Recent studies have suggested that tissue-specific cells may overcome their intrinsic lineage-restriction to de-differentiate or trans-differentiate upon exposure to a specific set of signals in vitro and in vivo [2-5]. Reprogramming of murine somatic cells in embryonic and adult fibroblast cultures to pluripotent ESC-like cells has recently been achieved by simultaneous viral transduction of four oncogenic transcription factors (i.e. Oct4, cMyc, Sox2 and Klf4) together [5]. With such proof-of-principle demonstration (which however uses irreversible genetic transformation), an ultimate practical utility of such reprogramming technology would require temporal and reversible manipulation by chemically defined factors. More recently, we have identified small molecules that can replace transcription factors to generate iPS cells from mouse neural progenitor cells [23] and primary fibroblasts transduced with only two genes, Oct4 and Klf4 (please see preliminary results). Hypothesis: Small molecules can be identified to improve reprogramming efficiency and replace certain transcription factors (e.g. reprogramming fibroblasts into iPS cells by only two genes, Oct4/Klf4, with synthetic small molecule BIX, which functionally replaces Sox2, as already demonstrated in the preliminary results). Under the Oct4/Klf4/BIX condition, additional small molecules could be identified that can improve reprogramming of fibroblasts into iPS cells, and some of such small molecules may also be able to replace the remaining Oct4 or Klf4 in conjunction with small molecule BIX to generate iPS cells. Such discovery would bring us one step closer to the ultimate chemical epigenetic reprogramming without any genetic manipulation. Approach: Cell-based phenotypic screens of synthetic small molecules and natural products have historically provided useful chemical ligands to modulate and study complex cellular processes, and recently provided a number of small molecules that can be used to selectively regulate stem cell fate [6]. Here we propose to develop and implement a high throughput screen of 100,000 diverse and discrete compounds in primary murine embryonic fibroblasts (MEFs) to identify small molecules that can induce/enhance programming MEF cells back to pluripotent iPS cells in conjunction with the viral transduction of the TWO factors, Oct4 and Klf4 (with much higher efficiencies: i.e. increased percentage of cells being reprogrammed, and shortened time required for inducing iPS cells). We will further examine whether identified compounds alone or in combinations can replace any of the remaining two factors. Furthermore, we will confirm and characterize their effects and activities via various in-depth cellular/biochemical assays, and carry out structure-activity- relationship (SAR) studies of the selected hit compounds to optimize their potency and specificity. Collectively, the studies described in this proposal will provide novel chemical tools for producing unlimited amount of (autologous) pluripotent cells from differentiated/lineage-restricted cells for various applications as well as studying the underlying molecular mechanisms of pluripotency and epigenetic regulations, and may ultimately facilitate development of small molecule therapeutics to stimulate tissue/organ regeneration in vivo. PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Pa Continuation Format Page
Collectively, the studies described in this proposal will provide novel chemical tools for producing unlimited amount of (autologous) pluripotent cells from somatic cells for various applications as well as studying the underlying molecular mechanisms of pluripotency and epigenetic regulations, and may ultimately facilitate development of small molecule therapeutics to stimulate tissue/organ regeneration in vivo. PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page Continuation Format Page
