Epigenetic Reprogramming of Somatic Cells by Nuclear Transfer, Chemical and Viral Inductions

Nuclear reprogramming, the process of returning a differentiated somatic cell to totipotency, can be achieved by somatic cell nuclear transfer (cloning), cell fusion and induction by expression of transcription factors.  Our lab has long been involved in reprogramming by cloning in the cattle model.  Cloned bovine embryos have similar expression profiles as embryos produced in vivo, suggesting significant and relatively complete nuclear reprogramming.  Cloned animals, however, have significant developmental problems.  We hypothesize that there are two stages of reprogramming.  The first stage occurs at the embryonic development when the epigenetic marks of somatic cells are erased.  The second stage of reprogramming occurs during tissue differentiation of the cloned embryos when epigenetic marks of each tissue are re-established.  Our data, together with those of others, suggest that the first stage of reprogramming occurs relatively completely by the mature oocyte.  Yet the second stage of reprogramming is relatively incomplete which causes clones’ developmental abnormalities. 

Induced pluripotent stem cells (iPSCs) generated by expression of four transcription factors have great promises for regenerative medicine in humans.  Small molecules offer the advantages of low cost without genomic modification and have been used to maintain pluripotency.  Because stem cells and cancer cells share many common features we investigated the possibility of reprogramming somatic cells with carcinogens at non-genotoxic levels.  Treating mouse B6/129 embryonic fibroblasts (MEFs) with 16 candidate chemicals resulted in colonies that were positive for both alkaline phosphatase and surface specific embryonic antigen-1 (SSEA-1), as well as the expression of 122 genes that are ESC-enriched.

Finally, we studied the mechanism of the LIF/Stat3 signaling pathway in iPSC generation.  For the first time, we demonstrated Jak/Stat3 activity plays a fundamental role in promoting pluripotency establishment at the epigenetic level. Specifically, by facilitating DNA demethylation/de novo methylation, and open-chromatin formation during late-stage reprogramming, activated Stat3 is essential in demethylation of Oct4 and Nanog regulatory elements in induced cells, which are marked by suppressed endogenous pluripotent gene expression.