Hematopoietic Stem Cell Quiescence Exposed Using Mitochondrial Membrane Potential

Abstract

Purpose of the Review:

Quiescence is a fundamental property of hematopoietic stem cells (HSCs). Despite the importance of quiescence in predicting the potency of HSCs, tools that measure routinely the degree of quiescence or select for quiescent HSCs have been lacking. This Commentary discusses recent findings that address this fundamental gap in the HSC tool box.

Recent findings:

Highly purified phenotypically-defined HSCs are heterogeneous in their mitochondrial membrane potential (MMP). The lowest MMP subsets are enriched in greatly quiescent HSCs with the highest potency within the purified HSC population. MMP provides an intrinsic probe to select HSC subsets with unique cell cycle properties and distinct stem cell potential. Using this approach, new and unanticipated metabolic properties of quiescent HSCs have been discovered. This methodology may improve the mechanistic understanding, of HSCs’ exit from and entry to, quiescence.

Summary:

Selecting HSCs using MMP is likely to lead to discoveries of new HSC properties, may improve the ex vivo maintenance of HSCs, and has implications for the clinic, including for improving HSC transplantations.

Commentary

Quiescence is a reversible state of growth arrest that is actively maintained in adult hematopoietic stem cells (HSCs) and which is vital to their biology ¹. Quiescence enables stem cells to sustain tissue homeostasis and allows for repair of defective/damaged tissue during their lifetime. By adopting a quiescent state, stem cells restrict their metabolism, and actively maintain low sensitivity to available nutrients. Quiescent HSCs are however fully apt to promptly mobilize resources to generate biomass when needed, for cell division. The stem cell niche is the site where HSCs are maintained in their quiescent state. Paradoxically, the depth of HSC quiescence determines HSC potency, optimally defined by HSCs’ capacity to fully reconstitute blood when transplanted in an animal where blood formation is compromised in defined in vivo transplantation settings. Quiescent stem cells are endowed with multipotency, although the daily production of blood is most likely sustained by lineage committed progenitors ². With age, quiescence is lost in a significant fraction of HSCs ^3–6. Loss of quiescence is at the core of the age-associated loss of stem cell regenerative capacity ^1,7. Loss of quiescence is also associated with increased load of HSC somatic mutations leading to clonal HSC dominance with age. This leads to loss of diversity in the pool of old stem cells that contribute to the blood formation that is associated with substantially diminished mature lineage output and its clinical consequences ^8,9. On the other hand, adopting a quiescent state in stem and progenitor cells is critical for generating leukemic stem cells (LSCs). Quiescence shields LSCs from therapies designed to target rapidly dividing cells, thereby fostering the recurrence of leukemia. Targeting quiescent leukemic stem cells is essential for a successful long-lasting leukemia therapy. Thus, measuring the depth of quiescence is critical for a mechanistic understanding of the reversible HSC exit from vs. entry to quiescence, and overall studies of healthy blood forming stem cell potential.

DNA nucleoside analogs and fluorescent dyes have been routinely used to measure HSC cycle progression vs. quiescence, however, they do not capture the depth of HSC quiescence ^10–12. These approaches, which may be useful for some types of evaluation, provide only a snapshot of cell division with correlative data on the HSC functional capacity. Quiescence of HSCs was best documented by the use of the GFP label retaining system that is based on the dilution of the fusion protein H2B-GFP (from Histone 2B fused with Green fluorescent protein) with each HSC division. This approach showed definitively that HSCs divide extremely rarely ^10,13. Mitochondrial membrane potential (MMP), the electric gradient across the mitochondrial inner membrane (or charge), ¹⁴, also measures HSC quiescence. The level of MMPs is reversely correlated with HSC potency ^11,15,16. The MMP heterogeneity segregates functionally distinct mouse and human HSC subsets, within highly purified phenotypically-defined primitive HSCs; thus, relative low levels of MMP is an intrinsic determinant of HSC quiescence ^6,15–21. Notably, the molecular signature of MMP-low and high HSCs is greatly similar to/overlapping with that of label-retention-defined dormant and activated HSCs ^11,22. While the degree of label retention is manifested only when HSCs are divided and physically separated, MMPs distinguish the quiescent (in G0) vs. cycling-primed (in G1) phase of the cell cycle in highly purified HSCs ¹¹. In addition, MMPs provide a probe, intrinsic to the cell in identifying quiescent subsets under homeostasis in both mouse and human HSCs, as well as in old and young HSCs, while label retention requires the use of transgenic mouse systems not easily adaptable to human HSCs ²³. MMP is a superior indicator of HSCs primed for activation than their cell cycle quiescence state defined by routine assays ²⁴. Therefore, selecting HSCs based on their intrinsic MMP is attractive for comparing the depth of quiescence of subsets in a pool of highly purified, yet heterogeneous, HSCs.

