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Defending the Christian Worlview, Creationism, and Intelligent Design » Molecular biology of the cell » Development biology » Origin of stem cells

Origin of stem cells

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1Origin of stem cells Empty Origin of stem cells Sun Feb 14, 2016 3:59 pm


Origin of stem cells

The Mysterious Embryonic Stem Cell 1

Stem cells are mysterious. They are cells that make or replenish other cells. For example, when you donate blood, your stem cells are in charge of replenishing your blood cells. Scientists have found stem cells throughout the body, each doing an assigned job. Some replenish blood cells, some replenish skin cells, and others repair heart tissue. These cells do not usually change jobs; they become specialists at making their particular cell types.
However, embryonic stem cells (ESC), those found in the eight- to ten-day-old embryo, are not specialists. They can make all the cells in the body, but how they are able to accomplish this feat is still unknown.

Researchers have been able to coax some of these specialized stem cells into reverting back to a stem cell that acts like an embryonic stem cell. These are call "induced pluripotent stem cells." As with embryonic stem cells, scientists are still seeking the keys to how these cells can develop into specific stem cell types. Interestingly, research on embryonic stem cells and induced pluripotent stem cells seems to indicate that stem cells are pre-programmed to do their thing, suggesting that this may be a case of an intelligently designed process. (See ENV, "A Piece from the Developmental Symphony").

Two compelling scientific articles this past month provide some hints of how stem cells are programmed. One study, in Molecular Cell, shows that embryonic stem cells will readily undergo apoptosis (cell death) if DNA is mis-copied, but once the embryonic stem cell gets assigned a job (i.e. differentiates), this sensitivity to apoptosis is "turned off." In other words, the cell has a layer of protection in place at just the point when a DNA error would spread throughout the entire organism, potentially causing irrevocable damage. And, just as conveniently, this heightened sensitivity to DNA damage is turned off at just the point when the cell starts to differentiate.
The key player in this process is the Bax protein. This protein is known to signal apoptosis, and is present in its active conformation in the ESC. Through a series of signals, Bax is "turned off" whenever the cell starts to differentiate. But during the delicate DNA replication process in the early embryo, the protein is active to ensure fundamental DNA errors are not perpetuated throughout the organism. Additionally, and adding yet another layer of complexity to this system, Bax is active, but other factors, including the location of the Bax protein, are in place to ensure that the cell does not undergo apoptosis prematurely.

A second study published in PLoS Genetics demonstrates that the level of DNA compaction affects stem cell differentiation. Scientists have long known that stem cells have loosely packed DNA compared to differentiated cells, which probably aids in DNA replication at the early embryonic stages. In eukaryotic cells, chromatin is involved in many DNA processes, including packing the DNA so that it fits inside the cell. The primary proteins in chromatin are called histones. Think of the histone/DNA complex as spools of yarn. The DNA wraps tightly around the histone so that the small spool, rather than the long DNA strand, can fit inside the cell. There are several families of histones, and only some of them are involved in the processes under consideration in this study. For the sake of simplicity, we will simply refer to them as histones. Please refer to the research article for a more detailed description of which histones are involved in which processes.

It seems that there is a coordinated link between DNA compaction and pluripotency. When one of the histones involved in DNA compaction was removed, the mouse embryonic stem cells did not differentiate properly. The stem cells did not get their assignments, as they normally would. The DNA must be loosely wound during the early embryonic stages, but as the embryonic stem cells differentiate (assigned to a specific cell type), histone levels increase and the DNA becomes more tightly wound around the histone.
The key protein players in the pluripotentcy process are Nanog and Oct4. Nanogand Oct4 are regulated via DNA methylation, and as this study found, the absence of certain histones, and therefore the presence of loosely wound DNA, keeps Oct4 "turned on." Usually Oct4 gets "turned off" as the cell transitions from a pluripotent stem cell to a differentiated stem cell. If Oct4 stays activated, then the ESC never undergoes differentiation.

The authors conclude:
Our results suggest a role of H1 [histone] and chromatin compaction in epigenetic regulation of the pluripotency gene Oct4, likely mediated through DNA methylation and histone modifications. To our knowledge, this represents a novel mechanistic link by which bulk chromatin compaction is directly linked to pluripotency, by participating in repression of the pluripotency genes.

This is not a simple case of cause-and-effect. Note that several factors are in place and each activates and inactivates in turn, as if programmed to do so.
In both of these studies, there are proteins that are "turned on" and "turned off" at just the right time so that the complicated ensemble of development processes can occur. These are epigenetic factors that affect the complex process of cell differentiation.

In any other context, we would consider this type of complexity, with a program that tells the components what do and when to do it, a hallmark of the most sophisticated engineering. Those kinds of instructions do not just arise in a kind of add-on or co-opted Darwinian method. The more we delve into the inner working of the cell, the more we see how complicated it is -- complicated in a way that suggests purpose and design.

In case you were wondering, by the way, the studies on embryonic stem cell apoptosis were performed with human embryonic stem cells. The stem cells came from a stem cell line at the University of Wisconsin. Human embryonic stem cell research is, of course, the subject of considerable moral controversy. Some people believe the human embryo should be accorded the same dignity as a human person, while others think that the human embryo, while it is human tissue, is not morally equivalent to a human person.
Even though this research does have compelling implications for intelligent design, we recognize that the methods by which the results were obtained, while legal in the United States, are very much open to question on ethical grounds. Furthermore, we recognize that scientific studies usually begin with animal systems before advancing to human systems, but the scientists in this study chose to investigate human systems. The scientists in the DNA and chromatin study, on the other hand, used mouse systems to derive their data.


2Origin of stem cells Empty Stem cells in the light of evolution Mon Feb 15, 2016 1:13 pm


Stem cells in the light of evolution 1


All organisms depend on stem cells for their survival. As a result, stem cells may be a prerequisite for the evolution of specific characteristics in organisms that include regeneration, multicellularity and coloniality. Stem cells have attracted the attention of biologists and medical scientists for a long time. These provide materials for regenerative medicine. We review in this paper, the link between modern stem cell research and early studies in ancient organisms. It also outlines details on stem cells in the light of evolution with an emphasis on their regeneration potential, coloniality and multicellularity. The information provided might be of use to molecular biologists, medical scientists and developmental biologists who are engaged in integrated research involving the stem cells.

