Dissecting direct reprogramming through integrative genomic analysis

Abstract

Somatic cells can be reprogrammed to a pluripotent state through the ectopic expression of defined transcription factors. Understanding the mechanism and kinetics of this transformation may shed light on the nature of developmental potency and suggest strategies with improved efficiency or safety. Here we report an integrative genomic analysis of reprogramming of mouse fibroblasts and B lymphocytes. Lineage-committed cells show a complex response to the ectopic expression involving induction of genes downstream of individual reprogramming factors. Fully reprogrammed cells show gene expression and epigenetic states that are highly similar to embryonic stem cells. In contrast, stable partially reprogrammed cell lines show reactivation of a distinctive subset of stem-cell-related genes, incomplete repression of lineage-specifying transcription factors, and DNA hypermethylation at pluripotency-related loci. These observations suggest that some cells may become trapped in partially reprogrammed states owing to incomplete repression of transcription factors, and that DNA de-methylation is an inefficient step in the transition to pluripotency. We demonstrate that RNA inhibition of transcription factors can facilitate reprogramming, and that treatment with DNA methyltransferase inhibitors can improve the overall efficiency of the reprogramming process. The ability to persuade fully differentiated (somatic) human cells into a pluripotent stem cell state reliably would be a great advance in regenerative medicine. Recent work in human and mouse cells showed that such reprogramming is possible, but current routes to iPS (induced pluripotent stem) cells are inefficient and the mechanisms involved are poorly understood. Now a genomic analysis of the reprogramming of murine fibroblasts and B lymphocytes, together with an analysis of the chromatin state and DNA methylation, throws light on the obstacles that prevent most cells from reprogramming. It seems that some cells become trapped in partially reprogrammed states due to incomplete repression of transcription factors, that transient RNA inhibition of transcription factors can aid reprogramming, and that treatment with DNA methyltransferase inhibitors can improve the efficiency of the reprogramming process. A genomic analysis of the reprogramming of murine fibroblasts and B lymphocytes was performed. It is shown that fully reprogrammed cells display gene expression and epigenetic states that are highly similar to embryonic stem cells. But in stable partially reprogrammed cell lines, there is reactivation of a distinct subset of stem cell-related genes and incomplete repression of lineage-specifying transcription factors.