Figure 6.

Differentiation and characterization of iPSC-derived RPGR-deficient retinal organoids

(A) Bright field image of an RPGR-deficient retinal organoid showing the brush border.

(B) Image of RPGR-deficient photoreceptors showing mitochondria-rich ISs (green), phalloidin delineated OLM (gray) and PHPR2+ OSs (red).

(C–F) Immunohistochemical analysis with RPGR N-terminal specific antibody (C, D, G, and H) and RPGR C-terminal specific antibody (E, F, I, and J). Images of control retinal organoids, showing typical punctate localization of RPGR (red) to the CC with both antibodies (C–F).

(G–J) Images of RPGR-deficient retinal organoids, showing less punctate staining for the constitutive variant of RPGR (G and H) and an absence of staining for the ORF15 isoform (I and J) in the region of the CC.

(K and L) Representative images of control and RPGR-deficient organoids showing phalloidin (magenta) localized to the OLM. Inset high magnification panels show no differences in staining pattern.

(M and N) Increased GFAP (green) staining in RPGR-deficient neuroepithelia, compared with control. OLM delineated with phalloidin (red) shows no difference.

(O and P) Images showing typical RHODOPSIN staining (green) localized to the OSs in both control and RPGR-deficient rod photoreceptors. Mis-localized RHODOPSIN (green) can also be seen in the cell bodies and processes of RPGR-deficient rods (P, white arrowheads). Nuclei were stained with DAPI (blue).

Scale bars, 5 μm (D, F, H, and J), 25 μm (B, C, E, G, I, and K–P) and 50 μm (A). IS, inner segment; CC, connecting cilia; ONL, outer nuclear layer; OLM, outer limiting membrane; OS, outer segment.