Fig. 5

Corneal organoids exhibited areas of conjunctiva and areas of CE and endothelium. A Bright-field images of developing corneal-like organoids derived from CB30 hiPSCs cultured for 35 (D35), 80 (D80), and 140 (D140) DIV. Scale bar: 300 µm in a–c; 1 mm in d; 2 mm in e. B Hematoxylin and eosin staining of corneal organoids at 140 DIV. Corneal organoids with stromal lumen (a, b, f). The dashed squared in (a) indicates magnified image in (b). Stratified surface epithelium (c) with collagen-filled stroma resembling native mouse cornea (d). Corneal organoids with fluid-filled stroma and an endothelium-like layer (e, g, h). Dashed squares in (e) indicate magnified images in (g, h). Scale bars: 300 µm in a, e; 50 µm in b, f–h; 25 µm in c, d. C Immunohistochemistry of the surface epithelium stained with cytokeratin 19 (CK19) (top panel). Scale bar: 10 µm. Transmission electron microscopy image of corneal organoids at 140 DIV showing corneal epithelial cells with apical microvilli (arrows), tight junctions between cells (arrowheads) and cell nuclei (N) (bottom panel). Scale bar: 5 µm. D RT-qPCR analysis of gene expression in mzOPCs at 30 DIV and in corneal organoids at 100 (CO d100) and 140 DIV (CO d140). Values were normalized to GAPDH. Data are presented as the mean ± SD (n = 3 independent experiments). Two-way ANOVA and Tukey’s post hoc tests were performed, and significant differences are represented with *p < 0.001 vs. OPC; ^#p < 0.001 CO d100 vs. CO d140. E Confocal images of immunostained corneal organoids at 140 DIV showing surface conjunctiva/limbal cells expressing CK5 (a, f, k), CK19 (b, g, l) and Na+/K+-ATPase (Na/K; k), corneal epithelial cells expressing PAX6 (h, m), cytokeratin 3 (CK3; n) and corneal endothelium expressing aquaporin-1 (AQP1) and N-CAD (o); and stroma containing cells positive for collagen type IV (COL IV; c, j), collagen type I (COL I; i) and vimentin (VIM) (d). The surface epithelium was delimited by COL IV and I, with p63 + cells on the apical side and some PAX6 + cells on the basal side (h, i, j). The cytoskeleton was stained with actin (e). Scale bars: 250 µm (a, b); 50 µm (c–e, k–n); 25 µm (f–j, o). Nuclei were stained with DAPI