (NoP or CFU-G) which differentiate into the neutrophilic lineage; eosinophil progenitors (EoP or CFU-Eo), cells that give rise to eosinophils; basophil/mast cell progenitors (BMCP) that give rise either to basophil progenitor cells (BaP or CFU-Ba) in the bone marrow or MCPs in the gastrointestinal mucosa; and finally monocyte progenitors (MoP or CFU-M) that develop toward monocyte lineages. In addition to the specific lineage progenitors, GMP cells can give rise to dendritic cells (DCs), which are professional antigen-presenting cells.. The common lymphoid progenitor (CLP) cells are capable of diferentiating into T cells, B cells, and natural killer (NK) cells. These multipotential CLP cells have been previously called colony-forming units–lymphoid (CFU-L) The NK cells are thought to be the prototype of T cells; they both possess similar capability to destroy other cells.Dendritic cells can also developed from CLP cells. Perhaps the easiest way to begin the histologic study of blood cell development is to refer to Figure 10.16 and Figure 10.19. Figure 10.19 shows the stages of blood cell
development in which characteristic cell types can be identified in the light microscope in a tissue section or bone marrow smear. Hemopoiesis is initiated in an apparently random manner when individual HSCs begin to differentiate into one of the lineage-restricted progenitor cells. Progenitorcells have surface receptors for specific cytokines and growth factors, including colony-stimulating factors (CSFs), that influence their proliferation and maturation into a specific lineage.



Development of Erythrocytes (Erythropoiesis)

Erythrocyte development starts from CMP cells that, under the influence of erythropoietin, IL-3, and IL-4, differentiate to MEP cells. Expression of transcription factor GATA-1is required for the terminal differentiation of MEP cells to definitive erythroid cell lineage. Under GATA-1 influence, MEP cells transform into erythropoietin-sensitive erythrocytecommitted progenitors (ErPs or CFU-E) that give rise to the proerythroblast.

The first microscopically recognizable precursor cell in erythropoiesis is called the proerythroblast.

The proerythroblast is a relatively large cell measuring 12 to 20 μm in diameter. It contains a large spherical nucleus with one or two visible nucleoli. The cytoplasm shows mild basophilia because of the presence of free ribosomes. Although recognizable, the proerythroblast is not easily identified in routine bone marrow smears.

The basophilic erythroblast is smaller than the proerythroblast, from which it arises by mitotic division. 

The nucleus of the basophilic erythroblast is smaller (10 to 16 μm in diameter) and progressively more heterochromatic with repeated mitoses. The cytoplasm shows strong basophilia because of the large number of free ribosomes (polyribosomes) that synthesize hemoglobin. The accumulation of hemoglobin in the cell gradually changes the staining reaction of the cytoplasm so that it begins to stain with eosin. At the stage when the cytoplasm displays both acidophilia, because of the staining of hemoglobin, and basophilia, because of the staining of the ribosomes, the cell is called a polychromatophilic erythroblast. 

The polychromatophilic erythroblast shows both acidophilic and basophilic staining of cytoplasm. 

The staining reactions of the polychromatophilic erythroblast may blend to give an overall gray or lilac color to the cytoplasm, or distinct pink (acidophilic) and purple (basophilic) regions may be resolved in the cytoplasm. The nucleus of the cell is smaller than that of the basophilic erythroblast, and coarse heterochromatin granules form a checkerboard pattern that helps identify this cell type. The orthochromatophilic erythroblast is recognized by its increased acidophilic cytoplasm and dense nucleus. The next named stage in erythropoiesis is the orthochromatophilic erythroblast (normoblast). This cell has a small, compact, densely stained nucleus. The cytoplasm is eosinophilic because of the large amount of hemoglobin (Fig. 10.20). It is only slightly larger than a mature erythrocyte. At this stage, the orthochromatophilic erythroblast is no longer capable of division.



The polychromatophilic erythrocyte has extruded its nucleus.


The orthochromatic erythroblast loses its nucleus by extruding it from the cell; it is then ready to pass into the blood sinusoids of the red bone marrow. Some polyribosomes that can still synthesize hemoglobin are retained in the cell. These polyribosomes impart a slight basophilia to the otherwise eosinophilic cells; for this reason, these new cells are called polychromatophilic erythrocytes (Fig. 10.21). The polyribosomes of the new erythrocytes can also be demonstrated with special stains that cause the polyribosomes to clump and form a reticular network. Consequently, polychromatophilic erythrocytes are also (and more commonly) called reticulocytes.

In normal blood, reticulocytes constitute about 1% to 2% of the total erythrocyte count. However, if increased numbers of erythrocytes enter the bloodstream (as during increased erythropoiesis to compensate for blood loss), the number of reticulocytes increases.




Kinetics of Erythropoiesis

Mitoses occur in proerythroblasts, basophilic erythroblasts, and polychromatophilic erythroblasts.

At each of these stages of development, the erythroblast divides several times. It takes about a week for the progeny of a newly formed basophilic erythroblast to reach the circulation. Nearly all erythrocytes are released into the circulation as soon as they are formed; bone marrow is not a storage site for erythrocytes. Erythrocyte formation and release are regulated by erythropoietin, a 34-kilodalton glycoprotein hormone synthesized and secreted by the kidney in response to decreased blood oxygen concentration. Erythropoietin acts on the specific receptors expressed on the surface of ErP.

Erythrocytes have a life span of about 120 days in humans.

When erythrocytes are about 4 months old, they become senescent. The macrophage system of the spleen, bone marrow, and liver phagocytoses and degrades the senescent erythrocytes. The heme and globin dissociate, and the globin is hydrolyzed to amino acids, which enter the metabolic pool for reuse. The iron on the heme is released, enters the iron-storage pool in the spleen in the form of hemosiderin or ferritin, and is stored for reuse in hemoglobin synthesis. The rest of the heme moiety of the hemoglobin molecule is partially degraded to bilirubin, bound to albumin, released into the bloodstream, and transported to the liver, where it is conjugated and excreted via the gallbladder as the bilirubin glucuronide of bile.

1) http://creation.com/serial-cell-differentiation-intricate-system-of-design
2) http://www.evolutionnews.org/2015/05/understanding_c_1095961.html
3) http://creation.com/life-is-in-the-blood

Development of Thrombocytes (Thrombopoiesis)

Each day bone marrow of a healthy adult produces about 1 x10^11 platelets, a number that can increase 10-fold in time of increased demand. The thrombocytopoiesis from the bone marrow progenitors is a complex process of cell divisions and differentiation that requires the support of interleukins, colony-stimulating factors, and hormones.

Thrombocytes (platelets) develop from a bipotent megakaryocyte/erythrocyte progenitor (MEP) cell that differentiates into megakariocyte-committed progenitor (MKP) cell and finally into a megakaryocyte.

Platelets are produced in the bone marrow from the same common myeloid progenitor (CMP) cells as the erythroid and myeloid series. Under the influence of granucolyte-macrophage colony-stimulating factor (GMCSF) and IL-3, CMP stem cell differentiates into a bipotent megakaryocyte/erythrocyte progenitor (MEP) cell. Further development proceeds toward a unipotent megakariocyte- committed progenitor (MKP) cell (or CFU-Meg), which further develops into the megakaryoblast. The megakaryoblast that develops from this MKP is a large cell (about 30  μm in diameter) with a nonlobed nucleus. No evidence of platelet formation is seen at this stage. Successive endomitoses occur in the megakaryoblast (i.e., chromosomes replicate), but neither karyokinesis nor cytokinesis occurs. Under stimulation by thrombopoietin, a 30-kilodalton glycoprotein hormone produced by liver and kidney, ploidy increases from 8n to 64n before chromosomal replication ceases. The cell then becomes a platelet-producing megakaryocyte, a cell measuring 50 to 70 μm in diameter with a complex multilobed
nucleus and scattered azurophilic granules. Both the nucleus and the cell increase in size in proportion to the ploidy of the cell. With the TEM, multiple centrioles and multiple Golgi apparatuses are also seen in these cells.
When bone marrow is examined in a smear, platelet fields are seen to fill much of the peripheral cytoplasm of the megakaryocyte. When examined with the TEM, the peripheral cytoplasm of the megakaryocyte appears to be divided into small compartments by invagination of the plasma membrane. As described above, these invaginations are the platelet demarcation channels (see Fig. 10.13).

Thrombocytopenia (a low blood platelet count) is an important clinical problem in the management of patients with immune-system disorders and cancer (i.e., leukemia). It increases the risk of bleeding and in cancer patients
often limits the dose of chemotherapeutic agents.

Development of Granulocytes (Granulopoiesis)

Granulocytes originate from the multipotential common myeloid progenitor (CMP) stem cell, which differentiates into granulocyte/monocyte progenitors (GMPs) under the influence of cytokines such as GM-CSF, granulocyte colony-stimulating factor (G-CSF), and IL-3. GM-CSF is a cytokine secreted by endothelial cells, T cells, macrophages, mast cells, and fibroblasts. It stimulates GMP cells to produce granulocytes (neutrophils, eosinophils, and basophils) and monocytes. The neutrophil progenitor (NoP) undergoes six morphologically identifiable stages in the process of maturation: myeloblast, promyelocyte, myelocyte, metamyelocyte, band cell, and mature neutrophil. Eosinophils and basophils undergo a morphologic maturation similar to that of neutrophils. GMP cells, when induced by GM-CSF, IL-3, and IL-5, differentiate to eosinophil progenitors (EoPs), and eventually mature to eosinophils. Lack of IL-5 causes the GMP cells to differentiate into basophil progenitors (BaPs), which produce basophils. One cannot differentiate eosinophilic or basophilic precursors from neutrophilic precursors morphologically in the light microscope until the cells reach the myelocytic stage when the specific granules appear.

Myeloblasts are the first recognizable cells that begin the process of granulopoiesis.

The myeloblast is the earliest microscopically recognizable neutrophil precursor cell in the bone marrow. It has a large, euchromatic, spherical nucleus with three to five nucleoli. It measures 14 to 20 μm in diameter and has a large nuclear-tocytoplasmic volume. The small amount of agranular cytoplasm stains intensely basophilic. A Golgi area is often seen where the cytoplasm is unstained. The myeloblast matures into a promyelocyte.

Promyelocytes are the only cells to produce azurophilic granules.

The promyelocyte has a large spherical nucleus with azurophilic (primary) granules in the cytoplasm. Azurophilic granules are produced only in promyelocytes; cells in subsequent stages of granulopoiesis do not make azurophilic granules. For this reason, the number of azurophilic granules is reduced with each division of the promyelocyte and its progeny. Promyelocytes do not exhibit subtypes. Recognition of the neutrophil, eosinophil, and basophil lines is possible only in the next stage—the myelocyte—when specific (secondary) and tertiary granules begin to form.

Myelocytes first exhibit specific granules.

Myelocytes begin with a more or less spherical nucleus that becomes increasingly heterochromatic and acquires a distinct indentation during subsequent divisions. Specific granules begin to emerge from the convex surface of the Golgi apparatus, whereas azurophilic granules are seen at the concave side. The significance of this separation is unclear. Myelocytes continue to divide and give rise to metamyelocytes.

The metamyelocyte is the stage at which neutrophil, eosinophil, and basophil lines can be clearly identified by the presence of numerous specific granules.

A few hundred granules are present in the cytoplasm of each metamyelocyte, and the specific granules of each variety outnumber the azurophilic granules. In the neutrophil, this ratio of specific to azurophilic granules is about 2 to 1. The nucleus becomes more heterochromatic, and the indentation deepens to form a kidney bean–shaped structure. Theoretically, the metamyelocyte stage in granulopoiesis is followed by the band stage and then the segmented stage. Although these stages are obvious in the neutrophil line, they are rarely if ever observed in the eosinophil and basophil lines in which the next easily recognized stages of development are the mature eosinophil and mature basophil, respectively.

