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

转录因子介导的核重编程实现了体细胞诱导多能干细胞（iPSC）的生成。这项技术开启了再生医学的新纪元。然而，其临床应用在很大程度上受到重编程效率低和培养过程中积累的遗传和表观遗传异常的限制。一些新的重编程方法已经被开发出来，以更有效和更安全的方法获得临床级的iPSCs。由于化学物质可以顺利进入细胞并且易于操作，化学重编程被认为是无转基因iPSCs最有前途的方法之一。自2013年首次发布化学重编程鸡尾酒以来，独立研究小组已经报道了一些化学鸡尾酒，用于生成小鼠化学iPSCs （mciPSCs）。然而，目前还没有一种用于人类ciPSCs的化学混合物。通过我们之前基于细胞的高通量筛选，我们确定了每种转录因子的1000个命中。在已批准的DFG项目中，我们确定咪唑吡啶是新型h3k4特异性去甲基化酶（KDM5）抑制剂。我们证明，先导化合物OCT4诱导化合物3 （O4I3）抑制H3K4的去甲基化，从而增强H3K4Me3在OCT4启动子处的占据，从而提高患者原代成纤维细胞的重编程效率。这一结果的一部分已经发表在《iScience》（细胞出版社）上。与海德堡大学共同授予O4I3相关专利，与其他学术实验室和公司的合作正在进行中。此外，基于2015年发表的先导化合物O4I2的代谢研究，我们成功制备了代谢稳定的衍生物（O4I4）。O4I3/4与SOX2、KLF4和MYC的结合实现了人成纤维细胞在没有外源OCT4的情况下的重编程。我正在申请更新项目，因为在剩下的几个月里很难回答开放性的问题，包括它的作用机制，以及O4Is与其他化学物质的结合是否可以产生hciPSCs。该更新项目的主要重点是开发用于生成hciPSCs的化学混合物。为了更深入地了解cskm介导的重编程中的遗传和表观遗传变化，将进行（生物素化）O4I4的蛋白质组学分析，转录组学分析的RNA-seq，使用ATAC-Seq评估染色质可及性，以及其他成熟的方法来确定O4I4的细胞靶点。我们的初步结果表明SOX2在CSKM重编程中是必不可少的。在进一步优化OCT4诱导化合物的基础上，我们拟合成筛选出的SOX2诱导化合物1和2 （S2I1/2）。我们将研究化学物质的各种组合，并在单细胞水平上分析细胞反应，以实现我们的主要目标，即人类体细胞中的化学重编程。

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.