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After the blastocyst stage, once an embryo implanted in endometrium (in case of rodent), the inner cell mass (ICM) of a fertilized embryo segregates into two layers: hypoblast and epiblast. The epiblast cells are the functional progenitors of soma and germ cells which later differentiate into three layers: definitive endoderm, mesoderm and ectoderm. Stem cells derived from epiblast are pluripotent. These cells are called epiblast-derived stem cells (EpiSCs) and have several different cellular and molecular characteristics with Embryonic Stem Cells (ESCs). [1] Pluripotency in EpiSCs is essentially different from that of embryonic stem cells. The pluripotency of EpiSCs is primed pluripotency: primed to differentiate into specific cell lineages. Naïve pluripotent stem cells (e.g. ESC) and primed pluripotent stem cells (e.g. EpiSC) not only sustain the ability to self-renew but also maintain the capacity to differentiate. [2] Since the cell status is primed to differentiate in EpiSCs, however, one copy of the X chromosome in XX cells (female cells) in EpiSCs is silenced (XaXi). EpiSCs is unable to colonize and is not available to be used to produce chimeras. Conversely, XX cells in ESCs are both active and can produce chimera when inserted into a blastocyst. Both ESC and EpiSC induce teratoma when injected in the test animals (scid mice) which proves pluripotency. EpiSC display several distinctive characteristics distinct from ESCs (table 1). The cellular status of human ESCs (hESCs) is similar to primed state mouse stem cells rather than naïve state. [3]
Table 1. Comparison of naïve and primed pluripotent states [2]
Property | Naive State | Primed State |
---|---|---|
Embryonic tissue | Early epiblast | Egg cylinder or embryonic disc |
Culture stem cell | Mouse ESCs | Mouse EpiSCs; primate "ESCs" |
Blastocyst chimaeras | Yes | No |
Teratomas | Yes | Yes |
Pluripotency factors | Oct4, Nanog, Sox2 (high levels), Klf2, Klf4 | Oct4, Nanog, Sox2 (low levels) |
Sox2/Oct4 dimerization | High | Low |
Naive markers | Active Pou5f1 distal enhancer | Active Pou5f1 proximal enhancer |
Specification markers | Absent | Fgf5, T |
Response to Lif/Stat3 | Self-renewal | None |
Response to Fgf/Erk | Differentiation | Self-renewal |
Clonogenicity | High | Low |
XX status | Both active | One X inactive |
Differentiating naïve pluripotent Stem cells into primed pluripotent stem cells (e.g. adding activin and fibroblast growth factor (FGF) in the culture medium) can be accomplished but reprogramming of primed cells into naïve cells is more difficult. Several approaches to reprogramming EpiSCs to achieve naïve pluripotency have been applied. One of those methods is overexpressing in the primed pluripotent stem cell a reprogramming factor Klf4, [4] or Sox2 and Klf4 (SK cocktail). [5] Klf4 alone can reset mouse primed cells, but SK is needed for human naive reset.
The reversion back to the naive state has also been achieved by suppressing the activity of the histone methyltransferase MLL1, also known as KMT2A. The inhibition of MLL1 via the small-molecule inhibitor MM-401 in EpiSCs showed increase in alkaline phosphatase staining as well as upregulation of "naive" markers such as Rex1 and downregulation of "primed" markers such as FGF5. Moreover, beyond the potency-state comparison, MLL1 inhibition was also shown to reactivate the silenced X-chromosome which is typically deactivated in post-implantation epiblast stem cells, suggesting an epigenetic reversion back to a more ground-level, naive state. What's more, some EpiSCs affected by the MLL1 inhibition-induced reversion were able to contribute to germline-competent chimeras, which had been considered as one of the most major differences between ESCs and EpiSCs. [6]
Scientists have been able to demonstrate the induction of EpiSC-like cells in vitro from mouse ESCs, which are referred to as Epiblast-like cells (EpiLC). [7] Many studies have used EpiLCs as suitable analogues for actual post-implantation derived epiblast stem cells, especially in attempts at reversion back to the "naive" state. Recently, overexpression of PR-domain Zinc Finger Protein 14 (PRDM14) in EpiLC was shown to cause a reversion back to an ESC-like state (with levels of Alkaline Phosphatase staining recovered to that observed in ESCs as well as more ESC-like cell morphology), with Klf2 being required for the mechanism to occur. [8] It has been proposed that PRDM14 induces this state by activating Klf2 via active demethylation recruitment of Oct-4; such technique has yet to be demonstrated in actual epiblast-derived EpiSCs.
In multicellular organisms, stem cells are undifferentiated or partially differentiated cells that can change into various types of cells and proliferate indefinitely to produce more of the same stem cell. They are the earliest type of cell in a cell lineage. They are found in both embryonic and adult organisms, but they have slightly different properties in each. They are usually distinguished from progenitor cells, which cannot divide indefinitely, and precursor or blast cells, which are usually committed to differentiating into one cell type.
Cellular differentiation is the process in which a stem cell changes from one type to a differentiated one. Usually, the cell changes to a more specialized type. Differentiation happens multiple times during the development of a multicellular organism as it changes from a simple zygote to a complex system of tissues and cell types. Differentiation continues in adulthood as adult stem cells divide and create fully differentiated daughter cells during tissue repair and during normal cell turnover. Some differentiation occurs in response to antigen exposure. Differentiation dramatically changes a cell's size, shape, membrane potential, metabolic activity, and responsiveness to signals. These changes are largely due to highly controlled modifications in gene expression and are the study of epigenetics. With a few exceptions, cellular differentiation almost never involves a change in the DNA sequence itself. However, metabolic composition does get altered quite dramatically where stem cells are characterized by abundant metabolites with highly unsaturated structures whose levels decrease upon differentiation. Thus, different cells can have very different physical characteristics despite having the same genome.
