Fig. 5.

Inhibition of PI3K increases _Δψ_m, reduces the reliance on glycolysis and facilitates neuronal differentiation of hDSCs. a hDSCs were cultured in the presence or absence of LY294002 (1 µM) for 4 days and JC-1 staining was performed to monitor _Δψ_m. Data shown are means ± SDs of 3 independent experiments with 3 different donors. An unpaired, two-tailed Student's t test was performed. b The bioenergetics of hDSCs cultured in hDSC stem cell medium in the presence or absence of LY294002 (1 µM) for 4 days were analyzed as described in the Materials and methods. Data shown are means ± SDs of 3 independent experiments with 3 different donors. An unpaired, two-tailed Student's t test was performed. c hDSCs were cultured in DMEM/10% FCS in the presence or absence of 1 or 100 µM LY294002 for 7 days. Neuron-like cells (red arrowheads) growing out of the hDSC sphere (dashed line) were observed by light microscopy (upper images; scale bars: 100 µm). TUJ1-positive neurons (red arrowheads) were calculated as percentage of the total cell number by evaluating more than 5 images per condition in 3 independent experiments (lower images; scale bars: 50 µm). An unpaired, two-tailed Student's t test was performed. Developing neuron-like cells following treatment with 100 µM LY294002 exhibited long dendrites (red and white arrows). d hDSCs treated with 100 µM of LY294002 were employed for patch clamp experiments. Averages for the maximal current density (pA/pF) were obtained by whole-cell currents in voltage-clamp mode; cells were held at −70 mV; step depolarization from −90 mV to +60 mV at 10-mV intervals was delivered. Representative traces of outward and inward currents are shown. Data were obtained from 3 independent experiments with 3 different donors