Dynamic Roles of Signaling Pathways in Maintaining Pluripotency of Mouse and Human Embryonic Stem Cells

Abstract

Culturing of mouse and human embryonic stem cells (ESCs) in vitro was a major breakthrough in the field of stem cell biology. These models gained popularity very soon mainly due to their pluripotency. Evidently, the ESCs of mouse and human origin share typical phenotypic responses due to their pluripotent nature, such as self-renewal capacity and potency. The conserved network of core transcription factors regulates these responses. However, significantly different signaling pathways and upstream transcriptional networks regulate expression and activity of these core pluripotency factors in ESCs of both the species. In fact, ample evidence shows that a pathway, which maintains pluripotency in mouse ESCs, promotes differentiation in human ESCs. In this review, we discuss the role of canonical signaling pathways implicated in regulation of pluripotency and differentiation particularly in mouse and human ESCs. We believe that understanding these distinct and at times—opposite mechanisms—is critical for the progress in the field of stem cell biology and regenerative medicine.

Introduction

Pluripotency refers to the ability of cells to differentiate into all lineages of an organism. Pluripotent cells can be directed to form any of the three germ layers—ectoderm, endoderm, and mesoderm by changing culture conditions and directing signaling pathways (Smith, 2009). The signaling pathways that maintain state of pluripotency may differ among species such as mice and humans. Interestingly, the core pluripotency factors remain to be the same, that is, Oct4, Sox2, and Nanog in both of these species (Schnerch et al., 2010). Other transcription factors such as Klf-4, c-Myc, and Tbx3 work along with these core factors to maintain pluripotent state (Storm et al., 2014).

Embryonic stem cells (ESCs) are pluripotent stem cells that are derived from the inner cell mass (ICM) of the developing blastocyst (Thomson et al., 1998; Zakrzewski et al., 2019). This is the stage after which lineage specification occurs in mammalian embryo development. There are two states of pluripotency—naive and primed. Naive state is similar to preimplantation conditions, whereas primed resembles postimplantation embryonic configuration (Weinberger et al., 2016). Mouse ESCs (mESCs) are “naïve” pluripotent cells, isolated ICM from an earlier preimplantation stage embryo. Epiblast stem cells (EpiSCs) are isolated from mouse postimplantation epiblasts that represent the primed state. Human ESCs (hESCs) have always been isolated from preimplantation embryos for ethical reasons. Yet, they resemble a primed state. Naive and primed states show certain dissimilarities in growth, gene expression, and differentiation potential (Kumari, 2016). It has been observed that the G1 phase is considerably shorter in ESCs—both human and mouse. However, the length of this phase increases as lineage commitment occurs (Pauklin and Vallier, 2013; Waisman et al., 2019).

Pluripotency mechanisms in mESCs and hESCs: same yet different?

Pluripotency of naive mESCs is maintained by adding leukemia inhibitory factor (LIF) and bone morphogenetic protein (BMP) to culture media. For culture of hESCs, basic fibroblast growth factor (bFGF or FGF2) and Activin/TGF-β are often included so as to promote pluripotency (Ma et al., 2016). Thus, the culture conditions for hESCs resemble mouse EpiSCs and not mESCs. The differences between extrinsic pluripotent signals of mESCs and hESCs may, in part, arise since these cells represent different states of pluripotency, that is, naive and primed (Kumari, 2016). Feeder cells, such as embryonic fibroblasts, which are often used in both mESC and hESC culture maintain pluripotency of the ESCs in a paracrine manner by secreting respective morphogens (Lee et al., 2004; Varzideh et al., 2023).

Remarkably, hESCs but not mESCs retain their ability to differentiate into trophoblasts (Cinkornpumin et al., 2020; Ginis et al., 2004). It has also been demonstrated that differentiation potential of mouse blastomeres can be expanded by manipulation of specific signaling pathways, namely, MAPK, Src, and Wnt. Such blastomeres exhibit expanded potential and are able to differentiate into ICM, as well as trophectoderm lineage (Yang et al., 2017). This highlights critical role of signaling pathways in regulating transition between the abovementioned stem cell states. It is fascinating to note that the activation of one pathway, that is, Wnt helps maintain pluripotency in mESCs, while the same pathway promotes differentiation in hESCs.

Regardless of the pathways regulating pluripotency, the same core pluripotency factors, namely, Oct4, Sox2, and Nanog work together to maintain the state of pluripotency by induction of several genes and also by activation of each another in mESCs and hESCs (Chen et al., 2008; Chew et al., 2005). In this review, we discuss the role of key signaling pathways implicated in the maintenance of pluripotency of mESCs and hESCs.

LIF Signaling Pathway

Evans and Kaufman (1981) were the pioneers as they established the in vitro conditions required for culturing of mESCs by coculturing them with feeder cells, that is, fibroblasts. Later, it was discovered that a cytokine named LIF, released by mouse embryonic fibroblasts, was responsible for maintenance of pluripotency in vitro. LIF initiates the downstream signaling cascade by heterodimerization of LIF receptor (LIFR) with the coreceptor, glycoprotein 130 (gp130). This activates JAK kinases which phosphorylate STAT3; phospho-STAT3 undergoes dimerization through their SH2 domains. The activated, dimerized STAT3 complex translocates to the nucleus to activate gene transcription (Huang et al., 2015). STAT3 regulates the expression of core pluripotency genes, namely, Oct4 and Nanog and along with them regulates expression of several other genes (Table 1).

Table 1.

Key Transcription Factors Involved in Regulation of Genes Associated with Pluripotency

Transcription factor/cofactor	Cell type	Experimental method	Number of binding sites	Key features	Reference
STAT3	mESCs	ChIP sequencing	2546	Co-occupied sites with Oct4, Sox2, and Nanog	Chen et al. (2008)
STAT3	mESCs	ChIP sequencing	948	Co-occupied sites with Oct4 and Nanog—22	Kidder et al. (2008)
cMyc (Myc)	mESCs	—	1459	Co-occupied sites with Oct4 and Nanog—32	Kidder et al. (2008)
SMAD1/5	mESCs	ChIP sequencing	562	Co-occupied sites with each other—127	Kidder et al. (2008)
SMAD4	mESCs	ChIP sequencing	2518	Co-occupied sites with each other—127	Kidder et al. (2008)
SMAD1/5	mESCs	ChIP sequencing	9327	Co-occupied sites with KLF4/5—3254	Morikawa et al. (2016)
NANOG, TCF3, SOX2, OCT4	hESCs	ChIP-STARR-seq	7948	Identified 7948 enhancers in close proximity to ESC TFs (NANOG, OCT4) and signaling pathway genes (TGF-β, FGF, WNT signaling)	Barakat et al. (2018)
P300/CBP	mESCs	ChIP and microarray	214	Direct targets of P300—Tbx3, Klf4, and Dax-1	Chitilian et al. (2014)
P53	hESCs	ChIP	3324	Untreated—3324, Differentiation—5550, Damage—2653	Akdemir et al. (2014)
Myc	hESCs	ChIP sequencing	—	Metabolic switch genes—14	Cliff et al. (2017)

ChIP, chromatin immunoprecipitation; FGF, fibroblast growth factor; hESCs, human embryonic stem cells; mESCs, mouse embryonic stem cells.

