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

用于生成人类化学诱导多能干细胞的化学品的鉴定、表征和优化

• 批准号: 321028751
• 负责人: Dr. Xinlai Cheng
• 金额: --
• 依托单位: Buchmann Institute for Molecular Life Sciences
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/321028751?language=en
• 关键词: Identification; characterization; optimization; chemicals; generation

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.

转录因子介导的核重编程实现了从体细胞产生诱导多能干细胞（iPSC）。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低重编程效率和培养过程中积累的遗传和表观遗传异常的限制。已经开发了几种新的重编程方法，以获得更有效和更安全的临床级iPSCs。由于化学物质可以顺利进入细胞并且易于操作，因此化学重编程被认为是最有前途的无转基因iPSC方法之一。自2013年以来，当第一个化学重编程的鸡尾酒被释放时，一些化学鸡尾酒已经被独立小组报道用于产生小鼠化学物质iPSC（mciPSC）。然而，针对人ciPSC的化学鸡尾酒还没有，通过我们之前基于细胞的高通量筛选，我们确定了每个转录因子的1000个命中。在批准的DFG项目中，我们鉴定了咪唑并吡啶类化合物作为新型H3 K4特异性脱甲基酶（KDM 5）抑制剂。我们证明了先导化合物，OCT 4诱导化合物3（O 4 I3），抑制H3 K4的去甲基化，从而增强H3 K4 Me 3在OCT 4启动子处的占据，导致患者原代成纤维细胞中重编程效率增加。该结果的一部分已发表在iScience（Cell Press）上。与海德堡大学共同授予了O 4 I3相关的专利，并与其他学术实验室和公司合作正在进行中。此外，基于2015年发表的先导化合物O 4 I2的代谢研究，我们成功地制备了代谢稳定的衍生物（O 4 I4）。O 4 I3/4与SOX 2、KLF 4和MYC的组合实现了人成纤维细胞的重编程，而没有外源性OCT 4。我正在申请一个更新项目，因为在剩下的几个月里，很难回答公开的问题，包括它的作用机制，以及O 4 Is与其他化学品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产生hciPSC的化学鸡尾酒。为了更深入地了解CSKM介导的重编程中的遗传和表观遗传改变，将进行（生物素化的）O 4 I4的蛋白质组学分析，用于转录组学分析的RNA-seq，使用ATAC-Seq评估染色质可及性，以及其他成熟的方法来鉴定O 4 I4的细胞靶点。我们的初步研究结果表明，SOX 2是必不可少的CSKM重编程。除了进一步优化OCT 4诱导化合物之外，我们还打算合成从我们的筛选中选择的SOX 2诱导化合物1和2（S2 I 1/2）。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，人体细胞中的化学重编程。