Tracing the Formation of Haematopoietic Stem Cells in Mouse Embryos by Single-cell Functional and RNA-Seq Analyses [single-cell]

Haematopoietic stem cells (HSCs) are derived early from embryonic precursor cells, such as haemogenic endothelial cells and pre-HSCs. However, the identity of precursor cells remains elusive due to their rareness, transience, and inability to be isolated efficiently. Here we employed potent surface markers to capture the nascent pre-HSCs at 30% purity, as rigorously validated by single-cell-initiated serial transplantation assay. Then we applied single-cell RNA-Seq technique to analyse five populations closely related to HSC formation: endothelial cells, CD45- and CD45+ pre-HSCs in E11 aorta-gonad-mesonephros (AGM) region, and mature HSCs in E12 and E14 foetal liver. In comparison, the pre-HSCs showed unique features in transcriptional machinery, apoptosis, metabolism state, signalling pathway, transcription factor network, and lncRNA expression pattern. Among signalling pathways enriched in pre-HSCs, the mTOR activation was uncovered indispensable for the emergence of HSCs but not haematopoietic progenitors from endothelial cells in vivo. By comparing with proximal populations without HSC potential, the core molecular signature of pre-HSCs was identified. Collectively, our work paves the way for dissection of complex molecular mechanisms regulating the step-wise generation of HSCs in vivo, informing future efforts to engineer HSCs for clinical application. RNA-Seq of 181 single-cell samples from 8 FACS sorted cell types: 1. endothelial cells (samples E11.0_EC_xxxx. CD31+ VE-cadherin+CD41-CD43-CD45-Ter119-); 2. T1 pre-HSCs (samples E11.0_T1_xxxx. CD31+CD45-CD41low c-Kit+CD201high); 3. T1 CD201- cells (samples E11.0_T1CD201neg_xxxx, CD31+CD45-CD41low c-Kit+CD201low/-) ; 4. T2 pre-HSCs (samples E11.0_T2_35xx. CD31+CD45+c-Kit+CD201high), 5. T2 CD41low (samples E11.0_T2_21xx, E11.0_T2_24xx and E11.0_T2_27xx. CD31+CD45+CD41low); 6. E12 HSCs (samples E12.5_FL_xxxx. Lin-Sca-1+Mac-1lowCD201+); 7. E14 HSCs (samples E14.5_FL_xxxx. CD45+CD150+CD48-CD201+); 8. Adult HSCs (samples Adult_HSC_xxxx. CD45+CD150+CD48-CD201+). ECs, T1 pre-HSCs, T1 CD201- cells, T2 pre-HSCs, T2 CD41low cells were sorted from E11 AGM region. Mature HSCs were sorted from E12 or E14 fetal liver and adult bone marrow.造血干细胞（Haematopoietic stem cells, HSCs）早期起源于胚胎前体细胞，例如生血内皮细胞（haemogenic endothelial cells）和前造血干细胞（pre-HSCs）。然而由于这类细胞数量稀少、存在时间短暂且难以高效分离，其具体身份仍未明确。本研究借助高效的表面标志物，以30%的纯度成功捕获了新生的前造血干细胞，并通过单细胞起始的连续移植实验进行了严格验证。随后，我们运用单细胞RNA测序（single-cell RNA-Seq）技术，分析了与造血干细胞形成密切相关的5类群体：E11主动脉-性腺-中肾（aorta-gonad-mesonephros, AGM）区域的内皮细胞、CD45阴性及CD45阳性的前造血干细胞，以及E12和E14胎肝中的成熟造血干细胞。对比分析显示，前造血干细胞在转录调控机制、凋亡过程、代谢状态、信号通路、转录因子网络以及长链非编码RNA（long non-coding RNA, lncRNA）的表达模式上均具有独特特征。在前造血干细胞富集的信号通路中，mTOR通路的激活被证实为体内内皮细胞向造血干细胞而非造血祖细胞分化所必需的条件。通过与不具备造血干细胞潜能的邻近细胞群体进行比较，我们鉴定出了前造血干细胞的核心分子特征。综上，本研究为解析体内调控造血干细胞逐步生成的复杂分子机制奠定了基础，可为未来开展临床级造血干细胞工程化构建的相关研究提供参考。本研究共对8个经荧光激活细胞分选（fluorescence-activated cell sorting, FACS）得到的细胞类群的181个单细胞样本进行了RNA测序，具体类群如下：1. 内皮细胞（样本命名格式为E11.0_EC_xxxx，表型为CD31+ VE-钙粘蛋白+CD41-CD43-CD45-Ter119-）；2. T1型前造血干细胞（样本命名格式为E11.0_T1_xxxx，表型为CD31+CD45-CD41low c-Kit+CD201high）；3. T1型CD201阴性细胞（样本命名格式为E11.0_T1CD201neg_xxxx，表型为CD31+CD45-CD41low c-Kit+CD201low/-）；4. T2型前造血干细胞（样本命名格式为E11.0_T2_35xx，表型为CD31+CD45+c-Kit+CD201high）；5. T2型CD41low细胞（样本命名格式为E11.0_T2_21xx、E11.0_T2_24xx及E11.0_T2_27xx，表型为CD31+CD45+CD41low）；6. E12期造血干细胞（样本命名格式为E12.5_FL_xxxx，表型为Lin-Sca-1+Mac-1lowCD201+）；7. E14期造血干细胞（样本命名格式为E14.5_FL_xxxx，表型为CD45+CD150+CD48-CD201+）；8. 成体造血干细胞（样本命名格式为Adult_HSC_xxxx，表型为CD45+CD150+CD48-CD201+）。其中内皮细胞、T1型前造血干细胞、T1型CD201阴性细胞、T2型前造血干细胞及T2型CD41low细胞均分选自E11期AGM区域；成熟造血干细胞则分别分选自E12或E14期胎肝以及成体骨髓。