In the neutrophil line, the band (stab) cell precedes development of the first distinct nuclear lobes.

The nucleus of the band (stab) cell is elongated and of nearly uniform width, giving it a horseshoelike appearance. Nuclear constrictions then develop in the band neutrophil and become more prominent until two to four nuclear lobes are recognized; the cell is then considered a mature neutrophil, also called a polymorphonuclear neutrophil or segmented neutrophil. Although the percentage of band cells in the circulation is almost always low (0% to 3%), it may increase in acute or chronic inflammation and infection.

Kinetics of Granulopoiesis

Granulopoesis in the bone marrow takes about two weeks.

The mitotic (proliferative) phase in granulopoiesis lasts about a week and stops at the late myelocyte stage. The postmitotic phase, characterized by cell differentiation—from metamyelocyte to mature granulocyte—also lasts about a week. The time it takes for half of the circulating segmented neutrophils to leave the peripheral blood is about 6 to 8 hours. Neutrophils leave the blood randomly—that is, a given neutrophil may circulate for only a few minutes or as long as 16 hours before entering the perivascular connective tissue (a measured half-life of circulating human neutrophils is only 8 to 12 hours). Neutrophils live for 1 to 2 days in the connective tissue, after which they are destroyed by apoptosis and are subsequently engulfed by macrophages. Also, large numbers of neutrophils are lost by migration into the lumen of the gastrointestinal tract from which they are discharged with the feces.

Bone marrow maintains a large reserve of fully functional neutrophils ready to replace or supplement circulating neutrophils at times of increased demand.

In normal conditions, the bone marrow produces more than 10^11 neutrophils each day. As a result of the release of neutrophils from the bone marrow, approximately 5 to 30 times as many mature and near-mature neutrophils are normally present in the bone marrow as are present in the circulation.

This bone marrow reserve pool constantly releases neutrophils into the circulation and is replenished by maturing cells. The reserve neutrophils can be released abruptly in response to inflammation, infection, or strenuous exercise. A reservoir of neutrophils is also present in the vascular compartment. This reserve consists of a freely circulating pool and a marginated pool, with the latter contained in small blood vessels. The neutrophils adhere to the endothelium much as they do before leaving the vasculature at sites of injury or infection (see page 275). The normally marginated neutrophils, however, loosely adhere to the endothelium through the action of selectin and can be recruited very quickly. They are in dynamic equilibrium with the circulating pool, which is approximately equal to the size of the marginated pool.

The size of the reserve pool in the bone marrow and in the vascular compartment depends on the rate of granulopoiesis, the life span of the neutrophils, and the rates of migration
into the bloodstream and connective tissue. The entire hemopoietic process is summarized in Table 10.3.

Transcription factors control the fate of hemopoietic cells, whereas cytokines and local mediators regulate all stages of hemopoiesis.

Intimate interactions between HSCs and their bone marrow microenvironment work toward redefining the identity and the differentiation pathways of these multipotential stem cells. Signaling molecules from a variety of bone marrow cells initiate intracellular pathways that ultimately target a select group of synergistic and inhibitory proteins known as transcription factors. They specifically bind to promoter or enhancer regions on DNA in the affected cell. By controlling transcription of the specific genes downstream, these transcription factors trigger a cascade of genetic changes that ultimately determines the course of the cells during differentiation. In addition to identifying the various intracellular transcription factors, recent studies have identified and begun to characterize numerous signaling molecules found in the bone marrow. These include glycoproteins that act as both circulating hormones and local mediators to regulate the progress of hemopoiesis and the rate of differentiation of other cell types (Table 10.4). Specific hormones such as erythropoietin or thrombopoietin, discussed in a previous section, regulate erythrocyte and thrombocyte development, respectively. Other factors, collectively called colony-stimulating factors (CSFs), are subclassified according to the specific cell or group of cells that they affect. Among the recently isolated and most completely characterized factors are several that stimulate granulocyte and monocyte formation, GM-CSF, G-CSF, and macrophage colony-stimulating factor (M-CSF). Interleukins, produced by lymphocytes, act on other leukocytes and their progenitors. IL-3 is a cytokine that appears to affect most progenitor cells and even terminally differentiated cells. Any particular cytokine may act at one or more stages in hemopoiesis, affecting cell division, differentiation, or cell function. These factors are synthesized by many different cell types, including kidney cells (erythropoietin), liver hepatocytes (thrombopoietin), T lymphocytes (IL-3), endothelial cells (IL-6), adventitial cells in the bone marrow (IL-7), and macrophages (the CSFs that affect granulocyte and macrophage development).


The isolation, characterization, manufacture, and clinical testing of cytokines (proteins and peptides that are signaling compounds) in the treatment of human disease is a major activity of the rapidly growing biotechnology industry. Several hemopoietic and lymphopoietic cytokines have been manufactured by recombinant DNA technology and are already used in clinical settings. These include recombinant erythropoietin, G-CSF, GM-CSF, and IL-3; others are under active development. GM-CSF (sargramostim, Leukine) is used clinically to stimulate production of WBCs following chemotherapy and to accelerate WBC recovery following bone marrow transplantation.


Development of Monocytes

The multipotential CMP stem cell also gives rise to the cells  that develop along the monocyte–macrophage pathway.

Monocytes are produced in the bone marrow from a GMP stem cell that can mature into a monocyte or another of the three granulocytic cell lines. In addition, GMP gives rise to dendritic cells. The proliferation and differentiation of CMP into committed GMP is controlled by IL-3. Further progression of monocyte progenitor (MoP) cell lineage depends on the continued presence of PU.1 and Egr-1 transcription factors and is stimulated by IL-3 and GM-CSF. The GMCSF also controls further differentiation into mature cells, which are then released into circulation. The transformation of MoPs to monocytes takes about 55 hours, and the monocytes remain in the circulation for only about 16 hours before emigrating to the tissues where they differentiate under influence of both GM-CSF and M-CSF into tissue macrophages. Their subsequent life span is not yet fully understood.

Development of Lymphocytes (Lymphopoiesis)

Development and lineage commitment of CLP cells depend on the expression of variety of transcription factors.

Although lymphocytes continuously proliferate in the peripheral lymphatic organs, the bone marrow remains the primary site of lymphopoiesis in humans. Members of the Ikaros family of transcription factors play major
roles in the differentiation of pluripotent HSCs toward common lymphoid progenitor (CLP) cells. Progeny of the CLP cells that express GATA-3 transcription factor are destined to become T lymphocytes. These cells that express GATA-3 leave the bone marrow as pre–T lymphocytes and travel to the thymus, where they complete their differentiation and thymic cell education (see Chapter 14, Lymphatic System). They then enter the circulation as long-lived, small T lymphocytes. Another transcription factor, Pax5, activates B-cell–specific genes in CLP cells destined to become B lymphocytes. In mammals, these cells originate in bursa-equivalent organs such as the bone marrow, gut-associated lymphatic tissue, and spleen. Although a number of transcription factors have been identified in the development of lymphoid cell lineages, little is known about factors that may influence development and lineage commitment of NK cells. NK cells most likely differentiate under the influence of IL-2 and IL-15 into immature pre–NK cells, and, after acquisition of NK–cell effector functions (ability to secrete interferon and cytotoxicity), become mature NK cells. The bone marrow is the main NK cell–producing organ. However, recent studies suggest that lymph nodes or fetal thymus may also contain NK-progenitor cells. Lymphocytes constitute as much as 30% of all nucleated cells in the bone marrow. The production and differentiation of lymphocytes are discussed in more detail in Chapter 14, Lymphatic System.

BONE MARROW

Red bone marrow lies entirely within the spaces of bone, medullary cavity of young long bones, and spaces of spongy bone.

Bone marrow consists of blood vessels, specialized units of blood vessels called sinusoids, and a spongelike network of hemopoietic cells (Fig. 10.22).



The bone marrow sinusoids provide the barrier between the hemopoietic compartment and the peripheral circulation. In sections, the cells in hemopoietic compartment appear to lie in “cords” between sinusoids or between sinusoids and bone. The sinusoid of red bone marrow is a unique vascular unit. It occupies the position normally occupied by a capillary; that is, it is interposed between arteries and veins. It is believed to be derived from vessels that have just nourished the cortical bone tissue. The sinusoids arise from these vessels at the corticomedullary junction. The sinusoid wall consists of an endothelial lining, a basal lamina, and an incomplete covering of adventitial cells. The endothelium is a simple squamous epithelium.
  The adventitial cell, also called a reticular cell, sends sheetlike extensions into the substance of the hemopoietic cords, which provide some support for the developing blood cells. In addition, adventitial cells produce reticular fibers. They also play a role in stimulating the differentiation of developing progenitor cells into blood cells by secreting several cytokines (e.g., CSFs, IL-5, IL-7). When blood cell formation and the passage of mature blood cells into the sinusoids are active, adventitial cells and the basal lamina become displaced by mature blood cells as they approach the endothelium to enter the sinusoid from the bone marrow cavity.

The bone marrow sinusoidal system is a closed circulation system; newly formed blood cells must penetrate the endothelium to enter the circulation.

As a maturing blood cell or a megakaryocyte process pushes against an endothelial cell, the abluminal plasma membrane is pressed against the luminal plasma membrane until they fuse, thus forming a transitory opening, or aperture. The migrating cell or the megakaryocyte process literally pierces the endothelial cell. Thus, migration across the bone marrow endothelium is a transcellular and not an intercellular event. Each blood cell must squeeze through an aperture to enter the lumen of a sinusoid. Similarly, a megakaryocyte process must protrude through an aperture so that the platelets can be released directly into the sinusoid lumen. The aperture is lined by the fused plasma membrane, thus maintaining the integrity of the endothelial cell during the transcellular passage. As the blood cell completes its passage through the aperture or the megakaryocyte that has extruded its platelets withdraws its process, the endothelial cell “repairs itself,” and the aperture disappears. In active red bone marrow, the cords of hemopoietic cells contain predominately developing blood cells and megakaryocytes. The cords also contain macrophages, mast cells, and some adipose cells. Although the cords of hemopoietic tissue appear to be unorganized, specific types of blood cells develop in nests or clusters. Each nest in which erythrocytes develop contains a macrophage. These nests are located near the sinusoid wall. Megakaryocytes are also located adjacent to the sinusoid wall, and they discharge their platelets directly into the sinusoid through apertures in the endothelium. Granulocytes develop in cell nests farther from the sinusoid wall. When mature, the granulocyte migrates to the sinusoid and enters the
bloodstream.

Bone marrow not active in blood cell formation contains predominately adipose cells, giving it the appearance of adipose tissue.

Inactive bone marrow is called yellow bone marrow. It is the chief form of bone marrow in the medullary cavity of bones in the adult that are no longer hemopoietically active,
such as the long bones of the arms, legs, fingers, and toes. In these bones, the red bone marrow has been replaced completely by fat. Even in hemopoietically active bone marrow in adult humans—such as that in the ribs, vertebrae, pelvis, and shoulder girdle—about half of the bone marrow space is occupied by adipose tissue and half by hemopoietic tissue. The yellow bone marrow retains its hemopoietic potential, however, and when necessary, as after severe loss of blood, it can revert to red bone marrow, both by extension of the hemopoietic tissue into the yellow bone marrow and by repopulation of the yellow bone marrow by circulating stem cells.



Red blood cells (RBCs)  erythrocytes

Unique red blood cells  3

Red blood cells (RBCs or erythrocytes) form the majority of the cells in the blood—and a quarter of all cells in the human body. They are unique among all others—in mammals, they have no nucleus and none of the usual energy-producing structures in the cell outside the nucleus. This is a design feature of mammals (creatures which, like us, suckle their young). Normally, a cellular nucleus carries the DNA which instructs the cell on how to perform its functions, including repair and reproduction, at the appropriate times. RBCs cannot do this because instead they are especially designed to carry oxygen, and in humans, having a nucleus would hinder this essential function. So the nucleus is lost after formation, leaving them with their characteristic biconcave shape. 
Two reasons have been suggested for this. First, the relative size of RBCs (6–8 µm diameter and just 2 µm thick)3 and capillaries (tiny blood vessels) is such that red blood cells often have to deform in order to squeeze through. A nucleus (about 6 µm on average4) could prevent passage of the cell and make it get stuck, blocking the circulation.

