Fig. 4: Validation of maturation strategy across hPSC-derived neuronal cultures.
a–f, GENtoniK treatment inducing synaptogenesis and spontaneous activity in cortical organoids. a, Representative images of immunofluorescent staining for SYN1 and MAP2 in day-60 organoids. b, Quantification of total SYN1 puncta per field (n = 8 cryosections randomly sampled from n = 20 organoids). c, Representative images of immunofluorescence staining for EGR-1 and MAP2 in unstimulated day-60 organoids. d, Quantification of EGR-1+ cells per field (n = 8 cryosections randomly sampled from n = 20 organoids). e, Representative confocal images with ROI traces of calcium imaging with Fluo-4 AM in whole day-60 cortical organoids that received treatment with DMSO (left) or GENtoniK (right). Traces show a 20-min time course captured at 0.2 ps. The red lines represent counted spikes. f, Quantification of total spikes per ROI in 20-min recordings of Fluo-4 AM calcium transients (n = 45 DMSO ROIs and 47 GENtoniK ROIs from 4 organoids per condition). g–m, GENtoniK promoting maturation of hPSC-derived SMNs. g, Representative high-content maturation assay images of ISL1/2+ SMNs (day 40 of hPSC differentiation). h–j, Quantification showing GENtoniK-improved KCl-induction of FOS+ cells (h), total neurite length (n = 12 for both conditions) (i) and nucleus area (n = 12 for both conditions) (j) in SMNs (n = 12 for DMSO and n = 11 for GENtoniK). k–m, GENtoniK treatment increasing firing rates and inducing spontaneous bursting activity on SMNs plated on high-density multielectrode arrays. k, Sample single-channel trace of GENtoniK-treated SMNs illustrating spike detection. l, Time-course analysis of average firing rates in SMNs plated on high-density microelectrode arrays (HD-MEAs), calculated from 60 s of activity in the 1/64th most active electrodes (n = 128 electrodes from 2-MEA probes). m, Representative 60-s spike rastergrams (top) and average firing rates (middle) of SMNs plated on HD-MEAs. Only GENtoniK-treated SMNs displayed spontaneous bursting events (orange bars). Bottom, array heatmap of a 4-s bursting event. In b, d, f, h–j and l, two-tailed Welch’s t-test was used. Asterisks indicate statistical significance. Mean values are represented by a black line. Errors represent s.e.m. Scale bars, 50 μm.