Role of LIF pathway in mESC regulation

STAT3 levels are crucial in the maintenance of pluripotency, with elevated STAT3 levels causing differentiation of the mESCs into the trophectoderm lineage. Activation of STAT3 through LIF leads to low Tfap2c expression (an inducer of trophectoderm differentiation). Hyperactivation of STAT3 may cause increased expression of Tfap2c which could override the self-renewing effect otherwise seen in mESCs (Tai et al., 2014). Hence, STAT3 activation is a key event determining the pluripotent state of mESCs.

Role in LIF pathway in hESC regulation

While LIF/JAK-STAT3 pathway is crucial for maintaining pluripotency of mESCs, it is dispensable for hESC maintenance although LIFR and the downstream signaling components are expressed in hESCs. It was seen that differentiation of hESCs will occur despite STAT3 activation. The ability of LIF to maintain mESCs in a state of pluripotency can be perhaps explained due to a specific role of this signaling pathway in mouse embryo development. Mouse embryos undergo LIF-mediated diapause which is the temporary arrest of mouse blastocyst development. Diapause does not occur in human blastocysts, thereby negating the need for LIF-mediated maintenance of pluripotency in case of hESCs (Dahéron et al., 2004).

Crosstalk of LIF with other pathways

In mESCs, along with STAT3, LIF also activates PI3K/Akt and ERK1/2 (ERK) pathways. PI3K/Akt pathway is involved in maintaining pluripotency. ERK pathway along with FGF pathway induces differentiation of mESCs while inhibiting their proliferation (Graf et al., 2011; Park et al., 2021). BMP enhances self-renewal and pluripotency of mESCs in the presence of LIF (Graf et al., 2011). Perhaps, this is mediated by BMP-induced inhibition of ERK pathway so as to prevent differentiation.

Most often, role of a signaling pathway in ESCs is deciphered using pharmacological inhibition of a specific pathway component (Table 2) (Lee et al., 2019). In fact, some of the previous studies have systematically screened and identified small molecule inhibitors of pathways that help to maintain pluripotency of mESCs or hESCs (Theunissen et al., 2014; Williams et al., 2017). Newly defined culture conditions free from feeders, serum, and cytokines have been established using a combination of small-chemical molecules, which inhibit ERK1/2 and glycogen synthase kinase 3 (GSK3). Under these conditions (2i), it is possible to maintain self-renewal of mESCs in the absence of LIF/STAT3 and BMP (Ohtsuka et al., 2015). Major signaling pathways which regulate pluripotency network in mESCs are depicted in Figure 1.

FIG. 1.

Main signaling pathways which regulate pluripotency network in mouse ESCs. LIF/JAK/STAT3 is critical as it maintains pluripotency in mouse ESCs by activating expression of core pluripotency genes, namely, Oct4, Sox2, and Nanog through STAT3-induced transcription. ESC, embryonic stem cell; LIF, leukemia inhibitory factor.

Table 2.

Small Molecule Inhibitors of Signaling Pathways Used for Regulation of Pluripotency

Name of the inhibitor	Concentration, μM	Specificity for signaling pathway component	Reference
LY294002	10	PI3K	Armstrong et al. (2006)
U0126	10 and 30	MEK1/2	Armstrong et al. (2006)
PD035901	1	MEK	Lin et al. (2009)
Sodium pyrrolidinethiocarbamate (PTDC)	50	NF-κB	Armstrong et al. (2006)
SB203580	—	P38	Chen et al. (2015)
SB431542	5	pSMAD2	Galvin et al. (2010)
A-83-01	0.5	Activin	Galvin et al. (2010)
XAV939	—	Wnt	Gattiglio et al. (2023)
CHIR99021	1 and 3	GSK3	Yang et al. (2017)
PD173074	0.1	FGF2	Yang et al. (2015)
Gö6983	—	Protein kinase C	Ware (2017)
Y-27632		ROCK	Emre et al. (2010)
WH-4-023	1	Src	Di Stefano et al. (2018)
WZ-4-145	1	Pan RTK	Theunissen et al. (2014)

GSK3, glycogen synthase kinase 3.

BMP and TGF-β/Activin/Nodal Signaling Pathways

Transforming growth factor-β (TGF-β) superfamily members, namely, TGF-β/Activin/Nodal and BMP play a critical role in development and are involved in pluripotency maintenance. TGF-β family ligands bind to specific type I or type II transmembrane receptors. This induces assembly of oligomeric complex composed of two type I and two type II receptors which further leads to phosphorylation of type 1 receptor (also known as Activin like kinase [ALK]) that acts as a ser/thr kinase to phosphorylate receptor SMADs. The phosphorylated SMAD proteins undergo homotrimerization. The trimeric complexes bind to the coregulatory SMAD—SMAD4. The activated SMAD complexes translocate to the nucleus and activate expression of specific genes.

BMP signaling pathway

BMP binding to BMP receptor leads to phosphorylation of SMAD1, SMAD5, and SMAD8. Activated SMAD complexes bind to co-SMAD—SMAD4, translocate to the nucleus to transcribe specific genes.

Role of BMP pathway in mESC regulation

BMP has been shown to regulate the expression of protein components of polycomb repressor complex to inhibit neural induction (Ong et al., 2023). BMP signaling results in SMAD complex-mediated activation of Inhibitor of Differentiation (Id) gene, preventing the differentiation of mESCs into the neuroectoderm (Ying et al., 2003). As mentioned above, BMP signaling and LIF/JAK-STAT signaling work together to maintain mESC pluripotency (Onishi et al., 2014; Varzideh et al., 2023). BMP4 activates the SMAD, PI3K/Akt, and Wnt/β-catenin pathways to maintain pluripotency (Lee et al., 2009). BMP4 also contributes to maintenance of mESC pluripotency by inhibition of the MAPK pathways induced by p38 and ERK. SB203580, a p38/MAPK inhibitor, could maintain pluripotency in mESCs lacking the BMP receptor, ALK3. This indicates that inhibition of a downstream signaling molecule can help achieve pluripotency, even if the cell is lacking functional receptors (Qi et al., 2004). Another study showed that BMP4 is essential for maintenance of genomic stability in mESCs (Wang et al., 2022).