Second, the shape and deformability of the red blood cell is optimized for the carrying and delivery of oxygen, and it maximizes the amount of hemoglobin that can be packed into the cell. Nevertheless birds, which have a very high oxygen requirement, do fine with nucleated RBCs, so there are other design features in birds that compensate for this.5

The system of the red blood cells giving oxygen to the cells of the tissues is reversed when the red blood cell reaches the lungs, where it gives up its carbon dioxide (though this is mostly carried by plasma6) and takes on a new load of oxygen. At rest, all the blood (5 litres in an adult) completes a circuit within a minute (spending 1 to 3 seconds in the capillaries). With exercise, circulation is as quick as every 10 seconds.7 Having a molecule such as hemoglobin which can handle oxygen so quickly and reversibly, when required, is amazing.

Role in disease

Blood diseases involving the red blood cells include:

Anemias (or anaemias) are diseases characterized by low oxygen transport capacity of the blood, because of low red cell count or some abnormality of the red blood cells or the hemoglobin.
Iron deficiency anemia is the most common anemia; it occurs when the dietary intake or absorption of iron is insufficient, and hemoglobin, which contains iron, cannot be formed
Sickle-cell disease is a genetic disease that results in abnormal hemoglobin molecules. When these release their oxygen lo ad in the tissues, they become insoluble, leading to mis-shaped red blood cells. These sickle shaped red cells are less deformable and viscoelastic meaning that they have become rigid and can cause blood vessel blockage, pain, strokes, and other tissue damage.
Thalassemia is a genetic disease that results in the production of an abnormal ratio of hemoglobin subunits.
Hereditary spherocytosis syndromes are a group of inherited disorders characterized by defects in the red blood cell's cell membrane, causing the cells to be small, sphere-shaped, and fragile instead of donut-shaped and flexible. These abnormal red blood cells are destroyed by the spleen. Several other hereditary disorders of the red blood cell membrane are known.^[47]
Pernicious anemia is an autoimmune disease wherein the body lacks intrinsic factor, required to absorb vitamin B₁₂ from food. Vitamin B₁₂ is needed for the production of hemoglobin.
Aplasticanemia is caused by the inability of the bone marrow to produce blood cells.
Pure red cell aplasia is caused by the inability of the bone marrow to produce only red blood cells.
How Did RBCs Evolve? 2

But blood cells go back, really far back. The first ones to evolve may have performed a mixed function of what white blood cells and red blood cells do now. Here's what the U.S. National Library of Medicine, National Institute of Health has to say: "The most primitive blood cell may have been a protohemocyte which was first involved in phagocytosis and nutrition. When metazoans (sponges) appeared [hundreds of millions of years ago], their "blood" cells, the archeocytes, were phagocytic. [Later] a progressive differentiation of several leukocytic types occurred. Differentiated cells appeared that distributed food and oxygen, thus erythrocytes evolved in certain marine or polychaete annelids [worms]."
In other words, the earliest ancestor of blood cells may have been one type of cell that provided both immunity and energy to the organism. Later, in what may have been sponges or marine worms, this “proto” blood cell evolved into several types of cells involved with immunity (WBCs) and other blood cells that distributed nutrients (plasma) and oxygen (RBCs). 

Bold assertions, but without going to give insights how this change could have happened at the molecular level. As usual, they just assert it may have happened. But how, remains a open question. 

Red Blood Cells: Centerpiece in the Evolution of the Vertebrate Circulatory System 1

All vertebrates except cold-water ice fish transport oxygen via hemoglobin packaged in red blood cells (RBCs).

Hemoglobins were the first of the oxygen  carriers .

1) http://az.oxfordjournals.org/content/amzoo/39/2/189.full.pdf
2) https://bloodcenterblog.stanford.edu/billie-rubin/how_did_rbcs_ev/
3) http://creation.com/life-is-in-the-blood

Platelets


Where is there an intermediate between the thrombocytes found in reptiles and birds and the platelets in mammals? What selective advantage do platelets provide and what brought the selective pressure to bear to create this complex system? It is a mystery of punctuated evolution. Readers of Scientia are invited to speculate. 2

Role in disease

Platelet disorders 1
Adapted from

The three broad categories of platelet disorders are "not enough"; "dysfunctional"; and "too many".

Thrombocytopenia
Immune thrombocytopenias (ITP) – formerly known as immune thrombocytopenia purpura and idiopathic thrombocytopenic purpura
Splenomegaly
Gaucher's disease
Familial thrombocytopenia
Chemotherapy
Babesiosis
Dengue
Onyalai
Thrombotic thrombocytopenic purpura
HELLP syndrome
Hemolytic-uremic syndrome
Drug-induced thrombocytopenic purpura (five known drugs – most problematic is heparin-induced thrombocytopenia (HIT)
Pregnancy associated
Neonatal alloimmune associated
Aplastic anemia
Transfusion associated
Pseudothrombocytopenia
Gilbert's Syndrome
Altered platelet function
Congenital
Disorders of adhesion
Bernard-Soulier syndrome
Disorders of activation
Disorders of granule amount or release
Hermansky-Pudlak Syndrome
Gray platelet syndrome
ADP Receptor defect
Decreased cyclooxygenase activity
Storage pool defects, acquired or congenital
Disorders of aggregation
Glanzmann's thrombasthenia
Wiskott–Aldrich syndrome
Acquired
Disorders of adhesion
Paroxysmal nocturnal hemoglobinuria
Asthma
Samter's triad (aspirin exacerbated respiratory disease/AERD)
Cancer
Malaria
Decreased cyclooxygenase activity
Thrombocytosis and thrombocythemia
Reactive
Chronic infection
Chronic inflammation
Malignancy
Hyposplenism (post-splenectomy)
Iron deficiency
Acute blood loss
Myeloproliferative neoplasms – platelets are both elevated and activated
Essential thrombocytosis
Polycythemia vera
Associated with other myeloid neoplasms
Congenital


Platelets have been mostly thought of as a fragment of a cell that stops bleeding and not much else. They can, also, produce damage because clotting can block arteries causing strokes and heart attacks. So, it is a surprise that recent research finds platelets have many other roles including being critical players in defense against microbes. In fact, they are able to find microbes and rapidly receive and secrete messages that stimulate immune defense. They have been found to be vital in calling for leukocytes and work with the immune cells to fight infections. They are, even, able to aid the very intelligent T cell when it communicates with B cells to enhance antigen responses. They are able to send complex signals and respond to signals from other cells. How can this little piece of cytoplasm, not even a full cell and without a nucleus, perform all of these functions? Does this research show platelet intelligence?

Platelets Do Much More

The easiest way for a microbe to enter the body is to follow the path of a trauma or foreign body. Following the opened area, microbes can slip into the tissue or the bloodstream. Therefore, as first responders to injury, it is natural that platelets could have both a central role in maintenance of blood flow and defense against invaders.


Injury immediately stimulates a blood clotting reaction that produces thrombin, which rapidly induces platelets to alter their shape and stop the flow of blood locally. Thrombin stimulates receptors on the platelets that attract connective tissue fibers (collagen, laminin, fibronectin, fibrinogen and other molecules). These fibers work with the platelets to plug damage with a scar.

In creatures much earlier in the evolutionary tree, such as very early vertebrates and invertebrates, there are specialized cells (hemocytes) that provide both blood flow and immune functions. In mammals, cells became more specialized into leukocytes, lymphocytes and platelets.


Mammals are the only creatures with platelets. It was thought that platelets dealt with only regulation of blood flow, leukocytes with inflammation and lymphocytes with regulation of immune function. Recently, it was noticed that platelets still have kept the capacity to help regulate immune responses. But, how can a cell without a nucleus do so much?

Basic Platelets

Platelets are small–2 to 4 μm (micron is 1/1000 of a millimeter)– and live for a week. Platelets are convex on both sides–shaped like a lens. They are manufactured by the large megakaryote blood cells in the bone marrow, which become 20 times larger when making platelets.
A hormone from the liver and kidneys, thombopoietin, is the signal to make more platelets. Each megakaryote makes 1 to 3 thousand platelets and a trillion platelets are manufactured daily. From the bone marrow, they flow into the blood stream. Some are stored in the spleen for emergency situations and released by signals from the sympathetic nervous system (fight and flight).
Platelets are born without a nucleus and their own DNA, but have many messenger RNAs that allow them to make proteins by themselves. The only DNA is in mitochondria.

Activation

When first formed, platelets look like little wrinkled brains. They have a large amount of extra membrane that is folded into channels, so that the platelet can change shape quickly. When activated by receptors, they go through dramatic shape change.

First, platelets attach to the broken vessels and then they become activated. When activated they produce many special receptors (using the ribosomes they already have, not DNA in a nucleus). Calcium increase triggers microtubules and actin to dramatically alter the scaffolding and the platelet produces many long arms that make it look like a small astrocyte or dendritic cell.

The shape change has been described in three stages. The first produces the multiple arms and legs. The second spreads out the body of the platelet and the third makes a thicker central body.The changes occur through an interaction of the microtubules and a change in the membrane with its invaginations. The motor of these changes is just below the membranes, which creates a dramatic increase in membrane surface and then produces the arms and legs. It is a remarkable process that occurs without stretching or adding lipids to the membrane.
The next phase is when the platelets join their arms and form aggregates. 

Releasing Granules

• δ- includes molecules that regulate blood flow—containing nucleotides such as ADP, amines such as histamine and serotonin, and ions such as Ca2and PO3–.
  
  
  
• α- has many different proteins and enzymes that attach to and kill microbes. They, also, have factors that increase clotting.
  
  
  
• λ-, lysosomal vesicles are filled with many enzymes that will alter the shape of the clot so that healing can occur.
  
  
  

To use these remarkable capacities, platelets change from the circular type that is well known, to amoeboid cells with pseudopods (arms) that can interact with microbes and even secrete reactive molecules that can kill microbes. The platelet uses a very large amount of specific molecules for various different microbes.

Platelets Have Many Sensory Receptors

There are more platelets than other blood cells and they have many pattern recognition receptors that often serve as the first responders to injury and invaders. When they recognize trouble, platelets deliberately move to the center of the trauma or infection and become the most numerous cells there. They specifically accumulate where there are microbe generated proteins and complement proteins. When the platelet receptor picks up specific microbe peptides, it rapidly changes its shape by altering the tubule scaffolding, stimulates calcium and releases some of its vesicles.


Platelets have chemokine receptors for all four of the major types (C, CC, CXC, CX3C), so they can be drawn from a great distance. Other mechanisms draw the platelets there—they respond to chemical signals related to collagen that, also, attract neutrophils. Importantly, platelets at the site release a class of molecule called kinocidins, that draw many other immune cells.
Platelets, also, have receptors for specific immune molecules such as IgG and IgA and IgE, CRP and other cytokines. Platelets receptors detect pathogen-associated molecular patterns (PAMPs) by using the well-known Toll-like receptors (TLR) and various types of cytokines in the IL-1 family. These responses form a cascade inside the cell even without a nucleus, including TNF and other major signals.

Platelets Use Complex Signaling

Like other intelligent larger cells, platelets engage in very complex signaling with many important Toll Like receptors (TLR), such as TLR1, TLR2, TLR4, TLR6, and TLR9. In different situations they use IL-6 and COX-2 in cross talk with other cells. It is too complex to relate here, but in many different situations, a vast number of different signals are used by this cell that has no nucleus. They respond differently to various subtypes of microbes and, then, transmit very specific cytokines from their large vocabulary. These cytokines cause other particular immune cells to activate in very precise ways.


