The Role Of Tfcp211 In The Self-renewal Of Mouse Embryonic Stem Cells

Embryonic stem cells (ES cells) are derived from the ICM (inner cell mass) of pre-implantation blastocysts. They can be maintained in culture indefinitely while retaining the capacity to generate nearly any type of cell in the body. Since the original derivation of mouse ES cells in 1981, culture conditions for sustaining pluripotency in vitro have been progressively refined. The cytokine leukemia inhibitory factor (LIF) could replace feeder cells and dual inhibition (2i) of glycogen synthase kinase 3 (Gsk3) and mitogen-activated protein kinase kinase (MEK) could enable robust ES cell propagation. When cultured using the 2i (CHIR99021 and PD0325901, referred to CHIR and PD03 in the text), ES cells display rather uniform marker expression.2i has been used to establish ESCs from different strains of mice and also rats. It is noteworthy that, while the triple combination of 2i/LIF appears optimal, mouse ES cells can be propagated by providing any two of these three components, implying same or complementary downstream targets to exert their self-renewal-promoting effect. However, no direct downstream connection has yet been established.LIF, CHIR and PD03 will activate LIF/STAT3, Wnt/β-catenin and suppress FGF/MEK/ERK signal pathway respectively in mouse ES cells. However, under feeder-free conditions, LIF alone is not sufficient to maintain self-renewal of ESCs derived from non-129 strains of mice such as C57BL/6, CBA and FVB, and addition of 2i is required. We performed DNA microarray analysis and found 2i can significantly induce Tfcp211 expression. Overexpressed Tfcp211 plus LIF can promote C57BL/6 ES cell self-renewal. Then, we found that CHIR, PD03 and 2i, all can upregualte Tfcp211 expression in 46C ES cells. We established an inducible Tfcp211 ES cell line and cultured them in serum-free condition (N2B27), we observed that ① most cell died or differentiated in N2B27, ② 95% ES cells maintain undifferentiated state in N2B27+CHIR, and ③ 70% ES cells still can propagate in N2B27+PD03. Taken together, these findings demonstrate that forced expression of Tfcp211 can recapitulate the effect of PD03, and partially that of CHIR, in promoting mouse ES cell self-renewal in the 2i condition.As LIF can be used to replace CHIR or PD03 in the presence of either in maintaining mouse ES cell self-renewal. We found that the expression of Tfcp211 was ① significantly downregulated after withdraw of LIF and ② and was blocked by LIF/STAT3 signaling pathway inhibitor when LIF was added in 46C ES cells. We then cultured the inducible Tfcp211 cells in serum condition without LIF and found that they ① maintained undifferentiated state, ② and had strong positive Alkaline phosphatase activity and Oct4 expression. In addition, elevated Tfcp2ll could support STAT3 knockout ES cell self-renewal. However, knockdown Tfcp211 with lentivirus impairs the self-renewal- promoting effect of LIF. These observations indicate that a high level of Tfcp211 is an important factor in liberating mouse ES cells from LIF dependence and in promoting self-renewal downstream of STAT3.Many transcriptional factors associated with ES cell pluripotency can reprogram Epiblast stem cell (EpiSCs) into ES cells, for example, STAT3 and its targets. We found that Tfcp211 expression is very low in EpiSCs and elevated Tfcp211 in EpiSCs is able to induce naive pluripotency. Next, we used Tfcp211 knockdown and overexpression cells and found that Nanog is a key downstream target of Tfcp211. Nanog-null ES cells overexpressing Tfcp211 differentiated in both N2B27+CHIR and serum culture conditions. Meanwhile, ① Elevated Nanog can rescue the differentiation induced by Tfcp211 shRNA, and ② Knockdown Nanog decrease the efficiency of Tfcp211 in reprogramming EpiSCs.Finally, we found that DNA-binding activity is essential for the self-renewal-promoting effect of Tfcp211, because the Tfcp211 mutant harboring Q214 and K216 mutation in DNA-binding reagion ① will abolish the role of Tfcp211 in mouse ES cells, and ② can’t sustain Nanog expression. In addition, we show that other members of the CP2 family do not function or respond in mouse ES cells in the same manner as Tfcp211.Summary, we conclude that Tfcp211 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.