Stem cell types in embryogenesis. At implantation, mouse blastocysts comprise three distinct cell types: the trophectoderm, the inner cell mass which produces the primitive endoderm, and the preimplantation epiblast. With specific in vitro culture conditions, three types of stem cells with similar potential for differentiation can be derived – ESC, trophoblast stem cells (TSCs), and extraembryonic endoderm stem cells (XEN). The TSCs and XEN are committed to form extraembryonic tissues only. After implantation, the blastocyst grows into a pre-gastrulation embryo from which pluripotent stem cells can be derived in mouse from the epiblast of early post-implantation embryos. These epiblast cells are like ESCs since they are pluripotent and differentiate into all germ layer tissues in vitro and into teratomas in vivo. They are progressively restricted in differentiation potential when allocated to the mesoderm and endoderm at the primitive streak but do retain by lineage analysis an ability to retain plasticity in cell fate. Tissue commitment occurs at early gastrulation (Reprinted by permission from Macmillan Publishers Ltd: Nature (Pera and Tam ), copyright 2010)
Two decades later the first embryonic human cell lines were derived by culturing the inner cell mass of the human blastocyst under the same conditions as those above required to derive mouse ESCs . Not until the mid-2000s was a standardized protocol developed for culturing with growth factors alone (fibroblast growth factor [FGF] + activin, as “Nodal”) to efficiently obtain human ESCs  without the feeder cell layer required to derive and maintain murine embryonic cell lines. Nodal is an important regulator of many processes in early embryonic development: Both Nodal and activin suppress the differentiation of human ESC, signaling through the same receptors. Currently LIF+ 2i (leukemia inhibitory factor and two kinase inhibitors) are the best growth factors to replace the requirement for the murine embryonic feeder layer .
The two most recent technologic innovations essential to realize the hype and promise of stem cells were (a) the derivation of pluripotent stem cells from the human embryo in 1998 [3, 6] and (b) the reprogramming of adult fully differentiated cells to induce numerous adult stem cells [7, 8], such as iPSC to recapitulate the pluripotency of an ASC to resemble that of a ESC.
Embryonic Stem Cells
ESCs make up the inner cell mass (ICM) of the preimplantation blastocyst which precedes any commitment to embryonic cell fates. ESC lines are obtained from enzymatic dispersion of the ICM and culturing under specific conditions. Once implantation occurs in the endometrium, mitotic division parallels lineage commitment on a molecular level, such that cells of the ICM and its derivative cell lines (epiblast stem cell or EpiSC) lose their pluripotency. Pluripotency is a transitory state for embryonic cells that exist for a brief window of development. Shortly after the onset of embryogenesis, the totipotent cells of the embryo become restricted in their developmental potential, becoming either extraembryonic tissues (such as the placenta and fetal extraembryonic membranes) or pluripotent progenitors which can form cells from the three primary germ cell layers (Fig. 11.1). Commitment is the process coupled to mitosis by which the progeny of a cell goes from pluripotent to unipotent and determined to one fate with only the set of genes to characterize it as a specific cell type switched on. Differentiation follows commitment so that the switched on genes are expressed and the cells appear morphologically distinct from its ancestors.
Stem cells derived from the embryonic germ cells can be maintained indefinitely in vitro in the pluripotent state and undifferentiated. The characteristics of “stemness” exclusive to cells in the ICM of the preimplantation blastocyst and their derived ESC lines include (1) expression of the four essential transcription factors (OCT4, Sox2, Klf4, and NANOG), (2) activity of both X chromosomes (Xa/Xa) in female cells (versus the normal developmental commitment to inactivate one X chromosome (Xi/Xa), and iii) a high activity in the maternal DLK1-DI03 locus.
The literature is full of the nomenclature of ESC subtypes which are obtained from blastocysts by either changing culture conditions or isolation from different original parent cell peri-implantation as opposed to preimplantation. These sources of stem cells are not pluripotent although they may share some features of ESCs and are not considered ESCs. For instance, EpiSCs can originate from epiblast of peri-implantation ICM or other stem cell (SC) lines from the trophoblast (TS) or extraembryonic endoderm (XEN), and they are not pluripotent. The ICM and epiblast of the mouse embryo, and presumably the human analogies, are dynamic cell populations whose interactions with the extraembryonic tissues surrounding them are crucial for cell-fate determination. Also, the facile interconversions of stem cells types may reflect an underlying heterogeneity within mouse ESC and EpiSC populations.
Different types of ESCs have a very plastic phenotype although a commonly shared genetic network of transcription factors maintains the pluripotent state. The analysis of human ESC cultures shows that they have high heterogeneity and there are subpopulations identified by flow cytometry. Cells with the greatest capacity to renew themselves express the highest level of pluripotency genes and are at the top of the pluripotency hierarchy . Many cells can co-express both lineage-specific genes and pluripotency genes; however, those with pluripotency genes only are at the top of pluripotency hierarchy. Importantly in stem cell biology, it is the balance of transcription factors which maintains the cell in the “stem state” capable of pluripotency. Extracellular signaling, propagated through intracellular signal-transduction pathways, maintains the genetic network that controls the “on and off” switch for pluripotency [10, 11]. The key signaling systems that maintain stem cells in their pluripotent state include transforming growth factor β (TGFβ), growth factors that signal through receptor tyrosine kinases (RTKs), WNTs, leukemia inhibitory factor (LIF), and JAK-STAT . The key signal in maintaining pluripotency is LIF/STAT3; conversely, it is the FGF/MAPK/TGFb/activin pathway that has been shown to induce differentiation and drive lineage and cellular commitment .