Keywords: Developmental biology, early organisms, natural selection, stem cell


Evolutionary biologist Theodosius Dobzhansky once wrote ‘Nothing in biology makes sense except in the light of evolution’1. The term ‘light of evolution’ was used earlier by biologist and statesman of science, Julian Huxley. In fact, the notion light of evolution originally came from Pierre Teilhard de Chardin, who was admired by Dobzhansky. The latter then went on to say that species diversity in planet Earth cannot be explained by the creation fairy tale because of the ecological complexity1. The question however is, whether or not the evolution of stem cell started with unicellular organism with its evolutionary linkage? The answer may be hidden behind the properties of the stem cell of self-renewal and differentiation. For example, single cell organisms have unique capabilities not only to renew by self but also perform differentiated role. On the contrary, stem cells in higher organisms have the ability to both self-renew through mitotic cell division and differentiate into a diverse range of specialized cell types.
Fossil evidence indicates the existence of uni-cellular prokaryotes on Earth over 3.5 billion years ago2. One of the foremost muticellular organisms was seaweed that came into existence about 1,300 million years ago3. So the existence of stem cells could be traced back to millions of years and these were developed through the process of natural selection. Accordingly, the appearance of stem cells could be viewed as fundamental in the lengthy evolutionary saga. In this review we have highlighted stem cells in the light of evolution. We have outlined details on stem cell multicellularity, regeneration and coloniality potential in the organismic and evolutionary perspectives.

Stem cells and multicellularity

The distribution of stem cells has been recorded throughout the animal and plant kingdoms, and all these organisms are known to be multicellular. However, during the development of multicellular organisms through natural selection, stem cells accompanied the evolutionary process, either in the mode of asexual or sexual reproduction4. In general, stem cells can be separated by two basic strategies2,4–7. One is asymmetric cell division and the other is stochastic differentiation. In the asymmetric cell division, there are mechanisms that might be described as invariant in which a stem cell gives rise through asymmetric cell division8 splitting into a stem daughter while the other undergoes differentiation. In the case of stem cell, due to asymmetric cell division, the cells first undergo a divide and produce one daughter like itself that maintains stem cell characteristics while the other programmed to differentiate into a non-stem cell fate leading to the path of differentiation. Such a division can be seen in single cell organisms and invertebrates. Asymmetric cell divisions occur during the development of Drosophila and Caenorhabditis elegans9. Similarly, multicellular organisms with a relatively few cell types can go though such splitting. In the multicellular hydra, head and foot can be regenerated in adult from a tiny piece of tissue mass from the body column10.
Stem cell follows another route of cell division which is ‘stochastic differentiation’. Here the stem cells make a combination of asymmetric divisions and symmetric ones. Finally, they produce either two stem cells (symmetric renewal), or two differentiated cells (symmetric differentiation). Some reports show that in olfactory epithelium11 and muscle12 stem cell follows the stochastic differentiation cell division.

Stem cells and regeneration potential

Regeneration has received renewed research interest in recent years. Stem cells have been extensively studied due to their regeneration potential. First, this property was shown by histologists in the 19th century who introduced an abstract term for cells which can specifically repair or regeneration potential. With the discovery of bone marrow cells in the 1950s, the haematopoietic stem cell concepts started to emerge13. Haematopoietic stem cells are accountable for the steady renewal of the blood cells while mesenchymal stem cells,  a group of stromal cells show multilineage segregation ability. Mesenchymal stem cells have been isolated from different multicellular organism like human4. The process in which a stem cell gives rise to daughter cells with definite probability of being either stem cells or committed progenitors is evident in a vast majority of mammalian self-renewing tissues. Generally, each stem cell division gives rise to a stem and a committed daughter at stable state. However, unevenness can be achieved on a population basis rather than individual cell division basis. Moreover, in some tissues there may be a range of cell behaviour with stem and progenitor cells at opposite ends of a spectrum instead of discrete stem and progenitor populations14–17. Nevertheless, great variability in the self-renewal process by a stem cell does occur18. In simple single cell organisms such as the amoeba for example, a simple cell division is equivalent to reproduction by which a new organism is created more frequently19. Stem cells of small rodents on the contrary are estimated to replicate about once in four weeks- for cats, it is once in per ten weeks and for higher primates once in 50 wk20. This is largely due to the intricacy of self-renewal process enhanced by natural selection.
In early animals, single cell carry out several physiological functions while serving as stem cell. A classical example is hydra where single epithelial cells appear to carryout several steady-state physiological functions while serving as stem cell21. These cells perform both the process of self-renewal and differentiation. Hydra belongs to the exclusively aquatic phylum of Cnidaria- these early branching-animals have survived for millions of years; they do not undergo ageing (senescence) hence biologically eternal. This natural everlasting characteristic can be attributed by the asexual mode of reproduction via budding- it simply requires a tiny tissue of stem cells with continuous self-renewal capability. Stem cell differentiation in the hydra is governed by co-ordinated actions of conserved signaling pathways. Hence, the hydra's stem cell represents a critical insight of general significance of its biology (i.e. cellular senescence, lineage programming and reprogramming, extrinsic signals in fate determination, tissue homeostasis) and the ultimate evolutionary origin22.
The role of genes in vertebrate regeneration has received great interest among the scientific community and studies have been targeted to cross-examine gene transcription and protein translation during different steps regeneration23–26. Maki et al23 reported that the expression of some genes namely Sox2, Klf4, andc-myc have been unregulated when it comes to regenerating potential. These three genes are in fact the most important induced pluripotent stem cell (iPSC) genes which can be unregulated in regenerating new lens and limb.
The zebrafish fins and Xenopus limbs have also become important models for the study of regeneration. In regenerating zebrafish fins, homologues of genes are related with the pluripotency and expressed to initiate the regeneration process27. Scientists have concluded that blastema cells in zebrafish fins and Xenopus limbs are not completely analogous to induced pluripotent mammalian stem cells but these tend to share some similarities in gene expression26. A study of zebrafish tail fins by Stewart et al28 has concluded that histone demethylase is necessary for regeneration by identifying targets of histone methylases and demonstrating histone modifications silence promoters of numerous genes involved in regeneration. The regulatory genes contain bivalent me(3)K4/me(3)K27 H3 histone modifications created by the concerted action of Polycomb (PcG) and Trithorax histone methyltransferases. During the evolutionary process, regeneration appears to be common among the adults of many non-vertebrate organisms. However, among the adult vertebrates, amphibians like the salamanders are unique in a way that they could regenerate limbs29,30. During the larval stage, the developing limb bud is poised of undifferentiated cells while the adult limb is composed of fully differentiated tissues. The limb stem cells appear to help in the regeneration process31. On the other hand, it has been reported that adult human has less potential of regeneration compared to other group of organisms. Nonetheless, some parts such as the skin fingertips, ribs, liver, kidney and heart have the capacity to regenerate and repair themselves to some extent where adult stem cells have been found in very low frequency32–34. It appears that the evolutionary process might have created the less occurrence of adult stem cell in humans.

