A method to investigate Pathological Variant load of Mitochondrial DNA in single human Mesenchyma stem cells

Mesenchymal stem cells (MSCs) are the parent cells to many cells of the musculoskeletal system including osteoblasts. Previous work has shown that in mouse models mitochondrial DNA (mtDNA) pathological variants lead to dysfunction of the respiratory chain of these osteoblasts and the premature development of age related osteoporosis. An increased rate of respiratory chain deficiency has also been observed in human osteoblasts. mtDNA mutates at an enhanced rate compared to nuclear DNA and has been linked to the ageing process in humans. 

We have developed and optimised an experimental pipeline to isolate single MSCs using flow cytometry before performing next generation sequencing to analyse the pathogenic variant load of 13 patients aged 22-88 to assess if pathogenic variants exist in mesenchymal stem cells and if there are age related changes.

Across all patients and all cells mtDNA pathological variants were present at similarly low levels. As per previous studies we found increased pathogenic variants within the mtDNA control region (D-Loop). However overall, there was no significant difference in the distribution of variants across the rest of the genome in all patients. Although a higher proportion of non-synonymous variants were seen this was not statistically significant.

We have shown that it is possible to isolate and sequence individual MSCs and observe mtDNA pathological variants. This gives a snapshot in time of the variant load for each patient. Due to low sample numbers compared to other studies investigate mtDNA in other cell types, we did not observe any age related increase in pathological variants. However this adds further evidence that clonally expanded, somatic mtDNA variants are common and could contribute to age related disease including osteoporosis.