Cyclin B1/CDK1 complex enhances reprogramming efficiency possibly via improving iPSC maturation

Abstract

Enhancing expression of interphase CDKs and cyclin D1 has been shown to increase somatic reprogramming efficiency, through promoting proliferation or overcoming p53-p21-induced G1 arrest. However, the functional role of cell cycle regulators on reprogramming could be contradictory, and there is no study regarding the role of mitotic CDK/cyclin on reprogramming. In this study, we investigated functional role of cyclin B1 and cyclin B1 associated CDK1 during human induced pluripotent stem cells (hiPSCs) formation in a p53 null background, thereby overcoming p53-p21 mediated cell arrest. Yamanaka factors (OKSM) and LIN28 in episomal vectors were applied to generate hiPSCs. We modulated cyclin B1 and CDK1 levels by overexpression, knockdown, and co-expression in human diploid fibroblast (HDF) primary cell line followed by iPSC reprogramming. Cyclin B1 overexpression significantly improved iPSC formation efficiency in human HDFs. Co-expression of cyclin B1 with low amount but not high amount of CDK1 also displayed a similar effect, whereas expression of cyclin B1 in CDK1 knockdown HDFs blocked iPSC formation. The results suggest that high expression of cyclin B1 is required to bind to intracellular CDK1 for its function on iPSC formation. Cyclin B1 also improved reprogramming efficiency in epithelial cancer cells with the ratio of AP+ iPS colonies was 56% in cyclin B1-expressing versus 41% in GFP-expressing cells. Cyclin B1 only slightly enhanced S and G2/M population at early stage of iPSC formation. Measurement of pluripotency markers from the pool of iPS colonies, we found that Oct4 and SSEA4 were highly expressed in both GFP- and cyclin B1-expressing iPSCs. By measuring TRA-1-60, one of the most specific markers of human pluripotent of stem cells, we noticed that relative proportion of TRA-1-60+ cells was similar in nascent iPSCs, but became 1.6-2.4 fold higher in cyclin B1-expressing iPSCs during subsequent re-plating (fold change was normalized to GFP-expressing cells), suggesting that cyclin B1 might have a role in inhibiting reversion of reprogramming. In summary, cyclin B1 and cyclin B1-CDK1 complex improve reprogramming efficiency. The underlying mechanism of whether and how cyclin B1 promoting maturation of reprogrammed cells is under investigation.