Stem cells and coloniality

Coloniality comprises large congregation of individuals in a place that includes the same species living together with a mutual advantage of self-protection. It is an important question in evolution that how group-living organisms assemble together and how the colonial origin came into existence that harbours over a million individuals breeding and living in proximity. Colonial behaviour itself is an evolutionary ambiguity because individuals pay fitness costs to breed in high densities35. However, the coloniality character has been recorded among some phyla, specifically the tunicates, cnidarians, entoprocta, ectoprocta and bryozoans36. Ascidians are one of the members of chordates that belong to the Tunicata phyla and they offer unique opportunities to investigate the biology of stem cells. At larval stage, the Ascidiains have a typical chordate body plan including notochord, dorsal hollow nerve tube and striated musculature. Subsequent to its swimming stage, the larvae settle and undergo extensive metamorphosis37. At that stage chordate characteristics are resorbed and ultimately end up as filter feeding sessile invertebrate mature animal. Due to its small size and rapid development, Ascidian larvae have been used widely as a model to study specification and differentiation events of developmental biology since they exhibit solitary and colonial forms. Solitary ones can reach up to 10 cm while the colonial variety can spread up to several meters2. The colonial ascidians have two developmental pathways to create an identical adult thus unique among chordates with regenerative capability and became an outstanding model for embryonic and stem cells research38.
One peculiar taxon known as Botryllid ascidian has become a model for allorecognition studies because of the allogeneic fusions revealing the evolutionary links between allorecognition, stem cell biology and ecology39. These colonial ascidians live in superficial sea water in all temperate zones around the world40. After hatching from their colony, Botryllus tadpoles larvae swim to surface where these attach and undergo metamorphosis resulting in the loss of chordate characteristics (tail, notochord, neural tube, and segmented musculature through the apoptosis)41. At the beginning of bud formation, the vesicle cells are morphologically undifferentiated; these stem cells can self-renew during asexual reproduction. These take part in organogenesis and gonad formation and these occur in clonemates in a vascular fused colony. So the genomes of circulating germline stem cells and somatic stem cells are the same and it is obvious that the individual tadpole is a target of natural selection. Colony fusion offers the opportunity for germline stem cells or somatic stem cells to move from one colony to a genetically distinct colony42.
Another example of coloniality is the hydrozoan colonies. This fauna is a member of the Cnidaria phyla. These colonies consist of multiple polyps connected together by tube like structure and all colonial include some polyps specialized for reproduction. The hydrozoan contains a population of migratory stem cells and the epithelial cells of the colonies serve as stem cell for some physiological process20,43. Presently, the hydrozoan colonies are point of attraction to study evolution44. However, the characteristic of coloniality in different organisms also proves the characteristic of multicellularity.

Stem cell evolution in branching vertebrates and mammals

In early branching vertebrates such as fish and amphibians, adult stem cells are within the organ, for example, retinal stem cells are found in the periphery of the retina while the ciliary marginal zone produces new neurons in retina throughout life. In these species, retina grows to keep pace with the enlargement of body. However, among higher vertebrates such as birds and mammals, when they reach adulthood, the retina stops growing so there appears to be no need for such a proliferative area with stem cells. A study suggests that a region similar to the ciliary marginal zone of fish and amphibians exists in the post-natal chick and adult mouse45.
In mammals, some evidence supports the properties of stem cell evolution. Due to larger size and longer life, larger mammals require more blood cells. As a matter of fact, the total number of human haematopoietic stem cells (HSCs) is equivalent to the total number of HSCs in cat and mouse. This fact strongly supports that the number of HSCs per animal is conserved in mammals46. After injury, active adult stem cells help to renew and regenerate the tissue. It is an essential physiological phenomenon in all mammals47. This example shows the evidence of conservation of active adult stem cell in mammals.

Stem cell and regulatory gene networks

How does the regulatory gene network perform behind the stem cell in the light of evolution? Various factors such as the microenvironment, signaling events and genetic characteristics are often associated with this property of stem cell48. Models like Clytia hemisphaerica are available to analyze how stem cell intrinsic factors are integrated with signaling events, and how the microenvironment of ‘stem cell niche’ maintains tissue homeostasis22. In the early branching-animal of Clytia, for example, nematoblasts occur between ectodermal epithelial cells within the ‘tentacle bulbs’ from where the tentacles grow. The tentacle bulbs are spherical outgrowths of bell margin of medusa. Spatial progression of nematoblast stages along the bulb axis is correlated with differential stem cell marker gene expression. Most of the cells at the base of the tentacle bulb express Clytia Piwi homologue gene but not a differentiation marker 49. In fact, the Piwi gene is a widely conserved stem cell marker throughout multicellular eukaryotes, and these cells might be considered as a population of stem cells50,51.
In several invertebrate groups, especially sponges and planarians, tissue plasticity and regeneration capacity based on stem cells are the common characteristics. These organisms harbour a Piwi gene, which is a conserved gene for regeneration and plasticity. Piwi homologues have been identified from freshwater sponge, Ephydatia fluviatilis, as candidate stem cell (archeocyte) markers52. Planarian regeneration is based upon totipotent stem cells, the neoblasts. Piwi, especially DjPiwi-1, has been identified from planarian stem cells during the regeneration process of the stem cell53. DjPiwi-1gene of planaria is a homologue of Drosophila piwi. This gene is a member of the PAZ-Piwi gene family and can be used as a marker for stem cell.
Would it be possible for a stem cell gene to change over evolutionary time? Computational tools and development of genomic resources could lend a hand to answer the ultimate evolutionary function of stem cells. In organisms such as the cnidarians, bilaterians and metazoans, Sox2 is known to date as one of the most conserved stem cell-specific genes54. Cnidarians and bilaterians were known to have diverged over 560 million years ago and the discovery of Sox2 in early branching metazoans suggests that there is a similarity of the regulator gene of stem cell potency that might have present in both groups. The presence of stem cell marker gene like Sox2 strongly supports the above evidence that appears to be highly conserved. However, Nanog or Oct 3/4 is present in metazoans55. On the other hand, there is another example of preserved regulatory gene of a stem cell like wnt gene. The wnt pathway has been recorded in the preservation of D. melanogaster germ, mammalian haematopoietic, gut, and hair follicle stem cells56–59. This wnt gene has been conserved in different stem cells during the process of evolution through natural selection.
As a matter of fact, stem cells and their niches evolved in the multicellular organisms and have many features that are conserved between vertebrates and Drosophila. One of the well characterized stem cell niches resides at the tip of the Drosophila ovary where it regulates germline stem cells. Subsequent types of stem cell, escort stem cells, also reside in this niche and interact closely with germline stem cells. These escort stem cells division provides one or two squamous cells that wrap each developing germ cell cyst and suggests how niche facilitates co-ordinated activity of these two types of stem cells. Gut stem cells are likely to be controlled by a niche that differs from the germline stem cells or escort stem cells niche in two respects. A niche cell might act as an anchor of the stem cell in position though it is not clear60. Further, the niche appears to repress most gut stem cells in a temporally and spatially regulated manner and most evidence suggests that multicellularism evolved separately. Nonetheless the piwi gene in Drosophila is more conserved61. It is evident that evolution enveloped systematic regulatory gene network in stem cell for self-renewal process formed by Oct4, Sox2, and Nanog, in particular, controlling embryonic stem (ES) cell pluripotency in mammals62 that are also more conserved.


