Fig. 6: HEC specification and haematopoietic cell emergence require stage-specific BMP and ROCK signalling modulation.
From: CD32 captures committed haemogenic endothelial cells during human embryonic development
a, ORA (significant BMP-related BP terms, Fisher exact test; FDR <0.05) on downregulated DEGs between DLL4+ versus CD32+ samples. Bars coloured by adjusted P value. b,c, Fold difference of CD32+ cell frequency in day 8 WNTd cells (b) and of CFC frequency post HEC cultures of cells obtained with BMP signalling modulation (c). Frequencies are shown relative to control. One-way ANOVA for all biological replicates (n = 3, independent, H9 hESCs), mean ± s.e.m. (for b, ***P = 0.0004; ****P < 0.0001; for c, control versus BMP4: *P = 0.0379; control versus BMPi: *P = 0.02170; BMP4 versus BMPi: ***P = 0.001). d,e, Flow cytometric analysis of CD32/DLL4 expression in day 8 WNTd cultures (d) and CD45/CD34 expression after HEC cultures of cells obtained with BMP signalling modulation (e). For d, gated on SSC/FSC/Live/CD34+CD43negCD184negCD73neg; for e, gated on SSC/FSC/Live. H9 hESCs, n = 3, independent. APC, allophycocyanin; PE, phycoerythrin; PE-Cy7, phycoerythrin-cyanine7; APC-Cy7, allophycocyanin-cyanine7. f, ORA (significant RHO-related Reactome Pathways terms, Fisher exact test; FDR <0.05) on downregulated DEGs between cells of cluster 11 versus clusters 0, 1 and 2. Bars are coloured by adjusted P value. g,h, Flow cytometric analysis of CD45/CD34 expression (g) and fold difference of CFC frequency after ROCK inhibition (h). Gated on SSC/FSC/Live. Frequencies are shown relative to control. One-tail paired Student’s t-test, non-parametric, for all biological replicates (H9 hESCs, n = 3, independent), mean ± s.e.m., *P = 0.0404. i, Model depicting the emergence of blood cells during human embryonic development. HECs are specified from mesodermal cells via VEGF and BMP signalling. HECs then undergo a NOTCH-dependent determination process that culminates with CD32 expression, which is followed by a cell cycle re-entry for the NOTCH-independent differentiation into blood cells. ECM, extracellular matrix.
