Using mitochondrial activity to select for potent human hematopoietic stem cells

Hematopoietic cell transplantation is a critical curative approach for many blood disorders. However, obtaining grafts with sufficient numbers of hematopoietic stem cells (HSCs) that maintain long-term engraftment remains challenging; this is due partly to metabolic modulations that restrict the potency of HSCs outside of their native environment. To address this, we focused on mitochondria. We found that human HSCs are heterogeneous in their mitochondrial activity as measured by mitochondrial membrane potential (MMP) even within the highly purified CD34¹CD38²CD45RA²CD90¹CD49f¹ HSC population. We further found that the most potent HSCs exhibit the lowest mitochondrial activity in the population. We showed that the frequency of long-term culture initiating cells in MMP-low is significantly greater than in MMP-high CD34¹CD38²CD45RA²CD90¹ (CD90¹) HSCs. Notably, these 2 populations were distinct in their long-term repopulating capacity when transplanted into immunodeficient mice. The level of chimerism 7 months posttransplantation was.50-fold higher in the blood of MMP-low relative to MMP-high CD90¹ HSC recipients. Although more than 90% of both HSC subsets were in G₀, MMP-low CD90¹ HSCs exhibited delayed cell-cycle priming profile relative to MMP-high HSCs. These functional differences were associated with distinct mitochondrial morphology; MMP-low in contrast to MMP-high HSCs contained fragmented mitochondria. Our findings suggest that the lowest MMP level selects for the most potent, likely dormant, stem cells within the highly purified HSC population. These results identify a new approach for isolating highly potent human HSCs for further clinical applications. They also implicate mitochondria in the intrinsic regulation of human HSC quiescence and potency.