Extended Data Fig. 7: A reproducible fetal HSC gene expression state identified by cNMF.

(a) Viable, lineage^- cells from mouse fetal liver and adult bone marrow were stained with antibodies against the indicated surface markers to identify hematopoietic progenitors (c-Kit⁺ Sca-1⁺) and HSCs (c-Kit⁺ Sca-1⁺ Flk2^- CD34^-). Both populations from both organ sites were then assessed for cell surface CD69 expression. Percent of parent population values are shown. (b) The percent of CD69+ cells in mouse fetal liver and bone marrow are compared for various HSPC immunophenotypes (gating strategy shown in a). Fetal livers from nine different fetal mice and bone marrow from six different adult mice (biologic replicates) were analyzed. Student’s two-tailed t-test was used to compare percent CD69+ cells in fetal liver and bone marrow, with * indicating p-value <0.05 and **** indicating p-value <0.0001; data are presented as mean +/- SD – p values are LSK 1.1E-8, HSC 0.03, MPP2 4E-8, MPP3 1E-9, MPP4 2E-10. (c) The indicated scRNA-seq datasets were analyzed by classifying cells based on both expression of CD69 and usage of the fetal-HSC-GEP. Enrichment of fetal-HSC-GEP usage was quantified and compared between CD69⁺ and CD69^- fractions, with p-values from two-sided Wilcoxon rank-sum testing and box plots indicating average and +/− 1 standard deviation. (d) In an independent scRNA-seq dataset²⁶ that contains human CD34⁺ cells from fetal liver and postnatal bone marrow up to early adulthood, a similar analysis as in (c) was performed demonstrating enrichment of fetal-HSC-GEP usage in fetal liver compared to early postnatal bone marrow, with p-values from two-sided Wilcoxon rank-sum and box plots indicating average and +/− 1 standard deviation. (e) In an independent scRNA-seq dataset⁹ that profiles human fetal liver and fetal bone marrow CD34⁺ cells, a similar analysis as in (c) was performed demonstrating no significant difference in usage of the fetal-HSC-GEP when comparing fetal liver and fetal bone marrow hematopoiesis, with p-values from two-sided Wilcoxon rank-sum testing. (f) In an independent scRNA-seq dataset⁶⁴ that captures mouse fetal liver and postnatal bone marrow c-Kit⁺ cells, a similar analysis as in (c) was performed demonstrating enrichment of fetal-HSC-GEP usage in mouse E13.5 fetal liver compared to adult mouse bone marrow, with p-values from two-sided Wilcoxon rank-sum testing. (g) Using fetal HSC and postnatal HSC DNA methylation data⁶³, the difference in methylation scoring surrounding the transcriptional start site of the top 200 genes contributing to the fetal-HSC-GEP is displayed, indicating greater DNA methylation postnatally, with p-values from two-sided Wilcoxon rank-sum testing.