Abstract
Our understanding on how selective mitochondrial autophagy, or mitophagy, can sustain the archetypal properties of stem cells is incomplete. PTEN-induced putative kinase 1 (PINK1) plays a key role in the maintenance of mitochondrial morphology and function and in the selective degradation of damaged mitochondria by mitophagy. Here, using embryonic fibroblasts from PINK1 gene-knockout (KO) mice, we evaluated whether mitophagy is a causal mechanism for the control of cell-fate plasticity and maintenance of pluripotency. Loss of PINK1-dependent mitophagy was sufficient to dramatically decrease the speed and efficiency of induced pluripotent stem cell (iPSC) reprogramming. Mitophagy-deficient iPSC colonies, which were characterized by a mixture of mature and immature mitochondria, seemed unstable, with a strong tendency to spontaneously differentiate and form heterogeneous populations of cells. Although mitophagy-deficient iPSC colonies normally expressed pluripotent markers, functional monitoring of cellular bioenergetics revealed an attenuated glycolysis in mitophagy-deficient iPSC cells. Targeted metabolomics showed a notable alteration in numerous glycolysis- and TCA-related metabolites in mitophagy-deficient iPSC cells, including a significant decrease in the intracellular levels of α-ketoglutarate -a key suppressor of the differentiation path in stem cells. Mitophagy-deficient iPSC colonies exhibited a notably reduced teratoma-initiating capacity, but fully retained their pluripotency and multi-germ layer differentiation capacity in vivo. PINK1-dependent mitophagy pathway is an important mitochondrial switch that determines the efficiency and quality of somatic reprogramming. Mitophagy-driven mitochondrial rejuvenation might contribute to the ability of iPSCs to suppress differentiation by directing bioenergetic transition and metabolome remodeling traits. These findings provide new insights into how mitophagy might influence the stem cell decisions to retain pluripotency or differentiate in tissue regeneration and aging, tumor growth, and regenerative medicine.
Highlights
Mitochondrial autophagy, or mitophagy, is a key cellular pathway for mitochondrial quality control that functions to clear mitochondria [1,2,3,4]
To test whether PINK1KO mouse embryonic fibroblasts (MEFs) constitute a useful model to dissect the role of mitophagy in the establishment of induced pluripotency, we first mimicked mitochondrial damage by experimentally depolarizing mitochondria with the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) and monitoring loss of MitoTracker staining after mitophagy stimulation [62, 67]
We transduced MEFs with OSK at a 1:1:1 ratio on day 0 and repeated the transduction up to four times, after which the regular media was replaced with standard mESC media supplemented with the knockout serum replacement (KSR)
Summary
Mitochondrial autophagy, or mitophagy, is a key cellular pathway for mitochondrial quality control that functions to clear mitochondria [1,2,3,4]. Mitochondria appear to play crucial roles during stemness factor-mediated nuclear reprogramming of somatic cells into induced pluripotent stem cells (iPSCs), a convenient “in a dish” model that allows a comprehensive understanding of stem cell biology. Functional metamorphosis of somatic oxidative phosphorylation into glycolytic metabolism plays a causal role in enabling the reprogramming process of acquisition and maintenance of stemness to occur [2535]. The establishment of induced pluripotency requires a transient and early energy-demanding metabolic state characterized by increased mitochondrial oxidative phosphorylation and hyperactive mitophagy [46, 47]. Because the unique metabolic state required to achieve cell plasticity is accompanied by significant temporal changes in mitochondrial function, composition, structure, and maturation, it might appear elementary to suggest that mitophagy is a prerequisite of induced pluripotency. Recent studies have shed light on how interlinked processes critical for mitochondrial health, including mitochondrial fragmentation and mitochondrial fission/fusion, significantly alter the efficiency and speed of induced pluripotency [48,49,50,51], but little information is available on the role of mitophagy in the acquisition and maintenance of stemness
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