Limited knowledge of cellular and molecular mechanisms underlying hematopoietic stem cell and multipotent progenitor (HSC/MPP) expansion within their native niche has impeded the application of stem cell-based therapies for hematological malignancies. Here, we constructed a spatiotemporal transcriptome map of mouse fetal liver (FL) as a platform for hypothesis generation and subsequent experimental validation of novel regulatory mechanisms. Single-cell transcriptomics revealed three transcriptionally heterogeneous HSC/MPP subsets, among which a CD93-enriched subset exhibited enhanced stem cell properties. Moreover, by employing integrative analysis of single-cell and spatial transcriptomics, we identified novel HSC/MPP ‘pocket-like’ units (HSC PLUS), composed of niche cells (hepatoblasts, stromal cells, endothelial cells, and macrophages) and enriched with growth factors. Unexpectedly, macrophages showed an 11-fold enrichment in the HSC PLUS. Functionally, macrophage–HSC/MPP co-culture assay and candidate molecule testing, respectively, validated the supportive role of macrophages and growth factors (MDK, PTN, and IGFBP5) in HSC/MPP expansion. Finally, cross-species analysis and functional validation showed conserved cell–cell interactions and expansion mechanisms but divergent transcriptome signatures between mouse and human FL HSCs/MPPs. Taken together, these results provide an essential resource for understanding HSC/MPP development in FL, and novel insight into functional HSC/MPP expansion ex vivo.

对造血干细胞和多能祖细胞（HSC/MPP）在其天然生态位内扩增的细胞和分子机制的了解有限，阻碍了基于干细胞的疗法在血液恶性肿瘤中的应用。在这里，我们构建了小鼠胎儿肝脏（FL）的时空转录组图谱，作为假设生成和后续新调控机制实验验证的平台。单细胞转录组学揭示了三个转录异质性 HSC/MPP 亚群，其中富含 CD93 的亚群表现出增强的干细胞特性。此外，通过采用单细胞和空间转录组学的综合分析，我们鉴定了新的HSC/MPP“口袋样”单位（HSC PLUS），由生态位细胞（成肝细胞、基质细胞、内皮细胞和巨噬细胞）组成）并富含生长因子。出乎意料的是，巨噬细胞在 HSC PLUS 中富集了 11 倍。在功能上，巨噬细胞-HSC/MPP共培养测定和候选分子测试分别验证了巨噬细胞和生长因子（MDK、PTN和IGFBP5）在HSC/MPP扩增中的支持作用。最后，跨物种分析和功能验证显示小鼠和人类 FL HSC/MPP 之间存在保守的细胞间相互作用和扩增机制，但转录组特征存在差异。总而言之，这些结果为了解 FL 中 HSC/MPP 的发育提供了重要的资源，并为离体功能性 HSC/MPP 扩增提供了新的见解。