Role of BMP pathway in hESC regulation

In hESCs, short-term BMP4 exposure can lead to induction of mesoderm, and long-term exposure aids in differentiation to trophoblast and extraembryonic endoderm (Zhang et al., 2008). BMP4 may act in a positive-feedback loop to promote the formation of extraembryonic endoderm tissue which in turn produces factors that can drive stem cell differentiation and proliferation. Hence, in hESC culture, inclusion of Noggin, a BMP signaling inhibitor, is commonly used, to prevent extraembryonic endoderm differentiation. However, treatment with Noggin can fail to maintain self-renewing capacity of hESCs and may give rise to cells that resemble neural precursor cells via BMP2 inhibition (Pera et al., 2004; Xu et al., 2002). Thus, BMP signaling is often blocked in hESC culture and promoted in mESC culture to achieve the same outcome—maintenance of pluripotency.

TGF-β/Activin/Nodal signaling pathway

TGF-β and Activin/Nodal pathways have common downstream effectors, that is, SMAD2 and SMAD3. Hence, these pathways are often considered to have similar functions in spite of their variable tissue-specific expression. SMAD2 and SMAD3 form a complex with SMAD4 to induce activation of specific genes upon translocation into the nucleus (Huang et al., 2015).

Role of TGF-β/Activin/Nodal pathway in mESC regulation

In mESCs, Activin/Nodal signaling is not essential for maintenance of pluripotency but is required to drive differentiation of mESCs into cells of the mesoderm lineage (Fei et al., 2010). Tapbp is a downstream target of Activin/Nodal signaling to allow mesoendoderm differentiation. Blocking of Activin/Nodal signaling by introducing SB431542, an inhibitor of SMAD2 signaling, resulted in activation of Id genes. As discussed above, Id genes are also the downstream targets of BMP signaling in mESCs. Thus, Nodal signaling may indirectly regulate BMP signaling activity in mESCs, possibly via SMAD7. Hence, in mESC culture, inclusion of Nodal inhibitors along with BMP, help in maintenance of their self-renewing capacity (Galvin et al., 2010). Interestingly, including Activin A along with an inhibitor of the ERK1/2 pathway maintained pluripotency of mESCs (Ashida et al., 2017).

Role of TGF-β/Activin/Nodal pathway in hESC regulation

In hESCs, activation SMAD2/3 via TGF-β/Activin/Nodal pathway is required for maintaining stemness and pluripotency (James et al., 2005) SMAD2/3 activates Nanog expression by binding the promoter of Nanog and inhibiting autocrine BMP signaling (Varzideh et al., 2023). Inhibition of TGF-β/Activin/Nodal signaling may cause differentiation into mesenchymal progenitor cells. Inhibition of the TGF-β/Activin/Nodal pathway by SB431542 during embryoid body formation resulted in expression of mesodermal markers like TBX5/6 (Mahmood et al., 2010). Activin also maintains hESCs in pluripotent state without feeder layer (Beattie et al., 2005).

Notably, low concentrations of Activin A (5 ng/mL) can sustain pluripotency and self-renewal of hESCs by inducing Nanog expression which blocks differentiation toward to neuroectoderm lineage. In fact, Activin A has been shown to be critical regulator of pluripotency of hESCs as it also induces expression of ligands such as FGF, Nodal while it inhibits BMP (Xiao et al., 2006). Activin-mediated activation of SMAD2 is essential in maintenance of pluripotency by targeting and activating Nanog expression. Nanog helps regulate expression of Oct4 (Sakaki-Yumoto et al., 2013). Nanog also lowers the transcriptional activity of SMAD2/3 by direct interaction so as to limit propensity of Activin/Nodal to induce mesoendoderm toward endodermal fate (Vallier et al., 2009; Xu et al., 2008). Yet, high concentrations of Activin A (50–100 ng/mL) decrease pluripotency and induce differentiation into endoderm (Varzideh et al., 2023). In fact, Activin A and BMP4 act in synergism to differentiate hESCs into definitive endodermal fate (Teo et al., 2012).

Hippo-YAP signaling pathway functions in early lineage differentiation of pluripotent stem cells, as a transcription co-factor, YAP binds to transcription factors such as TEA domain-containing (TEAD) proteins to regulate the expression of target genes. A regulatory complex composed of TAZ/YAP and TEADs, SMAD2/3 and Oct4 (called the TSO complex), helps maintain pluripotency in hESCs and represses mesoendoderm differentiation. Loss of this complex helps drive differentiation into the mesendoderm (Beyer et al., 2013). Thus, balance between TGF-β/Activin/Nodal pathway and others may determine the cell fate in hESCs (Ashida et al., 2017).

Key signaling pathways regulating pluripotency in hESCs are shown in Figure 2.

FIG. 2.

Key signaling pathways regulating pluripotency in hESCs. FGF binds to its receptor (FGFR) to activate several downstream pathways such as Ras/Raf/MEK/ERK and PI3K/Akt and thereby maintains pluripotency of hESCs. FGF, fibroblast growth factor; hESCs, human embryonic stem cells.

Crosstalk between TGF-β/Activin/Nodal and BMP signaling: role of SMAD4 and SMAD7

BMP signaling is indispensable for maintaining pluripotency of mESCs and dispensable for hESCs. Activin/Nodal signaling is necessary for pluripotency maintenance in hESCs, but dispensable for mESCs. While this generalization holds true, a study attempted to understand the role of SMAD4, the common downstream molecule in the TGF-β/Activin/Nodal and BMP signaling pathway, in hESCs. It was discovered that SMAD4 knockdown led to instability in hESCs and eventually, differentiation. Rapid differentiation seen in hESCs when treated with an inhibitor of TGF-β/Activin/Nodal signaling is dependent upon the presence of SMAD4. It was also observed that inhibition of TGF-β/Activin/Nodal receptor led to accumulation of SMAD1/5/8, the downstream molecules of BMP signaling. This implies that SMAD4 could be involved in negative regulation of BMP signaling pathway so as to maintain pluripotency (Avery et al., 2010). It has also been shown that in pluripotent mESCs, autocrine Nodal signaling negatively regulates BMP signaling through SMAD7 which causes downregulation of Id gene expression (Galvin et al., 2010).

