研究目的：红细胞是人体内数量最多的血细胞，也是最早出现的血细胞之一。前期对于人类红细胞发育等层面的研究多聚焦于成体，而对于人体胚胎发育早期红细胞的分子特征知之甚少。传统观点认为，红细胞是一种具有专性携氧功能的血细胞。不过也有研究数据表明，红细胞兼具了免疫调控的功能，特别是在机体处于感染或者肿瘤等应激状态下。然而，这种免疫调控功能是所有红细胞均具有，还是仅存在于部分红细胞群体仍不清晰。此外，对于具有免疫调控功能的红细胞的分子特征尚不明确。本研究通过收集贯穿人体多个发育位点（卵黄囊、胎肝、早产及足月儿脐带血和成体骨髓）的有核红细胞，并对其进行单细胞维度的研究，揭示了人胚发育早期有核红细胞的独特分子特征，解析了人类有核红细胞的功能异质性，并鉴定和验证了免疫调控红细胞亚群的存在。

研究方法：本研究以人类有核红细胞为研究对象，借助流式细胞分选术，收集了来自卵黄囊、胎肝、早产及足月儿脐带血和成体骨髓的有核红细胞，使用10x Genomics和STRT-seq平台完成建库和单细胞转录组测序，后续利用生物信息学的手段对其进行更加深入的下游分析。首先确定了有核红细胞的异质性，解析了各个细胞亚群的特征性基因和生物学通路，构建了有核红细胞分化的路径。随后，在细胞亚群水平，通过对不同发育位点的有核红细胞进行差异分析，揭示了其发育位点特异性。最后，对于免疫调控红细胞亚群，本研究从免疫相关分子通路、特征性基因、转录调控网络等层面进行了分析，并筛选了该亚群的细胞表面标志分子，通过原位多色免疫荧光染色和流式细胞术的方法确定了该细胞群体的存在。此外，通过与经典免疫细胞进行共培养实验，在细胞因子分泌、基因表达和蛋白质表达多个层面对其进行了解析。配体-受体对互作分析还鉴定出免疫调控红细胞与其他经典免疫细胞的互作分子对，并利用原位多色免疫荧光染色对其进行了验证。