Stem cell plasticity is critical to enable the body to respond to adapt to cellular loss or changing demands in cell production, tissue maintenance, remodeling, or regeneration in adults. Experimentally, data from mouse models differ from human stem cell discovery, since distinct phenotypic stem cells are experimentally accessible from all states of murine embryogenesis, suggesting that pluripotency is a continuum versus a transient window of time (Table 11.1). The various states of mouse stem cells suggest that these cells have reached different stages of developmental potential. There is more developmental equivalence demonstrated between human ESCs and mouse EpiSCs than other pluripotent cell types derived from the mouse embryo (Table 11.1). Unlike mouse ESCs, both human ESCs and mouse EpiSCs (1) require nodal or activin signals to maintain their pluripotent state, (2) neither requires LIF to maintain the pluripotent state, and (3) both grow poorly after dissociation into single cells. Notably there are differences in their cell-surface marker expression and gene expression.
Properties of various pluripotent cell populations in vitro
Type of stem cell
Response of factors
Nodal and/or activin
Mouse ES cells
Human ES cells
Definition of Stemness
The most studied stem cells are the ESCs, and the knowledge obtained from their discovery has directed the subsequent investigation of various types of nonembryonic stem cells, so a discussion of the definition of “stemness” is critical.
Investigators need to prove the following essential features to confirm “stemness” which includes the following properties and functional characteristics:
Phenotypically as small round cells they do not share a phenotype of any adult tissue but are able to generate de novo cells found in any adult tissue.
Expression of cell-surface markers of pluripotency from different stem cell origins including NANOG, Sox2, OCT4, and Klf4 (Table 11.1).
Activation of signal-transduction pathways to maintain “stemness” of which the key pathway signaling essential to maintain pluripotency is the LIF/STAT3 pathway.
Meta-analysis of microarray studies of human ESCs has led to a characterization of the human ESC transcriptome and epigenome . Similarly the characterization of gene expression of a large panel of human ESC lines has led to a characteristic transcriptional and epigenetic profile of the human ESC .
A characteristic proliferative state.
Functionality consistent with stem cells in vitro or in vivo.
In vitro ESC lines will grow as round colonies of very small cells dependent on LIF, a cytokine produced by the endometrium which allows blastocyst implantation , capable of expressing pluripotent markers (NANOG, SOX2, OCT4, and KLF4) and showing activity for the two enzymes telomerase and alkaline phosphatase. Cell surface markers include SSEA4 in humans, ABC transporters (ABCG2), and enzymes (alkaline phosphatase and telomerase TERT) .
In vivo developmental potential is assessed by a biologic assay of the ability of a cell to give rise to all cell types in the body. So stem cells are tested by injection into ectopic sites in host animals and true pluripotent cells form benign growths or teratomas, representing differentiated tissue from all three embryonic germ layers. A definitive test of in vivo pluripotency in animal models includes the generation of germline-competent chimeras at birth upon introducing stem cells into preimplantation embryos in foster mothers. The contribution of the stem cells to the chimera proves the capacity of the cells to differentiate to all tissue types and the functional capacity of the descendants of the ESCs.
Adult Stem Cells
Differentiated adult cells are present in all adult tissues, typically give rise to cells of an identical lineage, and are thus unipotent. In specific conditions such as Barrett’s metaplasia, they can transdifferentiate between lineages, such as evidenced by transdifferentiation of esophageal epithelial cells into intestinal mucin-secreting goblet cells.
In contrast, adult stem cells exist in the bone marrow and circulation or as residents in a specific tissue at a very low level. Endothelial progenitor cells for instance reside in a stem cell niche in the marrow and circulate in the blood at low levels (<0.01 % of circulating white cells) . Table 11.2 compares and contrasts ASCs to ESCs and iPCS. ASCs are partially lineage committed and have the capacity to give rise to all cell lineages from a single germ layer; thus, they are considered multipotent but still not pluripotent. For example, the adult hematopoietic stem cell is multipotent and can repopulate the bone marrow following bone marrow transplantation but is not traditionally thought of as capable of replacing other germ cell layers, such as endodermal or ectodermal lineages. Trauma, injury, or ischemia can induce the bone marrow-derived cells to home to the site of injury and their prevalence increases.
Comparison of the properties of various types of stem cells
Inner Cell Mass
Blastocyst of Embryo
BM, circulate and reside in tissue
Reprogrammed somatic cells
Risk of teratoma
Teratocarinoma even from 1 ESC
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