An improved method for the derivation of high quality iPSCs in the absence of c-Myc.

Induced pluripotent stem cells (iPSCs) hold tremendous potential for the development of new regenerative medicine therapies and the study of molecular mechanisms of pluripotency and development. However, reactivation of c-Myc, which results in tumor formation in chimeric mice, is a major roadblock in the translation of iPSCs into therapies. Although ectopic expression of c-Myc is not absolutely required for somatic reprogramming, in the absence of c-Myc, the overall efficiency of reprogramming is drastically reduced and the reprogramming time is increased. Subtle, abnormal epigenetic modifications in iPSCs derived in the absence of c-Myc have also been documented. Therefore, we developed a reprogramming method without c-Myc to generate high-quality iPSCs, a prerequisite to harnessing the full potential of iPSCs. In this study, we determined that serum replacement (SR)-based culture conditions dramatically increased the transcription factor-mediated reprogramming of mouse embryonic fibroblast cells (MEFs). The process was shortened to approximately 8 days when Oct4/Sox2/Klf4 (3F)-transduced MEFs were first cultured for 3 days under low serum conditions (LS protocol). The 3F-derived iPSCs that were generated by this method resembled mouse ES cells (mESCs) in morphology, gene expression, and in vitro differentiation. Finally, we observed that 3F-derived iPSC colonies were able to reach definite pluripotency in terms of molecular signatures when the catalytic function of c-Myc was tolerated. The 3F induction of pluripotency described here should facilitate the use of iPSCs and may also facilitate the mechanistic dissection of somatic reprogramming.