MMP segregation of HSC subsets revealed fundamental and new HSC properties ^6,15–19 (and reviewed in ^7,23,25). Using MMP, it was shown that in contrast to a long-held view, glycolysis is a source of energy in primed (towards activation) but not in quiescent HSCs ¹¹. This finding is in line with the notion that glucose fuels rapidly dividing, and not quiescent cells ^6,26. In addition, using this approach it was discovered, that lysosomes actively and dynamically regulate HSCs’ switch between quiescence and primed for activation ¹¹. These results are the opposite of what would have been anticipated from the known function of autophagy in HSCs ^6,27. Importantly, these findings contribute to a growing literature on the lysosomal platform as a fundamental regulator of cellular quiescence, including in stem cells beyond HSCs ^{7,23,25,28–31}. Lysosomal sequestration of damaged mitochondria, and potentially other cargo, eliminates unwanted damaged organelle/molecules in quiescent HSCs, modulates the generation of carbon mass, and contributes to priming stem cells ¹¹. In addition, lysosomal retention of mitochondria regulates HSCs’ MMP levels. Nonetheless, the precise molecular mechanism of lysosomal regulation of HSC quiescence and metabolic output during HSC priming have yet to be uncovered. These findings have major implications for the investigations of HSC as well as for the use of potent HSCs with balanced blood output in the clinic ³². They also raise the possibility that additional key properties of quiescent HSCs will be discovered. These include the function of a number of transcription factors that are specifically expressed in quiescent MMP-low HSCs ¹¹.

Although the use of MMP levels offers a unique view of quiescent HSCs, MMP do not inform on the extent of HSC quiescence over time or HSCs’ divisional history. Notably, for studies to generate meaningful data, MMP used in HSC comparative analyses should be clearly defined. In our studies the lowest 25% of MMP levels are compared to HSCs with the highest 25% of MMP levels ^11,24. Using different definitions of MMP-low vs. -high, and despite their similar data overall, investigators may reach different conclusions (see ^{11,15,16,19,24} (and commented in ^23,33). In addition, because loss of quiescence, under homeostasis, but not in response to stress, is associated with loss of stem cell function leading to stem cell exhaustion ¹³, the precise modulations of MMP in response to stressors, including to cultured milieu, and the mechanisms involved should be elucidated.

As low MMP levels predict quiescence, the mechanistic understanding of the regulation of mitochondrial function in quiescent HSCs, and the modulation of mitochondrial membrane potential with HSC priming, are critical. Lysosomal mitochondrial communications and cross regulations may be key in this process. Future HSC investigations using MMP subsets have the potential to yield additional transformative data.

• Mitochondrial membrane potential (MMP) provides an intrinsic probe to identify deeply quiescent HSCs and select for the most potent HSCs

• MMP-low HSCs are enriched for large and slow degrading lysosomes that dynamically regulate HSC potency

• Primed (MMP-high) but not quiescent (MMP-low) HSCs rely on glycolysis for their maintenance