Evolutionary theories are based on single gene homologies or cross-kingdom gene transfer assisting convergent evolution through biochemical processess in plants and animals63. Plants have been ignored focus as a resource for stem cell research because single cells from adult plants have the ability to make complete new adult plants6466. Despite this, plants and animals have some homology, both organisms evolved separately influenced by natural selection. The homology between the piwi gene in Drosophila, which controls germ line stem cells, and the ZWILLE gene in Arabidopsis, which controls the stem cells of the shoot meristem has led to the suggestion that “stemness” evolved in a single-celled ancestor, or plants and animals might have shared a multicellular ancestor5. In Metazoans, Drosophila, C. elegans, and all vertebrates, studies suggest that natural selection otherwise adhering to the principles of Mendel and Hardy-Weinberg and the mechanisms might have been due to fitness selection6770. However, during the evolutionary process, sequestration of cells in one conspecific animal from another is not the rule; many species tend to exist wherein genotypically distinct cells may intermix within a chimeric entity71,72. The ideas of stem cell-based organogenesis and stem cell-based regeneration are interrelated and developed through the evolutionarily selection process42. Therefore, stem cells are not only the entity of biological organization, accountable for the progress and the regeneration of tissue and organ systems, but also are units in the complex evolutionary process. However, more understanding between the relationships and dynamics of molecular signatures and gene regulatory circuits behind stem cell will lead us to know more about the stem cell in the light of evolution.

2) Mesenchymal stem cells, or MSCs, are multipotent stromal cells that can differentiate into a variety of cell types,[1] including: osteoblasts (bone cells),[2] chondrocytes (cartilage cells),[3] myocytes (muscle cells)[4] andadipocytes (fat cells).

3Origin of stem cells Empty Re: Origin of stem cells Mon Feb 15, 2016 3:30 pm


Regulation of Stem Cell Populations by microRNAs 1

microRNA Function in Pluripotent Stem Cells

Role of microRNAs in Embryonic Stem Cells (ESCs)

ESCs are derived from the inner cell mass of the blastocyst stage of the embryo and have been isolated in 1981 in mouse (mESC) [35] and in 1998 in human (hESC) [36]. They represent a powerful tool for developmental studies, in vitro diseases modeling and for potential cellular therapeutic in regenerative medicine. This is mainly due to the two important properties that defined them: pluripotency and self-renewal. ESCs are indeed pluripotent, they are able to differentiate into the three germ layers and give rise to cell types found in all tissues and organs of the body. They also possess an unlimited self-renewal capacity with continuous cell division in vitro. Undifferentiated ESCs display a particular abbreviated cell cycle profile critical for fast growth during early embryonic development [37]. The decision of an ESC to self-renew or differentiate is regulated by a complex set of factors, including transcription factors, chromatin modifications, signaling pathways and non coding RNAs [38]. The unique molecular program of ESCs needs to be conserved in order to maintain the undifferentiated pluripotent stage, whereas ESCs undergo major epigenetic and gene expression changes when cells are engaged in a differentiation process resulting in a massive transformation of cell phenotype. By their capacities to regulate simultaneously hundreds of targets, miRNAs represent good candidates for such rapid and large transformation.

A complex network of extrinsic and intrinsic factors in conjunction with chromatic remodeling is necessary to keep the ESC fate. A core network of transcription factors and RNA binding proteins has been defined in the past few years [39]. Among those, Oct4, Sox2 and Nanog, play a central role in the maintenance and acquisition of “stemness”, the ensemble of properties that define the stem cell fate. Various positive autoregulatory loops exist between those three transcription factors since each of them is able to bind to its own promoter and to the promoters of the two other members. Chromatin immunoprecipitation (ChiP) assays have revealed that they also control the transcription of many other players of the regulatory network of pluripotency, including Lin28, cMyc, Klf4, Tcf3 or Stat3 [39, 40]. Recently, a combination of a subset of those factors has been shown to reprogram somatic cells into pluripotent cells (induced pluripotent stem cells, iPSCs) possessing very similar properties to ESCs [41–43]. Interestingly, some of those stem cell core regulators are able to activate the promoters of several miRNAs in ESCs, including miR-290–295, miR-302/367 and miR-92 clusters [44].

In the past few years, miRNAs have appeared as central players in ESC self-renewal and differentiation. In this section, we will review our current knowledge of the role of miRNAs in ESC functions.

ESCs Display a Defined miRNA Signature

miRNA expression has been examined in mESCs and hESCs using cloning, qPCR, microarray and deep sequencing technologies by comparing undifferentiated ESCs to their differentiated counterparts [45–48]. Those experiments revealed that the global miRNA expression is low in ESCs compared to differentiated cells. Some of the ESC-enriched miRNAs are limited to ESCs, while other are more widely expressed but decrease significantly during differentiation. Thus, ESCs seem to be characterized by a unique miRNAs signature. We will use the term “ESC-enriched miRNA” in this review when referring to the miRNAs whose levels decrease as ESCs differentiate.

