Tracing the formation of hematopoietic stem cells in mouse embryos by single-cell functional and RNA-Seq analyses [10-cell]

Hematopoietic stem cells (HSCs) in adult are specified early from the endothelium-derived precursors (e.g., hemogenic endothelium, and pre-HSCs) in mouse mid-gestation embryos, the detailed process, however, is still largely unknown due to their rareness, transience, and current inability to prospectively isolate them efficiently . Here we developed a potent set of surface markers that could capture the earliest emerging HSCs, the CD45- pre-HSCs with high accuracy and purity, as rigorously and functionally verified by single-cell-initiated serial transplantation assays. Then we applied single-cell RNA-Seq technique to analyze five populations related to HSC formation: the CD45- (type 1) and CD45+ (type 2) pre-HSCs as well as endothelial cells in the E11 AGM region; and later mature HSCs in the E12 and E14 fetal livers. Compared to other cell populations, both type 1 and type 2 pre-HSCs have their unique signatures of transcription machinery, transcription factor network, signaling pathway, cell cycle status, metabolism state, and lncRNA expression patterns. Our work paves the way for dissection of the complex molecular mechanisms regulating the step-wise formation of HSCs from endothelial cells, thus informing future efforts on engineering HSCs for clinical application. RNA-Seq of 35 10-cell pooled samples from 5 FACS sorted cell types, i.e., endothelial cells (ECs), T1 and T2 pre-HSCs from E11 AGM region, as well as mature HSCs from E12 and E14 fetal liver成体造血干细胞（Hematopoietic stem cells, HSCs）早期起源于小鼠妊娠中期胚胎中源自内皮的前体细胞（例如生血内皮细胞、前造血干细胞（pre-HSCs）），然而由于此类细胞数量稀少、存在时间短暂，且目前尚无法高效实现前瞻性分离，其具体形成过程仍在很大程度上未被阐明。本研究开发了一套高效的表面标志物组合，可高精度、高纯度地捕获最早出现的造血干细胞——CD45阴性的前造血干细胞（pre-HSCs），该成果经单细胞起始的连续移植实验得到了严格的功能验证。随后，本研究运用单细胞RNA测序（single-cell RNA-Seq）技术分析了5类与造血干细胞形成相关的细胞群：E11时期主动脉-性腺-中肾区（aorta-gonad-mesonephros, AGM）的CD45阴性（1型）与CD45阳性（2型）前造血干细胞，以及内皮细胞；还包括E12、E14时期胎肝中的成熟造血干细胞。与其他细胞群相比，1型与2型前造血干细胞均具有独特的转录机器、转录因子网络、信号通路、细胞周期状态、代谢状态以及长链非编码RNA（long non-coding RNA, lncRNA）表达谱特征。本研究为解析从内皮细胞逐步形成造血干细胞的复杂分子机制铺平了道路，可为未来工程化制备造血干细胞用于临床应用提供理论支撑。本数据集包含35份10细胞混合样本的RNA测序（RNA-Seq）数据，样本来源于5种经荧光激活细胞分选（FACS）纯化的细胞类型，即E11 AGM区域的内皮细胞（ECs）、1型和2型前造血干细胞，以及E12、E14胎肝中的成熟造血干细胞。