基于tag-based单细胞转录组测序解析造血干细胞发育的可变剪接

The hematopoietic stem cell (HSC) of mice is transformed from a small fraction of hemogenic endothelial cells through endothelial-hematopoietic transition. The molecular regulation mechanism during the development of HSC has been an international research hotspot. Alternative splicing is an important mechanism for the diversity and complexity of transcriptomes and proteomes in organisms. It has been shown that it can participate in the fine regulation of embryonic hematopoietic development. In the preliminary work, we achieved the high-precision separation of single cells of many important types of cells during HSC development for the first time in the world. Recently, we used new surface markers to capture hemogenic endothelial cells. Based on the above important breakthroughs, this study intends to develop an alternative splicing recognition algorithm for tag-based sequencing data, combined with the single-cell tag-based sequencing data reported by the research group and the literature, to further analyze the alternative splicing dynamic expression profile and regulatory function during embryonic HSC development. This project is expected to provide an important theoretical basis for a comprehensive understanding of HSC development and guidance of HSC regeneration.

小鼠的造血干细胞（hematopoietic stem cell, HSC）由一小部分具有生血潜能的生血内皮细胞经过内皮-造血转化而来。HSC发育过程中的分子调控机制一直是国际上的研究热点。可变剪接是造成生物体内转录组和蛋白质组多样性和复杂性的重要机制，已有研究表明其可参与胚胎期造血发育的精细调节。前期工作中我们在国际上首次实现了HSC发育中多个重要类型细胞的单细胞高精度分离，近期又利用全新的表面标志对生血内皮细胞进行进准捕获。基于以上重要突破，本研究拟开发基于tag-based数据的可变剪接识别算法，结合本课题组自产及文献报道的单细胞tag-based测序数据，深入解析胚胎HSC发育过程中的可变剪接动态表达谱及调控功能。本项目有望为全面认识HSC发育和指导HSC再生提供重要的理论依据。

成体造血干细胞（HSC）被认为是在胚胎中期由生血内皮细胞（HEC）诞生的。由于HEC数量稀少且具有一过性等原因，具有HSC能力的HEC从未被严格鉴定和准确捕获，更不用说它们的真正血管前体。我们首先使用高精度的单细胞转录组测序方法，采用无偏方式分离相关的内皮细胞（EC）群体，并从胚胎天（E）9.5到E11.0以0.5天为间隔进行连续发育阶段取样。通过生物信息学分析，我们从主动脉-生殖腺-中肾（AGM）区域的主动脉中鉴定出两种分子上不同的动脉EC群体和具有HSC潜能的HEC。HEC数量在E10.0达到峰值，之后急剧减少。结合计算预测和体内功能验证，我们通过新构建的Neurl3-EGFP报告小鼠模型准确捕获具有HSC能力的HEC，并通过表面标记（Procr+Kit+CD44+，PK44）进一步富集。令人惊讶的是，单个HEC的培养中少量但稳定地观察到内皮-造血双潜能。值得注意的是，从E8.0开始的原始血管EC经历了两个命运选择步骤，成为HSC-primed HEC，即首先是动脉命运选择，然后是造血命运转化。综上所述，全面了解内皮细胞向HSC-primed HEC的进化和分子程序，将有助于未来在体外进行HSC的生产研究。