目的：造血干细胞（Hematopoietic stem cells，HSCs）处于造血级联的顶端，是造血系统建立和终身维持的基础。围产期造血发育作为胚胎HSCs和成体HSCs的关键过渡阶段鲜有研究。围产期还具有多位点造血的特点，HSCs同时存在于肝脏、脾脏、骨髓中，其组织间转录组及功能仍不明确，胚胎组织微环境细胞对于造血的支持作用也少有报道。肥大细胞起源于胚胎早期，是介导多种生物学功能的固有免疫细胞，能够合成和分泌5-羟色胺（5-hydroxytryptamine，5-HT）。5-HT是已知的造血支持因子，但肥大细胞、5-HT和HSCs间的关系仍是领域空白。本研究旨在通过高通量的10X Genomics单细胞转录组测序技术及体内外功能实验解析小鼠围产期造血特征，明确在此阶段特异出现的肥大细胞对于HSC的支持功能，并验证5-HT在该过程中的作用机制，以完善我们对于HSCs发育生物学及肥大细胞生物学的认识，为相关领域内研究提供新线索。

方法：1）利用流式细胞仪分选围产期（E18.5、PD1、PD2、PD5）C57BL/6J 小鼠骨髓、脾脏、肝脏的CD45⁺Lineage^-c-Kit⁺造血干/祖细胞并进行10X Genomics 3’端单细胞转录组文库的制备和测序，测序数据经质控处理、降维聚类及分群注释后，建立小鼠围产期多位点造血干/祖细胞（Hematopoietic stem and progenitor cells，HSPCs）的单细胞转录组图谱并找到肥大细胞特征群体及其表面标志分子。2）利用流式细胞仪分选并检测该肥大细胞特征群体数量及表面标志物的动态变化，借助瑞氏-吉姆萨、甲苯胺蓝、免疫荧光等染色方法明确该肥大细胞群体的形态特征和成熟身份，设计体外培养脱颗粒率测定实验验证其成熟功能。3）将既往发表文献中的多组织单细胞转录组数据与本文中PD1骨髓、脾脏肥大细胞进行数据整合、去批次、降维可视化，构建小鼠肥大细胞的跨组织转录组图谱。将本文中围产期造血组织肥大细胞的单细胞转录组单独进行亚群分析，基于差异表达基因分析、基因集打分等方法解析小鼠各组织肥大细胞的异质性及围产期造血组织肥大细胞的动态变化。4）利用免疫荧光染色技术确定围产期小鼠脾脏肥大细胞与HSCs的共定位关系；建立肥大细胞与HSCs的共培养实验体系，借助流式细胞术和移植实验证明围产期小鼠脾脏肥大细胞在体外共培养中对HSCs的支持作用，转录组测序数据分析富集到5-HT相关通路。5）利用酶联免疫吸附实验检测5-HT的表达水平，通过体外抑制剂实验验证围产期小鼠脾脏肥大细胞分泌的5-HT支持造血的作用。6）构建肥大细胞清除小鼠模型，利用流式细胞术、半固体集落形成实验、单细胞液体培养实验及移植实验检测及验证肥大细胞清除小鼠模型的围产期脾脏造血缺陷表型。7）通过流式细胞术、瑞氏-吉姆萨染色、免疫荧光技术及共培养实验探究人胚胎造血组织中肥大细胞的特征及功能。

结果：1）建立小鼠围产期多个造血位点HSPCs的单细胞转录组图谱，共包括围产期5个时间点、3个组织来源共133669个HSPCs，描绘了围产期各组织HSPCs成分的动态变化，发现围产期“一过性”出现的、骨髓脾脏特异性的肥大细胞特征群体。2）由单细胞转录组数据筛选得到肥大细胞特征群体的表面标志分子组合c-Kit⁺CD200R⁺，鉴定该群体具有典型的成熟肥大细胞形态，高表达ST2、CD63、CD16/32、Sca-1和Integrin-β7等分子，体外培养脱颗粒率高于成体腹膜腔肥大细胞证明其功能性。3）扩充现有小鼠肥大细胞的跨组织转录组图谱，包含新生小鼠骨髓、脾脏、肌肉、皮肤以及成体小鼠腹膜腔、皮肤等12种组织，描述了小鼠不同组织及发育阶段肥大细胞的转录组特征，发现新生小鼠骨髓、脾脏肥大细胞类似于结缔组织肥大细胞，同时也具有高增殖、高凋亡、高合成（组胺、5-HT、糜蛋白酶及细胞因子）的特点。4）围产期骨髓、脾脏肥大细胞亚群的动态变化及其不同时间点的转录组特征说明该肥大细胞群体在围产期早期处于增殖、迁移状态，中期处于耗能、蛋白合成阶段，晚期介导造血调控、炎症反应及凋亡。5）围产期小鼠脾脏肥大细胞与HSCs具有共定位关系，在体外共培养中肥大细胞能够扩增HSCs并维持其干性。6）与肥大细胞共培养的HSCs富集5-HT摄取通路，5-HT及其受体抑制剂特异性消除肥大细胞对HSCs的支持作用，证明围产期小鼠脾脏肥大细胞在体外通过5-HT维持HSCs功能。7）建立肥大细胞清除小鼠模型，主要验证了该模型小鼠围产期脾脏造血的缺陷：HSPCs数量减少、体外集落形成能力减弱、移植重建能力下降，还观察到体内清除肥大细胞的围产期小鼠脾脏中的5-HT的表达水平降低。8）发现人胚胎发育早期骨髓、脾脏及肝脏中存在肥大细胞，其动态变化及功能与小鼠类似。

