Figure 1.
Human iPSC differentiation into 3D lung organoids for modeling SARS-CoV-2 infection
(A) Representative phase-contrast image of lung organoids (LORGs) at 60 days (60D). Scale bar, 200 μm.
(B) H&E staining of 60D LORG showing alveolar-like morphology. Scale bars, 100 μm.
(C) Confocal images showing labeling for cells surrounding epithelial structures (ECAD, green) co-labeled with mesenchymal-cell-type marker vimentin (VIM, red). LORGs also show mesenchymal cells by immunolabeling with smooth muscle marker, smooth muscle actin (SMA, red), and acetylated tubulin (ACTUB, green). Scale bars, 100 μm; for enlarged images, 50 μm.
(D) Epithelial structures (ECAD, green) showing basal-like cells (P63, red) on basolateral surface and ciliated cells (FOXJ1, red) on the luminal side of the epithelium. Scale bars, 100 μm.
(E and F) Representative confocal images showing labeling for alveolar type 2 cells (SP-B, green and SP-C, red) co-labeled with TMPRSS2 (E, red) or SARS-CoV-2 receptor ACE2 (F, green). Scale bars, 100 μm; for enlarged images, 50 μm.
(G) LORGs express lung epithelial cell markers EPCAM, ECAD, and SPDEF. Mean ± SEM of n = 3 organoids cultured in different wells. ∗p < 0.05, ∗∗p < 0.01 by Student's t test.
(H) Expression analysis of proximal lung epithelial cell markers (SOX9, FOXJ1, FOXA1, FOXA2, SCB1A1, MUC5B, KRT5, KRT8, ASCL3, and TP63) and progenitor cell genes (NKX2.1 and SOX2) expressed in developing lungs. Mean ± SEM of n = 3 organoids cultured in different wells. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 by Student's t test.
(I) Analysis of lung alveolar type 1 cell genes AGER and HOPX. Mean ± SEM of n = 3 organoids cultured in different wells. ∗p < 0.05, ∗∗∗p < 0.001.
(J) Expression analysis of expression of lung alveolar type 2 cell genes SFTPB, SFTPC, ABCA3, and SLC34A2. Mean ± SEM of n = 3 organoids cultured in different wells. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
(K) qRT-PCR analysis of 60D LORG shows expression of ACE2, TMPRSS2, DPP4, and Furin. Data presented as mean ± SEM of n = 3 organoids cultured in different wells. ∗p < 0.05, ∗∗p < 0.01.
(L) Western blot analysis of 60D organoid showing protein levels for ECAD, SOX9, FOXJ1, MUC5AC, and alveolar type 2 cells (surfactant protein B [SP-B]), SARS-CoV-2 receptor (ACE2), and protease (TMPRSS2). n = 2 organoids.