MiR-302-Mediated Somatic Cell Reprogramming and Method for Generating Tumor-Free iPS Cells Using miR-302

Human induced pluripotent stem cells (iPSCs) by four factors have the risks of teratoma formation and potential tumorigenicity. To overcome this major hurdle, we examined the 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 all cell types and self-renewal with no risk of tumor formation. Despite overwhelming reports describing iPSC pluripotency, there have been no observations of tumor prevention mechanism that suppresses tumor formation similar to that in naturally occurring ESCs. The ESC-specific microRNA (miRNA), miR-302, regulates human iPSC tumorigenicity through co-suppression of both cyclin E-CDK2 and cyclin D-CDK4/6 cell cycle pathways during G1-S phase transition. 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-to-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). In addition to the prevention of stem cell tumorigenicity, the mechanism underlying miR-302-mediated iPSCs also includes the initiation of global genomic DNA methylation, activation of ESC-specific gene expression, and inhibition of developmental signaling. Overall, we have established an effective protocol to express the intronic miR-302 cluster, according to its own natural biogenesis mechanism to generate tumor-free iPSCs for use in biology and therapy.