Fig. 3
From: Autologous treatment for ALS with implication for broad neuroprotection
Mechanisms of neuroprotection by ALS iPSC-CM. a MTT assay for viability of MNs cultured with proteinase K-treated or heat-inactivated ALS iPSC-CM. ***P < 0.001, n = 4 for each condition. b MTT assay for viability of MNs that were cultured with 200 μg/ml H2O2 alone or co-treated with complete unfractionated ALS iPSC-CM, heparin-binding protein fraction, exosome fraction or HPB flow-through and exosome-free CM. *P < 0.05, **P < 0.01, n = 4 for each condition. c MTT assay for viability of MNs that were cultured with secretome of ALS iPSC-derived embryoid bodies (EBs). The viability was significantly decreased in the EB-CM group compared to that in the ALS iPSC-CM group (0.66 ± 0.02 vs 0.44 ± 0.05, P < 0.001). ***P < 0.001, n = 4 for each condition. d The effect of epigenetic modifiers on the neuroprotective activity of the iPSC secretome: ALS iPSCs were treated with the indicated inhibitors of pluripotency that influence epigenetics, and the activity of their CM was subsequently tested with H2O2-exposed ALS-derived MNPs, in quadruplicate MTT and Annexin V assays. EZH2 inhibitor GSK126 diminished the neuroprotective activity of the iPSC-CM with high statistical significance (viability: 0.64 ± 0.04 vs 0.47 ± 0.02, P < 0.01; apoptosis: 1.71 ± 0.17 vs 2.65 ± 0.23, P < 0.01), *P < 0.05, **P < 0.01, ***P < 0.001, n = 4 for each condition. e Levels of mitochondrial ROS. All MNs were stained with mitoSOX red and then the mean fluorescence intensity (MFI) was measured by a Guava Easycyte Flowcytometer. Autologous ALS iPSC-CM normalized mitochondrial activity of ALS-MNs statistically same as the known modifier, Cyclosporin A (CsA). Graphs show Mean ± SEM, n = 4. **P < 0.01. f The effects on inflammation were assayed by qRT-PCR. The indicated markers of inflammation were diminished by ALS iPSC-CM similarly to CsA. n = 3, ***P < 0.001, ns not statistically significant. n = 4, **P < 0.01, ***P < 0.001, ns not statistically significant. g The effect of CsA on MN viability and apoptosis. The viability of ALS-MNs as compared to healthy WTC-11 MNs was measured by MTT assay. The ratios of apoptotic to live MNs were assayed by Annexin V fluorescence in a plate reader. ALS-MNs were prone to cell death, as compared to WTC-11 MNs; ALS iPSC-CM, but not dF-CM or CsA, protected ALS-MNs from the ROS (mutant SOD1)-induced cell death