Although pluripotent stem cells hold great promise in the fields of human disease modeling and regenerative medicine, the molecular basis of Oct-4, Sox2, Klf4, and c-Myc (OSKM)-induced cellular reprogramming remains unclear. To investigate the molecular mechanisms involved in cellular reprogramming, we studied the immediate effects of expression of the OSKM reprogramming factors on mouse embryonic fibroblasts (MEFs) in this study. Induction of the OSKM reprogramming factors significantly altered primary MEF growth properties. Although MEFs not expressing the reprogramming factors underwent replicative senescence within 9-12 days in culture, MEFs expressing the four reprogramming factors proliferated continuously throughout the duration of the experiment, suggesting that the expression of the OSKM reprogramming factors inhibits or delays replicative senescence. Cell cycle progression by the reprogramming factors was accompanied by the accumulation of Cyclin D1 through the early stages of reprogramming in MEFs, leading us to hypothesize that it might play a positive role in cellular reprogramming. Consistent with this hypothesis, forced Cyclin D1 expression enhanced reprogramming if administered concomitant with expression of the OSKM reprogramming factors. Most importantly, unlike wild-type MEFs expressing reprogramming factors, the number of emerging alkaline phosphatase-positive cyclin D1-null colonies was significantly reduced and cyclin D1-null MEFs were unable to initiate mesenchymal-to-epithelial transition. Our studies demonstrate that cyclin D1 is an essential gene in the reprogramming process and that activation of cyclin D1 by reprogramming factors is an important process for somatic cell reprogramming.
