摘要

研究目的：

哺乳动物巨核细胞主要定居于胚胎期卵黄囊、肝脏和成年期骨髓、脾脏等器官中，执行血小板生成、免疫调节、微环境支持等多种重要功能。近期研究发现肺脏也是巨核细胞的重要定居器官，肺脏巨核细胞与同时期胎肝和骨髓巨核细胞的分子特征存在显著差异，在机体血小板产生和免疫调节中可能发挥重要作用。然而，目前关于肺脏巨核细胞的造血起源、发育规律、异质性及血小板产生等领域重要问题尚不清楚。因此，本研究通过谱系示踪小鼠模型明确肺脏巨核细胞的造血起源，并利用单细胞转录组测序结合生物信息学分析解析肺脏巨核细胞的转录组特征变化规律、异质性和产板路径，通过巨核细胞体内移植等实验手段验证肺脏巨核细胞的血小板产生能力和其产生的血小板分子特征，以期为肺脏巨核细胞的研究提供新的认知。

研究方法：

（1）利用Cdh5-CreER;Rosa26^tdTomato造血发育谱系示踪小鼠模型探究发育过程中肺脏巨核细胞的造血起源，同时通过原位免疫荧光、流式细胞分析术等方法解析小鼠肺脏巨核细胞的发育动态；（2）通过STRT-seq单细胞转录组测序技术对E11.5—8周共8个时间点肺脏巨核细胞进行转录组测序，绘制发育过程中肺脏巨核细胞的转录组分子图谱，并解析其分子特征的变化规律；（3）利用Seurat单细胞转录组分析流程及聚类分析等方法解析发育过程中肺脏巨核细胞的异质性，并比较其与肝脏巨核细胞的异质性差异；（4）通过差异基因比较和GO功能注释等分析方法解析巨核细胞免疫亚群的特性，并利用原位免疫荧光和流式细胞分析术鉴定巨核细胞免疫亚群；（5）通过差异基因比较和GO功能注释等分析方法识别巨核细胞微环境支持亚群的分子特征，并通过细胞间相互作用和受配体对分析挖掘其潜在功能；（6）利用倍体检测实验比较肺脏巨核细胞与肝脏巨核细胞的倍体化程度差异，通过拟时序分析解析肺脏、肝脏巨核细胞的产板路径及信号通路的差异；（7）通过生物信息学分析比较肺脏和肝脏巨核细胞血小板产生亚群的差异以揭示肺脏和肝脏巨核细胞的血小板产生功能差别，利用巨核细胞体内移植技术探究肺脏巨核细胞产生血小板能力及所产生的血小板的特性。

研究结果：

（1）肺脏巨核细胞由两波不同的造血起源共同贡献并在发育过程中持续存在

利用Cdh5-CreER;Rosa26^tdTomato造血发育谱系示踪小鼠模型分别标记卵黄囊造血和造血干细胞造血来源的血细胞并对肺脏中巨核细胞进行检测，结果显示卵黄囊造血和造血干细胞造血均能贡献肺脏巨核细胞。对不同发育时期的小鼠肺脏进行流式细胞分析术检测和原位免疫荧光染色，结果表明肺脏巨核细胞在发育过程中持续存在。通过分选出胚胎期和成年期肺脏巨核细胞分别进行体内移植探究两者血小板产生能力的差异，结果显示胚胎期肺脏巨核细胞的血小板产生能力高于成体期肺脏巨核细胞。

（2）随着发育不同生命时期肺脏巨核细胞转录组特征呈现动态变化

通过对不同生命时期小鼠肺脏巨核细胞进行单细胞转录组测序及生物信息学分析，结果表明肺脏巨核细胞的转录组特征随着发育存在一定的动态变化。小鼠出生后，血小板产生能力显著降低而免疫调节特征显著增强。同时，胚胎期的肺脏巨核细胞具有器官发育的特征，提示其在肺脏发育过程中可能发挥重要的功能。

（3）肺脏巨核细胞异质性特征与肝脏巨核细胞存在较大差异

整合不同发育时期肺脏和肝脏巨核细胞单细胞转录组测序数据，根据差异基因的表达情况鉴定到七个巨核细胞亚群。肺脏和肝脏巨核细胞的异质性亚群组成存在明显差异，肺脏巨核细胞以血小板产生亚群、微环境支持亚群和免疫特性的亚群为主。同时，肺脏巨核细胞的异质性亚群的比例随着发育存在动态变化，血小板产生亚群和微环境支持亚群主要分布于出生前，出生后以免疫亚群占主导。

（4）肺脏中存在抗原递呈特性和中性粒特性的两个免疫巨核细胞亚群

通过差异基因分析识别到具有抗原递呈特性和中性粒细胞特性的两个免疫巨核细胞亚群，并鉴定到CD206和CD177可以分别作为两个免疫亚群的表面标志分子。

（5）肺脏巨核细胞微环境支持亚群高表达调控肺脏发育的一系列基因

差异基因和基因集富集分析结果表明肺脏巨核细胞微环境支持亚群高表达调控肺脏发育的一系列基因，具有支持肺脏发育的潜在功能。细胞间相互作用分析提示肺脏巨核细胞微环境支持亚群与多种肺脏基质细胞存在潜在相互作用。

