Metabolic Control of Chromatin Accessibility Reverses Age Related Dysfunction in Human Hematopoietic Stem and Progenitor Cells

Aging impairs the function of human hematopoietic stem and progenitor cells (HSPCs) through coordinated changes in metabolism and chromatin. We investigated whether a metabolic pathway centered on succinate and α-ketoglutarate (αKG) influences chromatin accessibility, energy dependence, and stem cell health in human HSPCs. We characterized young (18–25 years) and older (> 70 years) human bone marrow (BM) CD34⁺ cells. Flow cytometry showed a shift toward early progenitors with age, but overall CFU output, especially erythroid colonies, declined. Single-cell differentiation assays (MEM) on purified CD45RA⁻CD38⁻CD34⁺CD133⁺CD90⁺ HSCs confirmed reduced clonogenic efficiency and decreased bi-lineage potential in aged cells. Alkaline comet assays indicated increased DNA damage in CD34+ cells as they aged. RNA-seq (hallmark analysis) revealed enrichment in p53/DNA damage, inflammatory responses (TNFα/IL-6/IL-2/IFNγ), Myc, and metabolic pathways (glycolysis, hypoxia, OXPHOS) in aged HSPCs. ATAC-seq showed globally increased chromatin accessibility, including at TSSs linked to these pathways. Although some chromatin-modifier transcripts differed (SMYD3 decreased, KAT6B decreased, PRDM5 increased), pathway analyses did not fully explain the widespread accessibility gains. Untargeted LC-MS displayed broad metabolite accumulation in aged HSPCs, with notably lower succinate levels, suggesting a succinate/αKG control point for chromatin regulation. To perturb this axis, we applied short ex vivo treatments (≤72 h). Dose–response testing (1–8 mM) showed that succinate (4 mM) increased CFU numbers, while αKG (4 mM) decreased them without affecting viability or immunophenotype. Western blots supported reciprocal chromatin effects: succinate increased H3K27me3 and decreased H3K27ac, while αKG had the opposite effect. In ATAC-seq, succinate reduced accessibility in aged HSPCs, whereas αKG increased accessibility in young HSPCs. Across sources (mPB, BM), succinate consistently enhanced clonogenicity. In contrast, αKG impaired it in young cells and had little detrimental effect in aged cells. MEM assays mirrored bulk results at the stem-enriched level: clonogenic efficiency and bi-lineage output were modulated by the metabolic treatment. Short treatments also impacted in vivo performance. In NSG xenografts, young DMSO controls outperformed aged controls in peripheral blood by weeks 9–12. Succinate pre-treatment restored aged PB engraftment to youthful levels, while αKG decreased young PB engraftment. Lineage composition differences at early time points merged by week 12. BM and spleen engraftment showed minor differences between groups, consistent with the model constraints for human hematopoiesis. Mechanistically, SCENITH showed that succinate shifted energy reliance toward glycolysis in bulk CD34⁺ and primitive CD34⁺CD133⁺CD90⁺ cells, resembling youthful dependence; αKG caused an opposite shift in young cells. Succinate increased HIF-2α protein, while αKG decreased it. Pharmacologic blockade of HIF-2α (PT2399) partially reduced succinate’s benefits on clonogenicity and DNA damage, indicating both HIF-2α–dependent and –independent effects consistent with dioxygenase cofactors linking metabolism to chromatin. Together, these data support a model in which succinate/αKG tuning regulates chromatin accessibility and energy status in human HSPCs, reducing DNA damage, increasing clonogenic potential (both bulk and single-cell), and enhancing early PB engraftment after brief ex vivo exposure. Limitations include pooled omics, NSG host constraints, and reliance on ATAC-seq/Western instead of locus-specific histone and cytosine maps. Future work will employ CUT&Tag (H3K27me3/H3K27ac), base-resolved 5mC/5hmC, and secondary transplants to assess durability and mechanism.