As with all mechanisms, sometimes they go awry. Platelets can mistakenly stimulate increased inappropriate reactions that create clots in the wrong places.

Amplification of Important Signals

The platelet has a unique amplification system for important signals with many different mechanisms. One response to staph aureus includes release ofδ granules with ADP and ATP and then a succession of stimulation by neighboring platelets releasing α granules with specific proteins and kinocidins. The microbe, then, attempts to stop the granule secretion and to break down the specific factors that are released.

Direct Attack on Microbes

Like the larger granulocyte cells such as neutrophils, platelet granules have many different proteins and peptides. Platelets have recently been found to release at least four distinct categories of anti microbe molecules in their granules.

• Kinocidins including CSCL4, CSCL7 and CCL5 – these can function as chemokines, that is attracting specific immune cells or directly killing microbes.
  
  
  
• Defensins including BD2 and Tb4
  
  
  
• Proteins made from breaking up other molecules
  
  
  
• Thrombocidins, which are made by enzymes cutting CXCL7
  
  
  

Kinocidins Are Very Complex Platelet Secreted Molecular Signals

Kinocidins are a complex large class of molecules. They include CSCL4, CSCL7 and CCL5, which can function as chemokines, attracting specific immune cells, as well as, directly killing microbes. Kinocidins are either full sized proteins, or pieces of proteins that are created by enzymes cutting the secreted proteins. They are similar to molecules housed in monocytes and macrophages. Their concentration is sensed by special thrombin receptors in the platelets that can increase the messenger RNA to make more. This increases the messenger RNA product by 30 to 100 times.


The major kinocidin is the peptide CXCL4, also known as platelet factor 4. It has a complex structure with different regions and effects—some to kill microbes, some to communicate with other cells. It has an interesting module that allows it to rapidly evolve to keep up with corresponding microbe evolution. These multiple parts are stabilized into a very specific structure.
Platelet kinocidins have unusual properties. They are released into the blood when triggered by microbe invasion. Those from the much larger immune cells are only used locally. Platelet kinocidins, also, serve as immune signals to recruit other cells, as well as stimulating a larger response. The kinocidins of larger immune cells don’t have these elaborate functions.



To make matters even more complex, the kinocidins attract specific phagocytes to fight inflammation, which produce enzymes that further break down these pieces into smaller pieces that are also able to attack microbes. In fact, microbes inadvertently produce enzymes that help produce these smaller molecules, some which are inactivated, but some ultimately attack the microbe itself.
The kinocidins are able to distinguish membranes of the microbes and the immune cells, often by the ability to bind to specific lipids that are in membranes. They are able to understand which to use at different levels of pH. They are able to choose among many small particles for many different microbes.

Platelet Kinocidins Can Attack Many Different Microbes

The platelet kinocidins are very effective against viruses, bacteria, fungus and protozoa. They have recently been shown to be critical in defense against HIV-1. s Other platelet factors have been shown to work against S aureus, strep, and others. They appear to limit strep heart infections.
Plasmodium falciparum, the cause of malaria, is greatly affected by platelet factors. It appears that CXCL4 is critical to human defense against malaria. The platelet factor can uniquely get into infected red blood cells. This is the major way that malaria can be killed in red blood cells. A larger number of platelets represents a greater fight with infection, whereas low platelet counts are caused by monocytes eating platelets.
Platelets have, also, been shown to be effective against many fungi—Candida, Aspergillus, Toxoplasma , Leishmania, and Schistosoma.

Other Platelet Mechanisms Against Microbes

Platelets make other chemicals against microbes. They can make hydrogen peroxide and other reactive oxygen particles. These are critical to combat worms.
Platelets don’t really eat microbes, but they do cover them so that others can eat them. They also form a surface for other complexes, such as the large thrombin protein complex that kills bacteria.

Platelets Call for Help

One of the most important capacities of platelets is recruiting and helping other larger immune cells. Unique platelet properties include surveillance of the surface of other immune cells, such as macrophages and liver cells. Platelets seem to be critical to finding infected macrophages.


Platelets are critical in helping neutrophils by actively stimulating receptors that bind to leukocytes and monocytes as well as binding to microbes, thereby connecting the two. This increases the ability of neutrophils to eat bacteria. These complexes built of platelets and leukocytes activate both cells and recruit more. They build larger complexes, called neutrophil extracellular traps, NETs, consisting of DNA strands from the neutrophil, histones and other fibers which attach to all types of proteins and help them to kill bacteria. This is a mechanism of killing bacteria without hurting the tissue.
Also, platelet kinocidins directly increase phagocytosis by neutrophils, which kill microbes inside the cell.


Platelets stimulate immune dendritic cells, which are the key cells that present antigens to T cells and B cells. Platelets rapidly connect with the microbe and aid the dendritic cells in getting antigens from them. When dendritic cells meet the platelets, they signal with a wide range of cytokines. These events greatly increase the specificity of the response against a particular microbe.


Platelets directly aid T cells by secreting very specific cytokines and kinocidins, which activate T cells. When viruses have invaded platelets, they signal to enhance the T cells that are required against that virus. This signaling helps the T cells to be ready to attack other infected cells as well. Of the many types of T cells, that both help and hurt different situations, the platelet is able to signal for the one that will enhance the specific response for a specific microbe. Signals from other cells including T cells stimulate many new receptors on the platelets, which brings neutrophils into the defense.
Platelets are able to help B cells build up an army to attack viruses.Without the platelet signals they are unable to perform. The platelets appear to be a necessary communication intermediary between T cells and B cells to help each other.

Microbes Strike Back

Some microbes are able to hijack platelet mechanisms and use activated platelets to seek out and attack other immune cells. They are, also, able to decrease the clotting effects of platelets and use the platelet itself for increased strength of attack. In one case, the microbe forms a matrix of fibrin and platelets to form an infected clot on a heart valve.

Platelet Intelligence

Platelets are critical to control blood loss, but also for many immune functions. They have a smaller, but very effective, repertoire of immune receptors and cytokines than the professional larger white blood cells. The fact that they are much smaller and more numerous is an advantage in traveling into difficult terrain. Recently, it was learned that this little cell, with no nucleus or DNA, but messenger RNA and mitochondria, is able to make large numbers of very complex molecules to fight a vast number of different microbes, infections, and injuries.


In fact, platelets are at the center of complex communication with a very large number of different cells. They are critical for the function of the leukocyte, dendritic cell, T cell, B cell and neutrophil and stay in constant communication with the vascular endothelial cell and red blood cells. They are able to make a very complex kinocidin protein, which can then be broken down into multiple very powerful specific small particles to fight many different microbes. Kinocidin, also, has a region that evolves along with the microbes to stay effective.
How can the small platelet, without a nucleus, be so intelligent?


1) https://en.wikipedia.org/wiki/Platelet#cite_note-Michelson.2C_Platelets.2C_2013.2C_p._vii-51
2) http://membercentral.aaas.org/blogs/scientia/invention-platelets

Macrophage

Role in disease

 1
Due to their role in phagocytosis, macrophages are involved in many diseases of the immune system. For example, they participate in the formation of granulomas, inflammatory lesions that may be caused by a large number of diseases. Some disorders, mostly rare, of ineffective phagocytosis and macrophage function have been described, for example.

As a host for intracellular pathogens
In their role as a phagocytic immune cell macrophages are responsible for engulfing pathogens to destroy them. Some pathogens subvert this process and instead live inside the macrophage. This provides an environment in which the pathogen is hidden from the immune system and allows it to replicate.

Diseases with this type of behaviour include tuberculosis (caused by Mycobacterium tuberculosis) and leishmaniasis (caused by Leishmania species).

Tuberculosis
Once engulfed by a macrophage, the causative agent of tuberculosis, Mycobacterium tuberculosis, avoids cellular defenses and uses the cell to replicate.

Leishmaniasis
Upon phagocytosis by a macrophage, the Leishmania parasite finds itself in a phagocytic vacuole. Under normal circumstances, this phagocytic vacuole would develop into a lysosome and its contents would be digested. Leishmania alter this process and avoid being destroyed; instead, they make a home inside the vacuole.

Chikungunya
Infection of macrophages in joints is associated with local inflammation during and after the acute phase of Chikungunya (caused by CHIKV or Chikungunya Virus).

Others
Adenovirus (most common cause of pink eye) can remain latent in a host macrophage, with continued viral shedding 6–18 months after initial infection.

Brucella spp. 
can remain latent in a macrophage via inhibition of phagosome–lysosome fusion; causes brucellosis (undulant fever).

Heart disease
Macrophages are the predominant cells involved in creating the progressive plaque lesions of atherosclerosis.Focal recruitment of macrophages occurs after the onset of acute myocardial infarction. These macrophages function to remove debris, apoptotic cells and to prepare for tissue regeneration.

HIV infection
Macrophages also play a role in Human Immunodeficiency Virus (HIV) infection. Like T cells, macrophages can be infected with HIV, and even become a reservoir of ongoing virus replication throughout the body. HIV can enter the macrophage through binding of gp120 to CD4 and second membrane receptor, CCR5 (a chemokine receptor). Both circulating monocytes and macrophages serve as a reservoir for the virus. Macrophages are better able resist infection by HIV-1 than CD4+ T cells, although susceptibility to HIV infection differs among macrophage subtypes.

Cancer
Macrophages contribute to tumor growth and progression. Attracted to oxygen-starved (hypoxic) and necrotic tumor cells they promote chronic inflammation. Inflammatory compounds such as Tumor necrosis factor (TNF)-alpha released by the macrophages activate the gene switch nuclear factor-kappa B. NF-κB then enters the nucleus of a tumor cell and turns on production of proteins that stop apoptosis and promote cell proliferation and inflammation. Moreover, macrophages serve as a source for many pro-angiogenic factors including vascular endothelial factor (VEGF), tumor necrosis factor-alpha (TNF-alpha), granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-1 and IL-6  contributing further to the tumor growth. Macrophages have been shown to infiltrate a number of tumors. Their number correlates with poor prognosis in certain cancers including cancers of breast, cervix, bladder and brain. Tumor-associated macrophages (TAMs) are thought to acquire an M2 phenotype, contributing to tumor growth and progression. Recent study findings suggest that by forcing IFN-α expression in tumor-infiltrating macrophages, it is possible to blunt their innate protumoral activity and reprogramme the tumor microenvironment toward more effective dendritic cell activation and immune effector cell cytotoxicity.

Obesity
Increased number of pro-inflammatory macrophages within obese adipose tissue contributes to obesity complications including insulin resistance and diabetes type 2.

1) https://en.wikipedia.org/wiki/Macrophage#Disease

Eosinophil

Eosinophil granulocytes, usually called eosinophils or eosinophiles (or, less commonly, acidophils), are a variety of white blood cell and one of the immune system components responsible for combating multicellular parasites and certain infections in vertebrates. Along with mast cells, they also control mechanisms associated with allergy and asthma. They are granulocytes that develop during hematopoiesis in the bone marrow before migrating into blood.

Role in disease

Eosinophilia can be idiopathic (primary) or, more commonly, secondary to another disease. In the Western World, allergic or atopic diseases are the most common causes, especially those of the respiratory or integumentary systems. In the developing world, parasites are the most common cause. A parasitic infection of nearly any bodily tissue can cause eosinophilia. Diseases that feature eosinophilia as a sign include the following:

Allergic disorders
Asthma
Hay fever
Drug allergies
Allergic skin diseases
Pemphigus
Dermatitis herpetiformis
Eosinophilic esophagitis
Eosinophilic gastroenteritis
Parasitic infections
Addison's disease
Some forms of malignancy
Acute lymphoblastic leukemia
Chronic myelogenous leukemia
Eosinophilic leukemia
Hodgkin lymphoma
Some forms of non-Hodgkin lymphoma
Systemic autoimmune diseases[4] (e.g., SLE, idiopathic eosinophilic synovitis)
Systemic mastocytosis
Some forms of vasculitis (e.g., Churg-Strauss syndrome
Cholesterol embolism (transiently)
Coccidioidomycosis (Valley fever), a fungal disease prominent in the US Southwest.
Human immunodeficiency virus infection
Interstitial nephropathy
Hyperimmunoglobulin E syndrome, an immune disorder characterized by high levels of serum IgE
Idiopathic hypereosinophilic syndrome.

