br Results br Discussion Derivation of foregut stem cells wi
Discussion Derivation of foregut stem liothyronine sodium with a strong proliferative capacity as well as the ability to self-renew represents an attractive cell source for multiple applications within the regenerative medicine field, including disease modeling, developmental studies, and cell-based therapies. Accordingly, our results describe a stepwise method to differentiate hPSCs into a multipotent population of foregut stem cells. Importantly, production of foregut cells has been reported previously (Green et al., 2011), but our study provides a culture system allowing the isolation, expansion, and differentiation of multipotent self-renewing foregut stem cells. Similarly, a recent study has shown that multipotent DE cells could be expanded in vitro (Cheng et al., 2012; Sneddon et al., 2012), yet these cells express a broad diversity of markers that render their embryonic identity difficult to establish. Furthermore, these studies relied on feeders, Matrigel, 3D culture conditions, or serum, any of which is not compatible with large-scale or clinical applications. Nevertheless, hFSCs resemble mouse anterior definitive endoderm cells isolated from mouse embryonic stem cells using the combination of reporter gene for HEX expression and the cell surface marker CXCR4 (Morrison et al., 2008). In agreement, hFSCs display a similar gene expression profile including the expression of HHEX and CXCR4 and share a similar capacity of differentiation toward liver and pancreas. However, hFSCs could also have the unique capacity to generate lung/thyroid cells while they have lost their capacity to generate gut cells. More importantly, hFSCs can be easily isolated from a diversity of hPSCs lines without the need for cell sorting and complex genetic modifications, thereby allowing the production of a near-homogenous population of cells with clinical value. Overall, the hFSC culture system addresses several important limitations associated with current methods available to isolate and to expand endodermal stem cells. hFSCs also share fundamental characteristics with their in vivo counterpart, including the expression of key markers such as FOXA2, CXCR4, HHEX, SOX17, and CERB. Nevertheless, the exact type of foregut cell described here is yet to be fully defined, as lineage tracing experiments have shown that foregut may contain only bipotential progenitors able to differentiate toward the hepatic and pancreatic lineages (Deutsch et al., 2001). However, the property of in vivo progenitors is likely to be dictated by their localization within the foregut and thus their surrounding environment. Moreover, the gut tube initially possesses a high degree of plasticity. Indeed, the hindgut domain, if taken at an early time point, is capable of producing liver and pancreatic bud structures when either juxtaposed against foregut cardiac mesoderm or placed in culture conditions with BMP and FGF (Bossard and Zaret, 2000; Wells and Melton, 2000). This suggests that during the early stages of gut formation, the entire gut epithelial sheet could be multipotent. Thus, the culture system described here could be less restrictive, enabling hFSCs to display the full range of their developmental plasticity. Self-renewing and multipotent adult stem cells could represent an advantageous source for the generation of large quantity of “safer” differentiated cells required for cellular therapy, because they could strongly reduce the risk of teratomas associated with pluripotent stem cells. However, it is important to underline that the isolation and serial passage of hFSCs did not improve the overall maturity of the end-stage population after differentiation, whether this be liver, lung, or pancreatic cells. Indeed, hepatocytes or pancreatic cells generated from either freshly derived foregut cells or P10 hFSCs still display a combination of adult and fetal characteristics and are not fully functional with regard to cytochrome P450 activity or insulin secretion, respectively. These results are in agreement with a broad number of studies that have demonstrated that fetal-like pancreatic/hepatic cells can be efficiently generated from hPSCs (Cho et al., 2012; Hannan et al., 2013; Rashid et al., 2010; Touboul et al., 2010) or from organ-specific progenitors (Cardinale et al., 2011; D’Amour et al., 2006; Deutsch et al., 2001; Morrison et al., 2008; Si-Tayeb et al., 2010; Sneddon et al., 2012; Sullivan et al., 2010; Van Haute et al., 2009; Wang et al., 2013; Zhao et al., 2009). Thus, the current report does not claim to solve this major challenge or even to improve existing protocol of differentiation. Our study only establishes that hFSCs are able to produce liver and pancreas cells similar to those generated from endodermal cells directly produced from hESCs (Cho et al., 2012; D’Amour et al., 2006; Hannan et al., 2013; Rashid et al., 2010; Si-Tayeb et al., 2010; Sneddon et al., 2012; Sullivan et al., 2010; Touboul et al., 2010; Van Haute et al., 2009; Zhao et al., 2009). The generation of fully functional cells from hPSCs remains a distant goal that will require the development of innovative approaches far beyond the scope of this work (Shan et al., 2013; Takebe et al., 2013).