Wnt通路调控不同分化潜能细胞iPS诱导进程的分子机制及应用

Induced pluripotent stem cells (iPS cells) were derived by forced expression of four transcription factors-Oct4, Sox2, Klf4 and c-Myc in differentiated somatic cells. iPS cells possess both the pluripotency similar to embryonic stem cells (ES cells) and advantage of convenient cell source which make them invaluable in regenerative medicine. But reprogramming is a time-consuming process with low efficiency and the mechanism is unclear, making the technique far from mature. We have established secondary reprogramming platform based on iPS mice (Cell Stem Cell,2009). In recent studies, we had found the differences existing between the reprogramming processes starting with hematopoietic cells and mouse embryonic fibroblasts which served as the most used starting cells for reprogramming study. And hematopoietic cells with different differentiation potential also varied in kinetics of iPS cell derivation. Through mRNA deep sequencing, we found the key components in Wnt signaling pathway such as β-catenin and Gsk-3β had different behaviors in different cells' reprogramming. And Gsk-3β inhibitor can dramatically increase the reprogramming efficiency of hematopoietic cells. In the following study, we'll investigate these key components in Wnt signaling with specific behaviors in different cells. Examine their function in starting cells and iPS cells as while as their impacts on the velocity and efficiency on iPS cell derivation utilizing gene manipulation strategies such as over expression, knockdown, site mutation, recue and so on. Based on these studies, we want to find different patterns of Wnt signaling in different cells. Understanding the relationship between Wnt signaling regulation and different reprogramming processes of different cells through interfere or construct these specific Wnt regulation patterns. Thus to search the reprogramming barriers starting with cells with low differentiation potential, provide new clues for understanding the molecular mechanism of reprogramming especially starting with hematopoietic cells possessing greatest clinic value, and promote the improvement and application of iPS technology.

诱导多能干细胞(iPS)类似胚胎干细胞并易于获得，最有潜力应用于再生医学治疗。然而重编程过程长时低效，机制不明是其应用瓶颈。申请人已成功建立基于iPS小鼠的二次重编程平台(Cell Stem Cell,2009)，基于该平台，近期研究发现造血细胞的重编程过程与成纤维细胞有所不同，不同分化潜能的造血细胞诱导进程也有差异，通过mRNA深度测序发现Wnt通路的关键分子β-catenin，Gsk-3β等在不同细胞重编程过程中的变化不同（见研究基础）。课题组拟针对这些特异变化的Wnt通路关键分子，通过基因功能研究检测它们在初始细胞和iPS中的作用及对重编程效率速率的影响，建立不同细胞中Wnt通路的作用模式和分子基础，通过对其作用模式的操作解析Wnt通路调控与不同细胞在重编程中表型的关系，寻找低分化潜能细胞中可能存在的限制因素，为理解重编程机制尤其是最具临床应用价值的造血细胞重编程机制提供科学依据。

诱导多能干细胞(iPS)类似胚胎干细胞并易于获得，最有潜力应用于再生医学治疗。然而重编程过程长时低效，机制不明是其应用瓶颈。本项目通过对不同造血细胞和成纤维细胞重编程的定性定量检测发现不同细胞具有不同表型，分析其在重编程早期的基因表达模式，发现Wnt信号通路在造血细胞中有明显激活，继而我们发现Wnt信号通路在不同细胞重编程中发挥不同功能，该通路主要转录因子Tcf蛋白家族在其中起到重要作用，通过激活Wnt通路可显著提高血细胞重编程效率，并可能在未来的临床应用中发挥作用，我们的研究从不同细胞类型的角度探究体细胞重编程的分子机制，为理解重编程机制尤其是最具临床应用价值的造血细胞重编程机制提供了科学依据。除此之外，我们通过结合胚胎发育以及肿瘤发生等与干细胞和重编程的关系发现并阐述了Obox1和Pkm2在重编程中的作用，对了解重编程的调控机制提供助力。