HD fibroblasts do not exhibit inclusion bodies, even upon cellular insults. (a), Fibroblasts induced to age in vitro by serial passaging (18 times), forced to exit cell cycle by contact inhibition, and then cultured for an additional 7 weeks, do not exhibit inclusion bodies. (b), Ctrl (Ctrl.19) or HD fibroblasts (HD.40) challenged with 1 mM H2O2 to induce oxidative stress do not exhibit inclusion bodies. (c), HD.40 fibroblasts transduced with CDM and a non-specific microRNA (miR-N.S.) to mimic reprogramming conditions but not neuronal induction, do not form inclusion bodies. The formation of inclusion bodies is present in all three lines reprogrammed from HD patients. (d), All three HD MSNs lines examined exhibit aggregated HTT inclusions (IBs) at post-induction day 30 (PID) analyzed by MW8 immunostaining. (e), Quantification with EM48 yields similar number of cells displaying inclusions to MW8 staining; Two-tailed student’s t-test, n = 4 biological replicates per group; ***P = 0.0003 df = 6. (f), The appearance of aggregated cytoplasmic HTT protein in HD.40 MSNs is detected as early as PID 14. By PID 21 HTT inclusions are numerous and after PID 28, inclusions are bigger and more defined, with little to no granules. Quantification of inclusion formation shows significant changes by PID 14; n = 3 biological replicates with each sample containing approximately 100 cells; Mean ± s.e.m. One-Way ANOVA (F6,17 = 65.47, P< 0.0001) with post hoc Tukey’s test (from left, n.s. = 0.19, **P = 0.0028, ***P = 3.2E-10. (f), Time course analysis with Ctrl.20 and HD.42 for the appearance of oxidative DNA damage phenotype by 8OH-dG staining. Significant differences between controls and HD MSNs is detected as early as PID 20, and continue to augment with time in culture. One-Way ANOVA (F9,190 = 30.86, P < 0.0001) with post hoc Tukey’s test (from left, n.s. = 0.99, n.s. = 0.34, **P = 0.0012, ***P = 5.8E-11, ***P = 1.0E-14; ***P < 0.001; **P < 0.01; n.s. = not significant. n = 20 cells per time point for each line. Mean ± s.d. a-c experiments were repeated independently twice and d, over 3 times. f-g experiments have not been independently repeated.