Unravelling human hematopoietic progenitor cell diversity through association with intrinsic regulatory factors

Summary Hematopoietic stem and progenitor cell (HSPC) transplantation is an essential therapy for hematological conditions, but finer definitions of human HSPC subsets with associated function could enable better tuning of grafts and more routine, lower-risk application. To deeply phenotype HSPCs, following a screen of 328 antigens, we quantified 41 surface proteins and functional regulators on millions of CD34+ and CD34− cells, spanning four primary human hematopoietic tissues: bone marrow, mobilized peripheral blood, cord blood, and fetal liver. We propose more granular definitions of HSPC subsets and provide new, detailed differentiation trajectories of erythroid and myeloid lineages. These aspects of our revised human hematopoietic model were validated with corresponding epigenetic analysis and in vitro clonal differentiation assays. Overall, we demonstrate the utility of using molecular regulators as surrogates for cellular identity and functional potential, providing a framework for description, prospective isolation, and cross-tissue comparison of HSPCs in humans.


Figure S1 :
Figure S1: Human cell surface molecule flow cytometry screen-related to Fig. 1.A) Gating strategy for HSPC populations.B) Median expression of molecules in the screen separated by HSPC populations.Sidebar colors same as in A).Arrows indicate markers called out in text.

Figure S2 :Fig. 1 .
Figure S2: Data preprocessing, leiden clustering and marker expression -related to Fig. 1.A) Gating strategy for viable CD45+ cells.B) Exemplative violin plots of CD38 expression of all mass cytometry data used in the study, separated by donor/tissue and colored by experimental batch before (top) and after (bottom) batch correction.Batch control samples used to calculate batch corrected boxed by dashed lines.C) Heatmap of median expression of leiden clusters, colored by metacluster annotation (top).D) UMAPs colored by protein expression (left) alongside violin plots of the same protein separated by manual gate (center) and metacluster (right) as in Fig. 1F.Diamond indicates median.

Figure S3 :
Figure S3: EMP populations sorting strategy and ATAC-seq quality controlrelated to Fig. 3. A) UMAP of BM cells in the erythroid trajectory with the addition of the MPPs clusters to the analysis.B) FACS strategy for EMP populations used in ATAC-seq and in vitro clonal differentiation assays.C) UMAP of BM cells in the erythroid trajectory, colored by metacluster (left) or projected FACS populations (right).D) Histogram of normalized Tn5 insertion profiles centered at transcription start sites (TSSs) for the indicated ATAC-seq libraries.E) Barplots of normalized TSS insertion scores across biological and technical replicates of cell populations.F) Histogram of insertion counts by fragment length for indicated ATAC libraries.G) UMAP previously published and annotated BM scATAC dataset with the projection of biological and technical replicates of ATAC-seq libraries.

Figure S4 :
Figure S4: Myeloid trajectories, sorting strategy, and ATAC-seq quality controlrelated to Fig. 4. A) Heatmap of first derivative of marker expression, showing rate of expression change, along the pDC pseudotime trajectory.B) Same as A) for monocyte trajectory.C) Same as A) for cDC trajectory.D) FACS strategy for myeloid population used in ATAC-seq and in vitro clonal differentiation in methylcellulose.E) UMAP of BM cells in the myeloid trajectory colored by metacluster (left) or projected FACS myeloid population (right).F) Histogram of normalized Tn5 insertion profiles centered at transcription start sites (TSSs) for the indicated ATAC-seq libraries.G) Barplots of normalized TSS insertion scores across biological and technical replicates.H) Histogram of insertion counts by fragment length for indicated ATAC libraries.I) UMAP of scATAC from healthy human BMMNCs colored by projections of biological and technical replicates of ATAC-seq libraries.

Figure S5 : 5 A
Figure S5: Additional analysis of mPB and BM -related to Fig. 5 A) Dotplot quantifying frequency of CD38-cells within CD34+ compartment by tissue/product.P-value derived from Wilcoxon rank sum test.B) Violin plots of marker expression by tissue/product.Diamond indicates median.C) Heatmap of median expression of metaclusters (top row colors), separated by tissue/product (second row colors).

Figure S6 : 6 A
Figure S6: FL is comprised of cells with phenotypic profiles unique from other hematopoietic tissues and products -related to Fig. 6 A) Barplot of frequency of CD34+ cells out of CD45+ cells in FL, separated by donor.B) UMAP of mononuclear CD45+ FL cells, colored by metacluster.C) UMAP colored by marker expression.D) Heatmap of median expression of leiden clusters, colored by metacluster annotation (top row).E) Frequency of FL metaclusters out of CD45+ cells.Dots indicate individual donors.Crossbar indicates mean.