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Fig. 4 | Stem Cell Research & Therapy

Fig. 4

From: Multiocular organoids from human induced pluripotent stem cells displayed retinal, corneal, and retinal pigment epithelium lineages

Fig. 4

Development and organization of the retinal layer within retinal organoids generated from CB30-derived mzOPCs. A (Left-middle) Bright-field images of retinal organoids derived from the CB30 hiPSC line at 140 DIV. The arrows indicate developing retinal tissue, and arrowheads indicate developing photoreceptor inner/outer segments. (Right) Hematoxylin and eosin staining of paraffin sections showing lamination of retinal tissue. Scale bars: 300 µm (left); 100 µm (middle); 50 µm (right). B RT-qPCR analysis of gene expression in mzOPCs at 30 DIV and retinal organoids at 70 (RO d70) and 140 DIV (RO d140). Values are 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 RO d70 vs. RO d140. C Confocal images of retinal organoid paraffin sections at 140 DIV immunostained with photoreceptor cell markers CRX, RECOV, rhodopsin (RHO), RG-opsin, and pericentrin (PERI) (a–d) corresponding to the outer nuclear layer (ONL); bipolar cell markers CHX10 and PKC-alpha and horizontal cell markers CALB (calbindin-28 K) (e–g) corresponding to the inner nuclear layer (INL); ganglion cell markers TUJ1 and PAX6 (i, j) corresponding to the ganglion cell layer (GCL); and synaptic markers SYNAP (synaptophysin) and vGLUT1 (k, l) corresponding to the outer plexiform layer (OPL). Scale bars: 100 µm in I, j; 25 µm in a–h, k, l. D Transmission electron microscopy analysis of retinal organoids at 140 DIV shows several retinal structures of connecting cilia (CC), inner segments (IS), mitochondria (mt), outer limiting membrane (OLM), outer segments (OS) and ribbon synapses (RS). Scale bars: 10 µm (a, b); 2 µm (c); 1 µm (d); 0.5 µm (e, f, g); 0.2 µm (h)

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