FGF Signaling Pathway

FGF is released by fibroblasts used as feeder cells in culture of hESCs. FGF can bind to four types of receptors—FGF1, FGF2, FGF3, FGF4 to initiate the signaling cascades (Han et al., 2013). Grb2 and FGF receptor substrate 2 activate downstream signaling pathways viz. PI3K/Akt and RAS/Raf/MEK/ERK.

Role of FGF pathway in mESCs

FGF/ERK signaling cascade plays a role in mESC differentiation, whereas it is important for promoting the self-renewing capacity of hESCs (Greber et al., 2010). FGF signaling contributes to transition of naïve mESC into a primed state (Raina et al., 2022). FGF also aids in the differentiation of naïve mESCs into epiblast and then into primitive endoderm cells (Kang et al., 2013). In this case, the transition of pluripotent cells to primitive endoderm depends upon the intensity of FGF pathway activation (Meharwade et al., 2023). It was shown that differentiation of blastocyst into primitive endoderm and epiblast does depend upon regulation of FGF/MAPK pathway, with excess FGF leading to a primitive endoderm fate and blockage of the pathway leading to formation of the epiblast (Yamanaka et al., 2010).

The discovery of Spautin-1 (Sp-1), a small molecule USP13 inhibitor, also indicated the contradictory role of FGF signaling pathway in maintaining the stemness of hESC versus mESC. Sp-1 inhibits the activity of USP-13 which is a Raf-1 deubiquitinating protein. Raf-1 is the mediator between external FGF stimulus and intracellular MEK/ERK signaling pathway. Inhibition of USP-13 by Sp-1 promoted stemness in naïve mESCs by inhibiting the FGF/MEK/ERK signaling pathway (Wang et al., 2021).

Role of ERK in mESCs

ERK1/2 (referred to as ERK hereafter) is critical for maintaining genomic stability in mESCs (Chen et al., 2015). However, it has also been reported that inhibiting ERK signaling via MEK inhibition enhances self-renewal and pluripotency of naïve mESCs (Ma et al., 2016). Subcellular localization of ERK is thought to be important in maintenance of pluripotency too, in which the nuclear translocation is prevented to maintain the mESCs in a pluripotent state (Hacohen Lev-Ran and Seger, 2022). Another study reported that ERK inhibits self-renewal of mESCs and induced endoderm differentiation (Hamilton and Brickman, 2014). A study revealed that heterodimers of Myc/MAX transcription factors inhibit ERK by regulation of DUSPs which are negative regulators of ERK activity (Chappell et al., 2013).

Role of FGF pathway in hESCs

In hESCs, bFGF (also known as FGF2) is routinely included in cultures to promote maintenance of pluripotency. bFGF signaling supports pluripotency, stemness, and hESCs self-renewal by activating the PI3K/Akt and ERK pathways (Varzideh et al., 2023). It has been proposed that bFGF maintains hESC pluripotency by activating ERK and NF-κB pathways which are activated by PI3K/Akt pathway (Armstrong et al., 2006). In hESCs, FGF induces neuroectoderm specification via ERK/PARP1/PAX6 axis (Yoo et al., 2011). Treatment of hESCs with FGF2 inhibitor leads to downregulation of Oct4 activity indicating that FGF2-mediated signaling regulates expression of Oct4 (Ding et al., 2010). The practice of including exogenous bFGF in hESC culture can be explained due to bFGFs ability to promote cell adhesion and form colonies that show reduced peripheral differentiation. bFGF may promote pluripotency by inhibiting expression of cell death genes (Eiselleova et al., 2009).

Crosstalk between BMP and ERK pathway

It has been shown that BMP signaling in mESCs leads to cross-activation of FGF pathway, inducing differentiation. BMP and FGF exerted opposite effects in the distribution of mesodermal cell fates in mouse epiblast cell line (Gattiglio et al., 2023).

Inhibition of MEK/ERK by small molecules has been shown to result in loss of pluripotency in hESCs (Li et al., 2007). Another study showed that inhibition of ERK activity prevents differentiation of hESCs into neuroectoderm and mesoderm. However, ERK inhibition is also necessary to block BMP4-induced differentiation into extraembryonic lineage. This could be due to the fact that ERK inhibits BMP signaling (Chen et al., 2017). Hence, inhibition of ERK activates BMP pathway; thus, cells differentiate toward extraembryonic endoderm (Na et al., 2010).

Wnt/β-Catenin Signaling Pathway

Wnt is a ligand which is involved in many cell proliferation and developmental processes. It binds to receptors, namely, Frizzled and LRP5/6, located on the cell surface. β-Catenin acts as the main effector protein of this pathway. Its stability is determined by the intracellular destruction complex composed of Axin, APC, CK1, and GSK3β. In the absence of Wnt ligand, β-catenin is phosphorylated by components of the destruction complex. Phospho-β-catenin is targeted for proteasomal degradation, causing its levels to drop. In the presence of Wnt ligand, destruction complex is relocalized to the cell membrane. This prevents phosphorylation and subsequent degradation of β-catenin. Hence, β-catenin levels increase in the presence of Wnt ligand. As a result, β-catenin is free to translocate to the nucleus and interact with TCF transcription factors to regulate the activity of various genes, including genes of the pluripotency network (Nusse and Clevers, 2017).

Role of Wnt pathway in mESCs

In case of mESCs, pluripotency depends upon retaining transcriptional activity of β-catenin (Miyabayashi et al., 2007). Hence, while culturing mESCs, GSK3β inhibitors are often included in the medium to promote self-renewing ability (Huang et al., 2015). GSK3β inhibition may promote pluripotency by maintaining expression of Oct4 (Li et al., 2012). The use of SB-216763, a GSK3β inhibitor, in mESC culture allowed for the maintenance of mESCs in their pluripotent state in the absence of LIF, when cultured with feeder layer of mouse embryonic fibroblasts (Kirby et al., 2012). In mESCs, it was seen that β-catenin buffers the repressive effect of TCF-3 to maintain pluripotency (Chen et al., 2017). Furthermore, blocking the interaction of β-catenin with TCF-1 may also help in maintaining the self-renewing capacity of mESCs as this interaction leads to differentiation of the cells (Chatterjee et al., 2015).