结论：本研究建立了小鼠围产期多个造血位点HSPCs的单细胞转录组图谱，描绘了围产期各组织HSPCs的动态变化，发现并鉴定了围产期脾脏、骨髓中特异“一过性”存在的成熟肥大细胞。数据扩充了现有小鼠肥大细胞的跨组织转录组图谱，提出现有肥大细胞分类的局限性，详细梳理了以新生小鼠骨髓、脾脏为主的各组织肥大细胞转录组特点。本研究发现了围产期小鼠脾脏肥大细胞与HSCs共定位，证明了其在体外共培养中能够通过造血支持因子5-HT扩增HSCs并维持其干性。肥大细胞清除模型小鼠围产期脾脏存在造血上游HSPCs的数量及功能缺陷，验证了肥大细胞在体内的造血支持作用，同时肥大细胞清除模型小鼠围产期脾脏中5-HT的表达水平降低提示该分子在体内的重要作用。初步鉴定了人胚胎造血组织中也存在一过性出现的成熟肥大细胞，其特征及功能与小鼠类似，说明肥大细胞对早期造血发育的参与具有物种间保守性。

Objective: Hematopoietic stem cells (HSCs) reside at the apex of the hematopoietic hierarchy and are fundamental for the establishment and lifelong maintenance of the hematopoietic system. Perinatal hematopoietic development, a critical transitional phase between embryonic HSCs and adult HSCs, remains underexplored. This period is also featured with multicentric hematopoiesis, with HSCs concurrently residing in the liver, spleen, and bone marrow, yet their inter-tissue transcriptomic profiles and functions are unclear. Additionally, the supportive role of embryonic tissue microenvironment cells in hematopoiesis is scarcely reported. Mast cells, originating early in embryonic development, are innate immune cells mediating various biological functions and can synthesize and secrete 5-hydroxytryptamine (5-HT). 5-HT is a known hematopoietic support factor, yet the relationship among mast cells, 5-HT, and HSCs remains an unexplored domain. This study aims to elucidate the multicentric hematopoiesis in perinatal mice using high-throughput single-cell transcriptomic sequencing technologies of 10X Genomics, to define the supportive role of mast cells characteristic of this stage on HSCs, and to validate 5-HT as a key factor in this process. This will enhance our understanding of HSC developmental biology and mast cell biology, offering new insights for research in these fields.

Methods: 1) Hematopoietic stem and progenitor cells (HSPCs) expressing CD45+Lineage-c-Kit+ from the perinatal (E18.5, PD1, PD2, PD5) bone marrow, spleen, and liver of C57BL/6J mice were sorting using flow cytometry. Subsequently, a 10X Genomics 3’ end single-cell transcriptomic library was prepared and sequenced. After quality control, dimensionality reduction, unsupervised clustering analysis, and cell cluster annotation, a single-cell transcriptomic atlas of perinatal multi-center HSPCs in mice was established. This atlas facilitated the identification of the characteristic population of mast cells and their surface markers. 2) Sort and monitor the dynamic changes in the quantity and surface markers of the characterized mast cell population. Morphological characteristics and the maturity of this mast cell population were confirmed through staining techniques such as Wright-Giemsa, toluidine blue, and immunofluorescence. An in vitro culture experiment was designed to measure the degranulation rate, validating the functional maturity of these mast cells. 3) Single-cell transcriptomic data from previously published multi-tissue studies were integrated with data from PD1 bone marrow and spleen mast cells in this study, employing batch effect removal and dimensionality reduction for visualization. This integration facilitated the construction of a cross-tissue transcriptomic atlas of mouse mast cells. Furthermore, sub-group analysis of single-cell transcriptomes of perinatal hematopoietic tissue mast cells was performed independently. This analysis utilized differential expressed gene (DEGs) analysis and gene set scoring methods to elucidate the heterogeneity of mast cells across tissues and the dynamic changes in perinatal hematopoietic tissue mast cells. 4) Using immunofluorescence staining techniques, the co-localization relationship of mast cells and HSCs in the spleen of perinatal mice was determined. A co-culture system involving mast cells and HSCs was set up, and through the application of flow cytometry, transcriptomic sequencing, and transplantation experiments, the supportive role of perinatal mouse spleen mast cells on HSCs in vitro was demonstrated. The functional enrichment analysis of the DEGs of transcriptomic sequencing data further enriched the 5-HT-related pathways. 5) Through ELISA assays and in vitro inhibitor experiments, the study validated that perinatal mouse splenic mast cells support hematopoiesis ex vivo via the secretion of 5-HT. 6) Construct a mast cell-depleted mouse model, and utilize flow cytometry, semi-solid clonal assays, single-cell liquid culture experiments, and transplantation experiments to detect and validate the perinatal splenic hematopoietic deficiency phenotype. 7) Exploring the characteristics and functions of mast cells in human embryonic hematopoietic tissues using flow cytometry, Wright-Giemsa staining, immunofluorescence techniques, and co-culture Experiments.

