Shoudong Ye

Embryonic stem cell self-renewal pathways converge on the transcription factor Tfcp2l1

Mouse embryonic stem cell (mESC) self‐renewal can be maintained by activation of the leukaemia inhibitory factor (LIF)/signal transducer and activator of transcription 3 (Stat3) signalling pathway or dual inhibition (2i) of glycogen synthase kinase 3 (Gsk3) and mitogen‐activated protein kinase kinase (MEK). Several downstream targets of the pathways involved have been identified that when individually overexpressed can partially support self‐renewal. However, none of these targets is shared among the involved pathways. Here, we show that the CP2 family transcription factor Tfcp2l1 is a common target in LIF/Stat3‐ and 2i‐mediated self‐renewal, and forced expression of Tfcp2l1 can recapitulate the self‐renewal‐promoting effect of LIF or either of the 2i components. In addition, Tfcp2l1 can reprogram post‐implantation epiblast stem cells to naïve pluripotent ESCs. Tfcp2l1 upregulates Nanog expression and promotes self‐renewal in a Nanog‐dependent manner. We conclude that Tfcp2l1 is at the intersection of LIF‐ and 2i‐mediated self‐renewal pathways and plays a critical role in maintaining ESC identity. Our study provides an expanded understanding of the current model of ground‐state pluripotency.

Modulation of β-catenin function maintains mouse epiblast stem cell and human embryonic stem cell self-renewal

Wnt/β-catenin signalling has a variety of roles in regulating stem cell fates. Its specific role in mouse epiblast stem cell self-renewal, however, remains poorly understood. Here we show that Wnt/β-catenin functions in both self-renewal and differentiation in mouse epiblast stem cells. Stabilization and nuclear translocation of β-catenin and its subsequent binding to T-cell factors induces differentiation. Conversely, retention of stabilized β-catenin in the cytoplasm maintains self-renewal. Cytoplasmic retention of β-catenin is effected by stabilization of Axin2, a downstream target of β-catenin, or by genetic modifications to β-catenin that prevent its nuclear translocation. We also find that human embryonic stem cell and mouse epiblast stem cell fates are regulated by β-catenin through similar mechanisms. Our results elucidate a new role for β-catenin in stem cell self-renewal that is independent of its transcriptional activity and will have broad implications in understanding the molecular regulation of stem cell fate.

Pleiotropy of Glycogen Synthase Kinase-3 Inhibition by CHIR99021 Promotes Self-Renewal of Embryonic Stem Cells from Refractory Mouse Strains

Background Inhibition of glycogen synthase kinase-3 (GSK-3) improves the efficiency of embryonic stem (ES) cell derivation from various strains of mice and rats, as well as dramatically promotes ES cell self-renewal potential. β-catenin has been reported to be involved in the maintenance of self-renewal of ES cells through TCF dependent and independent pathway. But the intrinsic difference between ES cell lines from different species and strains has not been characterized. Here, we dissect the mechanism of GSK-3 inhibition by CHIR99021 in mouse ES cells from refractory mouse strains. Methodology/Principal Findings We found that CHIR99021, a GSK-3 specific inhibitor, promotes self-renewal of ES cells from recalcitrant C57BL/6 (B6) and BALB/c mouse strains through stabilization of β-catenin and c-Myc protein levels. Stabilized β-catenin promoted ES self-renewal through two mechanisms. First, β-catenin translocated into the nucleus to maintain stem cell pluripotency in a lymphoid-enhancing factor/T-cell factor–independent manner. Second, β-catenin binds plasma membrane-localized E-cadherin, which ensures a compact, spherical morphology, a hallmark of ES cells. Further, elevated c-Myc protein levels did not contribute significantly to CH-mediated ES cell self-renewal. Instead, the role of c-Myc is dependent on its transformation activity and can be replaced by N-Myc but not L-Myc. β-catenin and c-Myc have similar effects on ES cells derived from both B6 and BALB/c mice. Conclusions/Significance Our data demonstrated that GSK-3 inhibition by CH promotes self-renewal of mouse ES cells with non-permissive genetic backgrounds by regulation of multiple signaling pathways. These findings would be useful to improve the availability of normally non-permissive mouse strains as research tools.

