Fig. 1

Unsupervised clustering of young and aged HSPCs revealed 15 clusters gathering mainly immature and to a lesser extend lineage-primed HSPCs. a Overview of the scRNA-seq sample preparation and analysis. Cells were isolated from bone marrow (BM) of young and aged mice and pooled to obtain 2 pools for each age. Pools of 2 and 3 BMs for both young and aged mice were analyzed. BM cells were FACS sorted to purify Lin⁻, Sca-1⁺, c-Kit⁺ (LSK) Flt3⁻ cells that defined the HSPCs. The four pools of HSPCs were processed using droplet-based single cell sequencing (10X Genomics) and multiple analyses were performed using bioinformatics tools to characterize aging effects. b UMAP plot of young and aged HSPCs (15,000 cells) analyzed using Seurat. Colors marked the 15 distinct clusters identified by unsupervised clustering and characterized with differential gene expression and gene set enrichment analyses. Each dot represents a cell. c Selected enrichment of our analysis (Gene Ontology, KEG and Reactome pathways Supplemental Table S3) for markers of each cluster and corresponding p values adjusted for multiple testing (padj). NA indicates non-relevant enrichment. d UMAP plots colored by expression of selected cluster markers. Cluster names are indicated in parenthesis. e Localization in the UMAP of LTHSCs, STHSCs, MPP2 and MPP3, identified by supervised classification. f Percentage of LTHSCs, STHSCs, MPP2 and MPP3 within the HSPC population, in each of the 15 clusters. g Percentage of computationally assigned cell cycle (G1/G0, S and G2/M) phases in each of the 15 clusters