A genome-wide siRNA screen in mESCs demonstrated that over 400 genes are involved in loss of pluripotency and beginning of differentiation. Most of these genes were shown to be involved in ERK and GSK3β pathways (Yang et al., 2012).

Role of Wnt pathway in hESCs

Role of Wnt signaling in pluripotency of hESCs is not yet clear. It has been reported that Oct4 represses endogenous Wnt/β-catenin signaling in hESCs. Activation of Wnt/β-catenin pathway leads to loss of pluripotent state and mesodermal differentiation (Davidson et al., 2012). Thus, it is speculated that hESCs' pluripotency maintenance depends upon blocking the transcriptional activity of β-catenin.

It has been shown that varying levels of endogenous Wnt signaling may lead to differential lineage specification of hESCs. Wnt^high cells are more likely to differentiate to form endodermal and cardiac cells, and Wnt^low cells differentiate to form neuroectodermal cells (Blauwkamp et al., 2012). It was observed that endogenous Wnt signals are responsible for the differentiation of hESCs in vitro. Endogenous Wnt signaling activates Fgf8, Wnt3, and Nodal, leading to induction of mesoderm. A study showed that Wnt inhibition is successful in preventing differentiation of hESCs. This may indicate that blocking of endogenous Wnt signaling is also critical in maintenance of hESC stemness (Kurek et al., 2014).

The role of the abovementioned core signaling pathways in regulation of pluripotency/lineage commitment of mESC and hESC is summarized in Figure 3.

FIG. 3.

Canonical signaling network that regulates pluripotency/lineage commitment in hESC and mouse ESC. Indirect regulation shown by dashed arrows, inhibition shown by blunt arrowheads.

Conclusion and Future Perspectives

It is interesting that two mammalian models of ESCs—mouse and human, have different, and sometimes, opposing mechanisms of activating the common transcription factors, which are conserved among both the species as core regulators of pluripotency network. Furthermore, signal intensity and/or duration determine distinct phenotypic differentiation outcomes in mESCs and hESCs. Why and how does activation of the same pathway maintain pluripotency in hESCs and promote differentiation in mESCs is a conundrum. Understanding this may provide greater insights into the early embryo events that occur during development and how they differ across mammalian species. Mouse EpiSCs, also known as primed state cells, respond to molecular signals much like hESCs. Hence, understanding the molecular “switch” that occurs in naive versus primed mESCs may help understand how and why hESCs and mESCs show a differential response to the same stimulus. Nevertheless, one cannot deny the possibility that dynamic role of signaling pathways in pluripotency is not only due to differences in stem cell states but also could be due to other considerations which remain to be discovered.

Footnotes

Acknowledgments

The authors thank the authorities at SVKM's NMIMS (Deemed-to-Be) University for their support.

Data Availability Statement

The datasets generated and analyzed during the current review are available from the corresponding author on reasonable request.

Authors' Contributions

A.O.: Methodology, data curation, and original draft preparation, S.M.M.: Conceptualization, original draft preparation, review and editing, final approval, and supervision.

Author Disclosure Statement

The authors declare they have no conflicting financial interests.

Funding Information

No funding was received for this article.

References

Akdemir

, Jain

, Allton

, et al. Genome-wide profiling reveals stimulus-specific functions of p53 during differentiation and DNA damage of human embryonic stem cells. Nucleic Acids Res, 2014; 42(1):205–223; doi: 10.1093/nar/gkt866

Armstrong

, Hughes

, Yung

, et al. The role of PI3K/AKT, MAPK/ERK and NFkappabeta signalling in the maintenance of human embryonic stem cell pluripotency and viability highlighted by transcriptional profiling and functional analysis. Hum Mol Genet, 2006; 15(11):1894–1913; doi: 10.1093/hmg/ddl112

Ashida

, Nakajima-Koyama

, Hirota

, et al. Activin A in combination with ERK1/2 MAPK pathway inhibition sustains propagation of mouse embryonic stem cells. Genes Cells, 2017; 22(2):189–202; doi: 10.1111/gtc.12467

Avery

, Zafarana

, Gokhale

, et al. The role of SMAD4 in human embryonic stem cell self-renewal and stem cell fate. Stem Cells, 2010; 28(5):863–873; doi: 10.1002/stem.409

Barakat

, Halbritter

, Zhang

, et al. Functional dissection of the enhancer repertoire in human embryonic stem cells. Cell Stem Cell, 2018; 23(2):276–288.e8; doi: 10.1016/j.stem.2018.06.014

Beattie

, Lopez

, Bucay

, et al. Activin A maintains pluripotency of human embryonic stem cells in the absence of feeder layers. Stem Cells, 2005; 23(4):489–495; doi: 10.1634/stemcells.2004-0279

Beyer

, Weiss

, Khomchuk

, et al. Switch enhancers interpret TGF-β and hippo signaling to control cell fate in human embryonic stem cells. Cell Rep, 2013; 5(6):1611–1624; doi: 10.1016/j.celrep.2013.11.021

Blauwkamp

, Nigam

, Ardehali

, et al. Endogenous Wnt signalling in human embryonic stem cells generates an equilibrium of distinct lineage-specified progenitors. Nat Commun, 2012; 3:1070; doi: 10.1038/ncomms2064

Bogliotti

, Wu

, Vilarino

, et al. Efficient derivation of stable primed pluripotent embryonic stem cells from bovine blastocysts. Proc Natl Acad Sci U S A, 2018; 115:2090–2095; doi: 10.1073/pnas.1716161115

10.

Chappell

, Sun

, Singh

, et al. MYC/MAX control ERK signaling and pluripotency by regulation of dual-specificity phosphatases 2 and 7. Genes Dev, 2013; 27(7):725–733; doi: 10.1101/gad.211300.112

11.

Chatterjee

, Saj

, Gocha

, et al. Inhibition of β-catenin–TCF1 interaction delays differentiation of mouse embryonic stem cells. J Cell Biol, 2015; 211(1):39–51; doi: 10.1083/jcb.201503017

12.

Chen

, Cheng

, Yen

, et al. Mechanisms of pluripotency maintenance in mouse embryonic stem cells. Cell Mol Life Sci, 2017; 74(10):1805–1817; doi: 10.1007/s00018-016-2438-0

13.

Chen

, Guo

, Zhang

, et al. Erk signaling is indispensable for genomic stability and self-renewal of mouse embryonic stem cells. Proc Natl Acad Sci U S A, 2015; 112(44):E5936–E5943; doi: 10.1073/pnas.1516319112

14.