STAT3 Phosphorylation at Tyrosine 705 and Serine 727 Differentially Regulates Mouse ESC Fates

STAT3 can be transcriptionally activated by phosphorylation of its tyrosine 705 or serine 727 residue. In mouse embryonic stem cells (mESCs), leukemia inhibitory factor (LIF) signaling maintains pluripotency by inducing JAK‐mediated phosphorylation of STAT3 Y705 (pY705). However, the function of phosphorylated S727 (pS727) in mESCs remains unclear. In this study, we examined the roles of STAT3 pY705 and pS727 in regulating mESC identities, using a small molecule‐based system to post‐translationally modulate the quantity of transgenic STAT3 in STAT3−/− mESCs. We demonstrated that pY705 is absolutely required for STAT3‐mediated mESC self‐renewal, while pS727 is dispensable, serving only to promote proliferation and optimal pluripotency. S727 phosphorylation is regulated directly by fibroblast growth factor/Erk signaling and crucial in the transition of mESCs from pluripotency to neuronal commitment. Loss of S727 phosphorylation resulted in significantly reduced neuronal differentiation potential, which could be recovered by a S727 phosphorylation mimic. Moreover, loss of pS727 sufficed LIF to reprogram epiblast stem cells to naïve pluripotency, suggesting a dynamic equilibrium of STAT3 pY705 and pS727 in the control of mESC fate. Stem Cells 2014;32:1149–1160

Depletion of Tcf3 and Lef1 maintains mouse embryonic stem cell self-renewal

ABSTRACT Mouse and rat embryonic stem cell (ESC) self-renewal can be maintained by dual inhibition of glycogen synthase kinase 3 (GSK3) and mitogen-activated protein kinase kinase (MEK). Inhibition of GSK3 promotes ESC self-renewal by abrogating T-cell factor 3 (TCF3)-mediated repression of the pluripotency network. How inhibition of MEK mediates ESC self-renewal, however, remains largely unknown. Here, we show that inhibition of MEK can significantly suppress lymphoid enhancer factor 1 (LEF1) expression in mouse ESCs. Knockdown or knockout of Lef1 partially mimics the self-renewal-promoting effect of MEK inhibitors. Moreover, depletion of both Tcf3 and Lef1 enables maintenance of undifferentiated mouse ESCs without exogenous factors, cytokines or inhibitors. Transcriptome resequencing analysis reveals that LEF1 is closely associated with endoderm specification in ESCs. Thus, our study adds support to the notion that the key to maintaining the ESC ground state is to shield ESCs from differentiative cues. Summary: Depletion of Lef1 and Tcf3 shows that ESCs could be shielded from differentiative cues to maintain the ESC ground state.

The transcription factor Gbx2 induces expression of Kruppel-like factor 4 to maintain and induce naive pluripotency of embryonic stem cells

The transcription factor Gbx2 (gastrulation brain homeobox 2) is a direct target of the LIF/STAT3 signaling pathway, maintains mouse embryonic stem cell (mESC) self-renewal, and facilitates mouse epiblast stem cell (mEpiSC) reprogramming to naïve pluripotency. However, the mechanism by which Gbx2 mediates its effects on pluripotency remains unknown. Here, using an RNA-Seq approach, we identified Klf4 (Kruppel-like factor 4) as a direct target of Gbx2. Functional studies indicated that Klf4 mediates the self-renewal–promoting effects of Gbx2, because knockdown of Klf4 expression abrogated the ability of Gbx2 to maintain the undifferentiated state of mESCs. We also found that Gbx2 largely depends on Klf4 to reprogram mEpiSCs to a mESC-like state. In summary, our study has uncovered a mechanism by which Gbx2 maintains and induces naïve pluripotency. These findings expand our understanding of the pluripotency control network and may inform the development of culture conditions for improved ESC maintenance and differentiation.

β-catenin coordinates with Jup and the TCF1/GATA6 axis to regulate human embryonic stem cell fate

β-catenin-mediated signaling has been extensively studied in regard to its role in the regulation of human embryonic stem cells (hESCs). However, the results are controversial and the mechanism by which β-catenin regulates the hESC fate remains unclear. Here, we report that β-catenin and γ-catenin are functionally redundant in mediating hESC adhesion and are required for embryoid body formation, but both genes are dispensable for hESC maintenance, as the undifferentiated state of β-catenin and γ-catenin double deficient hESCs can be maintained. Overexpression of β-catenin induces rapid hESC differentiation. Functional assays revealed that TCF1 plays a crucial role in hESC differentiation mediated by β-catenin. Forced expression of TCF1, but not other LEF1/TCF family members, resulted in hESC differentiation towards the definitive endoderm. Conversely, knockdown of TCF1 or inhibition of the interaction between TCF1 and β-catenin delayed hESC exit from pluripotency. Furthermore, we demonstrated that GATA6 plays a predominant role in TCF1-mediated hESC differentiation. Knockdown of GATA6 completely eliminated the effect of TCF1, while forced expression of GATA6 induced hESC differentiation. Our data thus reveal more detailed mechanisms for β-catenin in regulating hESC fate decisions and will expand our understanding of the self-renewal and differentiation circuitry in hESCs.

