Embryonic stem (ES) cells have the capacity to proliferate indefinitely in culture while maintaining the ability to differentiate to form any of the cells of the body. This unique combination of functions suggests that these cells could provide a potentially unlimited source of differentiated cells for the treatment of disease and aging. The ability of embryonic stem (ES) cells to give rise to all cells of the embryo and adult, in culture or within the context of the developing embryo, has been termed pluripotency. Pluripotency resides for a short period of time during the embryonic development of a mammal within the cells of the inner cell mass (ICM) of the blastocyst and the subsequent epiblast of the pre-gastrulation embryo, and is preserved in the primordial germ lineage during later development and into adulthood. In culture, pluripotent cells have been derived from all three embryonic populations. The Pluripotency of ESCs has attracted great attention for their potential use in tissue and cell therapy. However, the molecular and developmental mechanisms controlling Pluripotency and Differentiation of ESCs are largely unknown. Human ESC Pluripotency is regulated by a combination of Extrinsic and Intrinsic factors. Unlike Mouse, Extrinsic factor LIF (Leukemia Inhibitory Factor) is not sufficient to maintain Human ESC and
BMPs (Bone Morphogenic Proteins) cause rapid differentiation. Instead,
FGF (Fibroblast Growth Factor) signaling and a balance between
TGF-Beta (Transforming Growth Factor-Beta) /
Activin and BMP signaling are central to the self-renewal of Human ESCs. Intrinsic factors regulating Pluripotency in Human ESCs include a battery of transcription factors including
Oct4 (Octamer Binding Transcription Factor-4),
SOX2 (SRY (Sex Determining Region-Y) Box-2) and
Nanog (Ref.1 and 2).
TGF-Beta superfamily of ligands, which contains about 40 potential ligands in the Human genome, play a major role maintaining the Self- renewing Human ESCs. They signals through two main branches: the
SMAD1/5 (Sma and MAD (Mothers Against Decapentaplegic) Related Protein- 1/5) branch, which transduces on behalf of
GDF (Growth Differentiation Factor) ligands via the Type I receptors
ALK1 (Activin Receptor-Like Kinase-1),
ALK2 (Activin Receptor-Like Kinase-2),
ALK3 (Activin Receptor-Like Kinase-3) and
ALK6 (Activin Receptor-Like Kinase-6); and the
Nodal branch, which involves the activation of
SMAD2/3 (Sma and MAD (Mothers Against Decapentaplegic) Related Protein-2/3) via
ALK4 (Activin Receptor-Like Kinase-4), and
ALK5 (Activin Receptor-Like Kinase-5) and
ALK7 (Activin Receptor-Like Kinase-7). There are also two inhibitory
SMAD6 (Sma and MAD (Mothers Against Decapentaplegic) Related Protein-6), which selectively inhibits
SMAD7 (Sma and MAD (Mothers Against Decapentaplegic) Related Protein-7), which inhibits both branches of
TGF-Beta signaling, that provide a repressive input on the pathway. Upon activation by phosphorylation and association with a common
SMAD4 (Sma and MAD (Mothers Against Decapentaplegic) Related Protein-4), the receptor-activated SMADs translocate to the nucleus and, in concert with other transcription factors, regulate gene expression. Activation of the
Nodal branch through SMAD2/3 is associated with Pluripotency and is required for the maintenance of the undifferentiated state in Human ESCs. SMAD2/3 pathway is also required for positive regulation of several factors of
TGF-Beta signaling. These factors include
LEFTY1 (Left-right determination factor-1) and
LEFTY2 (Left-right determination factor-2).
Cripto encodes an
CFC co-receptor that is essential for responsivity to
LEFTY2 are both inhibitors of Nodal signaling. Expression of
LEFTY2 remain high in undifferentiated Human ESCs and reduce upon differentiation (Ref.3, 4 and 5).
In contrast to TGF-Beta/Activin/Nodal signaling, which promotes the maintenance of Pluripotent Human ESCs, BMP signaling is unable to support self-renewal and is associated with differentiation to Trophoblast or Extraembryonic Endoderm cells. In Human ESCs
BMP4 (Bone Morphogenic Protein-4) induces differentiation into Mesoderm and Ectoderm, whereas
BMP2 (Bone Morphogenic Protein-2) promotes Extraembryonic Endoderm differentiation. Repression of BMP signaling in Human ESCs by
FGF supports long term Self-renewal.
FGF2 (Fibroblast Growth Factor-2) is known as the best known factor promoting Self-renewal in Human ESCs. Exogenous
FGF2 is capable of maintaining Human ESCs in the absence of serum and feeder cells. Binding of
FGF to its receptor and Heparin leads to receptor autophosphorylation and activation of intracellular signaling cascades, including the Ras/ERK (Extracellular Signal-Regulated Kinase) pathway, the PLC-Gamma (Phospholipase-C-Gamma)/Ca2+ (Calcium) pathway, and the PI3K (Phosphoinositide 3-Kinase) pathway.
FGF2 promote self-renewal of Human ESCs by activating the PI3K pathway.
PI3K catalyzes the conversion of PIP2 (Phosphatidylinositol (3,4)-bisphosphate) to PIP3 (Phosphatidylinositol (3,4,5)-trisphosphate). The binding of PIP3 to the PH domain anchors
Akt to the Plasma membrane and allows its phosphorylation and activation by
PDK-1 (Phosphoinositide-Dependent Kinase-1). Activated
Akt promotes cell proliferation, survival, growth, and motility and is implicated in tumorogenicity. Survival of cell leads to Pluripotency.
