Mitochondrial Transfer to Host Cells from Ex Vivo Expanded Donor Hematopoietic Stem Cells

Mitochondria are highly dynamic organelles that orchestrate cell metabolism in complex ways by fission/fusion machinery, factors controlling biogenesis, ion transport, and autophagy/mitophagy. Mitochondrial dysfunction occurs not only in rare inherited disorders such as Leigh syndrome but also in various conditions including degenerative changes, metabolic syndromes, chronic inflammation, and cancers.

Mitochondrial DNA (mtDNA) transfer is an emerging mechanism that enables restoration of mitochondrial function in damaged cells or tissues. Recent evidence suggests that MSCs can be an efficient source of mitochondrial transfer. Thus, developing the technology to transfer mtDNA may be a promising strategy for the treatment of these conditions. However, other primary cells, such as hematopoietic stem cells (HSCs), have not been fully studied due to the lack of established technology to expand ex vivo. To test if mouse HSCs are capable of initiating mitochondrial transfer in the transplant setting we utilized mitochondrial-nuclear exchange (MNX) mice generated by assisted reproductive technology (Kesterson, 2016).

To evaluate mitochondrial transfer from donor HSCs to recipient cells, we utilized an ex vivo culture of mouse HSCs based on methodology recently reported (Wilkinson, 2019). First, we harvested bone marrow from young CD57BL6 mice and isolated HSCs to establish and confirm the reproducibility of ex vivo culture system. In our experiments, careful monitoring of cell morphology was required to time media changes utilizing the alternative culture media originally described. Moreover, we found that a stringent cKit+Sca1+lineage- (KSL) CD150+CD34- gate was required to sort an HSC population to avoid the contamination of hematopoietic progenitor cells. We observed comparable qualities of HSC expansion in vitro by flow cytometry as well as in vivo expansion of donor HSC (CD45.2BL6) in recipient mice (CD45.1UBC/GFP) with conditional transplant utilizing lethal irradiation.

To assess the mitochondrial transfer via donor HSCs, we used MNX mice with nuclei from C57BL/6J and mitochondria from the C3H/HeN strain, which allows detection of donor C3H mtDNA in C57 recipient cells. MNX mice have C57 immuno-phenotype and thus cells from MNX mice can be safely transplanted into the C57 hosts without triggering any immune response. Mitochondria in MNX mice inherit the bioenergetic economy of the C3H strain compared to the C57 strain. Thus, we hypothesized that the more resistant mitochondria of the donor MNX cells will have high chances of transfer into the C57 host cells. We assessed ex vivo HSC culture utilizing MNX mice (10 month old). As expected, MNX mice had increased in KSL and CD150+ HSC populations compared to young donors as well as in ex vivo culture. We then injected 5x10⁵ex vivo expanded MNX HSCs into lethally irradiated CD45.1 UBC/GFP recipient mice. We harvested recipient mice at 6 weeks to evaluate mitochondrial transfer from MNX donor to recipient, which is an early time point when hematopoietic engraftment should have stabilized. The chimerism in each tissue was very high, especially in the bone marrow. In parallel with this, we found that HPC/HSCs in the bone marrow were highly replaced with donor-derived cells and each population cell number was comparable with the non-transplant age- and sex-matched mice, which implies robust reconstitution capacities of donor cells in vivo. To investigate the potential of donor HSCs to transfer their mitochondria, we separated GFP- donor cells and GFP+ host cells via sorting from spleen and BM. The DNA analysis was available in sorted cells from spleen but not from BM that showed extremely low frequency recipient cells. The PCR after digestion with specific restriction enzymes showed that the donor C3H mtDNA was transferred to the GFP+ host cells. Interestingly, the reverse transfer of mtDNA from recipient to donor was also detected but to a much lower extent, which suggests bi-directionality of mitochondrial transfer.

In summary, we have evaluated and confirmed robust expansion of mouse HSCs ex vivo. This culture system, while requiring careful monitoring and optimization of culture constituents, has the potential for broad range of applications in HSC research. We have also established that using this technology enables us to demonstrate mitochondrial transfer between donor HSCs and recipient cells in the transplant setting.

Calvi:University of Rochester School of Medicine and Dentistry: Patents & Royalties: U.S. Patent No. 9,394,520; Massachusetts General Hospital and Harvard Medical School: Patents & Royalties: U.S. Patent No. 8,802,104 B2.