Chen

, Xu

, Yuan

, et al. Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell, 2008; 133(6):1106–1117; doi: 10.1016/j.cell.2008.04.043

15.

Chew

, Loh

, Zhang

, et al. Reciprocal transcriptional regulation of Pou5f1 and Sox2 via the Oct4/Sox2 complex in embryonic stem cells. Mol Cell Biol, 2005; 25(14):6031–6046; doi: 10.1128/MCB.25.14.6031-6046.2005

16.

Chitilian

, Thillainadesan

, Manias

, et al. Critical components of the pluripotency network are targets for the p300/CBP interacting protein (p/CIP) in embryonic stem cells. Stem Cells, 2014; 32(1):204–215; doi: 10.1002/stem.1564

17.

Cinkornpumin

, Kwon

, Guo

, et al. Naive human embryonic stem cells can give rise to cells with a trophoblast-like transcriptome and methylome. Stem Cell Reports, 2020; 15(1):198–213; doi: 10.1016/j.stemcr.2020.06.003

18.

Cliff

, Wu

, Boward

, et al. MYC controls human pluripotent stem cell fate decisions through regulation of metabolic flux. Cell Stem Cell, 2017; 21(4):502–516.e9; doi: 10.1016/j.stem.2017.08.018

19.

Dahéron

, Opitz

, Zaehres

, et al. LIF/STAT3 signaling fails to maintain self-renewal of human embryonic stem cells. Stem Cells, 2004; 22(5):770–778; doi: 10.1634/stemcells.22-5-770

20.

Davidson

, Adams

, Goodson

, et al. Wnt/β-catenin signaling promotes differentiation, not self-renewal, of human embryonic stem cells and is repressed by Oct4. Proc Natl Acad Sci U S A, 2012; 109(12):4485–4490; doi: 10.1073/pnas.1118777109

21.

Dekel

, Morey

, Hanna

, et al. Stabilization of hESCs in two distinct substates along the continuum of pluripotency. iScience, 2022; 25(12):105469; doi: 10.1016/j.isci.2022.105469

22.

Di Stefano

, Ueda

, Sabri

, et al. Reduced MEK inhibition preserves genomic stability in naive human embryonic stem cells. Nat Methods, 2018; 15(9):732–740; doi: 10.1038/s41592-018-0104-1

23.

Ding

VMY

, Ling

, Natarajan

, et al. FGF-2 modulates Wnt signaling in undifferentiated hESC and iPS cells through activated PI3-K/GSK3β signaling. J Cell Physiol, 2010; 225(2):417–428; doi: 10.1002/jcp.22214

24.

Eiselleova

, Matulka

, Kriz

, et al. A complex role for FGF-2 in self-renewal, survival, and adhesion of human embryonic stem cells. Stem Cells, 2009; 27(8):1847–1857; doi: 10.1002/stem.128

25.

Emre

, Vidal

, Elia

, et al. The ROCK inhibitor Y-27632 improves recovery of human embryonic stem cells after fluorescence-activated cell sorting with multiple cell surface markers. PLoS One, 2010; 5(8):e12148; doi: 10.1371/journal.pone.0012148

26.

Evans

, Kaufman

. Establishment in culture of pluripotential cells from mouse embryos. Nature, 1981; 292(5819):154–156; doi: 10.1038/292154a0

27.

Fei

, Zhu

, Xia

, et al. Smad2 mediates Activin/Nodal signaling in mesendoderm differentiation of mouse embryonic stem cells. Cell Res 2010;20(12):Article, 12; doi: 10.1038/cr.2010.158

28.

Galvin

, Travis

, Yee

, et al. Nodal signaling regulates the bone morphogenic protein pluripotency pathway in mouse embryonic stem cells. J Biol Chem, 2010; 285(26):19747–19756; doi: 10.1074/jbc.M109.077347

29.

Gattiglio

, Protzek

, Schröter

. Population-level antagonism between FGF and BMP signaling steers mesoderm differentiation in embryonic stem cells. Biol Open, 2023; 12(8):bio059941; doi: 10.1242/bio.059941

30.

Ginis

, Luo

, Miura

, et al. Differences between human and mouse embryonic stem cells. Dev Biol, 2004; 269(2):360–380; doi: 10.1016/j.ydbio.2003.12.034.

31.

Graf

, Casanova

, Cinelli

. The role of the leukemia inhibitory factor (LIF)—Pathway in derivation and maintenance of murine pluripotent stem cells. Genes (Basel), 2011; 2(1):280–297; doi: 10.3390/genes2010280

32.

Greber

, Wu

, Bernemann

, et al. Conserved and divergent roles of FGF signaling in mouse epiblast stem cells and human embryonic stem cells. Cell Stem Cell, 2010; 6(3):215–226; doi: 10.1016/j.stem.2010.01.003

33.

Hacohen Lev-Ran

, Seger

. Retention of ERK in the cytoplasm mediates the pluripotency of embryonic stem cells. Stem Cell Reports, 2022; 18(1):305–318; doi: 10.1016/j.stemcr.2022.11.017

34.

Hamilton

, Brickman

. Erk signaling suppresses embryonic stem cell self-renewal to specify endoderm. Cell Rep, 2014; 9(6):2056–2070; doi: 10.1016/j.celrep.2014.11.032

35.

Han

, Wang

, Hao

, et al. Molecular Mechanisms of Embryonic Stem Cell Pluripotency. In: Pluripotent Stem Cells. ( Bhartiya

, Lenka

. eds.) IntechOpen: 2013.

36.

Huang

, Ye

, Zhou

, et al. Molecular basis of embryonic stem cell self-renewal: From signaling pathways to pluripotency network. Cell Mol Life Sci, 2015; 72(9):1741–1757; doi: 10.1007/s00018-015-1833-2

37.

James

, Levine

, Besser

, et al. TGFbeta/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells. Development, 2005; 132(6):1273–1282; doi: 10.1242/dev.01706

38.

Kang

, Piliszek

, Artus

, et al. FGF4 is required for lineage restriction and salt-and-pepper distribution of primitive endoderm factors but not their initial expression in the mouse. Development, 2013; 140(2):267–279; doi: 10.1242/dev.084996

39.

Kidder

, Yang

, Palmer

. Stat3 and c-Myc genome-wide promoter occupancy in embryonic stem cells. PLoS One, 2008; 3(12):e3932; doi: 10.1371/journal.pone.0003932

40.