研究结果：（1）人类有核红细胞存在功能异质性亚群。将各个发育位点的有核红细胞进行整合分析，从中鉴定出四个特征分明的异质性亚群，包括三个行使经典红细胞功能的亚群和一个具有免疫特征的亚群。同时，尽管比例不同，但四个亚群在不同发育位点均存在。（2）免疫调控红细胞亚群拥有独特的分化路径。拟时序分析和拓扑结构分析发现，免疫调控红细胞拥有不同于经典红细胞的分化路径。经典红细胞的三个亚群距离较近，并且具有逐渐成熟的分化路径；而免疫调控红细胞存在独特的路径，与经典红细胞呈现出两个分化方向。（3）卵黄囊、胎肝和脐带血来源红细胞的独特分子特征。对来自不同发育位点的有核红细胞各亚群进行平行分析，发现在非成熟阶段，卵黄囊来源的有核红细胞高表达的差异基因多富集在胆固醇合成、有氧糖酵解等通路，胎肝有核红细胞主要富集了蛋白质合成相关通路，而脐带血来源有核红细胞则表现出相对较高的血红蛋白基因的表达；在成熟阶段，卵黄囊来源有核红细胞表现出特异性高表达的金属硫蛋白基因（MT2A, MT1G）和锌转运相关基因（SLC30A, SLC39A），并且兼具了高增殖的特征。（4）免疫调控红细胞兼具红系和免疫双重转录调控网络。转录因子活性分析发现，三群经典红细胞拥有着与各种成熟阶段相对应的转录因子富集，包括分化早期的MYC、GATA1、KLF1，分化末期的NFE2、TAL1等；免疫调控红细胞不仅富集了MYC、MYB、GATA1等红系相关转录因子，而且还特异性富集到MEF2C、SPI1、FOS、JUN等免疫相关转录因子，这些转录因子的靶基因同样富集到趋化因子信号通路、抗原处理和递呈等免疫相关通路。（5）免疫调控红细胞的鉴定。免疫应答的激活和细胞因子介导的信号通路相关基因和VSIR、TGFB1、TNFSF10在免疫调控红细胞亚群特异性高表达。通过筛选，发现CD63是免疫调控红细胞特异性最强且表达量最高的细胞表面标记分子，来自胎肝和脐带血样本的有核红细胞中也的确存在CD63阳性细胞亚群，比例约占GYPA⁺CD71⁺有核红细胞的2.58%和6.36%。原位免疫荧光染色同样发现了GYPA⁺CD63⁺细胞的存在，并且在蛋白水平发现这些双阳性细胞高表达HLA-E和TGF-b等。利用流式细胞术分选CD63阳性和CD63阴性红细胞，并将其与外周血单个核细胞进行共培养。相较于CD63阴性组，CD63阳性组CCL4、CXCL8、IFITM3等免疫相关基因表达显著增加，蛋白水平上发现IL-8、IL-6、TNF等在CD63阳性组同样显著增加。此外，将其与CD11b阳性的细胞进行共培养，发现CD63阳性红细胞能够显著促进CD11b阳性细胞TNF的表达。（6）各发育位点免疫调控红细胞的平行比较及其与免疫细胞的互作分析。利用各个发育位点免疫调控红细胞的特征性高表达基因进行相关性分析，发现卵黄囊、胎肝和脐带血来源的免疫调控红细胞距离较近，并且共享的基因数量也较多，而成体骨髓来源的免疫调控红细胞则距离三者较远，共享的基因数量也较少。基于各发育位点免疫调控红细胞的特异性高表达基因进行富集分析，发现卵黄囊免疫调控红细胞主要富集在低氧应答、胚胎器官发育等通路，胎肝免疫调控红细胞主要富集核糖体的生物合成、线粒体翻译等通路，脐带血免疫调控红细胞则富集在白细胞增殖、抗原加工和递呈等先天性免疫相关通路，而成体骨髓免疫调控红细胞富集的通路则包括淋巴细胞激活、T细胞活化的调控等继发性免疫相关通路。配体-受体对的互作分析结果显示，相对于经典红细胞，免疫调控红细胞与免疫细胞富集到的配体-受体对数量更多，强度更大。原位免疫荧光染色结果也对互作分析的结果进行了验证。在人胎肝切片中，CD63阳性的免疫调控红细胞与CD56阳性的NK细胞通过HLA-E-KLRC1互作，在空间上紧邻；与CD20阳性的B细胞，CD63阳性的免疫调控红细胞则通过MIF-CD74分子对进行互作，在空间位置上两类细胞也相互接触。

研究结论：本研究在单细胞维度整合分析了贯穿人体多个发育位点（卵黄囊、胎肝、早产及足月儿脐带血和成体骨髓）的有核红细胞，构建了人类有核红细胞从胚胎到成体的细胞和分子全景图，并对其分子表征和异质性进行了系统分析，为红细胞领域提供了非常有价值的数据资源。此外，体内外红细胞的对比分析还发现人胚胎干细胞分化而来的有核红细胞存在着代谢异常、蛋白质合成不足等缺陷，这些发现可以协助优化体外红细胞生成体系、提高体外体系产生红细胞的效率，为体外批量产生功能性红细胞奠定了理论基础。总之，本研究不仅揭示了人早期胚胎红细胞独特的分子特征和功能异质性，还成功鉴定和验证了免疫调控红细胞亚群的存在，开拓了有核红细胞的新功能研究领域。

