An inflammatory and quiescent HSC subpopulation expands with age in humans

Aging of the blood system impacts systemic health and can be traced to hematopoietic stem cells (HSCs). Despite multiple reports on human HSC aging, a unified map detailing their molecular age-related changes is lacking. We developed a consensus map of gene expression in HSCs by integrating seven single-cell datasets. This map reveals previously unappreciated heterogeneity within the HSC population. It also links inflammatory pathway activation (TNF/NF?B, AP-1) and quiescence within a single gene expression program. This program dominates an inflammatory HSC subpopulation that increases with age, highlighting a potential target for further experimental studies and anti-aging interventions. Overall design: Sternum bone marrow samples from young (<40 y.o.) and aged (>60 y.o.) individuals were analysed with scRNA-seq. Bone marrow cells from nine donors were collected from the iliac crest of patients undergoing cardiac surgery under an excess sample banking and sequencing protocol that covers all study procedures and was approved by the Institutional Review Board (IRB) of Mass General Brigham. Donors were confirmed negative for common CHIP mutations using targeted sequencing. Mononuclear cells were isolated using Ficoll or lymphoprep and cryopreserved in liquid nitrogen storage. Cells were thawed using standard procedures, and viable (DAPI negative) cells were sorted on a Sony SH800 flow cytometer. Next, 10,000-15,000 cells were loaded onto a 10x Genomics chip. Further processing was done using the recommended procedures for the 10x Genomics 3' v3.0, v3.1, or v4 chemistry. Libraries were sequenced on the NovaSeq SP 100 cycle with the following parameters (Read 1: 28 + Read 2: 75 + Index 1 (i7): 10 + Index 2 (i7): 10). Count matrices were generated using CellRanger v.7.1.0 with default settings and GRCh38 as the reference genome.