Extended Data Fig. 1: In vitro and in vivo potentials of Vwf-tdTomato⁻ Multi-HSCs and Vwf-tdTomato⁺ P-HSCs.

a, Representative flow cytometry profiles and gating strategy for sorting of BM Vwf-tdTomato⁻ and Vwf-tdTomato⁺ LSKGata1-eGFP⁻CD34⁻CD150⁺CD48⁻ cells for single cell transplantations. b, Frequency (%) of long-term (16–18 weeks post-transplantation) blood replenishment patterns in recipients reconstituted by a single LSKGata1-eGFP⁻CD34⁻CD150⁺CD48⁻ (also CD201⁺ in some experiments) Vwf-tdTomato⁻ HSC (n = 58; 13 experiments). The 3 blood patterns included as Vwf⁻ Multi-HSCs in the present studies are highlighted. c, Peripheral blood replenishment (mean % ± s.e.m.) in recipients reconstituted by a single LSKGata1-eGFP⁻CD34⁻CD150⁺CD48⁻ Vwf-tdTomato⁻ Multi-HSC (n = 39) or Vwf-tdTomato⁺ P-HSC (n = 30); 13 experiments. Phenotypic populations defined in Methods and Fig. 1a. d, Complete HSPC hierarchy by single Vwf-tdTomato⁺ P-HSCs (n = 9). Same mice as in Fig. 1a. Mean ± s.e.m. % contribution to each population. Orange: reconstituted in all mice. Grey: below detection level in all mice; mean of detection thresholds. Pink: reconstitution in some but not all mice (frequency of reconstituted mice is indicated in the upper left of each circle); mean of positive mice. Phenotypic populations defined in Methods and Fig. 1a. e, Frequency of wells with cell growth (mean ± s.e.m.) of the in vitro assay for granulocyte/macrophage (GM) and Mk lineage potentials shown in Fig. 1c. LT-HSC: 580 plated wells; ST-HSC: 660 wells; MPP2: 652 wells; MPP3: 720 wells; MPP4: 720 wells. Phenotypic populations defined in Methods and Fig. 1a. f, Unique case of a single Vwf-tdTomato⁺ P-HSC replenishing Vwf-tdTomato⁺ as well as Vwf-tdTomato⁻ P-HSCs but no Multi-HSCs. Left: Vwf-tdTomato and CD201 expression in LSKGata1-eGFP⁻CD150⁺CD48⁻CD45.2⁺ cells in the primary CD45.1 recipient of a single CD45.2 LSKGata1eGFP⁻CD34⁻CD150⁺CD48⁻CD201⁺ Vwf-tdTomato⁺ P-HSC. Middle: Blood reconstitution in the primary (1⁰) recipient at 22 weeks post-transplantation and in the secondary (2⁰) recipients (mean ± s.e.m.) 16 weeks after transplantation. CD201⁻Vwf-tdTomato⁻ n = 1; CD201⁺ Vwf-tdTomato⁻ n = 1; CD201⁻Vwf-tdTomato⁺ n = 2; CD201⁺ Vwf-tdTomato⁺ n = 2. Right: Interpretation of results regarding (non-) hierarchical replenishment of Multi-HSCs and P-HSCs. See Fig.1a for cell phenotypes and Supplementary Table 3.

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