Results: 1) Establish a single-cell transcriptomic atlas of perinatal mouse HSPCs across multiple centers, encompassing five time points and three tissue sources with a total of 133,669 HSPCs. This atlas illustrates the dynamic composition of HSPCs in different tissues during the perinatal period, revealing a “transient” population of mast cells specific to the bone marrow and spleen during this phase. 2) Using single-cell transcriptomic data, identify a characteristic combination of surface markers (c-Kit⁺CD200R⁺) on this group of mast cells, which exhibit typical features of mature mast cells. These cells highly express molecules such as ST2, CD63, CD16/32, Sca-1, and Integrin-β7. The higher degranulation rate in vitro, compared to mature peritoneal mast cells, confirms their functionality. 3) The existing cross-tissue transcriptomic atlas of mouse mast cells was expanded to include tissues from neonatal mice (bone marrow, spleen, muscle, and skin) and adult mice (peritoneal cavity and skin). This expanded analysis highlighted the transcriptomic features of mast cells across different tissues and developmental stages, revealing similarities between neonatal bone marrow and spleen mast cells and connective tissue mast cells, as well as characteristics such as high proliferation, apoptosis, and synthesis (histamine, serotonin, proteases, and cytokines). 4) The dynamic changes and transcriptional features of bone marrow and spleen mast cell sub-groups during the perinatal period indicate that these cells are in a state of proliferation and migration in early perinatal stages, shifting towards energy consumption and protein synthesis in the middle stages, and later mediating hematopoiesis regulation, inflammatory responses, and apoptosis. 5) Perinatal mouse spleen mast cells were found to co-localize with HSCs, and in vitro co-cultures demonstrated the ability of mast cells to expand HSCs while maintaining their stemness through the secretion of hematopoietic support factor 5-HT. 6) HSCs co-cultured with mast cells showed high expression of the 5-HT uptake pathway. The specific inhibition of 5-HT and its receptors eliminated the supportive effects of mast cells on HSCs, confirming that perinatal mouse spleen mast cells support hematopoiesis in vitro through 5-HT. 7) A mast cell depletion mouse model was established, mainly verifying a deficiency in perinatal spleen hematopoiesis: a decrease in HSPC numbers, reduced ex vivo colony-forming capacity, and diminished transplant reconstruction ability. Additionally, a reduction in 5-HT levels was observed in the spleens of perinatal mice following in vivo mast cell depletion. 8) It has been discovered that mast cells are present in the bone marrow, spleen, and liver during the early stages of human embryonic development, exhibiting dynamic changes and functions similar to those observed in mice.

Conclusions: This study successfully established a single-cell transcriptomic atlas of perinatal multi-center HSPCs in mice, detailing the dynamic changes in HSPCs across various tissues during the perinatal period and identifying a specific “transient” population of mature mast cells in the spleen and bone marrow. The data expanded the existing cross-tissue transcriptomic atlas of mouse mast cells, challenging current classifications of mast cells and thoroughly detailing the transcriptomic characteristics of various tissues, primarily new-born mouse bone marrow and spleen. This study revealed co-localization and in vitro co-culture hematopoietic support of perinatal mouse spleen mast cells with HSCs via 5-HT. The mast cell depletion model highlighted hematopoietic support defects in perinatal spleen, validating the in vivo role of mast cells in hematopoiesis, and suggested a critical role for 5-HT in vivo. Initial identification of transient mature mast cells in human embryonic hematopoietic tissues with similar characteristics and functions to mice indicates a conserved role of mast cells in early hematopoietic development across species.