Supplementary Figure 13: Heterogeneity of hiPSC-derived FD-SFTPC⁺ cells in single-cell analysis.

(a) Immunofluorescence images of several AT1 markers (PDPN, CAV1, AGER and HOPX) costained with GFP (SFTPC⁺ cells) in P3-AOs (n = 3 collected images). Error bars, 25 μm. (b) RT-qPCR of AT1 and AT2 marker expression in two distinct representative cell populations at single-cell resolution. “Positive” and “negative” indicate cell populations that were positive or negative for all AT1 markers of HOPX, PDPN, CAV1 and AGER, respectively. Error bars indicate the mean ± s.e.m. n = 13 and 15 single cells were analyzed as “positive” and “negative” population, respectively. *P < 0.05, **P < 0.01, N.S., not significant by two-tailed unpaired t-test (df = 26). (c) The component ratio of AT1 marker-based classification of P0, P2 and P5 FD-SFTPC⁺ cells. “High”, “moderate” and “low” indicate the cells expressing three/four, two and one/none of four AT1 markers (HOPX, PDPN, CAV1 and AGER), respectively. N.S., not significant by chi-squared test. (d) A 3D view of the PCA of 138 FD-SFTPC⁺ single cells based on 16,906 genes (GSE90813). (e) A 2D scattergram of the PCA depicted in (c).