Transflammation: It's Role in Direct Reprogramming_abstract

Project Title: NHLBI Progenitor Cell Biology Consortium -- Pilot Studies and number (RFP PCBC2012Pilot01)/ Transflammation: It's Role in Direct Reprogramming

Pilot Study Principal Investigator: John Cooke, M.D., Ph.D.

Hub Site Principal Investigator: John Cooke, M.D., Ph.D.

Awarded Organizations: Stanford University

Abstract:

As reported in the November 2012 issue of CELL, we have discovered a critical signaling pathway required for effective nuclear reprogramming of somatic cells to induced pluripotent stem cells (iPSCs). This discovery occurred when we were struggling to generate iPSCs using cell permeant peptide (CPP) versions of the “Yamanaka factors” (Oct4, Sox2, KLF4 and c-Myc). Our Yamanaka CPPs could bind in vitro to their cognate consensus sequence; could enter the cell nucleus; and could even rescue pluripotency in iPSCs in which a Yamanaka gene had been knocked down by shRNA. However, the Yamanaka CPPs were extraordinarily inefficient at inducing pluripotency in human fibroblasts. It occurred to us that perhaps the virus itself was important. When an irrelevant retroviral vector was added to medium containing the CPPs, efficient nuclear reprogramming was achieved. Ultimately, we determined that viral dsRNA stimulated Toll-like Receptor 3 (TLR3) to activate transcriptional pathways (mediated by NF-κB and IRF3) that caused global changes in epigenetic modifiers that placed the chromatin into an ‘open configuration’. The increase in epigenetic plasticity, documented by chromatin immunoprecipitation, permitted the CPPs to activate the network of core pluripotency genes (Lee et al., Cell. 151, 2012).

Our research mission is directed towards endothelial regeneration for the treatment of vascular diseases. With the recognition of the role of innate immunity in nuclear reprogramming, and its directed manipulation to favor an open chromatin state, we hypothesized that activation of TLR3, together with CPPs that drive endothelial cell (EC) specification, may induce the transdifferentiation of fibroblasts into ECs (“iECs”). Intriguingly, in preliminary data generated for this proposal, we have made another serendipitous discovery. Specifically, we have found that the TLR3 agonist Poly I:C, combined with exogenous endothelial growth factors, are sufficient to transdifferentiate human fibroblasts into ECs (in the absence of viral vectors or exogenous transcription factors). By increasing epigenetic plasticity, and by providing the correct environmental cues, it appears that we can attain effective transdifferentiation. Thus, by using small molecules alone, we propose to induce transdifferentiation of fibroblasts to endothelial cells.

This proposal describes a novel approach to transdifferentiation based on the recognition that TLR3 signaling induces epigenetic plasticity. A small molecule agonist of TLR3, combined with endothelial growth factors, appears to be sufficient to drive EC specification. Funding of this proposal will permit us to confirm this exciting preliminary result; to gain insight into mechanisms of transdifferentiation; to generate a small molecule strategy for therapeutic transdifferentiation that might be applied for direct reprogramming in vivo; and provide a strategy for other investigators to develop small-molecule based transdifferentiation of other cell types.