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Laminin (LN)-induced neurite growth is most extensive in the NF200+ve DRG neurons. Neurons were plated on poly-d-lysine (PL) or LN (50 µg/mL) in the presence or absence of nerve growth factor (NGF). At 24 h after plating, cells were fixed and immunostained for anti-CGRP (green) and anti-NF200 (red). (A–D) NF200+ve (red) neurons plated on LN show a robust increase in neurite growth (B, arrowhead), which was potentiated by the addition of NGF (D, red cell). CGRP+ve neurons (green) show only small amounts of neurite growth when plated on LN alone; however, plating them in the presence of NGF did stimulate increased neurite growth (D, green cell and arrowheads) that was not observed when plated on PL plus NGF. Scale bar, 50 µm.

Using normal human induced pluripotent stem cells (iPSCs) to model retinal development. Immunocytochemical analysis of iPSC-derived eyecup-like structures targeted against F-actin, SOX2, PAX6, OTX2, HuC/D, and recoverin (RCVRN). After 30 days of differentiation (D30), polarized neural epithelia (A, F-Actin in green) composed of proliferating cells (A, Ki67 in red) that are positive for the early retinal progenitor cell markers SOX2, PAX6, and OTX2 are present. After 60 days of differentiation (D60), PAX6 expression is restricted to OTX2-negative presumptive retinal pigment epithelium, whereas OTX2 is restricted to PAX6-negative photoreceptor precursor cells. After 100 days of differentiation (D100), eyecups are laminated with HuC/D-positiv ganglion cell–like neurons in the inner layer and recoverin-positive photoreceptor precursor cells in the outer layer. Insets depict individual fluorescent channels.  20× magnification. DAPI = 4'6-diamidino-2-phyenylindole.

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 CTGF drives iPSC differentiation into CECs. Representative image is provided showing cell morphology and CA4 expression at 0 ng/ml TWEAKR and 50 ng/ml CTGF. Shown are the means at each condition, while error bars represent the standard error of the mean (n = 9, **, p < .01, ***, p < .001). All scale bars represent 100 μm. Abbreviations: CEC, choroidal endothelial cell; CTGF, Connective tissue growth factor; DAPI, 4′,6-diamidino-2-phenylindole; TWEAKR, TNF-related weak inducer of apoptosis receptor.

 

Generating human iPSCs from a donor with normal ocular history: Pluripotent human TRA-1-60.All scale bars represent 100 μm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; CEC, choroidal endothelial cell; iPSC, induced pluripotent stem cell.

Identification of CEC differentiation candidates using the Taguchi L12 and factorial elimination screens. Representative image used to determine the percentage of CA4+ iPSC-derived CECs.All scale bars represent 100 μm. Abbreviations: CC, choriocapillaris; CEC, choroidal endothelial cell; CH, choroidal vasculature; CTGF, connective tissue growth factor; DAPI, 4′,6-diamidino-2-phenylindole; RPE, retinal pigment epithelium; TWEAKR, TNF-related weak inducer of apoptosis receptor; VEGFB, Vascular endothelial growth factor B.

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Immunocytochemical analysis using an antibody targeted against anti-acetylated tubulin (primary cilia marker) to stain dermal fibroblasts isolated from an unaffected control individual and a patient with molecularly confirmed MAK-associated RP. 

Light micrographs of wildtype, MAK morpholino-injected, and MAK morpholino and retinal MAK mRNA injected demonstrating normal overall morphology among groups. Histogram depicting the number of responses in the vision startle assay of wild-type, MAK morpholino-injected (MAK Mutant) and MAK morpholino/MAKRI mRNA injected (Mutant + MAKRI mRNA). MAK morphants had a significant decrease in average number of responses compared to wild-type fish (Wildtype vs MAK Mutant; p < 0.05). Injection of retinal MAK mRNA into MAK mutants partially rescued visual responses (i.e. no significant difference between wild-type and treatment groups).