Telomere Damage Maintains Hematopoietic Stem Cells (HSCs) in an Activated Metabolic State, Which Compromises Their Self-Renewal Capability

DNA damage and the attendant cellular responses of apoptosis, senescence, and altered differentiation are major drivers of hematopoietic stem cell (HSC) aging. A reservoir of persistent DNA damage signaling can derive from progressive telomere erosion, which occurs over the lifespan of humans. However, the molecular mechanisms by which telomere damage compromises HSC functions are largely unknown.

Here, though combined single-cell RNA-seq and functional studies of highly-purified c-Kit⁺Sca⁺Lin^-CD34^-flk2^-CD150⁺CD48^-CD41^-HSCs, we show that persistent telomeric damage does not activate programs of apoptosis or senescence but maintains HSCs in an activated metabolic state, which directly compromises their self-renewal capability.

To dissect the biological and molecular mechanisms by which persistent DNA damage affects HSC function we analyzed the HSC compartment of mice with short telomeres (G5/G6 TERT^ER/ER), which developed age-related defects. Immunophenotypic analysis of the HSC compartment showed that, compared with G0 TERT^ER/+ (G0) mice with intact telomeres (n=12), 2 month-old G5/G6 mice (n=17), had a significantly decreased number of HSCs (p<10^-3) that was associated with a decreased number of the lymphoid-biased MPP4 cells, and an increased number of both megakaryocyte-biased MPP2 and myeloid-biased MPP3 cells. HSC exhaustion and increased myeloid-to lymphoid output were reminiscent of stressed hematopoiesis and premature aging. G5/G6 HSCs exhibited a significant accumulation of telomere dysfunction-induced foci (p<10^-5) but did not display increased levels of apoptosis in steady-state conditions.

HSC exhaustion could result from apoptosis and/or senescence induced by telomere damage in HSCs entering the cell cycle or from an altered balance between self-renewal and differentiation. To distinguish between these two possibilities, we first investigated the effect of inducing young G5/G6 HSCs out of a homeostatic quiescent state. By tracking the real-time changes in the expression level of annexin V on HSCs induced to differentiate towards the myeloid lineage, we found that apoptosis was not the primary fate of G5/G6 HSCs upon entry into the cell cycle. Similarly, in vivo treatment with poly I:C induced the G5/G6 HSCs to enter into the cell cycle at the same rate as that of the G0 mice without inducing apoptosis. Transcriptomic analysis of poly I:C-treated G0 and G5/G6 HSCs, compared with vehicle-treated controls, revealed a significant enrichment of genes involved in the regulation of the cell cycle and platelet production, which is consistent with previous findings showing that megakaryocyte differentiation of HSCs occurs in response to poly I:C to replenish platelets that are lost during inflammatory insult. Importantly, we did not observe any significant change in gene expression between G0 and G5/G6 HSCs isolated from poly I:C-treated mice, which confirmed that telomeric damage did not limit HSCs' proliferation potential by activating programs of senescence or apoptosis.

Next, we evaluated the capability of single HSCs isolated from G0 or G5/G6 mice to either self-renew or differentiate. An evaluation of Numb inheritance and expression in G0 and G5/G6 HSCs (n=133 and n=113, respectively) induced to proliferate in vitro showed that G5/G6 HSCs had a 2-fold lower frequency of symmetric self-renewal division (p<10^-3) and a concomitant 2-fold higher frequency of symmetric commitment (p<10^-4). Accordingly, PB analysis revealed that the CD45.2-derived reconstitution was severely compromised in mice competitively transplanted with G5/G6 HSCs (0.26% vs 77%; p<10^-3). Single cell RNA-seq analysis of G0 and G5/G6 HSCs followed by the differential analysis of the clusters showed that 40% of G5/G6 HSCs were in an activated metabolic state associated with hyperactive OXPHOS and ROS signaling pathways, which are directly involved in HSC functional decline. Finally, we reactivated telomerase to investigate the possibility of restoring normal HSC function upon elimination of damage. Single cell RNA-seq and functional studies are ongoing to evaluate whether HSCs' activated metabolic state and compromised self-renewal capability are reversible processes.

This study challenges the concept that telomeric damage limits HSC's proliferative potential and offers unparalleled opportunities for unraveling regenerative strategies to ameliorate their decline.