Stem Cell Unit, Department of Genetics, Silberman Institute of Life Sciences, The Hebrew University, Jerusalem, Israel.
Nat Protoc. 2014 Mar;9(3):729-40. doi: 10.1038/nprot.2014.050. Epub 2014 Feb 27.
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.
人多能干细胞（hPSCs）的致瘤性被广泛认为是阻碍其在再生医学中应用的主要障碍。本方案描述了两种有效且强大的方法，可从单层培养物中化学消除起始肿瘤的 hPSCs。该方案详细说明了如何维持和分化 hPSCs，如何将化学抑制剂应用于 hPSCs 及其分化后代的培养物，以及如何使用体外和体内测定法评估所得细胞培养物的纯度。它还描述了如何挽救细胞毒性作用。消除和挽救测定可在 3-5 天内完成，体外评估需要再 1 天，体内评估需要长达 12 周以上。
