Figure 4. Detection of secreted sAPPα and total Aβ from single hiPSC-derived neural cells. Human iPSCs were differentiated to forebrain neural fates. At multiple time points of differentiation, cells were dissociated and plated in nanowells for 24 h. Cells were then imaged for a marker of living cells and sAPPα and Aβ detected using the experimental outline presented in Figure 1. The detection efficiency for single, living cells is shown for sAPPα (A) and Aβ (B) at different time points of differentiation and compared with the detection efficiency for 7WD4 cells over multiple experiments: iPSC differentiation d20–50 (n = 4), d51-d80 (n = 5), d81-d110 (n = 4), and 7WD4 (n = 13). Box shows minimum to maximum value and line shows the median value. **p < 0.01, Kruskal–Wallis multiple-comparisons test. C, D, Histogram distribution of SNR values of wells positive for sAPPα (C) and Aβ (D) for each range of differentiation time. For each bin, data from three independent experiments for each time point are combined. Histogram distribution of the relative frequency of sAPPα (E) and total Aβ (F) SNR printing intensity from wells with or without cells are shown (combined data from three representative experiments, each over 75 d of differentiation). G–I, Combined data from four experiments are shown for single, living cells differentiated to neuronal fates for 64–99 d. The intensity of sAPPα or Aβ secreted from three different groupings of single cells (sAPPα−/totalAβ+, sAPPα+/totalAβ−, sAPPα+/totalAβ+) are presented in G and H and the correlation of sAPPα and total Aβ from each group is presented in I. Black lines in G and H denote the mean value for each group. ****p < 0.0001, one-way ANOVA, Kruskal–Wallis multiple-comparisons tests.