Extended Data Fig. 8: Characterization of hSeO with SCN9A pathogenic variants.
From: Human assembloid model of the ascending neural sensory pathway
a. RT-qPCR analysis of SCN9A expression in control and SCN9A KO hSeO (Control, n = 6; SCN9A KO, n = 4 samples from 2 hiPS cell lines from 2 differentiation experiments, two-tailed unpaired t-test, **P = 0.0082) at day 48–69. b. RT-qPCR analysis of POU4F1 expression in control and SCN9A KO hSeO (Control, n = 6; SCN9A KO, n = 4 samples from 2 hiPS cell lines in 2 differentiation experiments, two-tailed unpaired t-test, n.s.; not significant) at day 48–69. c. Immunocytochemistry images for BRN3A in control and SCN9A KO hSeO. d. Quantification of the percentage of BRN3A+ cells in control and SCN9A KO hSeO (Control, n = 8; SCN9A KO, n = 6 samples from 2 hiPS cell lines from 2 differentiation experiments, two-tailed unpaired t-test, n.s.; not significant). e. Calcium imaging of control and SCN9A KO hSeO f. Quantification of the number of spontaneous calcium responses (Control, n = 28 cells from 5 organoids; SCN9A KO, n = 29 cells from 6 organoids, 2 hiPS cell lines from 2 differentiation experiments, two-tailed Mann-Whitney test, ***P = 0.0006) at day 64-82. g. Representative SCA correlograms between cells from hSeO–hSeO, hSeO–hdSpO, hSeO–hDiO, and hSeO–hCO for a control assembloid (top) and in a SCN9A KO assembloid (bottom). The histograms of peak time-shifts (below each correlograms panel) show peaks around 0 in the control, but not in the SCN9A KO assembloid. h. RT-qPCR analysis of POU4F1 expression in control and SCN9A T1464I hSeO (n = 4 samples from one hiPS cell line from 4 differentiation experiments, two-tailed unpaired t-test, n.s.; not significant) at day 25–31. i. Calcium imaging of control and SCN9A T1464I hSeO j. Quantification of the number of spontaneous calcium activity (CTL, n = 244 cells from 9 organoids; T1464I, n = 149 cells from 8 organoids, one hiPS cell line from 3 differentiation experiments, two-tailed Mann-Whitney test, ****P < 0.0001) at day 103–111. k. Representative SCA correlograms between cells from hSeO–hSeO, hSeO–hdSpO, hSeO–hDiO, and hSeO–hCO for a control assembloid (top) and in a SCN9A T1464I assembloid (bottom). Data shown as mean ± s.e.m. Gray dots indicate cells, large colored dots indicate organoids or qPCR samples in a, b, d, f, h, j. Each shape represents a hiPS cell line: Circle, 1205-4; Triangle, 1208-2; Square, KOLF2.1 J in a, b, d, f, h, j. Credits: e,i adapted from ref. 48, Springer Nature America, Inc.
