hPSC-derived microglia shape neuronal morphology and enhance network activity in vitro 175 5 regression or, when normality of the residuals was violated according to Shapiro-Wilk tests, Mann-Whitney U. P-values were corrected by applying Bonferroni correction for multiple comparisons (n = 8), both the p-value and adjusted p-values are reported. Similarly the effect of microglia addition on the functionality MEA measurements was determined, for this we compared parameters from with and without microglia at 12 weeks post plating. If the two-sided t-test on the amount of active electrodes showed a significant increase in activity, other parameters for activity were addressed with post-hoc analysis. These involved either Ordinary Least Square regression or, when normality of the residuals was violated according to Shapiro-Wilk tests, Mann-Whitney U. P-values were corrected by applying Bonferroni correction for multiple comparisons (n = 13), both the p-value and adjusted p-values are reported (Supplementary Table IV). Statistical analysis control vs leukodystrophy microglia-like cells Data sets comparing control vs. leukodystrophy microglia-like cell conditions, were all normally distributed, as determined by Shapiro-Wilk test. Sample size was either 4 cultures containing microglia-like cells from control lines against 8 from ALD lines, or 3 control lines against 5 of 4H leukodystrophy. Intrinsic microglia stress in ALD and 4H was determined by comparing numbers and roundness of microglia-like cells to controls. Two-sided T-Test were performed, and Fisher’s method was used to aggregate the intrinsic stress proxy metrics. Remaining microglia morphology measures and CD68+ percentage (for ALD) were compared by comparing the line averages, across all timepoints, of the controls to the leukodystrophy lines with T-Test using GraphPad Prism (v.10.2.0). To determine if the coculture morphology is changed upon microglia addition the four proxy measures: nuclear debris, mature neuron count (NeuN+) and number of axonal and dendritic segments were again used. Per line, one average from all timepoints, was created and two-sided T-Tests were used to compare control vs. leukodystrophy. Fisher’s method was again used to aggregate the proxy measures. If Fisher’s method was significant, post-hoc analysis was performed using the outcomes of the tests on the proxy metrics. Dendrite and axon morphology post-hoc analysis were performed using T-Test. P-values were corrected by applying Bonferroni correction for multiple comparisons (n = 8), both the p-value and adjusted p-values are reported. Active channels for control vs leukodystrophy microglia-like cell conditions were compared using the average of active channels across 7 to 12 weeks post plating, difference between Ctrl and ALD or Ctrl and 4H were addressed with T-Test, after checking for normality using Shapiro-Wilk test. No differences were found hence, no post-hoc testing was performed. In general, any significant parameters can be found in the
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