Identification of Potent Human Hematopoietic Stem Cells (HSCs) Using Mitochondrial Profile Towards Improving HSC Transplantation

Hematopoietic stem cell (HSC) transplantation remains the only cure for many blood and other disorders. However, achieving both sufficient progenitor cell numbers while maintaining long-term stem cell engraftment remains challenging. To address this, we explored metabolic properties that may modulate HSCs’ potency and quiescence and focused on mitochondrial activity of human HSCs. We found that human HSCs are heterogeneous in their mitochondrial membrane potential (MMP), a marker of mitochondrial activity. MMP profiles progressively shift towards lower levels in subpopulations with phenotypes of higher hematopoietic hierarchy (both non-mobilized peripheral blood, p<0.01 and CB, cord blood, p<0.001). Notably, even the most potent human CD34+CD38-CD90+CD45RA-CD49f+ (CD49f+) HSCs exhibited heterogeneous MMPs. These results are consistent with findings from our laboratory and others in murine HSCs (Liang, Arif et al., Cell Stem Cell, 2020, Sukumar et al, 2016, Vannini et al., 2016). We analyzed the functional activity of distinct subsets of MMP low and MMP high HSCs [25% lowest and highest MMP within CD34+CD38-CD90+CD45RA- (CD90+) HSCs] and showed that the frequency of LTC-IC in FACS-sorted MMP low HSCs is 1 in 7.75 as determined by limiting dilution and 7 times greater than MMP high HSCs (1 in 54.2) (p<0.01). Despite greatly reduced total number of colony forming cell generated from LTC-IC, the ratio of burst-forming unit-erythroid (BFU-E) to granulocyte/macrophage (G/M) colonies was 45 times higher in MMP high as compared to MMP low HSCs (p<0.01). In comparing in vivo repopulating capacity of these two fractions by transplanting 800 CB CD90+ HSCs of each subpopulation into sub-lethally irradiated immunodeficient NSG (NOD/SCID/IL2Rγnull) mice, we found the level of chimerism in peripheral blood was over 50 times greater in recipients of MMP low as compared to MMP high HSCs [16.7 (2.3-57.4) % vs. 0.33 (0.06-0.72) %; p<0.01, n=5] 7 months post transplantation. Unexpectedly, bone marrow of MMP high HSC recipients showed increased myeloid (CD33) vs. lymphoid (CD3 or CD19) lineage ratio (p<0.001). These results indicate greater stem cell potential of MMP low HSCs and a tendency to myeloid-biased lineage commitment in MMP high HSCs. We were surprised to find despite these differences in repopulation capacity in NSG mice, above 90% of both MMP low and MMP high HSCs were in G₀ phase of cell cycle as analyzed by Pyronin-Y/Hoechst staining. CDK6 expression, which is associated with HSC activation, was also undetectable by confocal microscopy in both fractions. To identify potential distinct kinetic of cell cycle entry, FACS-sorted single cells were cultured in serum free media containing SCF, TPO and Flt3. The division of each cell was monitored under microscopy every 12 hours for 6 days, followed by kinetic analysis [Only viable cells were scored; n= 98 (MMP low), n=104 (MMP high)]. The result revealed a slower kinetic of first cell division of MMP low as compared to MMP high HSCs. Both the mean time to complete first division (83.5 vs. 77.2 hrs; p<0.05) and the interval between the first and second division (39.0 vs. 35.8 hrs) were delayed for MMP low as compared to MMP high CD90+ HSCs. Furthermore, MMP high HSCs expressed higher level of CDK6 (p<0.01) after 34 hours’ culture, and expanded 3.5 times more (p=0.001) after 9 days’ culture as compared to MMP low HSCs. These results indicate that, upon cytokine priming quiescent MMP high HSCs exist G₀ phase more rapidly than MMP low HSCs. These functional differences were associated with distinct mitochondrial morphology as analyzed by mitochondrial specific probe TOM20. While total mitochondrial content remained similar (p>0.05), mitochondria of MMP low HSCs were fragmented to a greater extent as compared to MMP high HSCs (p<0.05). In addition, MMP low HSCs exhibited increased co-localization of DRP1 GTPase, that mediates mitochondrial fragmentation, to TOM20 as compared to MMP high CD90+ HSCs (p<0.01).

Altogether, these results suggest that highly primitive human HSCs are functionally segregated by their MMP levels. MMP low HSCs maintain quiescence ex vivo to a higher degree and exhibit greater stem cell potential, whereas MMP high HSCs are more primed for activation. These studies suggest that graded mitochondrial activity may be implicated in regulating human HSC quiescence and functional potency and provide insights towards improving human HSC transplantation.