Introduction Human embryonic stem cells HESCs can

Introduction
Human embryonic stem alzheimer\’s association (HESCs) can differentiate into the three embryonic germ layers and have the potential to develop into every cell in the human body (Thomson et al., 1998). In order to enable the clinical practice of HESC cell therapy it is essential to understand their differentiation course into the various cell types, and to generate appropriate differentiation protocols. Differentiation into the endoderm lineage is of special interest since it gives rise to both pancreatic and hepatic cells that have high clinical value (Murry and Keller, 2008). Since the isolation of HESCs, multiple differentiation protocols into diverse endoderm derivatives have been published, including pancreatic beta cells (D\’Amour et al., 2006; Jiang et al., 2007a, 2007b; Shim et al., 2007), hepatic cells (Roelandt et al., 2010; Si-Tayeb et al., 2010), lung alveolar epithelial cells (Van Vranken et al., 2007; Wang et al., 2007) and intestinal tissue (Spence et al., 2010). These differentiation protocols were mostly based on developmental cues that were discovered in animal models. Despite the tremendous progress in studying endoderm differentiation, the intermediate progenitor cells that play a significant role in the differentiation into mature endoderm derivative and their interactions are still largely unknown.
During early stages of development the inner cell mass (ICM), from which HESCs are derived, differentiates into a limited set of cell types: the primitive endoderm that will contribute to extra embryonic tissues, and the three embryonic germ layers – the ectoderm, mesoderm and endoderm. The three embryonic germ layers are generated through the complex process of gastrulation. The study of these early stages of human development has been demonstrated by several groups (reviewed in Murry and Keller, 2008). Previously we demonstrated via an embryoid body (EB) differentiation protocol, that HESCs initially differentiate into three distinct cell populations (Kopper et al., 2010). These cell populations could be isolated by specific cell surface markers. Molecular characterization determined that these cell populations represent the extra embryonic endoderm, primitive ectoderm and the mesendoderm.

Results
Previously, it was shown that three days after aggregation into embryoid bodies (EBs), HESCs differentiate into three major cell populations that correspond to the extra embryonic endoderm, primitive ectoderm and the mesendoderm (Kopper et al., 2010). The extra embryonic endoderm and mesendoderm cell lineages can be isolated using antibodies against erythropoietin receptor (EPOR) and N-CADHERIN (NCAD), respectively. In order to better characterize the mesendoderm cells we analyzed them using DNA micro-array, and screened the entire set of genes expressed in the NCAD+ cell population for additional uniquely expressed cell surface markers. We found that both CXCR4 and PDGFRA genes are exclusively expressed in NCAD+ cells (Fig. 1A), moreover, CXCR4 and PDGFRA are associated with early mouse endoderm and mesoderm cell lineages (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). Indeed, FACS analysis on 3-day old dissociated EBs with antibodies against CXCR4 or PDGFRA revealed the existence of both CXCR4+ and PDGFRA+ cell populations (Fig. 1B). When we double stained the EBs with antibodies against both CXCR4 and PDGFRA we could demonstrate that these cell surface markers segregate the EBs into four cell populations: CXCR4-/PDGFRA-; CXCR4+/PDGFRA-, CXCR4-/PDGFRA+ and a cell population which expresses both CXCR4 and PDGFRA (Fig. 1B). In order to further examine the identity of these cell populations we isolated them according to these surface markers using FACS. Following isolation, gene expression profile was performed for each of the populations. Correct sorting was verified by the expression levels of the marker genes PDGFRA and CXCR4 (Fig. 1C). Both the CXCR4+ cells and the CXCR4+/PDGFRA+ cells express endodermal genes such as KIT, HNF1B and HHEX (Gouon-Evans et al., 2006; Ott et al., 1991; Thomas et al., 1998). Interestingly, both the PDGFRA+ cells and the CXCR4+/PDGFRA+ cells express mesodermal genes such as RND3, EDNRB and HES1 (Goda et al., 2009; Masamizu et al., 2006; Welsh and O\’Brien, 2000) (Supplementary Table 1). These results support the hypothesis that the CXCR4+/PDGFRA+ cell population represent the mesendoderm lineage that is positive for both endodermal and mesodermal markers. In accordance with our hypothesis the mesendoderm cell population differentiates to endoderm (CXCR4+/PDGFRA-) or mesoderm (CXCR4-/PDGFRA+) that express only the endodermal or mesodermal markers, respectively. Moreover, we could demonstrate an up regulation of mesodermal markers such as T (Brachyury) and KDR (FLK-1) in the CXCR4-/PDGFRA+ cell population, suggesting that this cell population represents mesoderm progenitor cells (Supplementary Table 1).