Pluripotent stem cells are known to shift their mitochondrial metabolism upon differentiation, but the mechanisms underlying such metabolic rewiring are not fully understood. We hypothesized that during differentiation of human induced pluripotent stem cells (hiPSCs), mitochondria undergo mitophagy and are then replenished by the biogenesis of new mitochondria adapted to the metabolic needs of the differentiated cell. To evaluate mitophagy during iPSC differentiation, we performed live cell imaging of mitochondria and lysosomes in hiPSCs differentiating into vascular endothelial cells using confocal microscopy. We observed a burst of mitophagy during the initial phases of hiPSC differentiation into the endothelial lineage, followed by subsequent mitochondrial biogenesis as assessed by the mitochondrial biogenesis biosensor MitoTimer. Furthermore, hiPSCs undergoing differentiation showed greater mitochondrial oxidation of fatty acids and an increase in ATP levels as assessed by an ATP biosensor. We also found that during mitophagy, the mitochondrial phosphatase PGAM5 is cleaved in hiPSC-derived endothelial progenitor cells and in turn activates β-catenin-mediated transcription of the transcriptional coactivator PGC-1α, which upregulates mitochondrial biogenesis. These data suggest that mitophagy itself initiates the increase in mitochondrial biogenesis and oxidative metabolism through transcriptional changes during endothelial cell differentiation. In summary, these findings reveal a mitophagy-mediated mechanism for metabolic rewiring and maturation of differentiating cells via the β-catenin signaling pathway. We propose that such mitochondrial-nuclear cross talk during hiPSC differentiation could be leveraged to enhance the metabolic maturation of differentiated cells.

中文翻译：

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Mitophagy 介导诱导多能干细胞进行内皮分化的代谢重编程。

众所周知，多能干细胞在分化时会改变其线粒体代谢，但这种代谢重新布线的机制尚不完全清楚。我们假设在人类诱导多能干细胞 (hiPSC) 的分化过程中，线粒体经历线粒体自噬，然后通过适应分化细胞代谢需求的新线粒体的生物发生来补充。为了评估 iPSC 分化过程中的线粒体自噬，我们使用共聚焦显微镜对分化为血管内皮细胞的 hiPSC 中的线粒体和溶酶体进行了活细胞成像。我们在 hiPSC 分化为内皮谱系的初始阶段观察到线粒体自噬爆发，随后是线粒体生物发生生物传感器 MitoTimer 评估的随后的线粒体生物发生。此外，通过 ATP 生物传感器评估，经历分化的 hiPSC 显示出更大的脂肪酸线粒体氧化和 ATP 水平的增加。我们还发现，在线粒体自噬过程中，线粒体磷酸酶 PGAM5 在 hiPSC 衍生的内皮祖细胞中被切割，进而激活 β-连环蛋白介导的转录共激活因子 PGC-1α 的转录，从而上调线粒体生物发生。这些数据表明，线粒体自噬本身通过内皮细胞分化过程中的转录变化启动线粒体生物合成和氧化代谢的增加。总之，这些发现揭示了线粒体自噬介导的代谢重新布线和分化细胞通过 β-连环蛋白信号通路成熟的机制。