The Importance Of Being Physiologically Relevant: Molecular Methods To Identify Mitochondrial Transfer In Bone Marrow

Mitochondria are organelles that play a key role in cell metabolism and survival. We assume that mitochondria remain within their eukaryotic host cell, yet mitochondria move between cells; multiple in vitro and in vivo studies have now shown this. However, a comprehensive, tissue-wide, and physiologically relevant exploration of the cells, both cancerous and non-cancerous, that partake in mitochondrial transfer (MT) remains understudied, for cells of the bone marrow as for other tissue. Furthermore, molecular methods of MT identification, methods that leverage the self-contained mitochondrial genome and open the door to quantitative, reproducible, and high-throughput measurement of MT, are scarce.

This thesis comprises design of molecular techniques to determine the prevalence of intercellular MT in murine bone marrow cells, between haematopoietic cells of bone marrow transplants (BMTs), and the acute myeloid leukaemia (AML)-like C1498 line in a murine bone marrow cancer model. Adaptation in a subset of BMTs explored the effect of other cell populations, such as lineage negative (stem-like) cells, adherent (stromal-like) cells, and cells derived from compacted bone, as well as the effect of cell damage on MT. BMTs and the cancer cell line model were carried out on animal strains with at least one mitochondrial DNA (mtDNA) single nucleotide polymorphism (SNP) between cells to allow for MT detection.

MtDNA acquired from these models was examined in protocols designed, optimised, and employed to determine incidence of MT: Allele-Specific with a Blocking reagent (ASB) qPCR; Four-Primer and barcode ligation MinION amplicon sequencing; and Illumina miSeq amplicon deep sequencing. The latter of these could hypothetically identify MT resultant mtDNA sequences at a concentration of 1 in 10,000, although without the assurance of an additional CD45 nuclear control, which was found to be less sensitive.

Infrequent evidence that mitochondria could be transferred amongst the cells of the bone marrow was uncovered while utilising the most sensitive MT identification assay, Illumina sequencing. Although only preliminary, rare, that is, occurring in a minority of samples (5.8%-25%) but strong, that is displaying variant frequencies greater than 0.13 (0.2-0.5) indicative of MT, occurred in bone marrow cell populations, from standard haematopoietic, irradiation-damaged haematopoietic, and cancer cell line samples. Further investigation, preferably with an additional nuclear control SNP and more precise statistical methods, is needed to corroborate this infrequent MT signal in bone marrow cells.

The fundamental importance of mitochondria in biology and human health makes this research critical, as transfer of mitochondria is a proposed as a potential method of therapy resistance in cancers.