Metabolic Reprogramming, Autophagy, and Reactive Oxygen Species Are Necessary for Primordial Germ Cell Reprogramming into Pluripotency

Figure 8

(a) Number of reprogrammed PGCs in conditions of normoxia, normoxia and valproic acid (VPA), hypoxia, and hypoxia and VPA (5 mg/mL) for 7 days. VPA induces PGC reprogramming in normoxia but does not synergize with hypoxia. Asterisks show significance at . (b) EB formation and spontaneous differentiation of reprogrammed cells into cells of the three germ layers by VPA in normoxia (5 mg/mL). Markers of endoderm, ectoderm, and mesoderm are, respectively, albumin, AE1/AE3 cytokeratins, and vimentin. Scale bars correspond to 25 μm. (c) Confocal microscopy merge images for immunofluorescence against SSEA1 in green (PGCs), and HIF1α, HIF2α, cMyc, Klf4, and Nanog in red in nuclei of PGC cultures subjected to 5 mg/mL VPA for 48 h. Colocalization is seen as bright pink. Controls are the same as in Figure 2(b). Scale bars correspond to 25 μm. (d) Electron microscopy photographs showing VPA-induced mitophagy at day 3 (left) and day 6 (right). Autophagic vacuoles are labeled with red stars. M: mitochondria. N: nucleus. (e) Confocal microscopy merge image for immunofluorescence against SSEA1 in red (PGCs) and p62 autophagic vacuoles in green in cytoplasms of PGC cultures subjected to normoxia with 5 mg/mL VPA for 3 d. Nuclei are seen in blue with DAPI. Scale bars correspond to 25 μm.

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