Kirby

, Schott

, Noble

, et al. Glycogen synthase kinase 3 (GSK3) inhibitor, SB-216763, promotes pluripotency in mouse embryonic stem cells. PLoS One, 2012; 7(6):e39329; doi: 10.1371/journal.pone.0039329

41.

Kumari

. States of Pluripotency: Naïve and Primed Pluripotent Stem Cells. In: Pluripotent Stem Cells—From the Bench to the Clinic. ( Tomizawa

. ed.) InTech: 2016.

42.

Kurek

, Neagu

, Tastemel

, et al. Endogenous WNT signals mediate BMP-induced and spontaneous differentiation of epiblast stem cells and human embryonic stem cells. Stem Cell Reports, 2014; 4(1):114–128; doi: 10.1016/j.stemcr.2014.11.007

43.

Lee

, Park

, Jung

. Protein kinases and their inhibitors in pluripotent stem cell fate regulation. Stem Cells Int, 2019; 2019:1569740; doi: 10.1155/2019/1569740

44.

Lee

, Song

, Lee

, et al. Available human feeder cells for the maintenance of human embryonic stem cells. Reproduction, 2004; 128(6):727–735; doi: 10.1530/rep.1.00415

45.

Lee

, Lim

, Lee

, et al. Smad, PI3K/Akt, and Wnt-Dependent signaling pathways are involved in BMP-4-induced ESC self-renewal. Stem Cells, 2009; 27(8):1858–1868; doi: 10.1002/stem.124

46.

, Li

, Chen

. Oct4 was a novel target of Wnt signaling pathway. Mol Cell Biochem, 2012; 362(1):233–240; doi: 10.1007/s11010-011-1148-z

47.

, Wang

, et al. MEK/ERK signaling contributes to the maintenance of human embryonic stem cell self-renewal. Differentiation, 2007; 75(4):299–307; doi: 10.1111/j.1432-0436.2006.00143.x

48.

Lin

, Ambasudhan

, Yuan

, et al. A chemical platform for improved induction of human iPSCs. Nat Methods, 2009; 6:805–808; doi: 10.1038/nmeth.1393

49.

, Chen

. A dual role of Erk signaling in embryonic stem cells. Exp Hematol, 2016; 44(3):151–156; doi: 10.1016/j.exphem.2015.12.008

50.

Mahmood

, Harkness

, Schrøder

, et al. Enhanced differentiation of human embryonic stem cells to mesenchymal progenitors by inhibition of TGF-β/activin/nodal signaling using SB-431542. J Bone Miner Res, 2010; 25(6):1216–1233; doi: 10.1002/jbmr.34

51.

Meharwade

, Joumier

, Parisotto

, et al. Cross-activation of FGF, NODAL, and WNT pathways constrains BMP-signaling-mediated induction of the totipotent state in mouse embryonic stem cells. Cell Rep, 2023; 42(5):112438; doi: 10.1016/j.celrep.2023.112438

52.

Miyabayashi

, Teo

, Yamamoto

, et al. Wnt/beta-catenin/CBP signaling maintains long-term murine embryonic stem cell pluripotency. Proc Natl Acad Sci U S A, 2007; 104(13):5668–5673; doi: 10.1073/pnas.0701331104

53.

Morikawa

, Koinuma

, Mizutani

, et al. BMP sustains embryonic stem cell self-renewal through distinct functions of different Krüppel-like factors. Stem Cell Reports, 2016; 6(1):64–73; doi: 10.1016/j.stemcr.2015.12.004

54.

, Furue

, Andrews

. Inhibition of ERK1/2 prevents neural and mesendodermal differentiation and promotes human embryonic stem cell self-renewal. Stem Cell Res, 2010; 5(2):157–169; doi: 10.1016/j.scr.2010.06.002

55.

Nusse

, Clevers

. Wnt/β-catenin signaling, disease, and emerging therapeutic modalities. Cell, 2017; 169(6):985–999; doi: 10.1016/j.cell.2017.05.016

56.

Ohtsuka

, Nakai-Futatsugi

, Niwa

. LIF signal in mouse embryonic stem cells. JAKSTAT, 2015; 4(2):e1086520; doi: 10.1080/21623996.2015.1086520

57.

Ong

ALC

, Kokaji

, Kishi

, et al. Acquisition of neural fate by combination of BMP blockade and chromatin modification. iScience, 2023; 26(10):107887; doi: 10.1016/j.isci.2023.107887

58.

Onishi

, Tonge

, Nagy

, et al. Local BMP-SMAD1 signaling increases LIF receptor-dependent STAT3 responsiveness and primed-to-naive mouse pluripotent stem cell conversion frequency. Stem Cell Reports, 2014; 3(1):156–168; doi: 10.1016/j.stemcr.2014.04.019

59.

Park

, Huang

, Kwon

, et al. Cathepsin A regulates pluripotency, proliferation and differentiation in mouse embryonic stem cells. Cell Biochem Funct, 2021; 39(1):67–76; doi: 10.1002/cbf.3554

60.

Pauklin

, Vallier

. The cell-cycle state of stem cells determines cell fate propensity. Cell, 2013; 155(1):135–147; doi: 10.1016/j.cell.2013.08.031. Erratum in: Cell 2014;156(6):1338.

61.

Pera

, Andrade

, Houssami

, et al. Regulation of human embryonic stem cell differentiation by BMP-2 and its antagonist noggin. J Cell Sci, 2004; 117(7):1269–1280; doi: 10.1242/jcs.00970

62.

, Li

T-G

, Hao

, et al. BMP4 supports self-renewal of embryonic stem cells by inhibiting mitogen-activated protein kinase pathways. Proc Natl Acad Sci U S A, 2004; 101(16):6027–6032; doi: 10.1073/pnas.0401367101

63.

Raina

, Fabris

, Morelli

, et al. Intermittent ERK oscillations downstream of FGF in mouse embryonic stem cells. Development, 2022; 149(4):dev199710; doi: 10.1242/dev.199710

64.

Sakaki-Yumoto

, Liu

, Ramalho-Santos

, et al. Smad2 is essential for maintenance of the human and mouse primed pluripotent stem cell state. J Biol Chem, 2013; 288(25):18546–18560; doi: 10.1074/jbc.M112.446591

65.

Schnerch

, Cerdan

, Bhatia

. Distinguishing between mouse and human pluripotent stem cell regulation: The best laid plans of mice and men. Stem Cells, 2010; 28(3):419–430; doi: 10.1002/stem.298

66.

