Induced pluripotent stem (iPS) cells are a potential cell source for regenerative medicine. However, the tumorigenicity of iPS cells is a big concern for clinical application. In addition to the genetic manipulation of the reprogramming process and the greater risk of tumor formation, it is unclear whether iPS cells with normal development potential are still tumorigenic. Here, we investigated 3 mouse iPS cell lines, including one line that is able to generate full-term mice via tetraploid blastocyst complementation. We found that a small number of undifferentiated iPS cells could be steadily isolated and expanded after long-term differentiation of cells in vitro or in vivo. The residual undifferentiated iPS cells could be expanded and redifferentiated, and undifferentiated pluripotent stem cells could again be isolated after further rounds of differentiation, suggesting that residual undifferentiated iPS cells could not be eliminated by extended cell differentiation. The residual undifferentiated cells could form teratomas in vivo, indicating that they are a potential tumorigenic risk during transplantation. These findings prompt us to reconsider the strategies for solving the tumorigenic problem of iPS cells, not only focusing on improving the reprogramming process.
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