Future therapeutic applications of differentiated human embryonic stem cells (hESC) carry a risk of teratoma formation by contaminating undifferentiated hESC. We generated 10 monoclonal antibodies (mAbs) against surface antigens of undifferentiated hESC, showing strong reactivity against undifferentiated, but not differentiated hESC. The mAbs did not cross react with mouse fibroblasts and showed weak to no reactivity against human embryonal carcinoma cells. Notably, one antibody (mAb 84) is cytotoxic to undifferentiated hESC and NCCIT cells in a concentration-dependent, complementindependent manner. mAb 84 induced cell death of undifferentiated, but not differentiated hESC within 30 minutes of incubation, and immunoprecipitation of the mAb-antigen complex revealed that the antigen is podocalyxin-like protein-1. Importantly, we observed absence of tumor formation when hESC and NCCIT cells were treated with mAb 84 prior to transplantation into severe combined immunodeficiency mice. Our data indicate that mAb 84 may be useful in eliminating residual hESC from differentiated cells populations for clinical applications.

Human embryonic stem cells (hESCs) have the capacity to remain pluripotent and self-renew indefinitely. To discover novel players in the maintenance of hESCs, we have previously reported the generation of monoclonal antibodies that bind to cell surface markers on hESCs, and not to mouse embryonic stem cells or differentiated embryoid bodies. In this study, we have identified the antigen target of one such monoclonal antibody as the epithelial cell adhesion molecule (EpCAM). In undifferentiated hESCs, EpCAM is localized to Octamer 4 (OCT4)-positive pluripotent cells, and its expression is down-regulated upon differentiation. To further understand its biological function in hESCs, endogenous EpCAM expression was silenced using small interfering RNA. EpCAM knockdown had marginal negative effects on OCT4 and TRA-1-60 expression, however cell proliferation was decreased by >40%.Examination of lineage marker expression showed marked upregulation of endoderm and mesoderm genes in EpCAM-silenced cells, under both pluripotent and differentiating conditions. These results were validated using a hESC line whose EpCAM expression has been stably knocked down. Data from the stable line confirmed that downregulation of EpCAM decreases cell growth and increases gene expression in the endoderm and mesoderm lineages. In vivo, hESCs lacking EpCAM were able to form teratomas containing tissues representing the three germ layers, and gene expression analysis yielded marked increase in the endoderm marker alpha fetoprotein compared with control. Together these data demonstrate that EpCAM is a surface marker on undifferentiated hESCs and plays functional roles in proliferation and differentiation. STEM CELLS 2010;28:29-35 Disclosure of potential conflicts of interest is found at the end of this article.