（6）肺脏巨核细胞具有不依赖于多倍体化的独特产板方式

原位免疫荧光和巨核细胞倍体检测实验显示肺脏巨核细胞在整个发育时期均呈现低倍体特征，进一步通过提取肺脏和肝脏巨核细胞的增殖亚群、多倍体化亚群和产板亚群分别进行拟时序分析，我们发现肺脏巨核细胞与肝脏巨核细胞的成熟产板路径信号通路存在差异，提示肺脏巨核细胞具有不依赖于多倍体化的独特产板方式。

（7）肺脏和肝脏来源巨核细胞产生血小板的对比分析

肺脏和肝脏巨核细胞产板亚群的比较提示两者的产板能力和产生血小板的分子特征存在差异。分选出肺脏巨核细胞和肝脏巨核细胞分别进行体内移植血小板产生实验，结果显示虽然肺脏巨核细胞和肝脏巨核细胞均可以产生血小板，但肺脏巨核细胞产生血小板的数量少于肝脏巨核细胞。我们进一步对移植后的血小板进行检测，发现肺脏巨核细胞产生的血小板与肝脏巨核细胞产生的血小板分子特征存在差异。

研究结论：

在本研究中，我们围绕肺脏巨核细胞的造血起源、发育过程中转录组特征的动态变化以及细胞异质性进行探究，发现肺脏巨核细胞由卵黄囊造血和造血干细胞造血共同贡献并在发育过程中持续存在，同时肺脏巨核细胞在发育过程中经历明显的转录组分子特征和细胞异质性的变化，具体表现为血小板产生能力显著降低而免疫调节特征显著增强。细胞异质性分析识别出肺脏中存在抗原递呈特性和中性粒特性的两个免疫巨核细胞亚群，以及表达支持肺脏发育相关基因的肺脏巨核细胞微环境支持亚群。此外，我们研究了肺脏巨核细胞成熟产板路径及其产生的血小板的分子特征，发现肺脏巨核细胞具有不依赖于多倍体化的独特产板方式，且肺脏巨核细胞产生的血小板分子特征与肝脏巨核细胞产生的血小板存在差异。本研究为生理和病理条件下肺脏巨核细胞的研究提供了重要的数据参考，同时对于研究“器官特异的血小板”也具有重要的启示。

Abstract

Objective: Mammalian megakaryocytes primarily settle in the yolk sac and liver during embryonic life and in organs such as the bone marrow and spleen during adulthood. They perform various important functions such as platelet production, immune regulation, and microenvironmental support. Recent studies have revealed that megakaryocytes also reside in the lung, and their molecular characteristics differ significantly from those of fetal liver and bone marrow megakaryocytes. Lung megakaryocytes are enriched for a range of genetic sets for innate immune functions such as inflammatory response and immune response, suggesting that lung megakaryocytes may play an important role in immune regulation. However, current knowledge of lung megakaryocytes is limited to the traditional description of platelet production and molecular characteristics, leaving important questions unanswered about the hematopoietic origin, developmental patterns, heterogeneity, and platelet-producing regulatory mechanisms of lung megakaryocytes. In this research, a lineage-tracing mouse model was used to clarify the hematopoietic origin of lung megakaryocytes, and single-cell transcriptome sequencing was used to clarify their molecular characteristics and heterogeneity to offer new perspectives on lung megakaryocytes.

Methods:(1) To investigate the hematopoietic origin of lung megakaryocytes during development using the Cdh5-CreER;Rosa26^tdTomato lineage tracing mouse model, and also to resolve the developmental dynamics of lung megakaryocytes in mice by in situ immunofluorescence and flow cytometric analysis. (2) Transcriptome sequencing of lung megakaryocytes at eight time points from E11.5 to 8 weeks by STRT-seq single-cell transcriptome sequencing technology to map the transcriptome of lung megakaryocytes during development and to resolve the changes in their molecular characteristics. (3) To resolve the heterogeneity of lung megakaryocytes using the Seurat single cell transcriptome analysis process and cluster analysis, and to compare the differences in heterogeneity between them and liver megakaryocytes. (4) Characterization of megakaryocyte immune subpopulations by differential gene comparison and GO functional annotation, and identification of megakaryocyte immune subpopulations by in situ immunofluorescence and flow cytometric analysis. (5) Identifying the molecular characteristics of niche subpopulations by differential gene comparison and GO functional annotation, and mining their potential functions through intercellular interactions and recipient-ligand pair analysis. (6) To compare the differences in ploidy between lung megakaryocytes and liver megakaryocytes using ploidy detection assays, and to resolve the differences in platelet production pathways and platelet production signaling pathways in lung and liver megakaryocytes by pseudo-time analysis. (7) To compare the differences in platelet-producing subpopulations of lung and liver megakaryocytes by bioinformatics analysis to reveal the differences in platelet-producing functions between lung and liver megakaryocytes and to investigate the platelet-producing capacity of lung megakaryocytes and the properties of the platelets produced using the megakaryocyte in vivo transplantation technique.