Depending on the method used to analyze miRNA expression, the ESC lines and the differentiation protocols, small variations were observed. However all those studies share miRNAs described as ESC-enriched. The miR-290 family and miR-302 clusters account for the majority of all miRNAs expressed in undifferentiated mESCs. hESC-enriched miRNAs can be categorized in four major groups: miRNAs from the miR-302 cluster, miRNAs from the miR-17 family, miRNAs from the miR-371–373 cluster and miRNAs from C19MC (chromosome 19 miRNA cluster) [48]. Two additional families have been found enriched in hESCs in some studies: miR-130 and miR-200 [48]. The promoters of most of those miRNAs can be activated by Oct4, Sox2 and Nanog [44].

miRNAs whose expression increases during differentiation are also of importance since their low expression might keep ESCs in an undifferentiated state and will be discussed further later. The most studied among this group of miRNAs activated during differentiation are let-7, miR-145 (in human) and miR-134 (in mouse).

Majority of the hESC-enriched miRNA clusters are transcribed as polycistronic transcripts, suggesting that they share common upstream regulators and the same pattern of expression. Moreover, several of those ESC-enriched miRNAs have the same or a similar seed sequence, so can target a common group of mRNAs [48].
Global Disruption of Mature miRNAs

The fact that many miRNAs have the same seed sequence and that a single miRNA can target multiple mRNAs make difficult to study their function individually. Removal of all miRNAs can be achieved by deleting the genes encoding the enzymes involved in the processing of miRNAs, Drosha and Dicer, or their partners, such as DGCR8. Individual miRNAs can then be reintroduced as mimics to assess their functions. Homozygous Dicer1 knockout (KO) mice die early in the development [49] while conditional Dicer1 mutant mESCs are viable in culture but are defective in differentiation [50]. Dicer loss also leads to severe growth defects of mESCs and slightly prolongs G1 and G0 phases of their cell cycle [51]. In addition to its role in miRNA processing, Dicer has been shown to be involved in the biogenesis of endo-siRNAs and other small RNAs [52]. Therefore, the studies of DGCR8 KO in mice might better depict the functions of most miRNAs in mESCs. Similar to Dicer mutant, DGRC8 deficient mice are not viable, and DGCR8 KO mESCs exhibit a proliferation defect and fail to differentiate [53] Knockdown (KD) of Dicer or Drosha also dramatically attenuates cell division in hESCs and results in the formation of stem cells with high levels of stem cell factors, correlating with delayed differentiation [54]. Altogether those studies show that miRNAs are critical for ESC self-renewal and differentiation.
microRNAs Regulate ESC Proliferation

A tight regulation of stem cell division is primordial to sustain the self-renewal capacity of ESCs. Observed proliferation defects of Dicer, Drosha and DGCR8 mutant ESCs suggest that miRNAs are involved in the regulation of their cell cycle. ESCs exhibit a very specific expedited cell cycle due to a short transition from G1 to S phase [55]. Cell cycle checkpoints control progression through the phases of the cell cycle and are regulated by the sequential activation and inactivation of cyclin-dependent kinases (CDKs) by cyclins. However, contrary to somatic cells, ESCs express very low level of cell cycle inhibitors (p21cip1, p27Kip1 and p16INK4a) and exhibit an atypical cell cycle, in which the major point of regulation does not take place in the Restriction checkpoint [54, 56, 57].

Many of the defects in Dicer-deficient mouse ESCs can be reversed by transfection with members of the miR-290 cluster [58]. In accordance with this study, a screen performed to identify miRNAs that can rescue the proliferation defect observed in DGCR8 KO cells uncovered members of the miR-290 and miR-302 clusters as important mESC cell cycle regulators [53]. Those miRNAs were called ESCC (for ESC cell Cycle promoting) miRNAs. They share a common seed sequence, suggesting that ESCC miRNAs regulate a common set of genes. A search for their targets has revealed that they function by suppressing several key regulators of the Restriction checkpoint, thus enabling rapid proliferation of ESCs. Indeed those targets are inhibitors of the CyclinE/CDK2 pathway, known to regulate the G1/S transition and include p21, the Retinoblastoma like 2 protein (Rbl2) and Last2. ESCC miRNAs post-transcriptionally downregulate those inhibitors and increase CyclinE/CDK2 activity [53].

Interestingly, the promoters of miR-290 and miR-302 clusters are directly regulated by pluripotency factors and in turn ESCC miRNAs maintain the expression of the pluripotency factors by inhibiting their epigenetic silencing. For example miR-290 cluster in mice has been shown to target Rbl2 and decrease the expression of de novo DNA methyltransferases [53] Similarly, the proposed human ortholog for the mouse miR-290 family, miR-372, might regulate human Rbl2 [54].

Using a similar approach, miRNAs are also shown to be critical for human ESC self-renewal and proliferation [54]. Knocking-down Dicer or Drosha by lentivirus-delivered shRNA dramatically affected cell division in hESCs. Dicer and Drosha KD induced G1/S and G2/M transition delays compared to cells infected with lentivirus controls. Re-introducing ESC-enriched miRNAs as mature miRNA mimics into Dicer KD hESC showed that both miR-372 and miR-195 could partially rescue the cell cycle defect. Moreover, miR-195 overexpression in wild-type H1 hESCs was sufficient to increase cell proliferation. miR-195 alone was able to rescue the G2 defect in the Dicer-KD line by directly targeting WEE1 kinase, a negative regulator of the CyclinB/CDK complex in the G2/M transition. Introduction of miR-372 mimics dramatically reduced the levels of the G1/S transition inhibitor p21 in Dicer KD and overexpression of p21 affected hESC proliferation, suggesting that miR-372 regulates hESC cell cycle by modulating p21 expression [54] Another hESC-enriched miRNA, miR-92b, has also been shown to target p21 [54, 59]

Overall, these data suggest that miRNAs can cooperate in maintaining the proliferative capacity of ESCs and appear as major players in the control of embryonic stem cell division (Fig. 18.2).

