Figure 2. Time-resolved reference HSPC landscape at single-cell level.

(A) Experimental design for HSPC dynamics analysis with flow cytometry and scRNA-Seq. Table indicates specific time-point and the number of mice (replicates) used for Tom⁺ scRNA-Seq analysis, 2 mice in each time-point were used for the Tom^- fraction estimation. (B) UMAP projection of the integrated HSPC scRNA-Seq landscape (all Tom⁺ and Tom^- cells combined) with color-coded clusters. Outlier or aberrant clusters were removed for clarity (see Figure S2F,G). (C) Manual annotation of the landscape in B. Most differentiated clusters with clearly defined lineage markers are color-coded, intermediate undifferentiated states are shown in grey (Int prog), cluster containing HSCs is shown in pink. (D,E) Projection from B in grey, with embedded and color-coded immunophenotypic sub-populations from Nestorowa et al. data.⁷ Up to randomly selected 60 cells in each category are plotted. All cells are plotted in Figure S3A. (F) Projection from B in grey, with embedded and color-coded cKit⁺ progenitors, based on their output in lineage tracing in vitro cultures. Color-coded points correspond to cells harvested at day 2 with sufficient clonal information available at day 4 and day 6 of culture. Data from Weinreb et al.¹¹. (G) Projection from B in grey, with embedded and color-coded HSCs with no detected cellular output (inactive - childless) or contributing to haematopoiesis (active - parent) following 5-FU challenge in mice (data from Bowling et al.¹⁸). (H) Projection from B in grey, with Hoxb5-Tom⁺ cells harvested at indicated time-points shown in blue. Nestorowa et al.⁷ population definitions: LT-HSC – Lin-, cKit⁺, Sca1⁺, CD34^-, Flt3^-, MPP1 – Lin-, cKit⁺, Sca1⁺, Flt3^-, CD34⁺, CD150⁺, CD48^-, ST-HSC – Lin^-, cKit⁺, Sca1⁺, Flt3^-, CD34⁺, CD150^-, CD48^-, GMP Lin^-, cKit⁺, Sca1⁺, CD16/32⁺, CD34⁺, LMPP – Lin^-, cKit⁺, Sca1⁺, Flt3⁺, CD34⁺, MEP – Lin^-, cKit⁺, Sca1⁺, CD16/32^-, CD34^-, MPP3 – Lin^-, cKit⁺, Flt3^-, CD34⁺, CD150^-, CD48⁺, CMP – Lin^-, cKit⁺, Sca1⁺, CD16/32^-, CD34⁺.

Abbreviations: B prog - B cell progenitor, Bas - basophils, Bas/MC prog - Basophil and Mast Cell progenitors, DC prog - dendritic cell progenitors, Eos - eosinophils, Ery prog - erythroid progenitors, HSC - hematopoietic stem cells, Int prog - intermediate progenitors, Ly prog - lymphoid progenitors, Meg prog - megakaryocyte progenitors, Mono/DC prog - monocyte and dendritic cells progenitors, Neu prog - neutrophil progenitors, pDC - plasmacytoid dendritic cells