Results:

(1) Lung megakaryocytes are derived from dual hematopoietic origins and persist during development. The Cdh5-CreER;Rosa26^tdTomato lineage tracing mouse model was used to label blood cells from yolk sac hematopoiesis and hematopoietic stem cell hematopoiesis and to detect megakaryocytes in the lungs. Flow cytometry and in situ immunofluorescence staining of mouse lungs at different developmental stages showed that lung megakaryocytes persist during development. The difference in platelet production capacity between embryonic and adult lung megakaryocytes was investigated by sorting out embryonic and adult lung megakaryocytes for in vivo transplantation, and the results showed that embryonic lung megakaryocytes had a higher platelet production capacity than adult lung megakaryocytes.

(2) The transcriptome characteristics of lung megakaryocytes show a dynamic change with development. Single-cell transcriptome sequencing and bioinformatics analysis of mouse lung megakaryocytes at different time periods showed that the transcriptome characteristics of lung megakaryocytes showed a certain dynamic pattern with development. After birth, the platelet production capacity was significantly reduced, while the immunomodulatory characteristics were significantly enhanced. At the same time, embryonic lung megakaryocytes were shown to have organogenesis characteristics, suggesting that they may play an important function in lung development.

(3) The heterogeneous characteristics of lung megakaryocytes differ significantly from those of liver megakaryocytes. Single-cell transcriptome sequencing data of lung and liver megakaryocytes at different developmental periods were integrated, and seven megakaryocyte subpopulations were identified based on the expression of differential genes. The composition of heterogeneous subpopulations of lung and liver megakaryocytes was significantly different, with lung megakaryocytes dominated by subpopulations with platelet-producing, niche-supporting, and immune characteristics. At the same time, the proportion of heterogeneous subpopulations of lung megakaryocytes varied dynamically with development, with platelet-producing and niche-supporting subpopulations predominantly distributed before birth and the immune subpopulation predominating after birth.

(4) Two immune megakaryocyte subpopulations with antigen-presenting and neutrophilic properties are present in the lung. Two subsets of immune megakaryocytes with antigen-presenting and neutrophilic properties were identified by differential gene analysis, and CD206 and CD177 were identified as surface marker molecules for the two immune subsets, respectively.

(5) The lung megakaryocyte niche-supporting subpopulation has high expression of genes regulating lung development. Results of differential gene and gene set enrichment analyses suggest that the lung megakaryocyte niche-supporting subpopulation overexpresses a range of genes that regulate lung development and have the potential to support lung development. Intercellular interaction analysis suggested that the lung megakaryocyte niche-supporting subpopulation has potential interactions with a variety of lung stromal cells.

(6) Lung megakaryocytes have a platelet production mode that is not dependent on polyploidization. By performing ploidy assays on megakaryocytes, we found that lung megakaryocytes showed low ploidy characteristics throughout development. By extracting cyc subpopulations, polyploidization subpopulations, and platelet-producing subpopulations of lung and liver megakaryocytes separately for the pseudotime analysis, we found differences in the signaling pathways of platelet-producing pathways between lung megakaryocytes and liver megakaryocytes, suggesting that lung megakaryocytes have a platelet production mode that is not dependent on polyploidization.

(7) A comparative analysis of platelet production by lung and liver-derived megakaryocytes. A comparison of the platelet-producing subpopulations of lung and liver megakaryocytes suggested differences in the platelet-producing capacity and molecular characteristics of platelet production between the two. The results showed that although both lung and liver megakaryocytes could produce platelets, the number of platelets produced by lung megakaryocytes was less than that of liver megakaryocytes. We further examined the platelets after transplantation and found that the molecular characteristics of platelets produced by lung megakaryocytes differed from those of platelets produced by liver megakaryocytes.

Conclusion: In this study, we investigated the hematopoietic origin of lung megakaryocytes, the dynamic changes in transcriptome characteristics, and cellular heterogeneity during development, and found that lung megakaryocytes are contributed by both yolk sac hematopoiesis and hematopoietic stem cell hematopoiesis and persist during development. Furthermore, lung megakaryocytes undergo significant changes in molecular characteristics and cellular heterogeneity during development, as evidenced by a significant decrease in platelet production capacity and a significant increase in immune features. Cellular heterogeneity analysis identified two subpopulations of immune megakaryocytes with antigen-presenting and neutrophilic characteristics, as well as a lung megakaryocyte niche-supporting subpopulation that expresses genes relevant to lung development. In addition, we investigated the platelet production pathway of lung megakaryocytes and the molecular characteristics of the platelets they produce and found that lung megakaryocytes have a unique platelet production mode that is not dependent on polyploidization and that the molecular characteristics of platelets produced by lung megakaryocytes differ from those produced by liver megakaryocytes. This study provides important data for the study of lung megakaryocytes under physiological and pathological conditions, and also has important insights into the study of "organ-specific platelets".