Fig. 18.2
Role of miRNAs in ESC self-renewal, proliferation and differentiation
microRNAs Regulate ESC Differentiation

In addition to the proliferation defect, Dicer KO and DGCR8 KO mESCs fail to downregulate pluripotency factors upon differentiation [50, 53, 58, 60, 61]. Similarly, in hESCs the levels of Nanog, Oct4 and Sox2 are upregulated in Dicer- and Drosha-knockdown while most early differentiation markers fail to be expressed when cultured under differentiation-inducing conditions [54]. Re-introduction of ESCC miRNAs into Dicer and DGCR8 mutant mESC did not rescue the differentiation defect, suggesting that other miRNAs are involved in the maintenance of pluripotency and the induction of ESC differentiation [53, 54]. Several miRNAs have been reported to target the ESC transcriptional network and therefore be involved in silencing the self-renewal capacities of hESCs and mESCs during the early stages of their differentiation [62].

miR-145 is significantly upregulated upon differentiation of hESCs [48]. An increase of miR-145 represses the expression of pluripotency genes and facilitates differentiation, while the loss of miR-145 impairs differentiation and induces the expression of Oct4, Sox2, and Klf4 [63]. miR-145 controls ESC differentiation by directly targeting the stem cell factors, thereby silencing the self-renewal program. Interestingly, miR-145 promoter is repressed by OCT4 in hESCs, creating a double negative feedback loop [63].

In mESCs several miRNAs have been shown to promote differentiation by targeting genes encoding transcription factors involved in the maintenance of stem cell identity. miR-200c, miR-203 and miR-183 cooperate to repress Sox2 and Klf4 [64]. Upon retinoic-acid-induced differentiation of mESC, miR-134, miR-296 and miR-470 are up-regulated and target coding regions of Nanog, Oct4, and Sox2 [65].

When ESCs are engaged in a differentiation process, they need both to silence their self-renewal program and activate specific differentiated programs. It has recently been shown that let-7 is an important pro-differentiation factor that tightly controls the level of stem cell factors [66]. let-7 was one of the first miRNAs discovered for its role in the developmental timing of C. elegans [67]. pri-let-7 is transcribed in ESCs and pre-let-7 is found in their cytoplasm, however mature let-7 is not detected in undifferentiated ESCs while highly expressed in somatic cells. A study by Melton et al. revealed that let-7 can repress the mESC pluripotency program upon differentiation [66]. Re-introduction of mature let-7 family members into DGCR8 KO mESCs can rescue the differentiation defect by directly targeting transcripts of the self-renewal factors nMyc, Lin28 and Sal4. However let-7 family members had no effect when co-transfected with members of the ESCC miRNAs family, and let-7 did not induce differentiation in wild type mESCs. A model has been proposed in which let-7 and ESCC miRNA families oppose each other’s functions on ESC self-renewal: let-7 miRNAs repress pluripotency genes that are indirectly activated by ESCC miRNAs through an unknown target. Interestingly, as discussed earlier let-7 processing is negatively regulated by lin28 [31, 32] and lin28 expression is under the control of stem cell transcription factors cMyc, Oct4, Sox2 and Nanog [62]. Those results highlight how miRNAs are intricately integrated into the molecular network of pluripotency and are involved in switches crucial for cell fate decisions (Fig. 18.2).

While some miRNAs like miR-145, let-7 family or miR-200 family seem to reduce the pluripo-tency of ESCs, other miRNAs are involved in direct differentiation of ESCs toward a specialized lineages or terminally differentiated cell types. For example miR-133 and miR-1 are essential for the differentiation of ESCs into cardiomyocytes [68] and miR-9 promotes the differentiation into neuronal progenitors [69].
18.2.2 Role of microRNAs in Cellular Reprogramming

A huge breakthrough in the stem cell research field was achieved when Yamanaka group showed that it is possible to reprogram mouse embryonic fibroblasts into pluripotent cells, later called iPSCs, by ectopic expression of only four factors, Oct4, Sox2, Klf4 and cMyc (OSKM, Yamanaka factors) [43]. Omission of the oncogene cMyc from that cocktail still results in formation of iPSC colonies, though with a lower efficiency. This result has been repeated by several groups in human to reprogram various cell types from different tissues [42, 70, 71]. Besides the Yamanaka factors, another set of four factors can induce the generation of iPSCs, Oct4, Sox2, Lin28 and Nanog (OSLN, Thomson factors) [41]. Despite great efforts, the molecular mechanisms underlying the events of reprogramming remain mostly unknown. A growing numbers of studies are reporting an important role of miRNAs in reprogramming (Fig. 18.3a). This is not very surprising since, as mentioned earlier, miRNAs are critical for the balance between self-renewal and proliferation of ESCs.

Fig. 18.3
Functions of miRNAs in cellular reprogramming
Live cell monitoring of iPSC generation from human fibroblasts using miRNAs reporter vectors shows that miR-302s, the most abundant hESC miRNAs, are expressed during the early stage of the OSKM-induced reprogramming [72]. miRNA pro filing and qPCR analysis revealed that other ESC-enriched miRNAs are induced early during iPSC formation, including the miR-17 family [73]. This was expected since Oct4 and Sox2, two of the transcription factors used to induce reprogramming, can activate the promoters of miR-302 and miR-106 clusters [44, 62]. Fully reprogrammed iPSCs have a similar miRNA pro file than ESCs. However imperfectly reprogrammed mouse cells have been shown to inappropriately silence the Dlk1-Dio3 locus, containing about 50 miRNAs [74]. Despite expression of genes associated with pluripotency, cells with a silenced Dlk1-Dio3 locus contribute poorly to chimaeras and seem to have limited capacities to differentiate into certain type of tissue-specific cells. Moreover, recent studies suggest that iPSCs might retain a memory of the cell of origin they come from [75]. It would be interesting to determine if this memory could be linked to miRNA expression.

Disruption of miRNA maturation or function by knock-down of Drosha, Dicer or Ago2 using lentiviral vectors dramatically reduces the number of iPSC colonies induced by OSKM or OSK in mouse embryonic fibroblasts (MEF), suggesting that some miRNAs are essential for the reprogramming process [73].

Introduction of ESC-Enriched microRNAs Enhances Reprogramming

One of the first evidence of the involvement of miRNAs in the formation of iPSCs comes from a study by Judson et al. They demonstrated that several members of the miR-290 cluster can increase the efficiency of OSK-induced reprogramming of MEF to a similar ef fi ciency as OSKM. Interestingly, introduction of a miR-294 mimic did not enhance OSKM-induced reprogramming, suggesting that miR-294 acts as a downstream target of c-Myc, and that miR-290s can substitute for cMyc contribution in cellular reprogramming. Indeed cMyc can bind to the promoter region of the mir-290–295 cluster [76], and bioinformatic analysis suggest that miR-294 may regulate a subset of c-Myc target genes [77]. ESCC miRNAs can promote cell cycle progression in ESCs by targeting inhibitors of the G1/S transition like p21 [53, 54]. Moreover, it has been shown that cMyc can repress p21 expression by downregulating its transcription [78] or at the post-transcriptional level through members of the miR-17 family [79]. Several groups have also reported that p53 and its downstream effectors antagonize iPSC induction, and knock-down of p21 in mouse fibroblasts increases reprogramming ef fi ciency [80–82]. Therefore, inhibition of p21 by miRNA and subsequent activation of proliferation could partly explain why ESCC miRNAs enhance reprogramming efficiency (Fig. 18.3b). However, unlike with cMyc, a homogeneous population of fully reprogrammed colonies was observed with miR-294, suggesting that ESCC miRNAs also have functions independent of cMyc’s [76].