Mast cells

A mast cell (also known as a mastocyte or a labrocyte^[1]) is a type of white blood cell. Specifically, it is a type of granulocyte derived from the myeloid stem cell that is a part of the immune and neuroimmune systems and contains many granules rich in histamine and heparin. Although best known for their role in allergy and anaphylaxis, mast cells play an important protective role as well, being intimately involved in wound healing,angiogenesis, immune tolerance, defense against pathogens, and blood–brain barrier function

Role in disease

Parasitic infections

Mast cells are activated in response to infection by pathogenic parasites, such as certain helminths and protozoa, through IgE signaling.

Mast cell activation disorders are a spectrum of immune disorders that are unrelated to pathogenic infection and involve similar symptoms that arise from secreted mast cell intermediates, but differ slightly in their pathophysiology, treatment approach, and distinguishing symptoms. The classification of mast cell activation disorders was laid out in 2010.

Allergic disease

Allergies are mediated through IgE signaling which triggers mast cell degranulation.
Many forms of cutaneous and mucosal allergy are mediated in large part by mast cells; they play a central role in asthma, eczema, itch (from various causes), and allergic rhinitis and allergic conjunctivitis. Antihistamine drugs act by blocking histamine action on nerve endings. Cromoglicate-based drugs (sodium cromoglicate, nedocromil) block a calcium channel essential for mast cell degranulation, stabilizing the cell and preventing release of histamine and related mediators. Leukotriene antagonists (such as montelukastand zafirlukast) block the action of leukotriene mediators and are being used increasingly in allergic diseases.
Calcium triggers the secretion of histamine from mast cells after previous exposure to sodium fluoride. The secretory process can be divided into a fluoride-activation step and a calcium-induced secretory step. It was observed that the fluoride-activation step is accompanied by an elevation of cAMP levels within the cells. The attained high levels of cAMP persist during histamine release. It was further found that catecholamines do not markedly alter the fluoride-induced histamine release. It was also confirmed that the second, but not the first, step in sodium fluoride-induced histamine secretion is inhibited by theophylline. Vasodilation and increased permeability of capillaries are a result of both H1 and H2 receptor types.
Stimulation of histamine activates a histamine (H2)-sensitive adenylate cyclase of oxyntic cells, and there is a rapid increase in cellular [cAMP] that is involved in activation of H+ transport and other associated changes of oxyntic cells.

Anaphylaxis

In anaphylaxis (a severe systemic reaction to allergens, such as nuts, bee stings, or drugs), body-wide degranulation of mast cells leads to vasodilation and if severe, symptoms of life-threatening shock.
Histamine is a vasodilatory substance released during anaphylaxis.

Autoimmunity

Mast cells may be implicated in the pathology associated with autoimmune, inflammatory disorders of the joints. They have been shown to be involved in the recruitment of inflammatory cells to the joints (e.g., rheumatoid arthritis) and skin (e.g., bullous pemphigoid), and this activity is dependent on antibodies and complement components.

Mastocytosis and clonal disorders

Mastocytosis is a rare clonal mast cell disorder involving the presence of too many mast cells (mastocytes) and CD34+ mast cell precursors.^[26] Mutations in c-Kit are associated with mastocytosis.

Monoclonal disorders

Neoplastic disorders

Mastocytomas, or mast cell tumors, can secrete excessive quantities of degranulation products.^[20][21] They are often seen in dogs and cats.^[27] Other neoplastic disorders associated with mast cells include mast cell sarcoma and mast cell leukemia.

Mast cell activation syndrome

Mast cell activation syndrome (MCAS) is an idiopathic immune disorder that involves recurrent and excessive mast cell degranulation and which produces symptoms that are similar to other mast cell activation disorders.^[20][21] The syndrome is diagnosed based upon four sets of criteria involving treatment response, symptoms, a differential diagnosis, and biomarkers of mast cell degranulation.^[20][21]

Central nervous system

Unlike other hematopoietic cells, mast cells naturally occur in the brain where they interact with the neuroimmune system as part of the microbiota-gut-brain axis. In the human brain, mast cells are located in a number of structures which mediate visceral sensory (e.g., pain) or neuroendocrine functions – including the pituitary stalk, pineal gland, thalamus, and hypothalamus – or which are located at the blood–brain barrier or blood–cerebrospinal fluid barrier, including the area postrema, choroid plexus, and in the dural layer of the meninges near meningeal nociceptors. Mast cells serve the same general functions in the body and central nervous system, such as effecting or regulating allergic responses, innate and adaptive immunity, autoimmunity, and inflammation. Across systems, mast cells serve as the main effector cell through which pathogens can affect the brain-gut axis.
In the gastrointestinal tract, mucosal mast cells are located in close proximity to sensory nerve fibres, which communicate bidirectionally. When these mast cells initially degranulate, they release mediators (e.g., histamine, tryptase, and serotonin) which activate, sensitize, and upregulate membrane expression of nociceptors (i.e., TRPV1) on visceral afferent neurons via their receptors (respectively, HRH1, HRH2, HRH3, PAR2, 5-HT3);^[30] in turn, neurogenic inflammation, visceral hypersensitivity, and intestinal dysmotility(i.e., impaired peristalsis) result. Neuronal activation induces neuropeptide (substance P and calcitonin gene-related peptide) signaling to mast cells where they bind to their associated receptors and trigger degranulation of a distinct set of mediators (β-Hexosaminidase, cytokines, chemokines, PGD2, leukotrienes, and eoxins).

Autism

Research into an immunological contribution to autism suggests that autism spectrum disorder (ASD) children may present with "allergic-like" problems in the absence of elevated serum IgE and chronic urticaria, suggesting non-allergic mast cell activation in response to environmental and stress triggers. This mast cell activation could contribute to brain inflammation and neurodevelopmental problems.

Neutrophils


Neutrophil granulocytes (also known as neutrophils or occasionally neutrocytes) are the most abundant type of granulocytes and the most abundant (40% to 75%) type of white blood cells in most mammals. They form an essential part of the innate immune system. Functionality varies in different animals

Role in disease

Low neutrophil counts are termed neutropenia. This can be congenital (developed at or before birth) or it can develop later, as in the case of aplastic anemia or some kinds of leukemia. It can also be a side-effect of medication, most prominently chemotherapy. Neutropenia makes an individual highly susceptible to infections. It can also be the result of colonization by intracellular neutrophilic parasites.
In alpha 1-antitrypsin deficiency, the important neutrophil enzyme elastase is not adequately inhibited by alpha 1-antitrypsin, leading to excessive tissue damage in the presence of inflammation – the most prominent one being pulmonary emphysema.
In Familial Mediterranean fever (FMF), a mutation in the pyrin (or marenostrin) gene, which is expressed mainly in neutrophil granulocytes, leads to a constitutively active acute-phase response and causes attacks of fever, arthralgia, peritonitis, and – eventually –amyloidosis.^[28]
Decreases in neutrophil function have been linked to hyperglycemia. Dysfunction in the neutrophil biochemical pathway myeloperoxidase as well as reduced degranulation are associated with hyperglycemia.^[29]
The Absolute neutrophil count (ANC) is also used in diagnosis and prognosis. ANC is the gold standard for determining severity of neutropenia, and thus neutropenic fever. Any ANC < 1500 cells / mm³ is considered neutropenia, but <500 cells / mm³ is considered severe.^[30] There is also new research tying ANC to myocardial infarction as an aid in early diagnosis.

Complex Migration of Leukocytes 1

Leukocytes, immune cells also known as white blood cells, use very complex modes of travel to navigate the vastly different environments of the various human organs. A variety of immune cells, including T cells and neutrophils, travel throughout the body, in and out of these tissues. There are thousands of different factors in the tissues that affect travel in difficult environments, as well as many local cells that aid travel. The goal of the complex migration of leukocytes is to position the immune cell to fight infection. The navigation can be very tricky and involves a vast array of different mechanisms. It is not clear how cells are able to know how to do it. Like many other processes described in these posts, the complex migration of leukocytes involves elaborate back and forth communication between many cells and the vast language of cytokines and chemical factors.

All cells exposed to the outside world, such as the skin, the lungs and GI tract, come in contact with many toxins, injuries and microbes. The immune system takes on all of these challenges, and its cells must position themselves accordingly.


Usually, a small number of sentinel cells are present in any organ. But, when activated and alerted about danger, T cells, neutrophils and monocytes must travel to the site and into the organs. They need to be extremely active in their patrolling behavior in each different environment and, also, provide a very specific response upon arrival at the target. Their ability to migrate and rapidly enter the tissue is critical for the response.
At first the cells travel in the blood and then must grab onto the endothelial lining of the blood vessel using multiple adhesion molecules that allow rolling, tethering, and firm adherence. Then, they must travel between the endothelial cells and through the basement membrane into the extracellular space where they meet very different factors including chemical gradients that attract and repel them. They have to alter their shape as well as their direction many times. They can use their own actin-based propulsion to move, as well as travelling along non-cellular scaffolding structures. Once at the infected or damaged site they take stock and provide many repair functions.
Recently, new imaging devices have allowed the study of the navigation of live T cells and neutrophils, which has for the first time allowed fantastic research diretly observing these movements.

Migration

Individual cells have multiple different ways of movement for different three-dimensional situations. These are all provided by the operation of distinctive ways that actin-myosin motors, scaffolding structures, and moving tubules operate.




Vojtech.dostal
Amoeboid movement involves a round cell shape and a leading edge of the cell that moves. The pseudopod is a gliding form of movement that looks like an omoeba, maintaining the basic round shape, especially in the rear. Actin builds large molecules that rapidly move the leading edge forward into arms called pseudopods. Then other myosin motors contract in the middle of the cell. A large amount of different myosin motors in the back do not allow protrusion, which pushes movement forward.
Blebbing movement is another form that has contractions at the rear of the cell. This pushes cytoplasm forward forming round out-pouchings called blebs. This type of movement depends on outside adhesion.
Adhesions from the traveling cells attach to the lattice of the extracellular matrix allowing the cell to protrude forward. The attachment is by integrin molecules and a linking mechanism called the molecular clutch, which engages and disengages rapidly. This apparatus uses actin moving backwards and linking to integrin while the extra cellular matrix pushes the cell forward. Recent research shows that leukocytes walk stepwise with two feet below that alternatively form adhesions in different places. When the scaffolding proteins reach forward, these adhesions help push the cell.
Leukocytes (dendritic cells, neutrophils, and lymphocytes) use an actin protruding, integrin linking adhesion mode of movement some of the time and an independent form without attachment to extracellular structures other times. They can move along adhesive and non-adhesive surfaces at similar speeds.
When the tissue is very dense they


must make special actin-myosin contractions in the rear. Leukocytes are able to rapidly change their shape for different situations and travel quickly through many different environments.


In low-density matrices, they can use less myosin activity. In highly dense material they form another structure called pearl-chain nuclei. Here they change the shape of the nucleus to get through small crevices rather than the typical large oval nucleus. Very recently, research has shown that leukocytes can move using scaffolding either when stimulated by external signals, or spontaneously with out external direction. In this latter case, the cell determines a specific direction to move.

Neutrophils Travelling

Neutrophils clear debris and dangerous microbes from tissues. First they attach to the blood vessel near the inflammation on the other side and then they transit between the lining cells when immune cells outside the vessel signal to allow passage through the tight junctions.  After this, they are confronted with the basement membrane. They pass through this membrane, again, when signals from local cells allow it. 