Smith

. Design principles of pluripotency. EMBO Mol Med, 2009; 1(5):251–254; doi: 10.1002/emmm.200900035

67.

Storm

, Kumpfmueller

, Bone

, et al. Zscan4 is regulated by PI3-kinase and DNA-damaging agents and directly interacts with the transcriptional repressors LSD1 and CtBP2 in mouse embryonic stem cells. PLoS One, 2014; 9(3):e89821; doi: 10.1371/journal.pone.0089821

68.

Tai

C-I

, Schulze

, Ying

Q-L

. Stat3 signaling regulates embryonic stem cell fate in a dose-dependent manner. Biol Open, 2014; 3(10):958–965; doi: 10.1242/bio.20149514

69.

Teo

, Ali

, Wong

, et al. Activin and BMP4 synergistically promote formation of definitive endoderm in human embryonic stem cells. Stem Cells, 2012; 30(4):631–642; doi: 10.1002/stem.1022

70.

Theunissen

, Powell

, Wang

, et al. Systematic identification of culture conditions for induction and maintenance of naive human pluripotency. Cell Stem Cell, 2014; 15(4):471–487; doi: 10.1016/j.stem.2014.07.002. Erratum in: Cell Stem Cell 2014;15(4):523. Erratum in: Cell Stem Cell 2014;15(4)(4):524–526; doi: 10.1016/j.stem.2014.09.003

71.

Thomson

, Itskovitz-Eldor

, Shapiro

, et al. Embryonic stem cell lines derived from human blastocysts. Science, 1998; 282(5391):1145–1147. Erratum in: Science 1998;282(5391)(5395):1827; doi: 10.1126/science.282.5391.1145

72.

Vallier

, Mendjan

, Brown

, et al. Activin/Nodal signalling maintains pluripotency by controlling Nanog expression. Development, 2009; 136(8):1339–1349; doi: 10.1242/dev.033951

73.

Varzideh

, Gambardella

, Kansakar

, et al. Molecular mechanisms underlying pluripotency and self-renewal of embryonic stem cells. Int J Mol Sci, 2023; 24(9):8386; doi: 10.3390/ijms24098386

74.

Waisman

, Sevlever

, Elías Costa

, et al. Cell cycle dynamics of mouse embryonic stem cells in the ground state and during transition to formative pluripotency. Sci Rep, 2019; 9(1):8051; doi: 10.1038/s41598-019-44537-0

75.

Wang

, Zhao

, Liu

, et al. BMP4 preserves the developmental potential of mESCs through Ube2s- and Chmp4b-mediated chromosomal stability safeguarding. Protein Cell, 2022; 13(8):580–601; doi: 10.1007/s13238-021-00896-x

76.

Wang

, Wang

, Zhang

, et al. Inhibition of ubiquitin-specific protease 13-mediated degradation of Raf1 kinase by Spautin-1 has opposing effects in naïve and primed pluripotent stem cells. J Biol Chem, 2021; 297(5):101332; doi: 10.1016/j.jbc.2021.101332

77.

Ware

. Concise review: Lessons from naïve human pluripotent cells. Stem Cells, 2017; 35(1):35–41; doi: 10.1002/stem.2507

78.

Weinberger

, Ayyash

, Novershtern

, et al. Dynamic stem cell states: Naive to primed pluripotency in rodents and humans. Nat Rev Mol Cell Biol, 2016; 17(3):155–169; doi: 10.1038/nrm.2015.28

79.

Williams

CAC

, Gray

, Findlay

. A simple method to identify kinases that regulate embryonic stem cell pluripotency by high-throughput inhibitor screening. J Vis Exp, 2017; 123:55515; doi: 10.3791/55515

80.

Xiao

, Yuan

, Sharkis

. Activin A maintains self-renewal and regulates fibroblast growth factor, Wnt, and bone morphogenic protein pathways in human embryonic stem cells. Stem Cells, 2006; 24(6):1476–1486; doi: 10.1634/stemcells.2005-0299

81.

R-H

, Barron

, Gu

, et al. NANOG is a direct target of TGFβ/activin mediated SMAD signaling in human ES cells. Cell Stem Cell, 2008; 3(2):196–206; doi: 10.1016/j.stem.2008.07.001

82.

R-H

, Chen

, Li

, et al. BMP4 initiates human embryonic stem cell differentiation to trophoblast. Nat Biotech, 2002; 20(12):1261–1264; doi: 10.1038/nbt761

83.

Yamanaka

, Lanner

, Rossant

. FGF signal-dependent segregation of primitive endoderm and epiblast in the mouse blastocyst. Development, 2010; 137(5):715–724; doi: 10.1242/dev.043471

84.

Yang

, Ryan

, Wang

, et al. Establishment of mouse expanded potential stem cells. Nature, 2017; 550(7676):393–397; doi: 10.1038/nature24052

85.

Yang

, Kalkan

, Morrisroe

, et al. A genome-wide RNAi screen reveals MAP kinase phosphatases as key ERK pathway regulators during embryonic stem cell differentiation. PLoS Genet, 2012; 8(12):e1003112; doi: 10.1371/journal.pgen.1003112

86.

Yang

, Adachi

, Sheridan

, et al. Heightened potency of human pluripotent stem cell lines created by transient BMP4 exposure. Proc Natl Acad Sci U S A, 2015; 112(18):E2337–E2346; doi: 10.1073/pnas.1504778112

87.

Ying

, Nichols

, Chambers

, et al. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell, 2003; 115(3):281–292; doi: 10.1016/s0092-8674(03)00847-x

88.

Yoo

, Huang

, Zhang

, et al. Fibroblast growth factor regulates human neuroectoderm specification through ERK1/2-PARP-1 pathway. Stem Cells, 2011; 29(12):1975–1982; doi: 10.1002/stem.758

89.

Zakrzewski

, Dobrzyński

, Szymonowicz

. Stem cells: Past, present, and future. Stem Cell Res Ther, 2019; 10(1):68; doi: 10.1186/s13287-019-1165-5

90.

Zhang

, Yang

, Wu

, et al. MPP8 governs the activity of the LIF/STAT3 pathway and plays a crucial role in the differentiation of mouse embryonic stem cells. Cells, 2023; 12(16):2023; doi: 10.3390/cells12162023

91.

Zhang

, Li

, Tan

, et al. Short-term BMP-4 treatment initiates mesoderm induction in human embryonic stem cells. Blood, 2008; 111(4):1933–1941; doi: 10.1182/blood-2007-02-074120