Whereas the critical roles of innate lymphoid cells (ILCs) in adult are increasingly appreciated, their developmental hierarchy in early human fetus remains largely elusive. In this study, we sorted human hematopoietic stem/progenitor cells, lymphoid progenitors, putative ILC progenitor/precursors and mature ILCs in the fetal hematopoietic, lymphoid and non-lymphoid tissues, from 8 to 12 post-conception weeks, for single-cell RNA-sequencing, followed by computational analysis and functional validation at bulk and single-cell levels. We delineated the early phase of ILC lineage commitment from hematopoietic stem/progenitor cells, which mainly occurred in fetal liver and intestine. We further unveiled interleukin-3 receptor as a surface marker for the lymphoid progenitors in fetal liver with T, B, ILC and myeloid potentials, while IL-3RA^– lymphoid progenitors were predominantly B-lineage committed. Notably, we determined the heterogeneity and tissue distribution of each ILC subpopulation, revealing the proliferating characteristics shared by the precursors of each ILC subtype. Additionally, a novel unconventional ILC2 subpopulation (CRTH2^– CCR9⁺ ILC2) was identified in fetal thymus. Taken together, our study illuminates the precise cellular and molecular features underlying the stepwise formation of human fetal ILC hierarchy with remarkable spatiotemporal heterogeneity.

虽然先天性淋巴细胞 (ILC) 在成人中的关键作用越来越受到重视，但它们在早期人类胎儿中的发育层次在很大程度上仍然难以捉摸。在这项研究中，我们在受孕后 8 到 12 周对胎儿造血、淋巴和非淋巴组织中的人类造血干/祖细胞、淋巴祖细胞、推定的 ILC 祖/前体和成熟 ILC 进行分类，以获取单细胞 RNA - 测序，然后是体积和单细胞水平的计算分析和功能验证。我们从主要发生在胎儿肝脏和肠道的造血干/祖细胞中描述了 ILC 谱系定型的早期阶段。我们进一步揭示了白细胞介素 3 受体作为具有 T、B、ILC 和骨髓电位的胎儿肝脏淋巴祖细胞的表面标志物，而 IL-3RA^–淋巴祖细胞主要是 B 系。值得注意的是，我们确定了每个 ILC 亚群的异质性和组织分布，揭示了每个 ILC 亚型的前体共享的增殖特征。此外，在胎儿胸腺中发现了一个新的非常规 ILC2 亚群 (CRTH2 ^– CCR9 ⁺ ILC2)。总之，我们的研究阐明了人类胎儿 ILC 层次逐步形成的精确细胞和分子特征，具有显着的时空异质性。