The concept of many cells working together through complex communication was established with T cells in the the blood-brain barrier (see post on T cells in CSF ).  It now appears that this complex signaling for leukocyte travel occurs in all body tissues. Back and forth signaling includes endothelial cells, pericytes, astrocytes, microglia, neurons and factors from the extracellular matrix. The neurons regulate the blood flow and signal to all of these other cells as well as the traveling immune cells.
In fact, each bodily organ has complex communication with multiple special cells that are part of the process of leukocytes entering tissues. These include intelligent activity between endothelial cells (see post Intelligent Gut Epithelial Cell), pericytes that surround the vessels, perivascular macrophages just inside the tissue, immune mast cells and the basement membrane itself. Signals and factors from all of these different cells determine not only the entrance into the tissue but also the transit through it.

Basement Membrane and Pericytes

Small veins have basement membranes around them consisting of a very complex mesh of many long intertwined molecules. It holds together the wall of the blood vessel and generally stops cells like leukocytes from passing through. Inside the matrix are long cells called pericytes (many different sizes and densities in different regions) because they sit at the perimeter of the blood vessels.


Another type of cell that intermittently surrounds the blood vessels, are immune macrophages which lie just beyond the basement membrane in skin, muscle and the brain. They have been recently discovered to send signals that regulate the tight junctions between the cells lining the vessel. These signals open them and allow space for neutrophils. These local macrophages sitting right on the outside of the vessel, also, secrete other chemicals that attract the leukocyte and give direction to the migration into the tissue.
This all occurs by complex signaling between the endothelial cells, the cells around the vessels and the leukocytes that are travelling through. Mast cells that also sit near the vessels—immune cells filled with histamine to stimulate inflammation—sense microbes and secrete histamine but also send other important cytokines that stimulate inflammation.

Priming Leukocytes for Battle

In addition to aiding their movement into the tissue, signals from these cells inside the tissue to the leukocyte give specific instructions about the problems to be faced in the local tissue, the specific microbes, problems in traveling in the tissue, and issues in clearing debris. In response to these messages, the leukocyte changes shape and becomes polarized. It also produces specific proteins and integrins. These signals are different in each tissue.

Traveling In the Tissue

In the tissue, leukocytes are confronted with a wide variety of factors that hinder and aid their travels. These include factors in the extracellular matrix, factors from debris from damaged cells, a large number of cytokines (specific detailed signals with information) and chemokines (specific attracting or repelling functions).


In the tissue, neutrophils communicate with each other, much as microbe communities do. They secrete signals that aid each other to find their appropriate place and complete their mission. Meanwhile, the tissue cells secrete many different attracting molecules that form a gradient toward the critical region and can guide the leukocytes a very long distance. Leukocytes have very complex sensory receptors that allow them to understand different gradients.Leukocytes, also, rely on specific scaffolds in each tissue to make traveling easier—in lymph nodes, liver, lung, skin, and the brain.



Leukocytes know to leave the blood vessel in the direction of the infection. The new polarized shape helps the leukocyte to hone in the exact direction. They travel approximately 10um per minute and sometimes 30um per minute. When they arrive at the problem site they move more slowly. With communication among the leukocytes they gather in a cluster, until up to several hundred have arrived. In the case of injury, the cluster of leukocytes then walls off the area.


First, scouts move in to form a small group. They produce strong attracting signals to other leukocytes. This signal makes the polar shape even stronger and pulls a large number of cells in. Leukocytes relay these messages to cells that are more distant to increase specific direction of movement and speed. Some of these cells might die in the process, which produces other signals. In certain infections a process of leukocyte swarming occurs where a large amount of cells produce large aggregates. Then the leukocytes remodel the extracellular matrix. Later other immune cells, like monocytes, appear at the periphery.


Recent evidence confirms that migrating leukocytes use gradients, among several mechanisms, with higher densities closer to the site. One study showed that near a wound a hydrogen peroxide gradient was established extending a long ways into the tissue. Other studies have shown different tissues and injuries with a wide range of other chemical gradients. Some of these are produced in waves of different gradients. Leukocytes have to develop receptors for each of these.


Importantly, leukocytes are able to take in multiple competing signals and decide which are the most important. Each of these signals produces a different cascade of molecules within the cell to the nucleus. Some signals are telling the exact trouble spot and this will take precedence the closer they get to the site.
In some cases, there are repulsive signals, where the leukocytes take inflammation debris, turn around and travel in the opposite direction, back to the blood vessels and into lymph tissue and perhaps other organs. These leukocytes, then, stimulate a strong general signal from other parts of the body, which will sendl even more cells to travel to the trouble spot.

T Cells

T cells, perhaps the brains of the immune system (see post Intelligent T cells), use all of the previously mentioned strategies, but also additional ones, since they must find the antigen in a large region where there are few of the cells they are seeking. They need even better homing and movement strategies since the target cells might, also, be in the midst of very dense inflammation sites. They must systematically scan whole regions and therefore, cannot use simple attractive schemes.


Like leukocytes, T cells, also, polarize their shape after entering tissue. But, unlike leukocytes they change their shape often and frequently stop before again moving in a new direction during which they become round again. These shapes can help moving through specific types of extra cellular matrix.

When they encounter an antigen, they stop moving completely and form a synapse with the target cell, either infected or cancerous. By connecting with the cell for minutes to hours they are able to destroy it. Cytotoxic killer T cells destroy a target cell about every 6 hours. After the killing they resume travelling rapidly scanning for more target cells.
Killer T cells have many different strategies in different tissues. In one example, when killer T cells are chasing herpes simplex virus in the skin, they divided into two types of cells with different modes of movement. A CD4 type actively migrated throughout the dermal tissue, while a CD8 type changed its shape to that of a dendritic cell with many long arms and crawled in the epidermis very slowly between the cells. The CD4 type went into the blood, while the CD8 stayed in the epidermis, where it painstakingly found skin cells that had the virus antigen on its surface.

Several important signaling pathways for stimulation and inhibition affect T cells mobility, and these can impair its mobility as well as help it. In addition to other activation patterns of T cells, these signals greatly affect its activity. One signal increases the movement to the extent that it doesn’t form proper synapses with other immune dendritic cells, and therefore, doesn’t get the antigen information.


Another signal reduces the T cells speed of operation in the pancreas making the necessary contacts with other cells much less efficient. Another example is in the spleen where T cells can become exhausted causing very serious inflammatory disease.

Many Types of T Cell Travel

In tissues, T cells generally don’t use gradients for movement, since they are surveilling large tissues for a small amount of microbe antigen. Instead they commonly use a modification of the amoeboid random walk.


In the skin, the brain and in some tumors, they crawl along extra cellular matrix (ECM) structures, where there is cytokine and receptor communication between the T cell and the extra cellular matrix. Various fibers in the extra cellular matrix communicate with diverse T cells in each tissue. In some, back and forth communication changed both the extra cellular matrix itself and the shape of the T cells. The ECM can either help or hurt the travels of the T cells in different types of tissue.
Another type of random walk of the T cell observed in the brain is called Levy walks. In this type of travel there is amoeboid walk with intermittent high velocity runs that allow the T cell to rapidly get much further into the tissue. This type of walk is much better at finding hidden infected cells deep in the tissue. Special chemokine signals increase the speed of this type of movement increasing the ability to find the target cells.




Another mechanism of travel for the T cell is contacting other immune macrophages and dendritic cells that it meets along the way in the tissue.Contact with these other cells can be slow or rapid, where the T cell apparently gets information to continue its search by accumulating different signals.
Thus, T cells can use scaffolds of tissue and devise specific types of movement to help find the targets deep in specific tissues.
A complex technique involves the T cell accumulating and summing many different signals from a variety of cells along the way. An example of this occurs in autoimmune encephalomyelitis where multiple brief contacts with other cells help gather information for the T cells eventually effective search. To store this information, the T cell transfers a specific factor from the cytoplasm into its nucleus. The nucleus accumulates various signals from other cells and helps navigation. Gathering information by many brief contacts with cells along the way was also observed when chasing a cancer.

Cancer Cells Migration

Several other mechanisms of movement were recently described in cancer cells. To get through very tight spaces cancer cells stimulate a water flow that is like a sailboat mechanism. At the leading edge, the membrane takes in water and ions through channels called aquaporins. This flow of water is then pumped out the back of the cell, pushing the cell forward.


Another mode of travel in cancer cells involves “chasing” healthy cells. The ordinary cell alters its mode of travel to try to escape from the cancer cell, which increases the speed of both.
Recently, a third travel mode was crawling along blood vessels. 

Complex Migration of Leukocytes

As more is learned about cells of all types, what is striking is the extremely complex communication that pervades all physiology. During movement throughout the body, very complex communication occurs between a wide variety of cells, neurons, endothelial cells, basement membrane, pericytes that sit around blood vessels, and each type of immune cell. Each tissue has its own type of communication about the unique issues in traveling through and the language is universally complex.

With many different types of movement available, leukocytes, including T cells, are able to use a wide variety of different techniques to enter into the specific organs of the human body and travel to the critical site. Techniques include changing shapes and using the leading edges of the cell in different ways. Leukocytes can decide to suddenly swarm near a target. They use chemical gradients, cytokine signaling with cells and extracellular matrix. Cells are able to alter their shape and modes of transport as needed.


The leukocyte uses the back and forth communication with many other cells to analyze the best way to traverse the specific terrain as well as the specific response to the target problem. 
With so much sophistication in communication and in the adaptations to different tissues, how can anyone say that this is not an intelligent process? How can anyone think that leukocytes are not intelligent?


1) http://jonlieffmd.com/blog/complex-migration-of-leukocytes

Dendritic cell


Dendritic cells (DCs) are antigen-presenting cells (also known as accessory cells) of the mammalian immune system. Their main function is to process antigen material and present it on the cell surface to the T cells of the immune system. They act as messengers between the innate and the adaptive immune systems. Dendritic cells are present in those tissues that are in contact with the external environment, such as the skin (where there is a specialized dendritic cell type called the Langerhans cell) and the inner lining of the nose,lungs, stomach and intestines. They can also be found in an immature state in the blood. Once activated, they migrate to the lymph nodes where they interact with T cells and B cells to initiate and shape the adaptive immune response. At certain development stages they grow branched projections, the dendrites that give the cell its name (δένδρον or déndron being Greek for "tree"). While similar in appearance, these are structures distinct from the dendrites of neurons. Immature dendritic cells are also called veiled cells, as they possess large cytoplasmic 'veils' rather than dendrites.

Role in disease

HIV infection

HIV, which causes AIDS, can bind to dendritic cells via various receptors expressed on the cell. The best studied example is DC-SIGN (usually on MDC subset 1, but also on other subsets under certain conditions; since not all dendritic cell subsets express DC-SIGN, its exact role in sexual HIV-1 transmission is not clear). When the dendritic cell takes up HIV and then travels to the lymph node, the virus can be transferred to helper CD4+ T-cells,^[21] contributing to the developing infection. This infection of dendritic cells by HIV explains one mechanism by which the virus could persist after prolonged HAART. Many other viruses, such as the SARS virus seems to use DC-SIGN to 'hitchhike' to its target cells.^[22] However, most work with virus binding to DC-SIGN expressing cells has been conducted using in vitro derived cells such as moDCs. The physiological role of DC-SIGN in vivo is more difficult to ascertain.

Autoimmunity

Altered function of dendritic cells is also known to play a major or even key role in allergy and autoimmune diseases like lupus erythematosus and inflammatory bowel diseases (Crohn's disease and ulcerative colitis)[/ltr]

T cells 


T cells (also known as Thymocytes cells) or T lymphocytes are a type of lymphocyte (in turn, a type of white blood cell) that play a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T-cell receptor (TCR) on the cell surface. They are called T cells because they mature in the thymus^[1] (although some also mature in the tonsils). The several subsets of T cells each have a distinct function. The majority of human T cells rearrange their alpha/beta T cell receptors and are termed alpha beta T cells and are part of adaptive immune system. Specialized gamma delta T cells, which comprise a minority of T cells in the human body (more frequent in ruminants), have invariant TCR (with limited diversity), can effectively present antigens to other T cells^[3]and are considered to be part of the innate immune system.

