The tumorigenicity of human pluripotent stem cells (hPSCs) is widely acknowledged as a major obstacle that withholds their application in regenerative medicine. This protocol describes two efficient and robust ways to chemically eliminate the tumor-initiating hPSCs from monolayer culture. The protocol details how to maintain and differentiate hPSCs, how to apply chemical inhibitors to cultures of hPSCs and their differentiated progeny, and how to assess the purity of the resultant cell cultures using in vitro and in vivo assays. It also describes how to rescue the cytotoxic effect. The elimination and the rescue assay can be completed within 3-5 d, the in vitro assessment requires another day, and the in vivo assessment requires up to 12 additional weeks.
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acknowleDGMents We thank T. Golan-Lev for her assistance with graphic design, and M. Boehringer for critically reading the manuscript. N.B. is the Herbert Cohn Chair in Cancer Research. U.B.-D. is a Clore Fellow. The research was partly funded by a grant from Hoffmann La Roche-Yissum Collaboration, by the Israel Science Foundation (ISF) (grant no. 269/12) and by the ISF-Legacy Heritage Biomedical Science Partnership (grant no. 1252/12).