Single cell mRNA sequencing of de novo hematopoiesis from the fetal lung

Hemogenic endothelium, which gives rise to the earliest HSCs, is thought to be isolated to very early stages of development, peaking at E10.5 in mice. However, our data suggests there may be hemogenic endothelium capable of giving rise to de-novo HSPCs in the fetal lungs of mice at E17. We've adapted a platform to recapitulate the ex-vivo development of HSPCs from E17 lung endothelium. This sequencing experiment will help us to validate whether what we are seeing in culture is indeed endothelial to hematopoietic transition (EHT) and possibly identify the subset of endothelial cells that give rise to HSPCs. Overall design: E17 timed pregnant Wild Type C57BL6 mice are sacrificed at E17. The fetal lungs are isolated and digested with liberase into a single cell suspension. These cells are frozen down in 10% DMSO and replated on a thin later of diluted matrigel with HSPC media (containing mTPO, IL6, SCF, FGF, VEGF, Flt3). On D3, wells are washed with PBS and replaced with fresh media. On D4, D5 and D6, suspension cells are collected prepped for 10x. Adherent layers are digested with accutase, and sorted to enrich for live-VECAD+ cells, which are then prepped for 10x.

生血内皮细胞（hemogenic endothelium）是产生最早造血干细胞（hematopoietic stem cells, HSCs）的细胞类群，此前学界普遍认为其仅局限于发育极早期阶段，在小鼠胚胎发育第10.5天（E10.5）达到发育峰值。然而本研究数据显示，在小鼠胚胎发育第17天（E17）的胎肺中，或许存在能够新生造血干祖细胞（hematopoietic stem and progenitor cells, HSPCs）的生血内皮细胞。

本研究已改造适配一套实验平台，可重现E17胎肺内皮细胞体外生成HSPCs的发育过程。本次测序实验将用于验证体外培养体系中观察到的细胞转化过程是否确为内皮细胞向造血细胞转化（endothelial to hematopoietic transition, EHT），并有望筛选出可产生HSPCs的内皮细胞亚群。

整体实验设计：
选取已知受孕天数的野生型C57BL/6品系孕鼠，在胚胎发育第17天（E17）处死孕鼠。分离其胎肺，使用Liberase试剂消化为单细胞悬液。将细胞以10%二甲基亚砜（dimethyl sulfoxide, DMSO）作为冷冻保护剂冻存，随后铺于稀释的基质胶（matrigel）薄层上，使用HSPCs专用培养基（含mTPO、IL6、SCF、FGF、VEGF、Flt3）进行重铺培养。
培养至第3天（D3），采用磷酸盐缓冲液（phosphate buffered saline, PBS）洗涤培养孔并更换为新鲜培养基。分别于培养第4、5、6天收集悬浮细胞，用于10x Genomics单细胞测序文库制备。对贴壁细胞层使用Accutase消化，通过流式分选富集活细胞VECAD+阳性群体，随后同样制备10x Genomics单细胞测序文库。