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

Abstract

Today’s researchers generating induced pluripotent stem cells (iPS cells or iPSCs) usually consider their pluripotency rather than potential tumorigenicity. Oncogenic factors such as c-Myc and Klf4 are frequently used to boost the survival and proliferative rates of iPSCs, creating an inevitable problem of tumorigenicity that hinders the therapeutic usefulness of these iPSCs. To prevent stem cell tumorigenicity, we have examined mechanisms by which the cell cycle genes are regulated in embryonic stem cells (ESCs). Naturally, ESCs possess two unique stemness properties: pluripotent differentiation into almost all cell types and unlimited self-renewal without the 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 is no report of any tumor prevention mechanism in either ESCs or iPSCs. To this, our studies have revealed for the first time that an ESC-specific microRNA (miRNA), miR-302, regulates human iPSC tumorigenicity through cosuppression of both cyclin E-CDK2 and cyclin D-CDK4/6 cell cycle pathways during G1-S phase transition. Moreover, miR-302 also silences BMI-1, a cancer stem cell gene marker, to promote the expression of two senescence-associated tumor suppressor genes, p16Ink4a and p14/p19Arf. Together, the combinatory effects of inhibiting G1-S cell cycle transition and increasing p16/p14(p19) expression result in an attenuated cell cycle rate similar to that of 2-to-8-cell-stage embryonic cells in early zygotes (20–24 h/cycle), which is however slower than the fast proliferation rate of iPSCs induced by the 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 for the therapeutic use. In this chapter, we review the mechanism underlying miR-302-mediated tumor suppression and then demonstrate how to apply this mechanism to generate tumor-free iPSCs. The same strategy may also be used to prevent ESC tumorigenicity.