Fig. 4: scRNAseq analysis identifies a rare HSC-like population specific to embryo explant cultures.

a Limiting dilution transplantation of Em-Ex cells. One, two or three embryo equivalents were transplanted/recipient. Data pooled from three independent experiments. Left panel: Experimental schematic. Middle panel: %CD45.2⁺ PB of recipients at 16 weeks post-transplant. Each circle represents an individual recipient. Red circles highlight non-engrafted recipients. The number of mice engrafted/number of recipients at each cell dose is shown. Right panel: χ² analysis revealed a fit to the limiting dilution model (p-value = 0.717; t-test). The Log of the non-responding fraction is shown for each cell dose. b Representative FACS plots showing sorting strategy for CD31⁺CD45⁺c-Kit⁺CD41⁻ cells in YS-Ex and Em-Ex after 10 days of culture. 49 E8.5 concepti (5–7sp) were dissected, cultured and cells of interest collected by FACS. Post-sort purity check of sorted populations are shown. c Single cell global gene expression analysis of Em-Ex- and YS-Ex-CD31⁺CD45⁺c-Kit⁺CD41⁻ cells. Projection of single cell gene expression profiles onto tSNE1 (X-axis) versus tSNE2 (Y-axis) with Em-Ex cells in pink and YS-Ex cells in blue. X denotes the Em-Ex sub-population circled in red. (n = 3504 Em-Ex cells; n = 3037 YS-Ex cells) d Maturation stage analysis. Identified population was compared to available profile expression datasets on embryonic pre-HSC and HSC from Zhou et al.⁴². Assignment of cultured YS-Ex, Em-Ex and X-cells to embryonic hematopoietic populations. From the AGM: E11_EC (E11-Endothelial cells; CD31⁺VE-cadherin⁺CD41⁻CD43⁻CD45⁻Ter119⁻); E11_T1 (E11-pre-HSCs; CD31⁺CD45⁻CD41^lowc-Kit⁺ CD201^high); E11_T2_CD201neg (E11-pre-HSCs; CD31⁺CD45⁻CD41^lowc-Kit⁺CD201⁻); E11_ T2 (E11-AGM⁻CD31⁺CD45⁺CD41^low CD201^high); from the fetal liver, E12.5_FL (HSCs, Lin⁻Sca-1⁺Mac-1^lowCD201⁺; E14.5_FL (HSC, CD45⁺CD150⁺CD48⁻CD201⁺); and Adult HSC (BM-HSC, CD45⁺CD150⁺CD48⁻CD201⁺). e Volcano plot of differentially expressed genes between E11_T2 and E12.5_FL. Downregulated genes are shown in blue and upregulated in red, (FDR<0.05 and Log₂ fold change <−2 or >2, respectively. f Pattern of the 80 most differentially expressed genes in X-population. Source data are provided as a Source Data file