br Discussion Since the derivation of HESCs

Discussion Since the derivation of HESCs multiple differentiation protocols towards specific endodermal derivatives have been established (D\'Amour et al., 2006; Jiang et al., 2007a, 2007b; Shim et al., 2007; Roelandt et al., 2010; Si-Tayeb et al., 2010; Van Vranken et al., 2007; Wang et al., 2007; Spence et al., 2010). These differentiation protocols were based upon knowledge that was acquired through the study of animal developmental models. The derivation of HESCs provides an opportunity to explore the course of endoderm differentiation in a human in vitro model. In this work we have established a protocol that enables the dissection of the course of endodermal lineage differentiation. We characterized and isolated endoderm progenitor cells, studied their differentiation potential in defined conditions and compared it to normal endoderm development. Finally, we could demonstrate the effect of FGF2 on endoderm differentiation toward hepatocytes. The definitive endoderm is established during the gastrulation process. It was demonstrated in several animal models that the definitive endoderm differentiates from the mesendoderm – a bi-potent progenitor cell in the primitive streak region that can differentiate into both endoderm and mesoderm (Rodaway and Patient, 2001; Tada et al., 2005). We have previously demonstrated the existence of a mesendoderm cell population in early HEBs (Kopper et al., 2010). In the current study we used two cell surface markers, CXCR4 and PDGFRA, which specify endoderm and mesoderm, respectively (McGrath et al., 1999; Yusuf et al., 2005; Mizoguchi et al., 2008; Yang et al., 2008; Ataliotis et al., 1995; Mercola et al., 1990), and are co-expressed in the mesendoderm cell population (Fig. 1B). Using these cell surface markers we uncovered that in addition to the mesendoderm cell population (NCAD+/ CXCR4+/PDGFRA+), early HEBs contain two additional cell populations; CXCR4+/PDGFRA- and CXCR4-/PDGFRA+ cells. We suggest that these cell populations originate from the mesendoderm Cyt387 and correspond to the endoderm and mesoderm, respectively. Accordingly, we could demonstrate that both cell populations express genes such as PRDM1, GSC and LHX1 that are expressed in the primitive streak (PS) of gastrulation stage embryo (Fig. 2A) (Vincent et al., 2005; Norris et al., 2002; Shawlot and Behringer, 1995). Here we decided to focus on the endodermal cell lineage which is of great clinical importance. Thus, we confirmed the developmental identity of the CXCR4+ cells by demonstrating their endodermal differentiation potential. Once we recognized the definitive endoderm progenitor cells, we could compare their gene expression profile to other progenitor cells and sketch their unique gene signature (Fig. 2A). We evaluated the expression level of this gene signature over time and demonstrated that as expected from genes that specify a progenitor cell, most of them are down regulated upon differentiation (Fig. 3C). The endoderm progenitor cell gene signature includes both known endodermal genes such as HHEX, HNF1B, cKIT and CXCR4 and genes that were not shown to be related to the induction of the early endoderm. Among these genes we can find CXCR7 and LGR5, two receptors that can serve as additional definitive endoderm cell surface markers. Interestingly, LGR5, which is an orphan G-protein coupled receptor, serves as a marker for endodermal adult progenitor cells of the small intestine, colon and stomach (Barker et al., 2010). When we analyzed the endoderm progenitor cells on a single cell level we could demonstrate that high percentage of them are positive to SOX17 (Fig. 2B). The first stage of many endodermal differentiation protocols includes ActivinA treatment. In this work we demonstrated that low ActivinA concentration (15ng/ml) is sufficient to induce a considerable increase in the percentage of endoderm progenitor cells, and elevation in ActivinA concentration had no additional major effect on their percentage (Fig. 2C). The importance of TGF-beta signaling in endodermal differentiation was corroborated by the inhibition of the pathway in early HEBs, which resulted in down regulation of CXCR4 gene expression (11 fold reduction relative to control) (Fig. 2C).