Disorders

Deficiency

Main article: T cell deficiency

Causes of T cell deficiency include lymphocytopenia of T cells and/or defects on function of individual T cells. Complete insufficiency of T cell function can result from hereditary conditions such as severe combined immunodeficiency (SCID), Omenn syndrome, andCartilage-hair hypoplasia. Causes of partial insufficiencies of T cell function include acquired immune deficiency syndrome (AIDS), and hereditary conditions such as DiGeorge syndrome (DGS), chromosomal breakage syndromes (CBSs), and B-cell and T-cell combined disorders such as ataxia telangiectasia (AT) and Wiskott-Aldrich syndrome (WAS).
The main pathogens of concern in T cell deficiencies are intracellular pathogens, including Herpes simplex virus, Mycobacterium and Listeria. Also, fungal infections are also more common and severe in T cell deficiencies.

Natural killer cell

Natural killer cells or NK cells are a type of cytotoxic lymphocyte critical to the innate immune system. The role NK cells play is analogous to that of cytotoxic T cells in the vertebrate adaptive immune response. NK cells provide rapid responses to viral-infected cells, acting at around 3 days after infection, and respond to tumor formation. Typically, immune cells detect major histocompatibility complex (MHC) presented on infected cell surfaces, triggering cytokine release, causing lysis or apoptosis. NK cells are unique, however, as they have the ability to recognize stressed cells in the absence of antibodies and MHC, allowing for a much faster immune reaction. They were named "natural killers" because of the initial notion that they do not require activation to kill cells that are missing "self" markers of MHC class 1.^[1] This role is especially important because harmful cells that are missing MHC I markers cannot be detected and destroyed by other immune cells, such as T lymphocyte cells.
NK cells (belonging to the group of innate lymphoid cells) are defined as large granular lymphocytes (LGL) and constitute the third kind of cells differentiated from the common lymphoid progenitor-generating B and T lymphocytes.^[2] NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus, where they then enter into the circulation.^[3] NK cells differ from natural killer T cells (NKTs) phenotypically, by origin and by respective effector functions; often, NKT cell activity promotes NK cell activity by secreting IFNγ. In contrast to NKT cells, NK cells do not express T-cell antigen receptors (TCR) or pan T marker CD3 or surface immunoglobulins (Ig) B cell receptors, but they usually express the surface markers CD16 (FcγRIII) and CD56 in humans, NK1.1 or NK1.2 in C57BL/6 mice. The NKp46 cell surface marker constitutes, at the moment, another NK cell marker of preference being expressed in both humans, several strains of mice (including BALB/c mice) and in three common monkey species.^[4][5]
In addition to the knowledge that natural killer cells are effectors of innate immunity, recent research has uncovered information on both activating and inhibitory NK cell receptors which play important function roles including self tolerance and sustaining NK cell activity. NK cells also play a role in adaptive immune response,^[6] numerous experiments have worked to demonstrate their ability to readily adjust to the immediate environment and formulate antigen-specific immunological memory, fundamental for responding to secondary infections with the same antigen. The role of NK cells in both the innate and adaptive immune responses is becoming increasingly important in research using NK cell activity and potential cancer therapies.


Natural killer cell deficiency 1

The abnormality of NK cells, however, in certain cases represents the majority immunological defect. In aggregate, these conditions are termed NK cell deficiency.
Sequential production of interferon-g by NK1.11 T cells and natural killer cells is essential for the antimetastatic effect of a-galactosylceramide 2

1) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3917661/
2) https://www.researchgate.net/profile/Daniel_Pellicci/publication/11530930_Sequential_production_of_IFN-_by_NKT_cells_and_NK_cells_is_essential_for_the_anti-metastatic_effect_of_-galactosylceramide/links/54adc8710cf2828b29fcb472.pdf

Its telling that science has amongst many other things no compelling explanation of how blood, and hemolyth ( in insects ) evolved. Hematopoietic stem cells (HSCs) or hemocytoblasts are the stem cells that give rise to all the other blood cells through the process of haematopoiesis. Over ten blood cell types are required , all are essential. 
In adults, these stem cells are producted and located in the red bone marrow, which is contained in the core of most bones. 

Hematopoietic stem cells (HSCs) are self renewing: when they proliferate, at least some of their daughter cells remain as HSCs, so the pool of stem cells does not become depleted. The process of the development of different blood cells from HSCs to mature cells is called "hematopoeisis". 3

In developing embryos, blood formation occurs in aggregates of blood cells in the yolk sac called "blood islands. " 

What was the evolutionary driving force to produce yolk sacs ? Was blood of the mother not a pre-requisite to make yolk sacs ? So what came first, the yolk sac, or blood ? 

As development progresses, blood formation occurs in the spleen, liver, and lymph nodes.

Question: What was the evolutionary driving force to produce  the spleen, liver, and lymph nodes ? Was blood of the mother not a pre-requisite to make  the spleen, liver, and lymph nodes. ? So what came first, the  the mothers spleen, liver, and lymph nodes, or blood ? 

When bone marrow develops, it assumes the task of forming most of the blood cells for the body.

Question: What was the evolutionary driving force to produce  the bone marrow ? Was blood of the mother not a pre-requisite to make  the bone marrow ? So what came first, the  the mothers bone marrow, or blood ?

In a article from 2012, titled  Scientists unlock evolutionary secret of blood vessels 5 The process of building a closed circulation system is complicated biologically and, from an evolutionary perspective, time-consuming -- involving billions of years.  So, as always, they make up just so stories without a shred of evidence.  


During this lengthy process, new domains (parts of a protein that can evolve and function independently of each other) have been added progressively to key molecules.
The scientists focused on one specific domain known as UNE-S. UNE-S is part of SerRS, a type of tRNA synthetase in species with closed circulatory systems; tRNA synthesases are enzymes that help charge tRNA with the right amino acid to correctly translate genetic information from DNA to proteins.


The scientists found that UNE-S is essential for proper development of an embryo, containing a specific sequence or "nuclear localization signal" that directs SerRS to the cell nucleus. There, it affects the expression of a key regulator of new blood vessel growth.


As a stem cell matures, it undergoes changes in gene expression that limit the cell types that it can become and moves it closer to a specific cell type.

Question: How could the programming of this change of gene expression have evolved gradually, if the intermediate cell types have no function ?

Vasculogenesis is the formation of early vasculature which is laid down by genetic factors and originates in the blood islands of the embryonic yolk sac.

Role of the Epigenome in Vasculogenesis 1


Every cell in an individual has the same genetic makeup. Yet, there are a wide variety of cells in our tissues. This diversification results from the fact that only a subset of the total genome is expressed at any given point across space and time. What tells a cell that which subset of the genome would be expressed and when and how much? The epigenome. The ENCODE project, released in September 2012, proved that what we considered as the junk DNA was not junk after all.

The chromatin-remodeling enzyme BRG1 plays an essential role in primitive erythropoiesis and vascular development 

Brg1-containing SWI/SNF-like complexes, rather than Brm-containing complexes, play a crucial role in primitive erythropoiesis and in early vascular development.

SWI/SNF (SWItch/Sucrose Non-Fermentable)  is a nucleosome remodeling complex found in both eukaryotes and prokaryotes. In simpler terms, it is a group of proteins that associate to remodel the way DNA is packaged. It is composed of several proteins – products of the SWI and SNF genes (SWI1, SWI2/SNF2, SWI3, SWI5, SWI6) as well as other polypeptides.^[3] It possesses a DNA-stimulated ATPase activity and can destabilise histone-DNA interactions in reconstituted nucleosomes in an ATP-dependent manner, though the exact nature of this structural change is unknown.

Developmental processes require changes in gene expression to achieve cellular differentiation. Eukaryotes use chromatin-modifying factors to aid in the regulation of gene expression because large nuclear factors necessary for transcription cannot access DNA when it is tightly bound to histones in nucleosomes. Two main classes of chromatin-modifying factors achieve changes in chromatin structure and organization at individual genes. One class covalently modifies histone proteins to achieve a heritable epigenetic mark instructing further genetic regulation. The second class uses energy derived from ATP hydrolysis to alter the conformation or position of nucleosomes, thereby transiently making gene promoters accessible or inaccessible to large nuclear factors. Both classes of chromatin-modifying factors are necessary to achieve proper temporal and spatial patterns of gene expression in the embryo, and therefore play important roles in a number of developmental processes (de la Serna et al., 2006; Margueron et al., 2005).

The mammalian SWI/SNF-related chromatin-remodeling complexes comprise one major family of ATP-dependent chromatin-modifying factors. These large, multi-protein complexes use one of two different ATPases as their catalytic subunit: brahma (BRM, also known as SMARCA2) and brahma-related gene 1 (BRG1, also known as SMARCA4).

ATPases are a class of enzymes that catalyze the decomposition of ATP into ADP and a free phosphate ion.This dephosphorylation reaction releases energy, which the enzyme (in most cases) harnesses to drive other chemical reactions that would not otherwise occur. This process is widely used in all known forms of life.

Formation of blood and lymphatic vessels 6

Introduction

The vascular system is essential for the transport of oxygen, nutrients and waste to and from tissues. It also has other roles including regulating blood pressure, transporting hormones required for communication between different tissues and transporting cells of the immune system to the site of an immune response. The vascular system is one of the earliest systems to develop and function during vertebrate embryogenesis.

Vessel morphology

The vascular system is a tree-like network of arteries, veins and capillaries. Blood vessels are made up of endothelial cells, smooth muscle cells, connective tissue and perivascular cells as shown in Figure 1. Vascular development occurs through two processes; vasculogenesis and angiogenesis. Vasculogenesis leads to the formation of the first major embryonic blood vessels and results in formation of the primary vascular plexus in the yolk sac. The yolk sac is a membranous sac attached to the embryo that functions as the developmental circulatory system of the human embryo, before internal circulation begins. Once circulation begins, primary vessels are remodelled into arteries and veins in order to develop a functional, mature vascular system. This is known as angiogenesis.

Vasculogenesis  

Vasculogenesis is the de novo formation of blood vessels from endothelial cell precursors known as angioblasts. Angioblasts are generated during the first step of vasculogenesis. Most angioblasts are derived from progressive restriction of mesoderm cells to the endothelial lineage in response to signalling molecules such as 


IHH (Indian Hedgehog)
FGF2 (fibroblast growth factor)
BMPs (bone morphogenetic proteins)
and VEGF (vascular endothelial growth factor)

FGF-2 induces angioblast formation from mesoderm to promote vessel growth.

Most vertebrates species have three (Sonic hedgehog – Shh; Indian hedgehog – Ihh; and Desert hedgehog – Dhh), each with different expression patterns and functions, which likely helped promote the increasing complexity of vertebrates and their successful diversification. 7

Figure 2

Angiogenesis

Once the primary vascular complex has formed, angiogenesis causes vascular remodelling through the sprouting, branching and bridging of existing vessels and the growth of new vessels from existing vessels. This process requires endothelial cells to migrate, proliferate, establish junctions and apical-basal polarity, and deposit a stabilizing basement membrane. VEGF binding to VEGFR1 and 2 is one of the signals involved in causing this pruning and remodelling. VEGF is a key molecule required for vascular development, demonstrated by the fact that VEGF null mice can’t form blood vessels.
Branching of vessels during angiogenesis requires different endothelial cells to respond differently to common signals. For sprouting to occur, one endothelial cell must be selected for outward migration from the existing vessel, this cell is known as the tip cell. The tip cell then responds to a VEGF gradient by migrating up the gradient and away from the original vessel. The endothelial cells behind the tip cell form a sprout emerging from the original vessel. These cells are known as stalk cells and do not migrate independently, but follow the leading tip cell and eventually canalise to form vessels with a lumen. Stalk cells beside the tip cell release soluble VEGFR1, which competitively binds to VEGF in the VEGF gradient. This prevents it from binding to VEGFR1 on stalk cells and so preventing them from responding to this signal and migrating independently. The tip cell must eventually fuse with the target vessel to establish a circuit. After this active sprouting process, endothelial cells become quiescent by adopting a phenotype that promotes vessel integrity and stabilizes the vasculature.
Platelet-derived growth factor (PDGF)-BB and transforming growth factor (TGF)-β stimulate maturation and remodelling of vessels to produce a ‘mature’ vascular system. PDGF binds to PDGFR and TGF β binds to TGF βR to promote vessel maturation by stimulating smooth muscle cell migration and differentiation. These stimuli work together with artriovenous specification cues to facilitate proper development and enlargement of arteries and veins.
Binding of Angiopoietin1 to its receptor Tie2 causes differentiation, recruitment and interaction of perivascular cells such as pericytes and myofibroblast-like cells. Pericytes are found in close association with capillaries. They are embedded within the basement membrane of capillaries with their dendritic processes penetrating through to the endothelial cells, which have processes projecting back into the pericytes. Pericytes have been suggested to be involved in growth of new capillaries or vessel stability.

