Abstract
Pluripotent stem cells rely on glycolysis for their energy needs and shift their metabolism towards mitochondrial oxidative phosphorylation during differentiation in order to adapt to the metabolic needs of mature differentiated cells. Oxidative phosphorylation generates reactive oxygen species and can thus lead to collateral protein oxidation and damage. Excessive mitochondrial damage leads to mitochondrial autophagy or mitophagy during which mitochondria are marked for degradation. A key regulator of mitophagy is the outer mitochondrial membrane protein Mitofusin‐2 (Mfn2) which either directs mitochondria towards fusion with other mitochondria to optimize mitochondrial function or towards mitophagy. We hypothesized that differentiation of pluripotent stem cells would necessitate increased mitophagy to contain the oxidative damage due to enhanced oxidative phosphorylation.Human induced pluripotent stem cells (iPSCs) of human embryonic stem cells (hESCs) were treated with Bone Morphogenetic Protein 4 (BMP4) to induce differentiation to the mesodermal lineage. These cells exhibited an expected increase in the mesodermal transcription factor Brachyury. Cells were stained with MitoTracker to visualize mitochondria and assess mitochondrial network formation. We observed an increase in mitochondrial fusion during mesodermal differentiation. There was also a two‐fold increase in Mitofusin 2 protein levels with BMP4 treatment. The ratiometric fluorescent sensor mitoKeima which detects the pH of mitochondria and assesses mitophagy indicated significantly higher basal mitophagy after three days of BMP4 induced differentiation with an increase in the relative mitophagy index from 2.7 to 9.7 (p<0.01) within three days.These findings suggest that mitophagy and mitochondrial networking are critical Mitofusin‐2 mediated adaptive processes required for the metabolic reprogramming of differentiating stem cells.Support or Funding InformationNIH T32 Training Grant
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