The capacity to reprogram differentiated cells to an induced pluripotent state (iPS) is remarkable and of great utility for modeling diseases and screening for therapeutic agents, yet the earliest mechanisms that regulate this process remain largely unknown. To unravel the cascade of transcriptional events driving conversion of human fibroblasts to pluripotent cells, we are capitalizing on the rapid, efficient, and synchronous reprogramming typical of heterokaryons. In this system, mouse embryonic stem cells (mES) are fused in excess with human fibroblasts (hF) in non-dividing syncytia in which DNA replication does not occur and >80% of the ES signature is induced within 48 hours. The heterokaryon system is unique in its ability to monitor early and transient changes in chromatin accessibility as predictors of the expression of subsequent transcripts. Using global ATAC-seq (which detects regions of open chromatin) and global RNAseq (which detects expressed transcripts), we are resolving the cascade of regulators that lead to the expression of the pluripotency network of transcription factors Oct4, Sox2, KLF4 and c-Myc.
A major challenge in the field of nuclear reprogramming is attaining robust phenotypes. Heterokaryons recapitulate the sequential activation of gene networks typical of development and are applicable to a wide range of cell fate conversions. A major advantage of heterokaryons is that they enable regulation of cell fate switches in the context of the plethora of molecular constituents inherent in a cell. The cellular pathways we are identifying are key targets for therapeutic interventions and provide insights to improve the efficiency and safety of reprogramming cells to pluripotency (Bhutani, Blau, Nature, 2010)
, (Bhutani, Burns, Blau, Cell, 2011)
, (Brady, Blau, Nature Cell Biology, 2013)
, (Bhutani, Blau, FASEB J, 2012)
Applying insights from the newt
Inspired by the remarkable regenerative capacity of the newt, we identified and overcame a double barrier that in humans prevents the dedifferentiation of muscle cells required for such regeneration. This work continues in the lab.