How are arteries and veins specified?

A hierarchy of arteries, veins and capillaries is initially established through complementary gene expression of membrane bound ligands and receptors known as Ephrins and Ephs, respectively.  Ephrins are membrane bound ligands of which there are two types; type A is GPI linked (being anchored to the membrane by a glycosylphosphatidylinositol (GPI) linkage) and type B is a transmembrane ligand. Ephs are membrane bound receptors from a family of receptor tyrosine kinases which bind either A or B ligands. Binding of Ephs to Ephrins causes bidirectional signalling – each cell stimulates the other on contact. Ephrin B2 is expressed only in arteries and its receptor, EphB4, is expressed only in veins. When ephrin B2 on arterial endothelial cells makes contact with Eph4 on venous endothelial cells, fusion is permitted and angiogenic remodelling occurs. At sites without Eph4 and ephrinb2, fusion is forbidden.  
As shown in Figure 3, in Zebrafish the differential expression of Ephrin B2 and Ephb4 is established by Sonic hedgehog (Shh) expression from the notochord, which induces somite VEGF production. VEGF binds to its receptors VEGFR-2 and neuropilin-1, activating the Notch signaling pathway, which upregulates ephrinB2, specifying an artery. Less Notch stimulation in endothelial cells stimulates Ephb4 expression, specifying a vein. 

Figure 3

Vessel formation guidance

The branching and sprouting of vessels during angiogenesis is not random but involves path finding cues similar to those found in neuronal development, as described in Figures 4 and 5. In fact there is some evidence that nerves are required for the patterning and branching of blood vessels (see Mukouyama et al. 2002). Path-finding in vascular development involves the same signalling molecules as in neural development: Sprouty, Slit, Netrin and Semaphorins.

Figure 4

Figure 5

Lymphatic system

The lymphatic system transports lymph around the body in order to maintain fluid homeostasis. It also serves as a way of transporting cells of the immune system. The lymphatic vascular system is a highly branched network of capillaries and ducts that is present in most organs and tissues. The lymphatic system is made up of small capillaries which drain into precollecting vessels, then collecting vessels, and then into the thoracic duct or the right lymphatic trunk. Lymph then drains from here into the subclavian veins. Lymphatic vascular development requires transdifferentiation of venous endothelial cells into lymphatic endothelial cells, sprouting and maturation of lymphatic vessels, and separation of blood and lymphatic vasculature.
Lymphatic vessels stem from preexisting blood vessels. Lymphatic endothelial cells (LECs) differentiate from a subset of venous endothelial cells that expresses the transcription factor Prox1. PROX1 specifies lymphatic identity to blood endothelial cells. LECs bud and migrate away from veins to form the first lymphatic structures in regions where VEGF-c is supplied by the lateral mesoderm. In mice, reorganisation of lymphatic vasculature into capillaries, precollectors, and collecting lymphatic vessels starts at E15.5. Foxc2 and NFAT signalling pathways cooperate in establishing collecting lymphatic vessels. The Tie1 and Tie2 endothelial receptor tyrosine kinases also control lymphatic vascular development.

1) https://dreamerbiologist.wordpress.com/2013/04/12/role-of-the-epigenome-in-vasculogenesis/
2) https://www.sciencedaily.com/releases/2012/02/120221124810.htm
3) Boundless. “Development of Blood.” Boundless Anatomy and Physiology. Boundless, 21 Jul. 2015. Retrieved 04 Mar. 2016 from https://www.boundless.com/physiology/textbooks/boundless-anatomy-and-physiology-textbook/blood-17/development-of-blood-170/development-of-blood-848-10145/
4) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2459551/
5) https://www.sciencedaily.com/releases/2012/02/120221124810.htm
6) http://www.fastbleep.com/biology-notes/32/156/844
7) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4280113/

The requirement of various interdependent and irreducible complex organs and systems to make blood. 

https://reasonandscience.catsboard.com/t2295-hematopoiesis-the-mystery-of-blood-cell-and-vascular-formation#4866

Little attention has been given to the origin of blood. Hematopoiesis is the description of how blood cellular components form.  Blood is a fluid that circulates through the cardiovascular system.  Blood and the developing blood cells and their precursors are produced  in the bone marrow through pluripotential Hematopoietic stem cells (HSCs)  which give rise to all   ten different type of blood cells through the process of haematopoiesis  These blood cells are: macrophages, neutrophils, basophils, eosinophils, erythrocytes, dendritic cells, platelets, T cells, B cells, and natural killer cells.

Bruce Alberts and his colleagues noted: “Blood contains many types of cells with very different functions, ranging from the transport of oxygen to the production of antibodies. Some of these cells function entirely within the vascular system, while others use the vascular system only as a means of transport and perform their function elsewhere (1994, p. 1161).


1. Macrophages are essential components of the innate immune system. They are a type of white blood cell that engulfs and digests cellular debris, foreign substances, microbes, cancer cells, and anything else that does not have the types of proteins specific of healthy body cells on its surface in a process called phagocytosis.


2. Dendritic cells are needed to control B and T lymphocytes, and capture and process antigens, express lymphocyte co-stimulatory molecules, migrate to lymphoid organs and secrete cytokines to initiate immune responses.  


3. Neutrophils are a required type of immune cell that is one of the first cell types to travel to the site of an infection. Neutrophils help fight infection by ingesting microorganisms and releasing enzymes that kill the microorganisms. A neutrophil is a type of white blood cell


4. Basophils contain anticoagulant heparin, which prevents blood from clotting too quickly. They also contain the vasodilator histamine, which promotes blood flow to tissues.


5. Eosinophils effector functions include the production of cationic granule proteins and their release by degranulation, the production of reactive oxygen species such as hypobromite, superoxide, and peroxide, production of lipid mediators like the eicosanoids, enzymes, such as elastase. growth factors such as TGF beta, VEGF, and PDGF. etc....


6. Red blood cells ( erythrocytes ) are the most common type of blood cell and essential for the vertebrate organism's  delivering oxygen (O2) to the body tissues—via blood flow through the circulatory system 


7. Platelets  are an essential component of blood whose function (along with the coagulation factors) is to stop bleeding by clumping and clotting blood vessel injuries 


8. T cells are essential for human immunity. 


9. B cells are a type of white blood cell that makes antibodies. B lymphocytes are part of the immune system and develop from stem cells in the bone marrow. Primary B-cell immunodeficiencies (B-PID) constitute a heterogeneous group of immunodeficiencies characterized by defective production of antigen-specific antibodies and predisposition to recurrent and severe infections


10. Natural killer cells are a type of immune cell that has granules (small particles) with enzymes that can kill tumour cells or cells infected with a virus. A natural killer cell is a type of white blood cell. Also called NK cell and NK-LGL. They  are part of the innate immune defence against infection and cancer and are especially useful in combating certain viral pathogens




The process of developing the diverse blood cell repertoire from stem and progenitor cells termed hematopoiesis has been subject to considerable investigation. However, key steps in the complex process of hematopoiesis,
including hematopoietic stem cell generation during embryogenesis, hematopoietic stem, and progenitor cell expansion to accommodate physiological and pathological requirements, and mechanisms that ensure hematopoietic
stem and progenitor cell phenotypic integrity remain incompletely understood.

Recent studies indicate that hematopoietic stem cells (HSCs) also have the potential to differentiate into multiple non–blood cell lineages and contribute to the cellular regeneration of various tissues and multiple organs.

Blood is a bodily fluid in animals that delivers necessary substances such as nutrients and oxygen to the cells and transports metabolic waste products away from those same cells. In vertebrates, it is composed of blood cells suspended in blood plasma. Plasma, which constitutes 55% of blood fluid, is mostly water, and contains dissipated proteins, glucose, mineral ions, hormones, carbon dioxide (plasma being the main medium for excretory product transportation), and blood cells themselves. Albumin is the main protein in plasma, and it functions to regulate the colloidal osmotic pressure of blood. According to H.Glicksman, the regulation of blood pressure requires a irreducible complex system .
 The blood cells are mainly red blood cells, white blood cells  and platelets. The most abundant cells in vertebrate blood are red blood cells. These contain hemoglobin, an iron-containing protein, which facilitates oxygen transport by reversibly binding to this respiratory gas and greatly increasing its solubility in blood. In contrast, carbon dioxide is almost entirely transported extracellularly dissolved in plasma as bicarbonate ion. 

The bone marrow, which is the flexible tissue in the interior of bones, forms a suitable environment for stem cell survival, growth and development. It is composed of stromal cells and a microvascular network. Bones are an amazing example of design, present in all vertebrates. They have a huge advantage over man-made girders, in that they are constantly rebuilding and redesigning themselves to cope with changing stress directions.12 This involves a fine balance of the activity of bone-depositing cells (osteoblasts) and bone resorbing cells (osteoclasts).  It’s been recently shown that thyroid-stimulating hormone (TSH), best known for what its name says—stimulating the production of hormones in the thyroid gland—has an important role. It oversees both types of cells—without it, bones have osteoporosis in some parts (too little bone, so very weak), and are too dense in other patches. So both are essential. For bone formation, over 24 different proteins, cells, Vitamins, transcription factors etc. are required. So, we can confidently say, the production of blood requires a hudge number of different body parts, which are essential. Blood by its own has no function. The vascular system without blood has no function. Bones require a vascular system, which by their own are required to make blood. The vertebrate body is a hudge irreducible complex sytem, that could not have emerged gradually. Each multicellular organism had to emerge fully, in its interdependent form.   


Hemopoiesis (hematopoiesis) includes both erythropoiesis and leukopoiesis (development of red and white blood cells, respectively), as well as thrombopoiesis (development of platelets.




1) https://en.wikipedia.org/wiki/Hematopoietic_stem_cell
2) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2956013/
3) http://www.ncbi.nlm.nih.gov/pubmed/9521319
4) http://www.ncbi.nlm.nih.gov/pubmedhealth/PMHT0022058/
5) https://en.wikipedia.org/wiki/Basophil
6) https://en.wikipedia.org/wiki/Red_blood_cell
7) https://en.wikipedia.org/wiki/Platelet
8  http://www.tcells.org/beginners/tcells/
9) http://www.ncbi.nlm.nih.gov/pubmedhealth/PMHT0022043/
10) https://en.wikipedia.org/wiki/Blood
11) http://www.evolutionnews.org/2015/09/controlling_blo099611.html
12) http://creation.com/bone-building-perfect-protein-osteocalcin#r1
13) http://reasonandscience.heavenforum.org/t2296-origin-and-development-of-bones-osteogenesis?highlight=bones

T cells-Dendritic cells- Macrophages- Neutrophils-  Basophils- Red blood cells- Platelets are essential for blood. Irreducible complexity refutes evolution