Insights into novel regulators of human somatic cell reprogramming

Reprogramming of somatic cells generates induced pluripotent stem cells (iPSCs) that are invaluable resources in biomedical research. Transcriptional and epigenetic changes have been investigated to improve our understanding of reprogramming processes. Here, we extend the previous transcriptome studies by performing RNA-seq on cells defined by a combination of multiple cellular surface makers. We found that transcriptome changes during early induction of reprogramming are independent of the opening of compact chromatin by Yamanaka factors OCT4, SOX2, KLF4 and MYC. Our data also show that multiple cellular signaling pathways display a bi-phasic change: repression at the early stage and induction at the final stage of reprogramming. RNA-seq allowed us to uncover a switch in allelic specific expression during reprogramming, and to identify multiple spliced forms of genes uniquely expressed at progressive stages of reprogramming. In particular, we found a pluripotency-specific spliced form of CCNE1 that significantly enhances reprogramming. Our transcriptome data provide unique opportunities to understand human iPSC reprogramming.;Reprogramming to pluripotency is also accompanied by global genomic and epigenomic changes. Histone modification and DNA methylation states in iPSCs are highly similar to those in embryonic stem cells (ESCs). However, epigenetic differences still exist between iPSCs and ESCs. In particular, aberrant DNA methylation states found in iPSCs are a major concern for using iPSCs in a clinical setting. Thus, it is critical for future applications of iPSCs to find factors that regulate DNA methylation states in reprogramming. We asked whether non-coding RNAs that regulate both de novo methylation and demethylation would facilitate reprogramming. Through target prediction analysis and screening, we found that miR-29 family targets de novo DNA methyltransferases DNMT3A and DNMT3B, demethylases TET1, TET3 and TDG, as well as histone lysine demethylases such as KDM5B. The suppression of miR-29 family improved, while its overexpression decreased the efficiency of human somatic cell reprogramming. Through global DNA methylation and hydroxymethylation analysis, we found that DNA demethylation is a major event mediated by miR-29a in early reprogramming, and that iPSCs derived from miR-29a depleted fibroblasts are epigenetically more similar to established ESCs than control ones. Furthermore, miR29a treated iPSCs give rise to more complex three germ layer --- tissue differentiation as assessed by teratoma formation. Our findings uncover an important miRNA-based approach in generating iPSCs with altered epigenetic states.