Fig. 4: Single-cell RNA-sequencing identifies the transcriptional signatures of self-renewing HSCs emerging from clonal V⁺61⁺E⁺ hemogenic precursors in the AGM-EC niche in vitro.

a, b FACS analysis of the progeny of a single V⁺61⁺E⁺ hemogenic precursor from E11 AGM (42sp) following 4-day co-culture on AGM-EC, with a HSC phenotype (VE-Cad^low/−CD45⁺Gr1⁻F4/80⁻Sca1^hiEPCR^hi) and b mixed HSC and hematopoietic progenitor cell (HPC) phenotype. c, d UMAP projection of single-cell transcriptomes obtained from cells from a, b showing hematopoietic cluster (red box) expressing pan-hematopoietic marker Ptprc (CD45). e Expression of Runx1, HSC marker genes (Pdzk1ip1, Vwf, Procr, Fgd5), HPC-associated genes (Itgal, Cd48), and cell cycle genes (Cdkn1c, Mki67) in the hematopoietic cluster from c. f Expression of Runx1, Pdzk1ip1, and Itgal in the hematopoietic cluster from d. g Cell type classification of hematopoietic cells based on gene expression: HSC Type (expressing Runx1, Pdzk1ip1, and Vwf, not expressing Cd48 or Itgal) and HPC type (expressing Cd48 and/or Itgal). h Trajectory analysis using Monocle to order cells from the hematopoietic cluster in pseudotime, and gene expression heatmap over pseudotime of selected genes, including Notch pathway targets, differentially expressed during HSC differentiation to HPC. (See also Supplementary Fig. 8, Supplementary Data 4).