Data_Sheet_1_Hlf Expression Marks Early Emergence of Hematopoietic Stem Cell Precursors With Adult Repopulating Potential and Fate.ZIP

In the aorta-gonad-mesonephros (AGM) region of mouse embryos, pre-hematopoietic stem cells (pre-HSCs) are generated from rare and specialized hemogenic endothelial cells (HECs) via endothelial-to-hematopoietic transition, followed by maturation into bona fide hematopoietic stem cells (HSCs). As HECs also generate a lot of hematopoietic progenitors not fated to HSCs, powerful tools that are pre-HSC/HSC-specific become urgently critical. Here, using the gene knockin strategy, we firstly developed an Hlf-tdTomato reporter mouse model and detected Hlf-tdTomato expression exclusively in the hematopoietic cells including part of the immunophenotypic CD45– and CD45+ pre-HSCs in the embryonic day (E) 10.5 AGM region. By in vitro co-culture together with long-term transplantation assay stringent for HSC precursor identification, we further revealed that unlike the CD45– counterpart in which both Hlf-tdTomato-positive and negative sub-populations harbored HSC competence, the CD45+ E10.5 pre-HSCs existed exclusively in Hlf-tdTomato-positive cells. The result indicates that the cells should gain the expression of Hlf prior to or together with CD45 to give rise to functional HSCs. Furthermore, we constructed a novel Hlf-CreER mouse model and performed time-restricted genetic lineage tracing by a single dose induction at E9.5. We observed the labeling in E11.5 AGM precursors and their contribution to the immunophenotypic HSCs in fetal liver (FL). Importantly, these Hlf-labeled early cells contributed to and retained the size of the HSC pool in the bone marrow (BM), which continuously differentiated to maintain a balanced and long-term multi-lineage hematopoiesis in the adult. Therefore, we provided another valuable mouse model to specifically trace the fate of emerging HSCs during development.

在小鼠胚胎的主动脉-性腺-中肾（aorta-gonad-mesonephros, AGM）区域中，造血前体干细胞（pre-hematopoietic stem cells, pre-HSCs）由罕见且特化的生血内皮细胞（hemogenic endothelial cells, HECs）通过内皮-造血转化（endothelial-to-hematopoietic transition）生成，随后成熟为真正意义上的造血干细胞（hematopoietic stem cells, HSCs）。由于生血内皮细胞同时还会产生大量不定向分化为造血干细胞的造血祖细胞，因此开发特异性靶向pre-HSC/HSC的高效研究工具变得极为迫切。本研究通过基因敲入（gene knockin）技术，首先构建了Hlf-tdTomato报告基因小鼠模型，并在胚胎第10.5天（E10.5）的主动脉-性腺-中肾区域中，检测到Hlf-tdTomato的表达仅局限于造血细胞群中，包括部分免疫表型为CD45–和CD45+的pre-HSCs。通过体外共培养实验以及用于严格鉴定造血干细胞前体的长期移植实验，本研究进一步发现：与CD45–亚群不同，该亚群中Hlf-tdTomato阳性与阴性细胞群均具备造血干细胞潜能，而CD45+的E10.5 pre-HSCs仅存在于Hlf-tdTomato阳性细胞群中。该结果表明，细胞需在CD45表达之前或与CD45同时表达Hlf，才能分化为功能性造血干细胞。此外，本研究构建了新型Hlf-CreER小鼠模型，并通过在E9.5时期单次给药诱导，开展了时间限制性的遗传谱系示踪实验。我们在E11.5的主动脉-性腺-中肾前体细胞中检测到了标记信号，且这些标记细胞可分化为胎肝（fetal liver, FL）中的免疫表型造血干细胞。更为重要的是，这些被Hlf标记的早期细胞可参与并维持骨髓（bone marrow, BM）中的造血干细胞池规模，且可持续分化以维持成体小鼠体内长期、稳定的多系造血功能。综上，本研究提供了又一种可特异性追踪发育过程中新生造血干细胞命运的珍贵小鼠模型。