基于CRISPR/Cas9技术对人多能干细胞来源早期成体红系发育的关键分子调控机制的初步探索

Similar to hematopoiesis in embryos, hematopoietic stem/progenitor cells (HSPCs) (CD34+CD45+) could be derived from human pluripotent stem cells (hPSCs) in vitro. Before this, considerable definitive erythroblasts (EryD) are generated. We have reported that these erythroblasts generated before HSPCs (pre-HSPC-EryD) originate from mesoderm/endothelial hematopoiesis. In this study, the surface markers of precursors, progenitors and pre-HSPC-EryD in hPSC/AGM-S3 cocultures will be confirmed respectively. Important controlling genes will be exposed by single-cell qRT-PCR analysis. Then Cas9-hPSC lines with couple regulatory factors will be established based on CRISPR/Cas9 technology to identify the controlling genes’ function. The key signal pathways will be identified by adding small molecules and the production of pre-HSPC-EryD would be greatly improved. The biological characteristics of pre-HSPC-EryD will be compared to EryD derived from human cord blood CD34+ cells. The connection of precursor of pre-HSPC-EryD and endothelial cell will be explored. The foundation would be established for discovering the key regular molecules during its development process. The clinical application of erythroblasts derived from hPSCs will be promoted.

类似胚胎的造血发生过程，人多能干细胞（human pluripotent stem cells，hPSCs）经体外诱导分化可产生造血干/祖细胞（hematopoietic stem/progenitor cells，HSPCs)。在此之前有大量成体特性红细胞生成，我们揭示这些红细胞（命名为pre-HSPC-EryD）带有中胚层/内皮造血特征。本研究将精密界定pre-HSPC-EryD多个祖细胞阶段的表型分子，利用单细胞qRT-PCR分析揭示其发育过程中重要的调控基因。基于CRISPR/Cas9技术构建可调控hPSC株，验证目的基因的调控功能。利用小分子物质鉴定关键信号通路，有望大幅提高pre-HSPC-EryD产量。比较其与脐血CD34+细胞来源红细胞的生物学特性。本项目将探索pre-HSPC-EryD的起源，为阐明其发育的关键分子调控机制奠定基础，促进hPSCs来源红细胞的临床应用。

本研究利用10xGenetics单细胞测序分析，揭示H1/AGM-S3共培养体系很大程度模拟了体内胚胎早期发育时时造血的产生，为体外研究早期成体特性红细胞（命名为pre-HSPC-EryD）提供了很好的研究平台。通过本研究揭示了pre-HSPC-EryD的起源于生血中胚层，利用表型分子可精密界定其发育阶段，利用小分子物质确定发育的关键信号通路为Wnt。多维度优化培养条件，大幅提高pre-HSPC-EryD产量。与共培养体系其他多能造血祖细胞来源以及脐血CD34+细胞来源红细胞的生物学特性进行了比较，提示其很大程度上与EMPs相似。同时探索了基于CRISPR/Cas9技术构建可双示踪标记的hPSC株，摸索利用LncRNA慢病毒文库进行功能筛选。本项目有助于更好理解胚胎早期成体红细胞的发育，为促进hPSCs来源红细胞的临床应用奠定了一定基础。