Mechanism and method for generating tumor-free iPS cells using intronic microRNA miR-302 induction.

Today's researchers generating induced pluripotent stem cells (iPS cells or iPSCs) usually consider the pluripotency first, then, the potential tumorigenicity. Oncogenic factors such as c-Myc and Klf4 were frequently used to boost the survival and proliferative rates of iPSCs, creating the inevitable problem of tumorigenicity that hindered the therapeutic usefulness of the iPSCs. To prevent tumorigenecity in stem cells, we have examined mechanism(s) by which the cell cycle genes of embryonic cells were regulated. Naturally occurring embryonic stem cells (ESCs) possess two unique stemness properties: pluripotent differentiation into almost all cell types and self-renewal with no risk of tumor formation. These two features are also important for the use of ESCs or iPSCs in therapy. Currently, despite overwhelming reports describing iPSC pluripotency, there have been no observations of tumor prevention mechanism(s) that suppresses tumor formation similar to that in naturally occurring ESCs. Our recent studies have revealed, for the first time, a ESC-specific microRNA (miRNA), miR-302, which was responsible for regulating human iPSC tumorigenicity through co-suppression of both cyclin E-CDK2 and cyclin D-CDK4/6 cell cycle pathways during G1-S phase transition. Additionally, miR-302 also silenced BMI-1, a cancer stem cell marker gene, to promote the expression of two senescence-associated tumor suppressor genes, p16Ink4a and p14/p19Arf. Together, the combinatory effect of reducing G1-S cell cycle transition and increasing p16/p14(p19) expression resulted in a relatively attenuated cell cycle rate similar to that of 2-8-cell-stage embryonic cells in early mammalian zygotes (20-24 h/cycle), as compared to the fast proliferation rate of iPSCs induced by four defined factors Oct4-Sox2-Klf4-c-Myc (12-16 h/cycle). These findings provide a means to control iPSC tumorigenicity and improve the safety of iPSCs in the therapeutic use. In this chapter, we reviewed the mechanism underlying miR-302-mediated tumor suppression and then applied this mechanism to generate tumor-free iPSCs. The same strategy can also be used to prevent ESC tumorigenicity.