In contrast to the normal cells the requirement for

In contrast to the normal cells, the requirement for cell:cell contacts to promote survival was removed in the H7 and H14 adapted cells, implying the loss of niche dependence, as has also been reported elsewhere (Werbowetski-Ogilvie et al., 2009). Consequently, the functional significance of the observed increased motility of adapted order OTX-015 remains unclear. One possibility is that increased motility plays a role in the early stages of adaptation, allowing cells to make more contacts, but becomes superfluous once the adapted cells acquire further changes that render them cell contact independent. If this is the case, monitoring motility could provide a noninvasive approach for the early detection of culture adaptation.
Experimental Procedures
Author Contributions
Acknowledgments
Introduction Mouse embryonic stem cells (mESCs) and epiblast stem cells (EpiSCs) are stable, pluripotent cells derived from mouse blastocysts and early postimplantation embryo, respectively. Embryonic stem cells demonstrate characteristics of naive pluripotency, while, in contrast, EpiSCs are in a primed pluripotent state (Nichols and Smith, 2009). These cells, despite their temporal proximity in vivo, exhibit marked differences in their response to signals to maintain their pluripotency; mESC stability is governed by leukemia inhibitory factor (LIF) and bone morphogenic protein 4 (BMP4) signaling, while Activin A and basic fibroblast growth factor (bFGF) maintain EpiSCs. Conversion of mESCs to EpiSCs is primarily driven by culture in EpiSC-supportive conditions, specifically, by addition of the cytokines Activin A and bFGF (Guo et al., 2009). This conversion robustly yields culture-derived EpiSCs (CDEs). Similarly, conversion of EpiSCs to mESCs can be driven by activation of STAT3 (Bao et al., 2009; Onishi et al., 2012; van Oosten et al., 2012; Yang et al., 2010), the transcription factor directly downstream of mESC-supportive LIF signaling. Alternative methods of EpiSC conversion involve overexpression of transgenes (Festuccia et al., 2012; Gillich et al., 2012; Guo and Smith, 2010; Guo et al., 2009; Hall et al., 2009; Hanna et al., 2009; Yang et al., 2010) or culture on feeders (Bao et al., 2009; Bernemann et al., 2011; Greber et al., 2010; Han et al., 2010a; Sasaki et al., 2011; Zhou et al., 2010). Frequency of conversion remains low in most cases, with optimal conversion occurring at ∼10% efficiency when potent conversion-driving transcription factors, NANOG or KLF4, are overexpressed in combination with either optimal STAT3 activation (Yang et al., 2010) or orphan nuclear receptor, LRH-1 (Guo and Smith, 2010). Not surprisingly, STAT3-driven conversion of EpiSCs is critically limited by the inability of EpiSCs to robustly respond to LIF. We have previously demonstrated that EpiSCs regain LIF responsiveness upon control of the cellular microenvironment and local cell density using micropatterning (μP) (Onishi et al., 2012). This recovery of LIF responsiveness facilitates feeder-free conversion of EpiSCs at frequencies comparable to transgenic systems. We have previously demonstrated the importance of endogenous GP130 ligands in gaining LIF responsiveness (Davey et al., 2007). However, the observation that supplementing exogenous LIF to EpiSCs does not recover LIF responsiveness suggests the presence of additional signaling conversion barriers (Onishi et al., 2012). BMP signaling in the presence of LIF functions to maintain mESC pluripotency. However, when EpiSCs are exposed to BMP, SMAD1 activation induces their differentiation to both primitive endoderm and trophectoderm (Brons et al., 2007). Additionally, at higher concentrations (50 to 500 ng/ml), BMP drives the differentiation of EpiSCs to germ cells (Hayashi and Surani, 2009; Tesar et al., 2007). This behavior is consistent with that of human embryonic stem cells (hESCs); an observation further corroborated in other differentiation protocols involving BMP signaling (Vallier et al., 2009). The role of BMP in differentiation of EpiSCs is extensive and well defined. In contrast to its role in differentiation, here we demonstrate a role for BMP signaling in the upregulation of LIF signaling responsiveness and subsequent enhancement of the reprogramming efficiency of EpiSCs. The increase in LIF responsiveness of EpiSCs upon μP (Onishi et al., 2012) can be explained by an increase in local accumulation of BMP and GP130 (i.e., IL-6 family) ligands. Specifically, inhibition of endogenous BMP and GP130 signaling on μP results in the loss of LIF responsiveness. Additionally, in standard, nonpatterned culture, supplementation with both LIF and BMP4 increases LIF responsiveness by as much as 3-fold over addition of either LIF or BMP4 alone. We demonstrate that STAT3 and SMAD1, the respective downstream targets of LIF and BMP4, cooperate by directly binding and regulating transcription of the promoter region the gene encoding JAK-STAT pathway receptor LIF receptor (Lif-r). As is observed in neural precursors (Nakashima et al., 1999), the interaction between SMAD1 and STAT3 in EpiSCs is bridged by P300. We demonstrate that manipulation of P300 can tune the reinitiation of dormant JAK-STAT signaling in response to LIF and BMP, thereby affecting frequency of conversion. This demonstration of how signaling crosstalk changes the context and cell fate effects of BMP signaling may be indicative of conserved mechanisms in other systems and an important consideration for harnessing the full potential of pluripotent and multipotent stem cells.