Mitochondrial Activity Identifies a Transcriptionally and Functionally Distinct Subset of Aged HSCs with Lineage-Balanced Output

During ageing, the haematopoietic stem cell (HSC) pool expands numerically, but declines functionally. This functional decline is characterised by myeloid skewing and decreased long-term reconstitution potential and clinically manifests as anaemia, immune compromise and increased risk of clonal malignancy. We postulate that HSCs do not synchronously functionally decline with age, but instead represent a spectrum in which physiologically aged HSCs become dominant. In this study, we aimed to reveal properties that might identify physiologically young HSCs during chronological ageing and to exploit these in an attempt to rescue haematopoietic ageing. Young adult ((y), 2-3 months) and aged (old (o), >18 months) mouse HSCs were profiled and we observed a significant decrease (30-50%) in mitochondrial membrane potential (MMP) in oHSCs. Interestingly, a small fraction (15%) of oHSCs maintained a similar MMP to the bulk (70%) of yHSCs. We explored, initially at the transcriptional level, whether these MMPhigh HSCs are distinct from MMPlow HSCs. Strikingly, RNA sequencing of MMPhigh and MMPlow young and aged HSCs revealed that samples cluster by MMP over age. MMPhigh young and aged HSCs were characterised by upregulation of lymphoid and erythroid lineage markers, as well as RNA processing, MYC and E2F pathways. MMPlow young and aged HSCs were transcriptionally associated with ageing, inflammation and myeloid bias. Based on these results, we hypothesised that enhancing MMP in oHSCs might restore lineage-balanced peripheral blood (PB) output, used as a measure of functional improvement. To achieve this, we chemically enhanced MMP in oHSCs in vivo, using the mitochondrially-targeted drug mitoquinol (MQ). Interestingly, our initial experiments show that a 5-day treatment with MQ significantly shifted the B-cell/myeloid ratio in PB from 0.6 (aged) to 1.5 (MQ), in the direction of the ratio observed in young mice (2.9). This ratio change was not due to numerical depletion of myeloid cells, but due to restoration of B-cell numbers, including IgM+ B-cells commonly reduced with age. To assess whether the effect of MQ was due to HSC-intrinsic changes and could be sustained over time, HSCs were isolated from MQ-treated or untreated aged mice and transplanted into lethally irradiated recipients. Our experiments to date show that HSCs isolated from MQ-treated aged mice show superior engraftment and faster and greater B-cell reconstitution than HSCs from age-matched untreated animals, and that these improvements are stable over the 16-week assay. Based on our sequencing results indicating that RNA processing, MYC and E2F pathways are associated with MMPhigh HSCs, and previous work reporting a relationship between MMP and rate of mRNA transcription (das Neves et al., PLoS Biol. 2010; Johnston et al., PLoS Comput. Biol. 2012), we explored the possibility that MMP might orchestrate the observed changes by altering transcriptional rate of HSCs. We tested transcription rate in HSCs in vivo and found that yHSCs display a particularly high rate of mRNA transcription. Rate of transcription was significantly reduced in oHSCs compared to yHSCs, equivalent to their quantitative reduction in MMP. We could demonstrate a direct correlation between MMP and transcription rate in HSCs, by showing that MMPhigh sorted HSCs were transcribing nearly twice as fast as MMPlow sorted HSCs. Furthermore, in vivo injection with mitochondrial uncoupler CCCP caused a similar reduction in transcription rate of HSCs (>50%) as did conventional RNA Pol-II inhibitors (DRB, Flavopiridol), hereby demonstrating that transcription rate directly depends on MMP. This work indicates that mitochondrial state can separate HSCs with distinct transcriptional profiles linked to different cell fates. We speculate that changes in transcriptional profile arise from MMP-driven regulation of transcriptional rate in HSCs. This would open up the possibility that pharmacological manipulation of mitochondrial activity can alter transcriptional programs of HSCs with consequences for functionality. Disclosures No relevant conflicts of interest to declare.