Mitochondria are the power plants of the cell, the distant descendants of symbiotic bacteria that carry their own small circular genome, distinct from that of the cell nucleus. The mitochondrial genome is more prone to damage and less well repaired than the nuclear genome, and mitochondrial DNA mutations are thought to be important in aging. Deletion mutations can create broken mitochondria that outcompete undamaged peers to take over a cell, creating a small number of harmfully dysfunctional cells. Less severe point mutations are more commonplace, but evidence is contradictory regarding the degree to which this form of damage contributes to mitochondrial dysfunction in aging. Hence the value of generating a cell model of aging-like mitochondrial damage, to better enable studies of the dysfunction it generates.
The consequences of heteroplasmic mitochondrial mutations have been challenging to study as genome editing for mitochondrial DNA (mtDNA) is limited and there are few established tools to alter heteroplasmy in vitro. Model systems such as the "mtDNA mutator" mouse containing a mutant polymerase gamma implicate mtDNA changes in many aging phenotypes. However, this mouse model induces a large mix of genome alterations often with mtDNA depletion in cells, yielding much more disruption than the clonally expanded heteroplasmic mutation events that occur in usual aging in vivo. Much of our current knowledge regarding heteroplasmy comes from comparisons of primary cells from patients with mtDNA mutations to controls, often with low mutant heteroplasmy and unmatched nuclear genetics, or from immortal "cybrid" cells, which have a malignant pathophysiology and limit the capacity to study the impact of heteroplasmy on cell fate and viability.
Reprogramming somatic cells to pluripotency has been shown to reverse some markers of aging, and expression of reprogramming factors is proposed as a potential rejuvenating therapy. However, the impact of mtDNA heteroplasmy on this process has not been queried. Although heteroplasmy of pathogenic mtDNA variants is typically stable for differentiated cells in culture, multiple recent studies established that heteroplasmy shifts significantly with reprogramming of primary cells to induced pluripotent stem cells (iPSCs). However, beyond this single-measure characterization, the impact of altered heteroplasmy on cell function, and particularly on the capacity for rejuvenation remains unexplored. This is a key area to understand as critical roles are rapidly evolving for mitochondrial metabolism in both maintenance of pluripotency and stem cell differentiation.
We note that the differential segregation of mtDNA heteroplasmy following iPSC generation offers a novel opportunity to understand the impact of clonal increases or decreases in mtDNA heteroplasmy on cellular function. We hypothesize that iPSCs with increased mtDNA heteroplasmy have functional adaptations consistent with cellular aging. Thus, we generated iPSC colonies from three primary fibroblast lines with known heteroplasmy of deleterious mtDNA mutations and quantified heteroplasmy of these mutations in resultant clones. We report that resultant clones displayed a primary bimodal distribution of mutation heteroplasmy. We determined that high-level mtDNA deletion mutant iPSCs exhibit distinct growth properties, metabolic profiles, and altered differentiation capacity, with growth and metabolic shifts mirroring a key subset of changes observed in aging-induced cell and tissue dysfunction.
Link: https://doi.org/10.1111/acel.14402
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