Mitofusin agonists enhance long-term engraftment and potency of HSC cultures in vivo.

Umbilical cord blood (CBU) is a valuable source of hematopoietic stem cells (HSCs) due to its superior donor compatibility and lower incidence of graft-versus-host disease. However, its limited HSC content restricts its use in adult transplantation, necessitating new targets for ex vivo expansion and improved HSC potency. Mitofusin 2 (MFN2), a mitochondrial membrane fusion protein, is necessary for preserving HSC function and agonists of mitofusin activity have been characterized. We report that 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 acidification. Transcriptomic analysis implicates downregulation of MTOR signaling, and immunoprecipitation studies confirm a direct interaction between MFN2 and 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 functional expansion of human HSCs for potential therapeutic application.