Later, Li et al. proposed that miR-93 and miR-106b are key regulators of reprogramming activity. They found that they can enhance OSK and OSKM iPSC-induction in mouse by directly targeting p21 and TGF βR2 [73]. Ectopic expression in MEF by a retroviral vector of the miR-106a cluster (containing among others miR-20b) also increases reprogramming efficiency in OSK and OSKM iPSC-induction but with a greater effect with the three factors induction [83]. This result can be explained by the fact that cMyc can activate miR-106a cluster [38]. Members of the miR-302 cluster enhance reprogramming in both mouse and human, as well as miR-372 in human [76, 83, 84] and miR-130/301/721 in mouse [85].

In order to uncover the mechanisms behind this effect, a time course microarray analysis of the three factors plus or minus the miR-106a or miR-302 clusters have been performed in mouse [82]. This analysis showed that pathways changing at early time point during reprogramming with the addition of miR-106a cluster fall into three main groups: cell cycle, epigenetic modification and mesenchymal to epithelial transition (MET). Proteins belonging to those three groups were also found important miR-302 and miR-372 targets during OSK-induced reprogramming of human fibroblasts [84]. Direct targets include TGRβR2 and RHOC. Both are involved in MET. However miR-302a, b, c or d and 372 alone (without OSK reprogramming factors) were not able to induce the expression of epithelial markers [84]. The importance of MET in the reprogramming process has been highlighted recently and will be discussed in more details in the next section. Members of the miR-200 family have also been shown to facilitate the MET and improve reprogramming in mouse [86–88] (Fig. 18.3b).

Of note, all those studies were done with the Yamanaka factors. It will be interesting to see whether ESC-enriched miRNAs also enhance reprogramming induced by Thomson factors.

Other hESC-enriched miRNAs have been identified, but their potential role in reprogramming has not been investigated yet. In particular, some miRNAs of the C19MC cluster, containing miR-515 and miR-520 families, have different seed sequences than miRNAs enhancing iPSC formation, like miR-302, miR-372, miR-200 and miR-106. It will be critical in the future to test the function of these other hESC-enriched miRNAs in iPSC induction.
Inhibition of Tissue-Specific microRNAs Promotes Formation of iPSCs

Several studies came to the same conclusion that ESC-enriched miRNAs can enhance human and mouse reprogramming by targeting proteins involved in cell cycle, epigenetic modification and MET. miRNAs having a negative effect on those pathways have been shown to inhibit iPSC formation. During the dedifferentiation of somatic cells, important changes need to occur in their molecular signature : they have to acquire ESC-like signature but also have to down-regulate the tissue specific signature. miR-21 and miR-29a are the most abundant miRNAs in mouse fibroblasts and are downregulated during reprogramming by more than 50 %. It has recently been shown that inhibition of miR-21 and miR-29a using miR antagomirs enhances reprogramming efficiency through p53 downregulation [89]. miR-34 is also a target of p53 early during iPSC formation and constitutes a barrier for somatic cells reprogramming since genetic ablation of miR-34 in mice significantly promotes iPSC generation [90]. Moreover, cMyc can repress let7 family members indirectly through upregulation of lin28. Opposite effects of let-7 and ESCC miRNAs prompted researchers to test whether inhibition of let-7 has an effect on reprogramming. Indeed, antisens inhibitors of let7 modestly enhance reprogramming efficiency of MEF induced by OSK or OSKM [66].
Introduction of microRNAs Can Induce Reprogramming Without Other Factors

It was reported previously that introduction of a polycistronic cassette expressing miR-302a–b-c-d was sufficient to generate cells highly resembling to hESC from cancer cell lines and human hair follicule cells [91, 92]. Those cells, named miR-iPSC re-expressed hESC stem cell factors, their global gene expression was very close to hESC’s and they were able to differentiate into various lineages. However iPSC isolation and characterization were not well described and incomplete.

More recently, two independent groups have convincingly shown that human and mouse iPSCs can be derived from fibroblasts without the requirement of exogenous transcription factors by adding microRNAs [93, 94]. Anokye-Danso et al. demonstrated that lentiviral expression of the miR-302/367 cluster is able to reprogram MEF and human foreskin and dermal fibroblasts in a very rapid and efficient way [93]. miR-302/367-iPSC display similar self-renewal and pluripotency characteristic to OSKM-iPSC. miR-367, which has a different seed sequence than miR-302s, is required for reprogramming. Moreover low level of the histone deacetylase HDAC2 is also required, confirming the importance of chromatin modeling in iPSC reprogramming. Until now, the generation of iPSC from somatic cells was a very slow and inefficient process. In Anokye-Danso et al. study, not only miR-302/367 cluster improves the temporal kinetics of iPSC colony apparition, but also increases the efficiency by two orders of magnitude compared to existing protocols, when using similar viral titers. With a percentage approaching 10 % of human fibroblasts generating iPSCs, this method could be used in large-scale iPSC formation. The authors propose that such high efficiency could be explained by the nature of miRNAs themselves since a single miRNA can target hundreds of mRNAs simultaneously, hence coordinating several pathways and allowing a major phenotype change of the identity of the cell. miRNA derived-iPSCs have been called mi-iPSCs.

