Mitofusin agonists improve the long-term repopulating activity of human cord blood HSCs after ex vivo culture

Umbilical cord blood (CBU) is a valuable source of hematopoietic stem cells (HSCs) due to lower incidence of graft-versus-host disease. However, limited HSC counts restrict use in adults, necessitating improved methods for ex vivo expansion and improved HSC function. Mitofusin 2 (MFN2), a mitochondrial membrane fusion protein, is necessary for preserving HSC function and mitofusin agonists have been characterized. We report ex vivo culture of CBU HSCs with mitofusin agonists (MAs) enhances long-term repopulating activity by over five-fold in both primary and secondary transplantation assays without changes of total nucleated cells or phenotypic HSCs. Mechanistically, MA-treated HSCs show suppressed protein synthesis, increased autophagic flux, and elevated lysosomal mass. Transcriptomic analysis implicates downregulation of MTOR signaling, and immunoprecipitation studies suggest MFN2 interacts with MTOR. These data support a model in which fusion-competent MFN2 sequesters MTOR, promoting a catabolic state that preserves HSC potency. Our findings suggest a novel MFN2-MTOR regulatory axis that enhances the function of human HSCs cultured ex vivo for potential therapeutic application.