The Role of Mitofusin 2‐Mediated Mitophagy and Compensatory Mitochondrial Biogenesis in the Differentiation of Human Pluripotent Stem Cells

Abstract

BackgroundInduced pluripotent stem cells have emerged as a critical new tool to model diseases and generate differentiated cells used for regenerative therapies. Stem cells undergo metabolic reprogramming as they transition from a predominately glycolytic state when they are pluripotent towards enhanced mitochondrial oxidative phosphorylation as they differentiate. The GTPase Mitofusin 2 (Mfn2) is a key mediator of mitochondrial dynamics, regulating mitochondrial fusion as well as the autophagic degradation of dysfunctional mitochondria (mitophagy). However, the role of Mfn2 in regulating the bioenergetics of differentiating cells is not fully understood. We hypothesized that Mfn2‐mediated mitophagy and the compensatory biogenesis of new mitochondria are required for the differentiation of pluripotent stem cells.Methods and ResultsHuman induced pluripotent stem cells were differentiated towards an endothelial lineage and were analyzed at day 0 (pluripotent), day 2 (mesodermal), and day 7 (differentiated endothelial cells). In the early stages of differentiation, autophagy increased three‐fold as assessed by the ratiometric fluorescent autophagy sensor Keima (p=0.0074) and increased levels of phospho‐Mfn2, a mediator of mitophagy. When Mfn2 was depleted, autophagy decreased 67%, indicating that this was a predominantly Mfn2‐dependent process. Subsequent to mitophagy, mitochondrial mass increased at days four through seven as measured by immunofluorescence and mitochondrial DNA levels. Mitochondrial biogenesis was assessed by the mitochondrial construct MitoTimer which exhibits time dependent shifts in fluorescence. After cells were fully differentiated, biogenesis decreased to approximately half (p=0.008). To determine if this burst of mitophagy and subsequent mitochondrial biogenesis plays a role in the shift in metabolism towards mitochondrial ATP, we quantified the ATP:ADP ratio using the biosensor Perceval HR. We observed that intracellular ATP levels increased seven‐fold with differentiation (p<0.001). However, Mfn2 depletion markedly attenuated this increase (p=0.0056). We also investigated whether mitophagy would regulate cell differentiation in a feedback manner. The mitochondrial phosphatase PGAM5, which is cleaved during mitophagy and modulates the Wnt pathway, was increased after the induction of mitophagy on day 4 of differentiation (p=.04).ConclusionMitophagy and compensatory mitochondrial biogenesis are Mfn2‐mediated adaptive processes which are required for the metabolic reprogramming of differentiating human pluripotent stem cells and can regulate the differentiation process via a novel feedback mechanism.Support or Funding InformationNIH R01‐HL126516, NIH P01‐HL060678, NIH 5T32HL007829This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.