Enhanced intercellular transfer of mitochondria from nuclear respiratory factor 1 (NRF1)-primed mesenchymal stem cells: towards creation of superior mitochondrial delivery hubs
Mitochondrial dysfunction is a pervasive hallmark of diverse diseases. In endothelial cells (ECs), oxidative stress, bioenergetic failure, and dysregulated mitochondrial dynamics (fusion-fission, mitophagy) damage the endothelium and promote vascular pathologies such as diabetes, atherosclerosis, and aging. Mitochondrial augmentation, via direct transplantation of isolated mitochondria or cell-to-cell transfer of the organelle, has emerged as a strategy to restore mitochondrial function in metabolically compromised cells. We recently established that overexpressing nuclear respiratory factor 1 (NRF1), a driver of mitochondrial biogenesis, in mesenchymal stem cells (MSCs) increases mitochondrial content and preserves mitochondrial function under senescence-inducing stress. Here, we advance NRF1-primed MSCs as enhanced mitochondrial hubs for intercellular mitochondrial delivery to cells undergoing mitochondrial dysfunction. We hypothesized that NRF1 overexpression engages mitochondrial transfer machinery, thereby enhancing both tunneling nanotube (TNT)- and extracellular vesicle (EV)-mediated mitochondrial transfer to stressed ECs, improving EC mitochondrial fitness and health. mRNA-mediated NRF1 priming of MSCs increased expression of proteins involved in mitochondrial motility and transfer, enhanced TNT formation, and increased production of mitochondria-containing EVs. Single-cell RNA sequencing (scRNA-seq) results show that NRF1 priming shifted MSCs into distinct transcriptional states, with NRF1-enriched clusters exhibiting coordinated upregulation of cell-adhesion/cytoskeletal connectivity programs and vesicle-fusion/trafficking pathways, features consistent with enhanced structural coupling and secretory transfer capacity. NRF1 priming increased TNT-like F-actin intercellular bridges in direct co-culture and elevated mitochondria-containing EV transfer in transwell assays, demonstrating augmented mitochondrial delivery through both contact-dependent and contact-independent routes. Consequently, recipient ECs displayed reduced mitochondrial ROS, preserved membrane potential, improved oxidative phosphorylation and ATP production, rebalanced mitochondrial dynamics of fusion-fission and mitophagy. NRF1-primed MSCs further attenuated oxidative stress-induced EC senescence and apoptosis. Together, these findings identify NRF1 activation as a mechanism to reprogram MSCs into high-capacity mitochondrial donors and support NRF1-driven mitochondrial hub engineering as a strategy to strengthen mitochondrial transfer-based therapies for diseases characterized by mitochondrial dysfunction.