Objectives: Erythroid cells are the most abundant hematopoietic cells in the human body and one of the earliest emergence blood cells. Previous studies of the development of human erythroid cells mainly focus on the adult, but little is known about the molecular characteristics of erythroid cells at the early stage of human embryonic development. Although erythroid cells are conventionally defined as the oxygen carriers, emerging studies have suggested that erythroid cells can play an important role in immunomodulatory, especially when the body is under stress conditions, such as infection or tumor. However, it is unclear whether all nucleated erythroid precursors, or just specific subsets, possess these immunomodulatory functions, and the molecular characteristics of these immunomodulatory erythroid cells remains unknown. In this study, we conducted single-cell RNA-sequencing (scRNA-seq) analysis of human erythroid precursors collected from multiple sites (Yolk Sac, YS; Fetal Liver, FL; Preterm Umbilical Cord Blood, PT-UCB; term UCB and adult Bone Marrow, BM) during ontogeny. We revealed the unique molecular characteristics of the human embryonic erythroid precursors and dissected the functional heterogeneity of erythroid precursors. We also identified and verified the existence of an immunomodulatory-erythroid subset.

Methods: In this study, human erythroid precursors were collected by fluorescence-activated cell sorting (FACS) from YS, FL, PT-UCB, term UCB and adult BM. 10x Genomics and STRT-seq platforms were used to construct the library and perform single cell transcriptome sequencing, followed by further bioinformatics analysis. Firstly, we determined the heterogeneity of human erythroid precursors, the feature genes and biological pathways of each subset, and constructed the potential differentiation trajectory of the human erythroid precursors. Subsequently, we performed comparative analysis within each cluster of cells originating from different developmental stages, which uncovered the molecular changes of erythroid precursors that occur in different developmental stages. Finally, immunomodulatory-erythroid subset was analyzed from the aspects of immune-related molecular pathways, feature genes, transcriptional regulatory networks. We screened the cell surface markers and determined the existence of immunomodulatory-erythroid subset, further confirmed by in situ multicolor immunohistochemistry (IHC) staining and flow cytometry. By co-culture with classical immune cells, we determined the immunomodulatory functions of immune-prone erythroid through cytokine secretion, gene expression and protein expression assays. Interaction analysis also identified interacting molecular pairs between immune-prone erythroid and other classical immune cells, which was verified by in situ IHC staining.

