Extended Data Fig. 4. Rett syndrome fusion organoids from a second patient hiPSC line exhibit neural network irregularities in calcium indicator measurements.

a, Immunohistochemical analyses of isogenic Cx and GE organoids from a second Rett syndrome patient hiPSC line (harboring a 1461A>G missense mutation, indicated by “II”) reveals either the presence (iCtrl-II) or absence (Mut-II) of MECP2 expression. Representative images from n=2 independent experiments and 6 imaged sections. b, Mut-II Cx+GE fusions contain hyperexcitable neurons as indicated by the red boxed regions in the bottom ΔF/F colorized amplitude plot and spike plot. These plots show trains of repeatedly firing Ca²⁺ transients with short interspike intervals that are not present in iCtrl-II Cx+GE (top plots). c, There is no discernible change in synchronization of calcium transients between Mut and iCtrl as reflected in the clustergrams. d, The hyperexcitable phenotype in Mut-II Cx+GE fusions is reflected in the pooled data both by significant increases in multispiking neurons and decreases in mean and median interpeak intervals. Pooled data quantifications, n = 10 iCtrl-II and n =6 Mut-II fusion organoids, where each n is an independently generated organoid. Two-sided Mann-Whitney tests were used, *P = 0.0071 for the proportion of multispiking neurons, **P = 0.0047 for the mean interspike interval, **P = 0.0017 for the median interspike interval, ns = not significant. Plot in d displays the full distribution of individual data points with dotted lines indicating the median and quartile values.