The reprogramming of ordinary somatic cells into induced pluripotent stem cells, capable in principle of then generating any other type of cell, was a major advance for cell biology and its application to medicine. It is still sufficiently recent for the implications and uses still to be a work in progress. One of the more interesting observations to emerge from the recent years of experimentation is that this reprogramming appears to erase some aspects of mitochondrial aging. Take fibroblasts with damaged mitochondria from a skin sample from an aged individual, reprogram them to generate a population of induced pluripotent stem cells, differentiate those stem cells into a new set of fibroblasts, and the resulting cell population has dramatically improved mitochondrial function. One possibility is that reprogramming triggers some aspects of the comprehensive repair programs that take place very early in embryonic development, wiping away as much of the parental molecular damage as possible. Parents are old and babies are born young, so something of this ilk must be hidden away somewhere in the repertoire of cellular behavior. That isn't to say it can be usefully applied in adults, of course: there are any number of vital, intricate structures in our organs, the brain particularly, that would probably be fatally disrupted by the operation of such a program. Time will tell.
Is this apparent mitochondrial rejuvenation actually mitochondrial rejuvenation, however? Is it fixing the all-important damage to mitochondrial DNA, for example? Every cell has hundreds of mitochondria, the descendants of ancient symbiotic bacteria, complete with a leftover fragment of the original DNA that still encodes a range of necessary proteins used in mitochondrial functions. Mitochondria still divide like bacteria to make up their numbers, even though they are treated just like any other cellular component and broken down for recycling when damaged. Their most important function is the generation of energy store molecules to power cellular operations, but this process produces oxidizing molecules as a side-effect. They damage the cellular machinery they react with, and the most vulnerable target is the mitochondrial DNA right next door. Most oxidative damage to proteins and DNA in cells is rapidly repaired, but mitochondrial DNA isn't as well protected as the DNA in the cell nucleus. Further, some forms of mitochondrial DNA damage, such as large deletions, can produce mutant mitochondria that are both dysfunction and resistant to being culled by cellular quality control mechanisms. They quickly outcompete the normal mitochondria, and a cell taken over in this way becomes dsyfunctional itself, carrying out a range of bad behavior that contributes to the progression of aging. Thus mitochondrial DNA damage is an important topic; if researchers observe what looks like mitochondrial rejuvenation, then the quality of the mitochondrial DNA is a key question.
The authors of this commentary discuss a paper published earlier this year that argues against repair of mitochondrial DNA in the course of cellular reprogramming. If confirmed that means that a potential shortcut to allow cell therapies to better treat the diseases of aging may not in fact exist: dealing with mitochondrial DNA damage when using a patient's own cells is still required, one way or another. The favored method is that outlined in the SENS proposals, using gene therapy to move critical mitochondrial genes into the cell nucleus. There are other possible approaches, though none of those seem to be as far along towards clinical application. While one door closes, another opens, however. As pointed out below, the preservation of mitochondrial damage might indicate that reprogramming as it presently stands, in which only a tiny number of cells are successfully converted, may be a good way amplify rare mutations in cell samples. That in turn might help with the still challenging task of putting reliable numbers to the degree to which mitochondrial DNA is damaged in old cells.
The process of cellular reprogramming is believed to be able to "turn back the developmental clock" by allowing somatic cells to acquire a state that is normally associated only with embryonic stem cells (ESCs). Indeed, human induced pluripotent stem cells (iPSCs) can be obtained from aged individuals and still show the key properties of ESCs, including self-renewal, elongated telomeres, and round-shaped mitochondria with underdeveloped cristae. However, it remained to be determined whether reprogramming to pluripotency could actually erase aging-associated signatures and thus represent a rejuvenation route. A new paper now clearly demonstrates that iPSCs not only do not erase the signs of aging but, due to their clonal origin, may even reveal aging-related defects in the mitochondrial DNA (mtDNA) that were not detectable in the whole parental tissues.
Using iPSCs derived from both skin fibroblasts and peripheral blood mononuclear cells (PBMCs) researchers have shown that all iPSCs exhibited mtDNA mutations that could not be observed in the whole-tissue DNA extracts of the parental cells. These mutations were originally considered as negative by-products of reprogramming as a consequence of oxidative stress-mediated genomic damage. However, it was demonstrated that also skin fibroblasts grown as individual clones exhibit mtDNA mutations that are not seen in the pooled fibroblast population. Hence, individual cloned fibroblasts and iPSCs may both represent the progeny of a single parental fibroblast cell, thereby enabling the detection of mtDNA mutations that were already present in the original fibroblast population but remained undetectable due to their relatively low presence. Several studies indicate that mtDNA mutations, including large-scale deletions, increase with aging. In accordance, researchers detected increased presence of mtDNA mutations in fibroblasts and iPSCs derived from aged individuals compared to young individuals. Moreover, the identified mutations in somatic cells and derived iPSCs were mostly located in coding genes, while ESCs displayed mtDNA variants primarily within the non-coding D-loop. This gives further support to the notions that the majority of mtDNA alterations seen in adults is of somatic rather than embryonic origin.
An important point to be addressed was the functional consequence of the detected mtDNA mutations. The presence of mtDNA alterations that were not seen in the pooled parental fibroblasts were previously found to not cause major bioenergetic defects, as all generated iPSCs could efficiently undergo the extensive metabolic shift that is associated with cellular reprogramming. However, detailed analyses unveil diminished metabolic function in iPSCs carrying high heteroplasmic mtDNA mutations. Hence, in order to correctly employ patient-derived iPSCs for disease modeling and therapeutic studies, it will be imperative to include the detection of mtDNA integrity as part of the basic characterization toolkit. This will be especially relevant when dealing with patients of advanced age who may harbor increased amount of mtDNA mutations. Overall, this work strongly confirms that, in addition to nuclear genome integrity, mitochondrial genome integrity will become a key parameter to investigate for all medical applications of iPSCs. Furthermore, it highlights the strength of single-cell studies, which may reveal the real biological variability that pooled population studies have so far prevented to be identified. In conclusion, in order to allow faithful and meaningful discoveries, future analysis of iPSCs and their derivatives should not shy away from mitochondrial genome monitoring and single-cell technology.