Dye-Independent Methods Reveal Elevated Mitochondrial Mass in Hematopoietic Stem Cells

Abstract

Hematopoietic stem cells (HSCs) produce most cellular energy through glycolysis rather than through mitochondrial respiration. Consistent with this notion, mitochondrial mass has been reported to be low in HSCs. However, we found that staining with MitoTracker Green, a commonly used dye to measure mitochondrial content, leads to artefactually low fluorescence specifically in HSCs because of dye efflux. Using mtDNA quantification, enumeration of mitochondrial nucleoids, and fluorescence intensity of a genetically encoded mitochondrial reporter, we unequivocally show here that HSCs and multipotential progenitors (MPPs) have higher mitochondrial mass than lineage-committed progenitors and mature cells. Despite similar mitochondrial mass, respiratory capacity of MPPs exceeds that of HSCs. Furthermore, although elevated mitophagy has been invoked to explain low mitochondrial mass in HSCs, we observed that mitochondrial turnover capacity is comparatively low in HSCs. We propose that the role of mitochondria in HSC biology may have to be revisited in light of these findings.

Keywords: efflux; hematopoiesis; hematopoietic stem cells; mitochondria; mitochondrial respiration; mitochondrial turnover; mitotracker.

Figure 1. High mitochondrial mass in HSCs

a. MTG MFI of mouse BM hematopoietic populations normalized to HSCs (mean ± s.e.m., n=4). b. Fluorescence histogram (left) of mouse BM stained with MTG and relative mtDNA:nDNA ratio (right) within the 10% MTG^lo and MTG^hi fractions normalized to MTG^hi cells (mean ± s.e.m., n=5). c. Flow cytometric profile of mouse BM (green) and HSCs (black) stained with MTG in the absence (left) or presence (right) of VP. d. MTG MFI of mouse BM hematopoietic populations in the presence of VP normalized to HSCs (mean ± s.e.m., n=4). e. Relative mtDNA:nDNA ratio within the 10% MTG_VP^lo and MTG_VP^hi BM fractions stained in the presence of VP, normalized to MTG^hi (mean ± s.e.m., n=4). f. HSC frequency within the 10% MTG^lo and MTG^hi mouse BM fractions stained with MTG in the absence or presence of VP (mean ± s.e.m., n=6). g. Donor contribution of BM MTG^lo and MTG^hi fractions stained in the absence (left) or presence (right) of VP in the PB of recipients 16 weeks after competitive transplantation (mean ± s.e.m., n>15 recipients pooled from three independent transplants). h. Fluorescence histogram of BM (left) from mitoDendra2 mice and relative mtDNA:nDNA ratio (right) within the 10% Dendra2^lo, Dendra2^mid and Dendra2^hi fractions normalized to Dendra2^hi (mean ± s.e.m., n>3). i. Dendra2 MFI of BM populations of mitoDendra2 mice normalized to HSCs (mean ± s.e.m., n=3). j. HSC frequency within the 10% Dendra2^lo and Dendra2^hi BM fractions of mitoDendra2 mice (mean ± s.e.m., n=5). k. Donor contribution of BM Dendra2^lo and Dendra2^hi fractions in the PB of recipients 16 weeks after competitive transplantation (left) or serial transplantation (right) (mean ± s.e.m, n≥9 recipients pooled from two independent transplants). l. Relative mtDNA:nDNA ratio in mouse BM hematopoietic populations (mean ± s.e.m., n≥4) normalized to HSCs. m. Representative images of mitochondrial nucleoids visualized with immunostaining for Tfam in mouse BM hematopoietic populations (scale bar, 5.01μM). n. Quantification of mitochondrial nucleoids (mean, n≥17 cells pooled from three independent experiments, scale bar 5.01μm). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n.s.- not significant. See also Figures S1 and S2.

Figure 2. Metabolism and mitochondrial turnover

a. Real time analysis (left) of mitochondrial oxygen consumption (OCR) during mitochondrial stress test and quantitative analysis (right) of basal respiration, ATP production and maximal respiration in HSCs (LSKCD48^-), MPPs (LSKCD48+) and CPs (mean ± s.e.m., n=5). b. Relative mtDNA:nDNA ratio in HSCs from young and old mice normalized to young (mean ± s.e.m., n=3) c. Effect of mitophagy inducer CCCP and autophagy inhibitor CQ on the relative mtDNA:nDNA ratio of 3T3 and d. hematopoietic populations normalized to untreated (mean ± s.e.m., n˃3). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n.s.- not significant.