Rejuvenation of aged hematopoietic stem cells and niche via mitochondrial engineering. - Abstract Hematopoietic stem cell (HSC) transplantation (HSCT) and HSC based gene and cell therapies hold curative potential for diverse hematologic and immunologic disorders. However, donor age remains a critical determinant of transplant success. Aging drives profound HSC dysfunction, manifested by diminished regenerative potential, lineage skewing toward myelopoiesis, and an increased burden of clonal hematopoiesis (CH) associated mutations. These defects heighten the risk of graft failure, donor-derived leukemia, and poor survival after HSCT. Aging-associated HSC decline is driven in large by mitochondrial dysfunction. Aged HSCs exhibit impaired mitophagy, aberrant oxidative phosphorylation, and disrupted bioenergetics, changes that are further amplified by chronic inflammation and remodeling of the bone marrow (BM) niche. The fate and function of HSCs are shaped not only by their intrinsic metabolic state but also by their communication with niche partners, particularly mesenchymal stromal cells (MSCs). Our recent work demonstrated that HSPC-BM niche interactions are metabolically coupled via Connexin-43 dependent mitochondrial transfer, preserving HSC survival and niche function. Moreover, targeted mitochondrial supplementation into HSC preserves mitochondrial integrity, enhances metabolic adaptability, and maintains self-renewal during stress hematopoiesis, highlighting the importance of both mitochondrial quality and intercellular mitochondrial communication in hematopoietic regeneration. Our preliminary data show that mitochondrial transfer from HSPCs to MSCs markedly declines with age, compromising MSC bioenergetics and weakening their ability to sustain balanced hematopoiesis. We further identified that supplementation of aged HSCs with young mitochondria rescues mitochondrial membrane potential, restores AcH4K16 polarity, and rejuvenates transcriptome of aged HSC. Together, these findings highlight mitochondrial quality and transfer as central drivers of hematopoietic aging and compelling therapeutic targets. This proposal seeks to define the mechanistic basis and therapeutic potential of mitochondrial engineering to rejuvenate aging hematopoiesis. In Aim 1, we will test whether heterochronic mitochondrial supplementation and augmentation of mitochondrial function restore the youthful potential of aged HSCs by rewiring metabolic flux, enhancing autophagy, and resetting epigenetic programs. In Aim 2, we will dissect how HSPC-MSC mitochondrial transfer governs niche metabolism, trophic function, and the competitive fitness of aged versus CH-mutant HSPCs. By integrating metabolic, epigenetic, and intercellular communication pathways, this work addresses fundamental drivers of hematopoietic aging. The anticipated outcomes will provide mechanistic insight into how restoring mitochondrial quality and transfer can normalize aged HSC function and niche support. Ultimately, this study lay the foundation for innovative, clinically translatable strategies to improve HSCT outcomes in older patients and donors, while reducing risks of clonal dominance and age associated hematologic disease.
