Identification of two distinct human hematopoietic stem cell subsets and their age-dependent dynamics by single-cell multi-omics

Abstract Hematopoietic stem cells (HSCs) sustain lifelong blood production due to their unique capacities for self-renewal and multilineage differentiation. These properties decline with age, shifting from balanced lympho-myeloid output in youth and adults to impaired self-renewal and myeloid bias in the elderly, contributing to age-associated immune dysfunction and blood disorders. Although both intrinsic and extrinsic factors have been implicated in HSC aging, the molecular mechanisms underlying these declines remain elusive. HSC heterogeneity further complicates this picture. Mouse studies have revealed multilineage HSCs (multi-HSCs) and myeloid-biased HSCs (myeloid-HSCs), with myeloid-HSCs increasing with age and contributing to aged phenotypes. However, whether similar subsets exist in humans and influence aging hematopoiesis remains unknown. Defining molecular trajectories of human HSCs and linking them to function and heterogeneity would be a critical step toward addressing these gaps. Single cell sequencing techniques have advanced HSC biology by revealing pronounced transcriptional and epigenetic heterogeneity within phenotypically purified HSCs. However, scRNA-seq or scATAC-seq alone is unable to explore the gene-chromatin regulatory machinery intimately determining cell fate. To close this gap, we applied single-cell multi-omics sequencing (scMulti-omics-seq, RNA-seq, and ATAC-seq in the same cell) to human CD34⁺ HSPCs from umbilical cord blood and bone marrow donors of varying ages (<18, 20-50, >65 yrs). Unbiased clustering identified two distinct HSC subsets. One subset showed high expression of myeloid-specific transcription factors and their motif activities (CEBPB, MYB), inflammatory genes (NFKB1, PELI1), and enriched pathways such as myeloid differentiation, immune response, and senescence; thus, termed myeloid-HSCs. The other subset expressed higher levels of HLF, IGF1R, tumor-suppressor genes (NKAIN2, INPP4B), and enriched pathways related to lymphocyte differentiation, stem cell division, and cell polarity; termed multipotent HSCs (multi-HSCs). Transcriptional and epigenetic signature analysis indicated multi-HSC enrichment in stemness and dormancy, while myeloid-HSCs were enriched in low-output and Mk-bias. Cell surface markers are being validated for functional assays, providing the first evidence of lineage-biased HSC subsets in humans. Profiling across age groups showed stepwise reductions in gene expression and chromatin accessibility from cord blood to adulthood, with partial reversal in elderly donors. In contrast, proportions of HSCs/MPPs in G1/G0 phase rise from cord blood to adults and decline in the elderly. Both transcriptional and epigenetic HSC signatures declined progressively with age. Notably, myeloid-HSCs increased dramatically from 0.5% in cord blood to 33.7% in elderly marrow, with reciprocal decreases in multi-HSCs. Gene regulatory network analyses are underway to define age-dependent regulatory mechanisms. Rejuvenating the aged blood system has potential to improve health in the elderly. Notably, ex-vivo cultured HSCs commonly share declined self-renewal and myeloid bias with aged HSCs. We recently developed a culture method that overcomes these shortages and enables the expansion of human long-term HSCs with balanced myeloid/lymphoid output. We next tested our culture condition for rejuvenation by culturing human aged CD34⁺ HSPCs for eight days. scMulti-omics-seq analysis showed that rejuvenated HSCs/MPPs contained >30% multi-HSCs but minimal myeloid-HSCs; and are enriched with gene sets associated with high-output, stemness and multilineage potential. In contrast, uncultured aged counterparts have >30% myeloid-HSCs and <10% multi-HSCs; and enriched with dormancy, Mk-bias and low output. Clustering analysis indicated rejuvenated cells were closer to young and distant from old counterparts. Preliminary xenograft transplantation data showed that, at 16 weeks post-transplantation, mice in the rejuvenated group exhibited higher human CD45⁺ chimerism in peripheral blood, compared to minimal engraftment observed in mice receiving uncultured aged cells. In summary, our study provides the first lifespan-resolved human HSC single-cell multi-omics atlas, reveals age-dependent molecular and subset dynamics, and demonstrates ex vivo rejuvenation potential. The identification of human myeloid-HSCs and multi-HSCs extends the concept of co-existing lineage-heterogeneous HSC subsets from mouse to man.