Fig 2: Mature iPSC-derived neuronal cultures express functional NMDA receptors (NMDARs). XCL-4 derived mature cultures (30–39 days in maturation media) were loaded with the calcium indicator Fluo-4 for 30 min and then washed with HBBSS5.5 media, followed by the addition of glycine (final concentration of 100 µM) prior to imaging. Using a time series protocol at 40× magnification, two baseline images were taken prior to the addition of vehicle (HBBSS5.5), or a final concentration of 300 µM NMDA. For some cultures, the NMDAR antagonist MK-801 at a final concentration of 10 µM was added after additional images were taken. (A–C) Representative montages are shown. Fluo-4 time series images were analyzed using MetaMorph. Individual cells were identified and Fluo-4 fluorescence intensity at each time point was measured. The intensity of cells at each time point were averaged together. The first two time points were averaged as the baseline intensity, and all data were normalized to the corresponding baseline. (D) Data from the montage shown in panel (C) were graphed using SigmaPlot 14.5. One-way repeated measures ANOVA and Bonferroni tests were performed using SigmaPlot 14.5. Data are represented as mean + SEM. ***P < 0.001. n = 21 cells per condition. (E) A representative trace is shown of an NMDAR-mediated electrically evoked postsynaptic current from XCL-4 derived mature cells in the presence of the GABAA receptor antagonist, picrotoxin (final concentration: 50 µM), and the AMPA receptor antagonist, NBQX (final concentration: 5 µM), before (black) and after (red) application of the NMDAR antagonist, APV (final concentration: 50 µM).

Fig 3: NMDAR antagonist MK-801 rescues KS network phenotypes. a MK-801 (1 µM) effect on KSMOS neuronal network activity (DIV 28). After 90 min, Naspm was added. Graph showing the effect of MK-801 (1 µM) treatment on the neuronal network burst frequency for CMOS and KSMOS derived neuronal network 20 min after application. The values are normalized to the nontreated (NT) condition. b Representative raster plot showing the activity exhibited by KS patient-derived neuronal network (KSMOS) grown on MEAs either nontreated or after one week of treatment with MK-801. Six second of raw data showing a burst recorded from a representative channel are shown for the NT and the treated conditions. c–g Quantification of network properties as indicated, n = 8 for CMOS; n = 6 for KSMOS and n = 6 for KSMOS treated with MK-801. h Canonical scores plots based on discriminant analyses for control (i.e., C1, C2, CMOS, and CCRISPR), KSMOS and KSMOS treated with MK-801 (84% correct classification). Discriminant functions are based on using the following network activity parameters: firing rate, network burst rate, network burst duration, percentage of spike outside network burst and coefficient of variability of the inter-burst interval. Group envelopes (ellipses) are centered on the group centroids, n = 50 for controls; n = 6 for KSMOS and KSMOS treated with MK-801. Pie charts visualize accuracy of discriminant analyses functions by showing the relative distribution of lines per a-priory group after reverse testing for group identity. Data represent means ± SEM. *P < 0.05, ***P < 0.0005, ****P < 0.0001, one-way ANOVA test and post hoc Bonferroni correction was performed between conditions. Source data is available as a Source Data file

Fig 4: Immunofluorescent confocal imaging and characterization of mature human neuronal cultures. (A–D) XCL-4 derived mature cultures (28–37 days in maturation media) were fixed, permeabilized, and stained for the mature excitatory neuron markers ß-tubulin III [TuJ1; green; (A,B)] and microtubule-associated protein 2 [MAP2; red; (A,D)], the obligate NMDAR subunit, NMDAR1 [red; (B)], the presynaptic marker synaptotagmin 1 [Syt1; red; (C)], the postsynaptic marker postsynaptic density protein 95 [PSD-95; green; (C)], and the astrocytic marker glial fibrillary acidic protein [GFAP; green; (D)]. All cultures were counterstained with the nuclear stain DAPI [blue; (A–D)].

Fig 5: NMDA receptor subunit 1 is upregulated in KS patient-derived neurons. a Representative western blots and graph showing the NR1 protein levels in control and KS iNeurons at DIV 28. Values of KSMOS are normalized to CMOS and values of KSCRISPR are normalized to CCRISPR (n = 3). b ChIP assay of H3K9me2 followed by promoter-specific (BDNF_pr4, GRIN1_pr2, and PPIA_pr2) qPCR performed in iNeurons from CMOS, KSMOS, CCRISPR, and KSCRISPR (n = 4 for mosaic lines and n = 3 for crispr lines). c Representative images showing NR1 expression and quantification for CMOS, KSMOS, CCRISPR, and KSCRISPR (n = 12). d Schematic representation of methodology to obtain the AMPA and NMDA ratio. e Representative traces and quantification of NMDA/AMPA ratio for CMOS, KSMOS, CCRISPR, and KSCRISPR (n = 8). Blue bars indicate light stimulus. Scale bar 100 ms, 10 pA. Data represent means ± SEM. *P < 0.05, **P < 0.005, ***P < 0.0005, one-way ANOVA test and post hoc Bonferroni correction was performed between conditions. Source data is available as a Source Data file