Metabolic Activity Distinguish Reserve and Primed HSCs

A population of reserve HSCs has been reported to be in a deep-quiescent (or dormant) state and function as a back-up population of HSCs to support life-time hematopoiesis (Li and Clevers, 2010; Wilson et al., 2008). Currently, characterization of reserve HSCs is mainly based on cell cycle quiescence, however, the metabolic state in reserve HSCs is yet to be defined. Here, we show that reserve HSCs maintain not only a quiescent state but also an overall low metabolic activity whereas the primed HSCs maintain still a quiescent state but primed at metabolic state. First, we used CD49b(Benveniste et al., 2010) to further separate conventional long-term (LT) HSCs (CD34^-Flk2^-Lineage^-Sca-1⁺c-Kit⁺)(Yang et al., 2005) into CD49b^lo and CD49b^hi subpopulations and confirmed their enrichment with previously identified dormant (Scl-H2B-GFP label retaining cells, LRCs), thus we termed CD49b^lo and CD49b^hi subpopulations as candidates of reserve and primed HSCs. We then determined the cell cycle frequencies of reserve, primed, and ST-HSCs (Cd34⁺Flk2^-LSK) respectively as once in 3 months, 3-4 weeks, and 3-4 days. Functionally, Reserve HSCs had on average 3.5-fold higher functional capacity compared with primed HSCs. RNA-seq analysis revealed that reserve HSC predominantly expressed a list of imprinting genes that associate with growth-restriction functions; primed HSCs expressed relatively-high number of genes involving in mitochondria fusion, organization, and function, while ST-HSCs expressed genes reflecting an active cycling state. Conducting metabolic assays, we found that reserve HSCs not only maintain quiescence but also maintain overall low metabolic activity in both glycolysis and mitochondrial capacity. In contrast, primed HSCs are in a quiescent state, but with their metabolic state primed as evidenced by an increased glycolytic activity and mitochondrial potential for subsequent active proliferating state in ST-HSCs. Intriguingly, our data suggests that the functionality of reserve HSCs correlates to metabolic state rather than cell cycle.