Fig. 5: CD32 identifies a NOTCH-independent HEC state.
From: CD32 captures committed haemogenic endothelial cells during human embryonic development
a, UMAP of single-cell data of day 8 WNTd CD34+CD43negCD184negCD73neg cells. H1 hESCs, n = 1. Cells clustered at resolution 0.6. RUNX1-expressing clusters highlighted by a dashed black line. ECs, endothelial cells; EndoMT, endothelial-to-mesenchyme transition; M phase, mitotic phase. b, UMAP of the single-cell trajectory performed by Monocle3 on clusters 0, 1, 2, 11, 16 and 17 displaying RUNX1 as a differential marker. c, GSEA (significantly enriched GO terms, adjusted P value <0.05) on FC pre-ranked genes from the comparison between cluster 11 and clusters 0, 1 and 2 (top) or cluster 11 and clusters 16 and 17 (bottom). Upregulated genes are shown in red, downregulated in blue. NES, normalized enrichment score; ECM, extracellular matrix. d, UMAP (top) and donut charts (bottom) showing the cells in clusters 0, 1 and 2, cluster 11, and clusters 16 and 17 coloured according to the cell cycle phase. G1 phase, yellow; S phase, purple; G2/M phase, green. e, Pseudotime kinetics of the expression variation of FCGR2B and HES1, HEY1 and HEY2 along the clusters with differential RUNX1 expression. Cells coloured by cluster identity. Lines denote relative average expression of each gene in pseudotime. f, Flow cytometric analysis of CD45 and CD34 after HEC culture of CD32+/neg treated with DMSO, as control, or γ-secretase inhibitor L-685,458 (γSi) to block NOTCH signalling. Gated on SSC/FSC/Live. n = 3, independent. APC-Cy7, allophycocyanin-cyanine7. g, A barplot showing the frequency of CD45+ cells derived from CD32+ and CD32neg fraction treated with DMSO (in petroleum) or γ-secretase inhibitor L-685,458 (γSi, in white) to block NOTCH signalling. One-way ANOVA for all biological replicates (n = 3), mean ± s.e.m. (*P = 0.0456; NS, not significant, P = 0.9623). h, Flow cytometric analysis of CD45/CD34 after HEC cultures. of wild-type H1 hESCs (left) and H1 hESCs expressing 3× short hairpin RNAs (shRNAs) against FCGR2B (referred to as CD32 knock-down, KD, right), gated on SSC/FSC/Live/GFP+ cells.
