MicroRNA and Small Molecule Reprogramming to Pluripotency
Abstract:
This pilot project proposal is based upon exciting preliminary studies that have emerged as part of the UPenn-Seattle PCBC UO1 consortium. Studies from the laboratory of Ed Morrisey (UO1 PI) and in collaboration with Jon Epstein (co-PI) have demonstrated that highly efficient reprogramming of mouse and human fibroblasts to iPS cells can be accomplished without the use of any of the Yamanaka factors. This has been accomplished by expression of the miR302 cluster in combination with an Hdac inhibitor. The exciting implication of these results is that efficient reprogramming may be possible without the use of viral vectors or genomic integration, thus paving the way for translational studies in humans. In this pilot project, we propose to demonstrate feasibility of efficient reprogramming of human cells using miR mimics and small molecules, and to further elucidate the mechanism of action of this reprogramming method.
One of the limitations of induced pluripotent stem cell reprogramming is the relative inefficiency of the process. Our preliminary data shows that expression of the miR302/367 cluster along with valproic acid rapidly and efficiently reprograms both human and murine somatic cells to an iPS state in the absence of Oct4/Sox2/Klf4/Myc. Reprogramming is two orders of magnitude more efficient than standard Oct4/Sox2/Klf4/Myc reprogramming. miR302/367 iPS cells display similar characteristics as Oct4/Sox2/Klf4/Myc-iPS cells including the expression of pluripotent marker genes, teratoma formation, efficient generation of high percentage chimeric mice, and evidence for germline contribution. Moreover, we demonstrate that suppression of Hdac2, which is specifically degraded by valproic acid, is required for miR302/367 mediated reprogramming. Preliminary studies indicate that miR mimics can replace miR302/367 viral expression constructs to mediate reprogramming. These data demonstrate the potent and cooperative role of miRNAs and Hdac mediated pathways in pluripotent stem cell reprogramming, and pave the way for the translational use of iPS cells in humans.
Copyright ©2013 NHLBI Progenitor Cell Biology Consortium.