Akt activation is required for the efficient expression of
ECM (Extracellular Matrix) proteins. Role of
PI3K in maintaining Human ESC self-renewal requires further investigation. A role for
PI3K has been proposed in Human ESC survival, where it mediates the survival activity of
Neurotrophins, signaling through
TRK Receptors (Tyrosine Kinase Receptors) (Ref.3, 6, 7 and 8).
WNTs (Wingless-Type MMTV Integration Site Family Members) proteins are also believed to play an important role in controlling ESC maintenance. Canonical WNT signaling involves the binding of
WNT ligands to the
Frizzled receptors. This, in turn, activates
Dsh (Dishevelled), which displaces
GSK-3Beta (Glycogen Synthase Kinase-3Beta) from the
APC (Adenomatous Polyposis Coli) /
AXIN (Axis Inhibitor) complex, preventing Ubiquitin-mediated degradation of
Ctnn-Beta (Catenin-Beta). Subsequently
Ctnn-Beta accumulates and translocates into the nucleus where it associates with
TCF (T Cell Factor)/
LEF (Lymphoid Enhancer Factor) proteins to activate transcription of
WNT target genes. WNT signaling is known to be involved in regulating the proliferation of Stem cells, including Intestinal and Hematopoietic cells,and the Self-renewal of Hematopoietic stem cells. Components of the WNT signaling pathway are present in Human ESCs, although levels of different receptors varied between undifferentiated and differentiated populations. WNT is believed to stimulate Human ESC proliferation as well as differentiation (Ref.3, 9 and 10).
S1P (Sphingosine-1-Phosphate), a bioactive Lysophospholipid, also supports Human ESC self-renewal.
S1P signals both extracellularly, through
EDG (Endothelial Differentiation Gene) receptors coupled to
G-Proteins, and intracellularly by undefined mechanisms.
S1P has been implicated in a diverse range of biological processes, including cell growth, differentiation, migration and apoptosis in many different cell types. Because the prevention of apoptosis is a common self-renewal mechanism, S1P potentially aids the self-renewal process in Human ESCs cells.
PDGF (Platelet Derived-Growth Factor) has also been implicated in the prevention of apoptosis.
PDGF promotes intracellular S1P signaling by activating
SphK (Sphingosine Kinase), which in turn converts Sphingosine to
S1P. The combination of Extracellular
S1P supports Human ESC self-renewal (Ref.11 and 12).
The undifferentiated state of Human ESCs is also maintained by the action of transcription factors some of which are ESC-specific and common between Human and Mouse ESCs. Major transcription factors regulating Pluripotency include
Oct4SOX2Nanog. The best-characterized gene of these is
Oct4, which functions to maintain Pluripotency both in vivo and in vitro.
Oct4 is a POU domain transcription factor that is specifically expressed in all Pluripotent cells. There is scarce knowledge concerning the upstream factors that regulate the expression of the key transcription factors. The expression of
Oct3/4 is regulated by a proximal enhancer and promoter in the Epiblast and by a distal enhancer and promoter at all other stages in the Pluripotent cell lineage. Limited downstream target genes of
Oct3/4 have been identified, out of which
FGF4 is the most common target.
SOX2, a Sry-related transcription factor, activates the transcription of target genes such as
Oct4, in cooperation with
SOX2 expression is controlled by
SOX2, suggesting a positive feedback mechanism that is related to the maintenance of ESC self-renewal. Two regulatory elements, SRR1 and SRR2, have been found in the area of the
SOX2 gene in undifferentiated ES cells. In the presence of these two regulatory elements, a high level of gene expression is achieved through synergistic activity. The partnership between
SOX2 is involved in the expression of the
UTF1 (Undifferentiated embryonic cell Transcription Factor-1) gene found to encode an ESC-specific co-activator. The binding sites for both factors are located in the second intron of the
UTF1 gene with no intervening spaces. Thus,
Oct3/4SOX2 partnership is indispensable in the maintenance of Pluripotency.Stellar, a gene with similar expression to
Oct3/4 has recently been identified. Its expression is mainly restricted to Pluripotent Human ESCs, Premeiotic Germ cell tumors, and Testicular Germ cell tumors (Ref.13, 14 and 15).
Nanog is another member of the group of transcription factors whose functions are deemed essential for the process of Self-renewal in Human ESCs.
Nanog is a NK2-family homeobox transcription factor and it acts by transcriptional activation, achieved by binding to homeobox domains in downstream target genes. Analogous to
Nanog expression is high in Human ESCs and is down regulated as cells differentiate. Transcription of
Nanog is regulated by the binding of
SOX2 to the
Nanog co-occupy the promoters of a large range of genes, many of which encode developmentally important transcription factors.
Nanog together occupy a minimum of 353 of Human ESC genes to maintain Pluripotency. Thus, Human ESCs exhibit a number of signaling pathways involved in Self-renewal and Pluripotency that are interdependent and display a range of cross-talk mechanisms. Deciphering these pathways holds promise for generating improved methodologies for maintenance and proliferation of Pluripotent Human ESCs in vitro. Together, the understanding of the exogenous and endogenous factors determining cell fate will facilitate the use of these cells in cell-based therapies and will allow understanding of early developmental processes (Ref.2, 16, 17 and 18).