Multiome-based identification of HSC subtype markers shows that ageing, but not inflammatory stress, increases HSC platelet bias

Individual haematopoietic stem cells (HSCs) display heterogeneous capacities to reconstitute the major blood lineages, including platelets, erythrocytes, myeloid cells, B cells, and T cells. Single-cell transplantation assays have revealed that the HSC compartment contains lineage-restricted subtypes capable of reconstituting only specific lineages, such as platelets, alongside true multi-lineage HSCs. The proportion of these lineage-restricted HSCs increases with age, contributing to declined immunity and potentially to clonal haematopoiesis. To date, their identification and isolation have primarily relied on transgenic reporters, such as the Vwf-EGFP mouse line, which offer only moderate accuracy. In this study, we profiled highly purified adult mouse long-term HSCs (LT-HSCs; LSK CD48⁻ CD150⁺ CD34⁻) using the DOGMAseq platform, which enables simultaneous measurement of transcriptome (RNA-seq), chromatin accessibility (ATAC-seq), and surface protein expression (CITE-seq). We subsequently identified candidate clusters corresponding to platelet-biased and multi-lineage HSCs, and developed a fluorescence-activated cell sorting (FACS) strategy to enrich these populations. This strategy employs four surface markers detectable with commercially available antibodies and achieves higher enrichment specificity than the Vwf-EGFP reporter.