FIGURE 3.

Hlf-tdTomato enriches functional CD45⁺ pre-HSCs in the E10.5 AGM region. (A) Representative FACS plots showing the proportion of Hlf-tdTomato⁺ and Hlf-tdTomato^– cells in the CD31⁺CD45⁺Kit⁺CD201^hi population in the AGM region of E10.5 Hlf-tdTomato embryos. Data are representative of five independent experiments. (B) Representative FACS histogram showing the fluorescence intensity of tdTomato in the CD31⁺CD45^–Kit⁺CD201^hi and CD31⁺CD45⁺Kit⁺CD201^hi populations in the AGM region of E10.5 Hlf-tdTomato embryos. Values of mean fluorescence intensity of tdTomato in the Hlf-tdTomato⁺ cells are indicated. Data are representative of five independent experiments. (C) Representative morphology of hematopoietic cells generated from Hlf-tdTomato⁺ cells and Hlf-tdTomato^– cells within the CD31⁺CD45⁺Kit⁺CD201^hi population. Scale bar, 100 μm. (D) Graph showing the frequency of Lin^–CD45⁺CD201⁺Sca-1⁺ cells generated from the indicated populations from the E10.5 AGM region after co-culture measured by FACS analysis. Data are shown as means ± SEM from five independent experiments. (E) Representative FACS plots showing the expression of Hlf-tdTomato and Kit in Lin^–CD45⁺CD201⁺Sca-1⁺ cells that are generated from the indicated populations of the E10.5 AGM region after co-culture. Data are representative of five independent experiments. (F) Cell sorting strategy for the populations from the AGM region of E10.5 Hlf-tdTomato embryos for co-culture/transplantation assay. The cell populations sorted for functional assay are denoted by colored boxes. (G) Representative FACS plots of blood chimerism of donor-derived (CD45.1⁺CD45.2⁺) cells in CD45⁺ cells at 16 weeks post-transplantation. (H) Graphs showing the blood chimerism in CD45⁺ cells of each of all the recipients from 4 to 16 weeks post-transplantation. The recipients were transplanted with the culture products of CD31⁺Kit⁺CD45⁺Hlf-tdTomato⁺ (triangle, left) or CD31⁺Kit⁺CD45⁺Hlf-tdTomato^– (square, right) cells from the AGM region of E10.5 Hlf-tdTomato embryos. Numbers of repopulated/total recipients are shown in the brackets. For each transplantation assay, cells from a total of 15 Hlf-tdTomato positive embryos belonging to three independent litters (biological repeats) were sampled and cultured as donor cells, and five recipients were used. Each line represents a recipient and each color represents an independent litter (biological repeat). (I) Representative FACS plots showing proportions of donor cells and each hematopoietic lineage at 16 weeks post-transplantation. Myeloid cells (Gr1/Mac-1⁺), B cells (B220⁺), and T cells (CD3⁺) in peripheral blood are shown. (J) Bar plot showing the percentages of donor contribution to granulocytes/monocytes (GM, red), B cells (green), and T cells (blue) in the peripheral blood at 16 weeks post-transplantation. (K) Graph showing donor contribution to the bone marrow hematopoietic stem progenitor cells (Lin^–Sca-1⁺Kit⁺, LSK) of two successfully repopulated recipients at 16 weeks post-transplantation.

Next, Hlf-tdTomato⁺ and Hlf-tdTomato^– cells within the CD31⁺Kit⁺CD45⁺ population from the E10.5 AGM region were, respectively, isolated and co-cultured with OP9-DL1 for 6 days, then transplantation assay was performed with the culture products. Of note, multi-lineage repopulation in peripheral blood and multiple organs (BM, spleen, and thymus) at 16 weeks post-transplantation was only detected in the recipients of Hlf-tdTomato⁺ group, consistent with the detection of the chimerism in the immunophenotypic HSCs and hematopoietic progenitors in BM (Figures 3F–K and Supplementary Figures 3C,D). Therefore, Hlf-tdTomato expression further enriched the HSC-competence in E10.5 CD45⁺ population. The finding also indicated that if a cell expresses CD45 prior to Hlf at this early stage, it has lost the possibility of specification toward HSCs.