Shortly after this work was published, the Miyoshi et al. reprogrammed human and mouse multipotent adipose stromal cells as well as human dermal fibroblasts into pluripotent stem cells using seven miRNAs: 200c, 302a, 302b, 302c, 302d, 369-3p and 369-5p [94]. miRNAs were introduced by four transfections of mature double-stranded miRNAs within the first 8 days of reprogramming. The efficiency of generating mouse mi-iPSCs was similar to that seen in the original report of Yamanaka using OSKM induction in MEF. However the efficiency was considerably lower in human mi-iPSCs generated from human fibroblasts. More repeated transfections during the course of reprogramming might increase the efficiency of iPSC formation. Nonetheless, this study brings proof of principle that iPSCs can be obtained with miRNAs without the need for genomic integration of foreign DNA and might hold significant potential for both biomedical research and regenerative medicine. The miRNAs used in the Miyoshi et al. study belong to three families of miRNAs. The use of members of the miR-302 family confirmed previous studies showing that miR-302s can enhance OSK-induced iPSC formation or generate iPSC without other stem cell factors. miR-302 family appears as the most important miRNA family involved in reprogramming from human cells. As mentioned before, the promoter of miR-302 cluster is directly activated by Oct4 [44] and miR-302 have been shown to facilitate the MET during dedifferentiation of fibroblasts [83, 84]. We can wonder if miR-302 could be the equivalent of Oct4 in reprogramming since in any combination of stem cell factors, Oct4 is necessary for iPSC generation. It will be interesting to determine whether a combination of miRNA without miR-302 could also induce iPSC formation and whether miR-372 could replace miR-302s since they share the same seed sequence. Contrarily to the Anokye-Danso et al. study, miR-367 was not required to induce the reprogramming, but could be replaced by miR-200c, a miRNA important for MET, and members of the miR-369 family. Target prediction softwares suggest that miR-369s could also be involved in MET. Moreover, interestingly miR-369-3 is one of the few miRNAs that can up-regulate the translation of its target mRNAs on cell cycle arrest [29]. miR-302s, 367, 200c and 369 have different seed sequences, so both Anokye-Danso et al. and Miyoshi et al. protocols are likely to induce reprogramming through targeting of different mRNAs and pathways. Further studies should tell which combination of mature miRNAs is the best one and when each miRNA is involved during the course of iPSC formation. This would allow to determinate the best cocktail and timing of miRNA introduction in order to reach the maximum efficiency. It will also be interesting to investigate whether miR-induced reprogramming follows the same steps as OSKM or OSLN-induced reprogramming.

miRNAs can be powerful tools for reprogramming and consequently for therapeutic applications since they avoid integration of factors into the genome and can be used for large scale production of iPSCs.
18.2.3 Role of microRNAs in Cell Fate Transitions

Mesenchymal to Epithelial Transition

The epithelial-to-mesenchymal transition (MET) is the set of coordinated changes in cell-cell and cell-matrix interactions leading to loss of mesenchymal features and acquisition of epithelial characteristics. MET has been shown to play a pivotal role during embryonic development and its reverse process, the epithelial to mesenchymal transition (EMT), is important for cancer progression and invasion [95]. The process of reprogramming of fibroblasts resembles MET since it consists of transformation from single layer of adherent cells into tightly packed clusters of round ESC-like cells. MET seems to be a hallmark of the initiation phase characterized by an increase of epithelial-associated genes and a decrease of mesenchymal factors [96] siRNA against epithelial markers, in particular E-cadherin, totally inhibit the formation of iPSCs [86]. Therefore MET appears as a crucial step of fibroblasts dedifferentiation. Signaling pathways involved in the regulation of MET affect the efficiency of reprogramming and several miRNAs can regulate reprogramming by targeting proteins involved in the MET (Fig. 18.3b). As mentioned, members of the miR-200 family synergize with OSKM or other miRNAs to promote MEF reprogramming via regulation of MET by downregulating mesenchymal markers such as Zeb1 and Zeb2 [86–88, 94, 97, 98] Moreover miR-106a, miR-106b, miR-17, miR-93, and miR-302 cluster function in reprogramming is dependent of the fact that they all target TGFβR2, resulting in an increase of E-cadherin expression during fibroblast reprogramming [73, 83, 84]

The epithelial-to-mesenchymal transition (MET) is the set of coordinated changes in cell-cell and cell-matrix interactions leading to loss of mesenchymal features and acquisition of epithelial characteristics. MET has been shown to play a pivotal role during embryonic development and its reverse process, the epithelial to mesenchymal transition (EMT), is important for cancer progression and invasion [95]. The process of reprogramming of fibroblasts resembles MET since it consists of transformation from single layer of adherent cells into tightly packed clusters of round ESC-like cells. MET seems to be a hallmark of the initiation phase characterized by an increase of epithelial-associated genes and a decrease of mesenchymal factors [96] siRNA against epithelial markers, in particular E-cadherin, totally inhibit the formation of iPSCs [86]. Therefore MET appears as a crucial step of fibroblasts dedifferentiation. Signaling pathways involved in the regulation of MET affect the efficiency of reprogramming and several miRNAs can regulate reprogramming by targeting proteins involved in the MET (Fig. 18.3b). As mentioned, members of the miR-200 family synergize with OSKM or other miRNAs to promote MEF reprogramming via regulation of MET by downregulating mesenchymal markers such as Zeb1 and Zeb2 [86–88, 94, 97, 98] Moreover miR-106a, miR-106b, miR-17, miR-93, and miR-302 cluster function in reprogramming is dependent of the fact that they all target TGFβR2, resulting in an increase of E-cadherin expression during fibroblast reprogramming [73, 83, 84]

Fibroblasts are mesenchymal cells, however iPSCs have also been generated from other cell types and not all cells have to go through MET during reprogramming. A question comes to mind: would the miRNAs shown to enhance or induce reprogramming through MET activation have the same effect on reprogramming of epithelial somatic cells such as keratinocytes.
Transition Between Different Pluripotent States

It has been shown recently that expression of specific miRNAs can define the developmental state of ESCs and iPSCs [48]. hESCs are likely to be the in vitro equivalent of mouse epiblast stem cells (EpiSCs), derived from the post-implantation epiblast stage, while mESCs are derived from the inner cell mass of pre-implanted embryos and represent an earlier stage of embryonic development [99]. Low concentrations of sodium butyrate, a HDAC inhibitor, can induce hESCs to go back to an earlier developmental stage [100]. This method constitutes a useful tool to study the expression of miRNAs in early steps of human development. miR-372 cluster is expressed at higher levels in butyrate-treated hESCs than in hESCs while miR-302 cluster expression was slightly lower [48]. It would be important to analyze miR-302 and miR-372 expression levels in newly derived hESCs that might represent an earlier state of development. miR-302 cluster was expressed at considerably higher levels in EpiSCs than in mESCs [48]. miRNAs can be good indicators of the state of pluripotency, in particular miR-302 could be used as a marker for the epiblast stage in mouse. Moreover, it will be interesting to assess whether overexpression of miR-372 in hESCs can make them regress to an earlier developmental stage and whether over-expression of miR-302 in mESCs can in the contrary differentiate them toward an EpiSClike stage.


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