We have based our selection scheme on classic markers that

We have based our selection scheme on classic markers that have been extensively characterized and found to be exclusively expressed on only certain cell types in the mouse embryo. The stage-specific embryonic Caspase-3/7 Inhibitor (SSEA), first described in 1978 by Solter and Knowles, are well-known highly specific markers (SSEA1 and SSEA3) for undifferentiated mouse ESCs (Solter and Knowles, 1978) or VYS endoderm cells (Fox et al., 1984), respectively. In addition, the cell surface marker epithelial cell adhesion molecule (EpCAM) has been recently described as exclusively expressed on definitive endoderm cells after e9.5 (Sherwood et al., 2007), is known to be expressed in fetal pancreas (Cirulli et al., 1995), and has a role in the morphological development of the pancreas (Cirulli et al., 1998). Prior to e9.5, EpCAM is also expressed on inner cell mass (ICM) and VYS cells as well as ESCs, which are derived from ICM cells. We therefore reasoned that if we initially removed ESCs and VYS cells present in ESC-differentiated cultures by sequential negative selection steps, using magnetic activated cell sorting (MACS) employing anti-SSEA1 and anti-SSEA3 antibodies, we could then positively select from the remaining SSEA1–/SSEA3– population only those cells expressing EpCAM, which should represent highly purified definitive endoderm cells. In this paper, we present the results of a unique three-stage selection scheme to purify definitive endoderm cells, the in vitro characterization of selected cells, and results of in vivo growth demonstrating their inability to form teratomas or tumor growths. In addition, we present surprising results that suggest a novel way to further differentiate and/or select from purified definitive endoderm cells only those precursor cells expressing PDX1, the hallmark for posterior foregut/pancreatic-committed gut endoderm.
Results and discussion
Conclusion We have outlined a simple procedure for eliminating the tumorigenic potential from differentiated ESC populations of both mouse and human origin. MACS-sorted SSEA1–/SSEA3–/EpCAM+ cells never formed teratomas. Significantly, the enriched cells have limited proliferative capacity in vivo and are lineage-restricted; the gene expression pattern of sorted cells indicates posterior foregut regional endoderm specification. Freshly sorted cells can be amplified and differentiated in vitro to express key pancreatic progenitor proteins including PROX1 and SOX17. The sorted definitive endoderm population includes or gives rise in vivo to PDX1+ putative FGSC cells, which are capable of extensive proliferation and express a repertoire of genes indicative of endoderm-committed progenitor cells. Nongenetic methods to remove teratoma-forming potential that also enable isolation of desirable cell types may reduce therapeutic risks of using ESC-derived populations.
Materials and methods
Acknowledgments
Disclosure statement
Introduction Human induced pluripotent stem cell (hiPSC) lines can be maintained in their undifferentiated state and yet be differentiated into various cell types, providing a resource of considerable promise for the future of in vitro disease modeling and drug discovery. For example, disease-specific hiPSC lines from patients with spinal muscular atrophy (Ebert et al., 2009), familial dysautonomia (Lee et al., 2009), and amyotrophic lateral sclerosis (Dimos et al., 2008), all demonstrated disease phenotype in differentiated neurons. In addition, hiPSC-derived hepatocytes (Sullivan et al., 2010; Guguen-Guillouzo et al., 2010) and cardiomyocytes (Yokoo et al., 2009; Tanaka et al., 2009; Zwi et al., 2009) have applications for drug safety evaluation, bridging the gap between in vitro studies with heterologous cell lines and human clinical trials. Furthermore, hiPSC lines derived from patients carrying specific genetic diseases provide a novel and exciting platform to study drug/disease interactions, an approach that has not been previously possible.