TFCP2L1 represses multiple lineage commitment of mouse embryonic stem cells through MTA1 and LEF1

ABSTRACT TFCP2L1 is a transcription factor that is crucial for self-renewal of mouse embryonic stem cells (mESCs). How TFCP2L1 maintains the pluripotent state of mESCs, however, remains unknown. Here, we show that knockdown of Tfcp2l1 in mESCs induces the expression of endoderm, mesoderm and trophectoderm markers. Functional analysis of mutant forms of TFCP2L1 revealed that TFCP2L1 depends on its N-terminus and CP2-like domain to maintain the undifferentiated state of mESCs. The N-terminus of TFCP2L1 is mainly associated with the suppression of mesoderm and trophectoderm differentiation, while the CP2-like domain is closely related to the suppression of endoderm commitment. Further studies showed that MTA1 directly interacts with TFCP2L1 and is indispensable for the TFCP2L1-mediated self-renewal-promoting effect and endoderm-inhibiting action. TFCP2L1-mediated suppression of mesoderm and trophectoderm differentiation, however, seems to be due to downregulation of Lef1 expression. Our study thus provides an expanded understanding of the function of TFCP2L1 and the pluripotency regulation network of ESCs. Summary: TFCP2L1 interacts with MTA1 to inhibit endoderm specification, while suppressing mesoderm and trophectoderm differentiation, in part, through downregulation of Lef1 expression.

Inhibition of Wnt/β-catenin signaling by IWR1 induces expression of Foxd3 to promote mouse epiblast stem cell self-renewal

Inhibition of Wnt/β-catenin signaling facilitates the derivation of mouse epiblast stem cells (EpiSCs), as well as dramatically promotes EpiSC self-renewal. The specific mechanism, however, is still unclear. Here, we showed that IWR1, a Wnt/β-catenin signaling inhibitor, allowed long-term self-renewal of EpiSCs in serum medium in combination with ROCK inhibitor Y27632. Through transcriptome data analysis, we arrived at a set of candidate transcription factors induced by IWR1. Among these, Forkhead box D3 (Foxd3) was most abundant. Forced expression of Foxd3 could recapitulate the self-renewal-promoting effect of IWR1 in EpiSCs. Conversely, knockdown of Foxd3 profoundly compromised responsiveness to IWR1, causing extinction of pluripotency markers and emergence of differentiation phenotype. Foxd3 thus is necessary and sufficient to mediate self-renewal downstream of Wnt/β-catenin signaling inhibitor. These findings highlight an important role for Foxd3 in regulating EpiSCs and will expand current understanding of the primed pluripotency.

Tfcp2l1 safeguards the maintenance of human embryonic stem cell self-renewal†

Tfcp2l1 is a transcription factor critical for mouse embryonic stem cell (mESC) maintenance. However, its role in human ESCs (hESCs) remains unclear. Here, we investigated the role of Tfcp2l1 in controlling hESC activity and showed that Tfcp2l1 is functionally important in the maintenance of hESC identity. Tfcp2l1 expression is highly enriched in hESCs and dramatically decreases upon differentiation. Forced expression of Tfcp2l1 promoted hESC self‐renewal. Functional analysis of the mutant forms of Tfcp2l1 revealed that both the CP2‐ and SAM‐like domains are indispensable for Tfcp2l1 to maintain the undifferentiated state of hESCs. Notably, the CP2‐like domain is closely related to the suppression of definitive endoderm and mesoderm commitment. Accordingly, knockdown of Tfcp2l1 significantly induced differentiation preferentially into definitive endoderm and mesoderm. Further studies found that inhibition of Wnt/β‐catenin signaling pathway by IWR1 is able to eliminate the differentiation caused by Tfcp2l1 downregulation. Taken together, these findings reveal the unique and crucial role of Tfcp2l1 in the determination of hESC fate and will expand our understanding of the self‐renewal and differentiation circuitry in hESCs.