Functional and Molecular Heterogeneity of Aging Hematopoietic Stem Cells

Background: Aging hematopoiesis is characterized by increased numbers of hematopoietic stem cells (HSCs) that exhibit impaired self-renewal and long-term reconstitution potential. We previously demonstrated that differences in surface expression of c-Kit in young mouse HSCs could identify distinct functional HSC subsets, with c-Kit^hi HSCs exhibiting reduced self-renewal and megakaryocytic biased differentiation. We therefore sought to determine if similar functional and molecular differences may be observed in old HSCs.

Methods: We evaluated c-Kit^hi and c-Kit^lo subsets of HSCs (CD34-CD150+LSK) from young (8-12 wk), and old (18-20 mo) C57BL/6J mice. For competitive transplantation assays, 250 double sorted c-Kit^hi or c-Kit^lo HSCs from aged CD45.2 mice were mixed with 5x10⁵ unfractionated bone marrow mononuclear cells (BMMCs) from young CD45.1 mice. In parallel, we generated the chromatin accessibility and transcriptional profiles of c-Kit^hi and c-Kit^lo HSCs from young and old mice.

Results: We first performed competitive transplants of old c-Kit^hi and c-Kit^lo HSCs into young recipients. These studies demonstrated age-related myeloid bias exhibited by both HSC subsets. In addition, similar to young HSCs, old c-Kit^hi HSCs exhibit significantly reduced long-term reconstitution capacity compared to old c-Kit^lo HSCs. Next, to evaluate age-related functional differences, we performed equal competitive transplants in which equal numbers of c-Kit^hi or c-Kit^lo old and young HSCs were transplanted into the same young recipient. These studies demonstrated that c-Kit^hi HSCs exhibit similar reconstituting capacity independent of age. In contrast, old c-Kit^lo HSCs exhibited significantly reduced long-term reconstitution compared to their young counterparts, though with better-preserved self-renewal capacity, as reflected in their self-renewal quotients (SRQ). However, when we quantified the capacity of transplanted HSCs to give rise to downstream committed progenitors and mature hematopoietic cells, calculated as their differentiation quotient (DQ), old c-Kit^lo HSCs exhibited markedly reduced DQ compared to their young counterparts. In contrast, c-kit^hi recipients showed that old c-Kit^hi HSCs gave rise to mature cell lineages more efficiently.

To investigate molecular mechanisms driving heterogeneity in HSC aging, we evaluated the transcriptional profiles of young and old HSCs. Old c-Kit^lo HSCs showed significant enrichment of genes associated with inflammation, interferon responses, and the quiescent state in comparison to old c-Kit^hi HSCs. On the other hand, old c-Kit^hi HSCs were enriched for cell cycle related and MYC target genes compared to old c-Kit^lo HSCs and young HSCs. Old c-Kit^hi HSCs also exhibited increased expression of genes associated with high cell output state and high mitochondrial membrane potential, which is associated with a more active HSC cell state.

To investigate the potential role of chromatin accessibility in determining the observed transcriptional changes, we performed ATAC-seq of young and old HSC subsets. Old HSCs exhibited more open chromatin peaks than young HSCs, with the number of peaks decreasing in the order of old c-Kit^lo > old c-Kit^hi > young c-Kit^hi > young c-Kit^lo HSCs. Evaluation of predicted transcription factor binding sites showed enrichment for CTCF and PU.1 sites in young c-Kit^lo and c-Kit^hi HSCs, respectively, with PU.1 associated with high cell output states. In contrast, old c-Kit^lo and c-Kit^hi HSCs were enriched for ERG and CTCF sites, respectively, with predicted binding sites enriched in genes associated with low and high cell output, respectively. These data suggest that differential ERG binding of quiescence and cellular output-related gene signatures regulate functional differences between old c-Kit^lo and old c-Kit^hi HSCs.

Conclusion: Overall, our studies demonstrate functional heterogeneity among old HSCs and identify a novel strategy to identify old HSCs with preserved self-renewal and long-term reconstitution capacity. Identifying and prospectively fractionating old HSCs offers a novel approach for investigating the molecular mechanisms underlying HSC aging.

No relevant conflicts of interest to declare.