Figure 5. iPSCs from an 84-year-old woman acquired embryonic-like mitochondrial ultra-structure and functionality.

(A) Transmission electron microscopy (TEM) analysis showing mitochondrial ultra-structural morphology in fibroblasts and in pluripotent stem cells. HFF1, neonatal fibroblasts; NFH2, dermal fibroblasts from an 84-year-old woman; NFH17 from an 80-year-old man; NFH18 from an 80-year-old woman; H9, female hESC line; iPS4, iPSC line derived from HFF1 fibroblasts; OiPS3 and OiPS6, iPSC lines generated from NFH2 fibroblasts. Scale bars, 500 nm. (B–C) Measurement of long and short mitochondrial diameters in NFH2 fibroblasts and in two NFH2-derived iPSC lines (OiPS3 and OiPS6). 50 mitochondria were measured for each sample using the EMMENU4 software (Fastscan, TVIPS). Upon reprogramming the long diameter becomes shorter while the short diameter increases, suggesting that the organelles tend to acquire a round-like shape. Error bars indicate the standard error of means (SEM). (D) Mitochondrial membrane potential (MMP) was assessed employing the fluorescent dye TMRE in live cells to obtain a relative measurement of mitochondria functionality in terms of energy coupling. All human fibroblasts exhibited low basal MMP. The fluorescent intensity appeared increased in undifferentiated hESCs and iPSCs, while differentiated cells at the borders of pluripotent stem cell colonies showed reduction of basal MMP signal. Thus, the mitochondrial functionality of aged iPSCs appeared similar of that of young iPSCs and hESCs. The presence of mitochondrial hyper-polarization, suggestive of reduced ATP consumption, in all pluripotent stem cells is in agreement with the distinctive metabolic features of undifferentiated stem cells which rely more on glycolytic rather than oxidative-based energy generation. Scale bars, 50 µm.