PurposePrevious studies have demonstrated the ability of retinal cells derived from human embryonic stem cells (hESCs) to survive, integrate into the host retina, and mediate light responses in murine mouse models. Our aim is to determine whether these cells can also survive and integrate into the retina of a nonhuman primate, Saimiri sciureus, following transplantation into the subretinal space.MethodshESCs were differentiated toward retinal neuronal fates using our previously published technique and cultured for 60 to 70 days. Differentiated cells were further treated with 20 μM N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) for a period of 5 days immediately prior to subretinal transplantation. Differentiated cells were labeled with a lentivirus expressing GFP. One million cells (10,000 cells/μL) were injected into the submacular space into a squirrel monkey eye, using an ab externo technique.ResultsRetCam imaging demonstrated the presence and survival of human donor cells 3 months after transplantation in the S. sciureus eye. Injected cells consolidated in the temporal macula. GFP+ axonal projections were observed to emanate from the central consolidation of cells at 1 month, with some projecting into the optic nerve by 3 months after transplantation.ConclusionsHuman ES cell-derived retinal neurons injected into the submacular space of a squirrel monkey survive at least 3 months postinjection without immunosuppression. Some donor cells appeared to integrate into the host inner retina, and numerous donor axonal projections were noted throughout, with some projecting into the optic nerve.Translational RelevanceThese data illustrate the feasibility of hESC-derived retinal cell replacement in the nonhuman primate eye.

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Sorsby Fundus Dystrophy (SFD) is a rare form of macular degeneration that is clinically similar to age-related macular degeneration (AMD). SFD results from mutations found in the tissue inhibitor of metalloproteinase 3 (TIMP3) gene. TIMP3 is secreted by the retinal pigmented epithelium (RPE) into the underlying Bruch’s membrane (BrM), and it plays a critical role in maintaining extracellular matrix (ECM) homeostasis. A characteristic feature of post-mortem SFD globes is a thick layer of sub-RPE deposits overlying a disorganized BrM. Although likely central to the pathogenesis of SFD, no animal models have reproduced this phenotype. We generated induced pluripotent stem cell (iPSC)-derived RPE lines from SFD family members with the S204C TIMP3 mutation and observed that SFD RPE have highly dysregulated ECM and form large basal deposits by ~30 days in culture. The sub-RPE deposits are similar in ultrastructure and composition when compared to SFD family member globes. Mutant TIMP3 correction by CRISPR-Cas9 gene editing in SFD iPSC RPE cells resulted in the reversal of sub-RPE calcium deposition. We found that SFD TIMP3 has decreased inhibition of secreted matrix metalloproteinases. ECM dysfunction substantially impacts cellular metabolism. Targeted metabolomics data showed that intracellular 4-hydroxyproline, a major breakdown product of collagen, is significantly elevated in SFD RPE. Further, SFD RPE also has decreased intracellular reduced glutathione and is more vulnerable to oxidative stress. These findings suggest that key elements of SFD pathology can be recapitulated in culture which may lead to insights into disease mechanisms and potential treatments.Significance StatementThis study demonstrates that retinal pigmented epithelial (RPE) cells generated from patients with Sorsby Fundus Dystrophy (SFD) produce highly dysregulated extracellular matrices. SFD RPE form large basal deposits in culture that are similar in composition to what is observed in donated SFD post-mortem globes from family members. Further, SFD RPE demonstrate high levels of 4-hydroxyproline, a major breakdown product of collagen. SFD RPE are also more vulnerable to oxidative stress. Our studies indicate that key elements of SFD pathology can be recapitulated in culture, and ECM dysregulation may lead to metabolic changes detrimental to RPE health.

PurposeStandard techniques for the treatment of congenital lower eyelid entropion may not yield suitable outcomes or may result in overcorrection if disinsertion of the lower eyelid retractors is not the primary cause. Herein, we propose and evaluate a technique using subciliary rotating sutures combined with a modified Hotz procedure for repair of lower eyelid congenital entropion that addresses these concerns.MethodsA retrospective chart review was conducted of all patients who underwent lower eyelid congenital entropion repair by a single surgeon using subciliary rotating sutures combined with a modified Hotz procedure between 2016 and 2020. Study variables included patient demographics, follow-up period, postoperative complications, operative success, and recurrence.ResultsTwelve patients (19 eyelids) met the study inclusion criteria. The mean patient age was 7.1 ± 6.1 years (range, 0.2–22 years). Nine of the patients were female (75%) and 3 were male (25%). The distribution of eyelids was 8 right (42%) and 11 left (58%). The mean follow-up time was 19.5 ± 15 (range 2.5–45) months. There were two eyelids (11%) that had entropion recurrence after initial repair in patients with concomitant compound disease processes. Repeated repair resulted in success with no recurrence at last follow-up. Overall, the described entropion repair technique was successful and without recurrence in 17 eyelids (89%). There were no cases of ectropion, lid retraction, or other complications.ConclusionsSubciliary rotating sutures combined with a modified Hotz procedure are effective for correction of congenital lower eyelid entropion. As the technique does not manipulate the posterior layer of the lower eyelid retractors, it may be useful for when retractor reinsertion does not yield adequate improvement and may also reduce the risk of eyelid retraction and overcorrection in particular cases.