Results: (1) Determining the functional heterogeneous of human erythroid precursors. Four distinct heterogeneous subsets, including three classical (C1-C3) and one immune-prone subset (C4), were identified by integration analysis. Cells from each cluster were present throughout the developmental stages examined at varying frequencies. (2) Immunomodulatory-erythroid subset has unique differentiation pathways. Pseudotime analysis and topological structure analysis showed that immunomodulatory-erythroid subset has different differentiation pathway from classical erythroid precursors. The three classical erythroid subsets were close to each other, and matured progressively from C1 to C3, the immune erythroid subset (C4) showed a divergent developmental trajectory. (3) Unique molecular characteristics of erythroid precursors derived from YS, FL and UCB. Parallel analysis of the subsets of erythroid precursors from different developmental stages showed that the cholesterol synthesis and aerobic glycolysis were enriched in YS C1 cells, the FL C1 cells showed strong signals for protein synthesis, whereas UCB C1 cells exhibited more abundant expression of globin genes. In the mature stage, YS C3 cells were characterized by expression of metallothionein genes MT2A, MT1G and MT1H, as well as the zinc transporter genes ZnT (SLC30A) and ZIP (SLC39A). YS C3 cells also showed high proliferation characteristics. (4) Immunomodulatory-erythroid cells coupled with dual erythroid and immune regulatory networks. The analysis of transcription factor activity showed that MYC, GATA1 and KLF1 were downregulated along the erythroid maturation, whereas NFE2 and TAL1 were sequentially activated to promote terminal erythroid maturation in three classical erythroid subsets. Immunomodulatory-erythroid cells not only shared the core erythroid lineage regulons of GATA1, MYB and MYC, but also highly enriched in immune-related regulons such as GATA2, MEF2C, SPI1, FOS, and JUN. The target genes of these immune-related regulons were enriched in categories such as chemokine signaling pathway and antigen processing and presentation. Genes associated with activation of immune response and cytokine-mediated signaling pathway, as well as VSIR, TGFB1, TNFSF10 were specifically highly expressed in immunomodulatory-erythroid cells. Through screening for surface markers, CD63 was highest and most specifically expressed in immunomodulatory-erythroid cells. CD63 positive erythroid subsets were exited in the erythroid cells from FL and UCB, accounting for about 2.58% and 6.36% of the GYPA⁺CD71⁺ erythroid precursors. Expression of CD63 on GYPA⁺ erythroid precursors was also detected by IHC staining and they also expressed the immune signature proteins, such as HLA-E and TGF-b, which were detected in GYPA⁺CD63⁺ cells by IHC staining. (5) Immunomodulatory-erythroid cells exert immunomodulatory functions. CD63⁺ and CD63⁻ erythroid precursors were sorted by FACS and co-cultured with peripheral blood mononuclear cells (PBMCs). Compared with GYPA⁺CD71⁺CD63⁻-PBMC culture, the expressions of CCL4, CXCL8, IFITM3 and other immune-related genes were highly expressed in the GYPA⁺CD71⁺CD63⁺-PBMC culture. The levels of IL-8, IL-6 and TNF were also significantly increased in CD63 positive group. In addition, enhanced TNF expression in CD11b⁺ cells were observed after co-culture with GYPA⁺CD71⁺CD63⁺ cells. (6) Comparative analysis of immunomodulatory-erythroid cells at the distinct development stages and the interaction analysis between immunomodulatory-erythroid cells and immune cells. Correlation analysis was conducted based on expression of the signature genes in corresponding immunomodulatory-erythroid cells. It was found that a greater resemblance of immunomodulatory-erythroid cells among the YS, FL and neonatal UCB stages compared with the features of the corresponding population in adult BM. Enrichment analysis based on the higher expression of stage specific genes revealed that YS stage-specific genes were associated with hypoxia and embryonic organ development, those in FL were associated with active ribosomal and mitochondrial activities, whereas those in UCB displayed enhanced immune properties through enrichment of the leukocyte proliferation and the antigen receptor-mediated signaling pathway, those in adult BM associated with adaptive immunomodulatory functions were evident in the immunomodulatory-erythroid counterparts, such as the regulation of lymphocyte activation and T cell activation. Interaction analysis showed that more significantly enriched pairs and stronger signal intensities were detected among immunomodulatory-erythroid and immune cells than between classical-erythroid and immune cells across all developmental stages, which were also verified by in situ IHC staining, we found the physical juxtaposition of HLA-E⁺GYPA⁺CD63⁺ immunomodulatory-erythroid cells and KLRC1⁺CD56⁺ NK cells, MIF⁺GYPA⁺CD63⁺ immunomodulatory-erythroid cells and CD74⁺CD20⁺ B cells in FL tissue sections.

Conclusions: In this study, integrated, single-cell transcriptomic studies of erythroid precursors from the human YS, FL, preterm UCB, term UCB and adult BM, constructed a cellular and molecular panorama of human erythroid precursors from embryo to adult, this systematic and comprehensive molecular characterization and heterogeneity analyses provides a valuable data resource and important insights into erythropoiesis. In addition, the comparative analysis of in vitro and in vivo erythroid cells also found that insufficient protein synthesis and multiple aberrant metabolic and biological processes were detected in hESC-derived cells during erythroid maturation. These findings could improve the in vitro erythropoietic system and enhance the efficiency of the erythroid cells production from in vitro system. In conclusion, this study not only revealed the unique molecular characteristics and the functional heterogeneity of human erythroid precursors, but also successfully identified and verified an immunomodulatory-erythroid subset throughout human ontogenesis, bring a new field of functional research on nucleated erythroid cells.