Characterization of Primary Cilia Formation in Human ESC-Derived Retinal Organoids.

Objectives: Primary cilia are conserved organelles found in polarized mammalian cells that regulate neuronal growth, migration, and differentiation. Proper cilia formation is essential during eye development. Our previous reports found that both amacrine and retinal ganglion cells (RGCs) contain primary cilia in primate and rodent retinas. However, whether primary cilia are present in the inner retina of human retinal organoids remains unknown. The purpose of this study is to characterize the primary cilia distribution in human embryonic stem cell (hESC-derived retinal organoid development. Materials and Methods: Retinal organoids were differentiated from a hESC line, harvested at various developmental timepoints (day 44-day 266), and immunostained with antibodies for primary cilia, including Arl13b (for the axoneme), AC3, and Centrin3 (for the basal body). AP2α, Prox1, GAD67, Calretinin, GFAP, PKCα, and Chx10 antibodies as well as Brn3b-promoted tdTomato expression were used to visualize retinal cell types. Results: A group of ciliated cells were present in the inner aspects of retinal organoids from day 44 to day 266 in culture. Ciliated Chx10-positive retinal progenitor cells, GFAP-positive astrocytes, and PKCα-positive rod-bipolar cells were detected later during development (day 176 to day 266). Ciliation persisted during all stages of retinal developmental in AP2α-positive amacrine cells, but it was decreased in Brn3b-positive retinal ganglion cells (RGCs) at later time points. Additionally, AC3-positive astrocytes significantly decreased during the later stages of organoid formation. Conclusions: Amacrine cells in retinal organoids retain cilia throughout development, whereas RGC ciliation gradually and progressively decreases with organoid maturation.

Evaluation of the Algerbrush II rotating burr as a tool for inducing ocular surface failure in a mouse model.

Purpose: The Algerbrush II has been widely used to induce corneal and limbal injuries in animal models. The extent of injury varies with the duration of exposure, pressure from the placement of the burr, and the size of the burr. However, no study has explored the correlation between the duration of exposure and the severity of injury in mouse model with corneal and limbal stem cell deficiency (LSCD) induced using the Algerbrush II. Therefore, this study aimed to evaluate the variations in the severity of corneal and limbal injury with different durations of the Algerbrush II application. Methods: The entire cornea and limbus of C57BL/6 mice were injured for 30-45 s, 60-75 s, 90-120 s, and 3-4 min. Photography and slit-lamp examination was performed on days 0, 2, 4, and 7, followed by hematoxylin & eosin, periodic acid-Schiff, and immunohistochemical staining. Statistical analysis was performed using one way ANOVA analysis. Results: A duration of 30-45 s of injury was found to be sufficient to induce superficial corneal and limbal epithelial debridement and re-epithelialization was completed in all eyes by day 7; however, clinical signs of LSCD were not observed in all mice. Increasing the exposure time to 90-120 s resulted in central 2+ corneal opacity with limbal and paracentral corneal neovascularization. All eyes injured for 3-4 min displayed clinical signs of LSCD, such as persistent epithelial defects on day 7 after the injury, central corneal neovascularization, and 2.2+ diffuse corneal opacity. Histological signs of LSCD, including goblet cell metaplasia and K13 expression on the corneal surface, were observed in all injured eyes. Conclusions: Our findings suggest that the duration of injury is an important factor influencing the severity of LSCD in a murine model of injury. A 1-mm rotating burr was found to be more effective for keratectomy and pigment release, whereas a 0.5-mm burr was more suitable for corneal epithelial debridement.

The Use of Panitumumab-IRDye800CW in a Novel Murine Model for Conjunctival Squamous Cell Carcinoma.

Purpose: Conjunctival squamous cell carcinoma (SCC) is a sight-threatening ocular surface malignancy with the primary treatment modality being surgical resection. To evaluate surgical imaging modalities to improve surgical resection, we established a novel murine model for conjunctival SCC to demonstrate the utility of panitumumab-IRDye800, a fluorescently labeled anti-epidermal growth factor receptor (EGFR) antibody. Methods: NOD-scid IL2Rgammanull (NSG) mice received subconjunctival injection of UM-SCC-1 or SCC-9, head and neck SCC cell lines. On tumor growth, mice were injected with Panitumumab-IRDye800CW, and imaged with a small animal imaging system and optical coherence tomography (OCT). Immunohistochemistry for SCC markers were used to confirm tumor origin. Results: Seventy-five percent (N = 4) of the UM-SCC-1 group developed aggressive, rapidly growing tumors that were P40 and EGFR positive within two weeks of inoculation. The SCC-9 tumors failed to demonstrate any growth (N = 4). Ocular tumors demonstrated high fluorescence levels with a tumor to background ratio of 3.8. Conclusions: Subconjunctival injections are an appropriate technique to create in vivo models for assessing treatment modalities and novel therapies in conjunctival SCC. Translational Relevance: This model demonstrates Panitumumab-IRDye800CW's utility in the ophthalmic setting and suggests that clinical trials may be warranted.