Figure 3.

Siponimod/BAF312 effect on decreasing apoptosis in human-induced pluripotent stem cells and neural stem progenitor cells (hiPSCs-NSCs/NCPs) using the IncuCyte live-cell analysis system. hiPSCs-NSCs/NPCs cells were seeded in 96-well plates and treated with or without TNFα (20 ng/mL) under varying concentrations of siponimod/BAF312. Then, 5 µM of a caspase-3/7 reagent was added to each well to visualize the numbers of hiPSCs-NSCs/NPCs cells undergoing apoptosis. Cells were then imaged for ~6 days. Data were analyzed using IncuCyte analysis software (v2020C) to detect and quantify the number of green (apoptotic) cells per image. (A) TNFα dose response curve. TNFα dose-dependently (5, 10, 20, 40 ng/mL) increased apoptosis of hiPSCs-NSCs/NPCs cells (blue line) compared to medium alone without TNFα (red line). (B) Siponimod/BAF312 effect on decreasing apoptosis. Under the treatment of TNFα (20 ng/mL) (blue line), siponimod/BAF312 dose-dependently (0.1, 0.5, 1.0, 5.0 nM, except lowest dose of 0.01nM) decreased apoptosis of hiPSCs-NSCs/NPCs cells (green lines) compared to vehicle alone (TNFα 20 ng/mL, blue line) without siponimod/BAF312. (C) Enlarged scale to show statistical significance for all doses except the lowest dose of 0.01 nM that was sustained up to 5.8 days. These experiments were repeated three times with similar results. The data from one representative experiment are shown. Student’s t-test (2 tail) statistical analysis was used in these experiments. p values were set to ≤0.05 for statistical significance.