Embryonic stem cells (ESCs) are pluripotent stem cells derived from the inner cell mass of a blastocyst, an early-stage pre-implantation embryo. Human embryos reach the blastocyst stage 4–5 days post fertilization, at which time they consist of 50–150 cells. Isolating the inner cell mass (embryoblast) using immunosurgery results in destruction of the blastocyst, a process which raises ethical issues, including whether or not embryos at the pre-implantation stage have the same moral considerations as embryos in the post-implantation stage of development.
Embryoid bodies (EBs) are three-dimensional aggregates formed by pluripotent stem cells. These include embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC)
Oct-4, also known as POU5F1, is a protein that in humans is encoded by the POU5F1 gene. Oct-4 is a homeodomain transcription factor of the POU family. It is critically involved in the self-renewal of undifferentiated embryonic stem cells. As such, it is frequently used as a marker for undifferentiated cells. Oct-4 expression must be closely regulated; too much or too little will cause differentiation of the cells.
Homeobox protein NANOG(hNanog) is a transcriptional factor that helps embryonic stem cells (ESCs) maintain pluripotency by suppressing cell determination factors. hNanog is encoded in humans by the NANOG gene. Several types of cancer are associated with NANOG.
In biology, reprogramming refers to erasure and remodeling of epigenetic marks, such as DNA methylation, during mammalian development or in cell culture. Such control is also often associated with alternative covalent modifications of histones.
In biology and medicine, stem cell genomics is the analysis of the genomes of stem cells. Currently, this field is rapidly expanding due to the dramatic decrease in the cost of sequencing genomes. The study of stem cell genomics has wide reaching implications in the study of stem cell biology and possible therapeutic usages of stem cells.
In developmental biology, the cells that give rise to the gametes are often set aside during embryonic cleavage. During development, these cells will differentiate into primordial germ cells, migrate to the location of the gonad, and form the germline of the animal.
Induced pluripotent stem cells are a type of pluripotent stem cell that can be generated directly from a somatic cell. The iPSC technology was pioneered by Shinya Yamanaka and Kazutoshi Takahashi in Kyoto, Japan, who together showed in 2006 that the introduction of four specific genes, collectively known as Yamanaka factors, encoding transcription factors could convert somatic cells into pluripotent stem cells. Shinya Yamanaka was awarded the 2012 Nobel Prize along with Sir John Gurdon "for the discovery that mature cells can be reprogrammed to become pluripotent."
SRY -box 2, also known as SOX2, is a transcription factor that is essential for maintaining self-renewal, or pluripotency, of undifferentiated embryonic stem cells. Sox2 has a critical role in maintenance of embryonic and neural stem cells.
Shinya Yamanaka is a Japanese stem cell researcher and a Nobel Prize laureate. He is a professor and the director emeritus of Center for iPS Cell Research and Application, Kyoto University; as a senior investigator at the UCSF-affiliated Gladstone Institutes in San Francisco, California; and as a professor of anatomy at University of California, San Francisco (UCSF). Yamanaka is also a past president of the International Society for Stem Cell Research (ISSCR).
Forkhead box D3 also known as FOXD3 is a forkhead protein that in humans is encoded by the FOXD3 gene.
A mesenchymal–epithelial transition (MET) is a reversible biological process that involves the transition from motile, multipolar or spindle-shaped mesenchymal cells to planar arrays of polarized cells called epithelia. MET is the reverse process of epithelial–mesenchymal transition (EMT) and it has been shown to occur in normal development, induced pluripotent stem cell reprogramming, cancer metastasis and wound healing.
Cell potency is a cell's ability to differentiate into other cell types. The more cell types a cell can differentiate into, the greater its potency. Potency is also described as the gene activation potential within a cell, which like a continuum, begins with totipotency to designate a cell with the most differentiation potential, pluripotency, multipotency, oligopotency, and finally unipotency.
Embryonic stem cells are capable of self-renewing and differentiating to the desired fate depending on their position in the body. Stem cell homeostasis is maintained through epigenetic mechanisms that are highly dynamic in regulating the chromatin structure as well as specific gene transcription programs. Epigenetics has been used to refer to changes in gene expression, which are heritable through modifications not affecting the DNA sequence.
Induced stem cells (iSC) are stem cells derived from somatic, reproductive, pluripotent or other cell types by deliberate epigenetic reprogramming. They are classified as either totipotent (iTC), pluripotent (iPSC) or progenitor or unipotent – (iUSC) according to their developmental potential and degree of dedifferentiation. Progenitors are obtained by so-called direct reprogramming or directed differentiation and are also called induced somatic stem cells.
Tsix is a non-coding RNA gene that is antisense to the Xist RNA. Tsix binds Xist during X chromosome inactivation. The name Tsix comes from the reverse of Xist, which stands for X-inactive specific transcript.
F-box protein 15 also known as Fbx15 is a protein that in humans is encoded by the FBXO15 gene.
JacobH. Hanna is a Palestinian Arab-Israeli biologist who is working as a professor in the Department of Molecular Genetics at the Weizmann Institute of Science in Rehovot, Israel. An expert in embryonic stem cell research, he is most recognized for developing the first bona fide synthetic embryo models from stem cells in the petri dish in mice and humans.