Identification, characterization and optimization of chemicals for the generation of human chemically induced pluripotent stem cells

点击翻译按钮获取中文摘要

Transcription factor-mediated nuclear reprogramming achieves the generation of induced pluripotent stem cells (iPSC) from somatic cells. This technology opens a new era in regenerative medicine. However, its clinical application is largely limited by low reprogramming efficiency and genetic and epigenetic abnormality accumulated during the cultivation. Several new reprogramming methods have been developed for a more efficient and safer approach to clinical-grade iPSCs. Because chemicals are smoothly accessible to cells and can be easily manipulated, chemical reprogramming is considered one of the most promising ways to transgene-free iPSCs. Since 2013, when the first cocktail for chemical reprogramming was released, a few chemical cocktails have been reported by independent groups for the generation of mouse chemicals iPSCs (mciPSCs). However, a chemical cocktail for human ciPSCs is not available yet.Through our previous cell-based high throughput screening, we determined 1000 hits of each transcription factor. In the granted DFG project, we identified imidazopyridines as novel H3K4-specific demethylase (KDM5) inhibitors. We demonstrated that the lead compound, OCT4 inducing compound 3 (O4I3), inhibits the demethylation of H3K4 and thereby enhances the occupation of H3K4Me3 at the promoter of OCT4, leading to increased reprogramming efficiency in patient primary fibroblasts. Part of this result has been published in iScience (Cell Press). A patent related to O4I3 was granted together with Heidelberg University, and the cooperation with other academic labs and companies is ongoing. Moreover, based on the metabolic study of O4I2, a lead compound published in 2015, we successfully produced a metabolic stable derivative (O4I4). The combination of O4I3/4 with SOX2, KLF4, and MYC achieved the reprogramming of human fibroblasts without exogenous OCT4. I am applying for a renewal project, because in the remaining months it is difficult to answer the open questions, including its mechanism of action and whether the combination of O4Is with other chemicals can generate hciPSCs. The major focus of this renewal project is the development of a chemical cocktail for the generation of hciPSCs. To get a deeper insight into the genetic and epigenetic alternation in CSKM-mediated reprogramming, proteomic analysis of (biotinylated) O4I4, RNA-seq for transcriptomics analysis, evaluation of chromatin accessibility using ATAC-Seq, and other well-established methods will be performed to identify cellular targets of O4I4. Our preliminary result showed that SOX2 is indispensable for CSKM reprogramming. In addition to further optimization of OCT4 inducing compounds, we intend to synthesize SOX2 inducing compound 1 and 2 (S2I1/2) selected from our screening. We will examine various combinations of chemicals and analyze cellular responses at the single-cell level to achieve our major aim, chemical reprogramming in human somatic cells.