PURPOSE: The development of primary human retinal pigmented epithelium (hRPE) for clinical transplantation purposes on biodegradable scaffolds is indispensable. We hereby report the results of the subretinal implantation of hRPE cells on nanofibrous membranes in minipigs. METHODS: The hRPEs were collected from human cadaver donor eyes and cultivated on ultrathin nanofibrous carriers prepared via the electrospinning of poly(L-lactide-co-DL-lactide) (PDLLA). "Libechov" minipigs (12-36 months old) were used in the study, supported by preoperative tacrolimus immunosuppressive therapy. The subretinal implantation of the hRPE-nanofibrous carrier was conducted using general anesthesia via a custom-made injector during standard three-port 23-gauge vitrectomy, followed by silicone oil endotamponade. The observational period lasted 1, 2, 6 and 8 weeks, and included in vivo optical coherence tomography (OCT) of the retina, as well as post mortem immunohistochemistry using the following antibodies: HNAA and STEM121 (human cell markers); Bestrophin and CRALBP (hRPE cell markers); peanut agglutining (PNA) (cone photoreceptor marker); PKCα (rod bipolar marker); Vimentin, GFAP (macroglial markers); and Iba1 (microglial marker). RESULTS: The hRPEs assumed cobblestone morphology, persistent pigmentation and measurable trans-epithelial electrical resistance on the nanofibrous PDLLA carrier. The surgical delivery of the implants in the subretinal space of the immunosuppressed minipigs was successfully achieved and monitored by fundus imaging and OCT. The implanted hRPEs were positive for HNAA and STEM121 and were located between the minipig's neuroretina and RPE layers at week 2 post-implantation, which was gradually attenuated until week 8. The neuroretina over the implants showed rosette or hypertrophic reaction at week 6. The implanted cells expressed the typical RPE marker bestrophin throughout the whole observation period, and a gradual diminishing of the CRALBP expression in the area of implantation at week 8 post-implantation was observed. The transplanted hRPEs appeared not to form a confluent layer and were less capable of keeping the inner and outer retinal segments intact. The cone photoreceptors adjacent to the implant scaffold were unchanged initially, but underwent a gradual change in structure after hRPE implantation; the retina above and below the implant appeared relatively healthy. The glial reaction of the transplanted and host retina showed Vimentin and GFAP positivity from week 1 onward. Microglial activation appeared in the retinal area of the transplant early after the surgery, which seemed to move into the transplant area over time. CONCLUSIONS: The differentiated hRPEs can serve as an alternative cell source for RPE replacement in animal studies. These cells can be cultivated on nanofibrous PDLLA and implanted subretinally into minipigs using standard 23-gauge vitrectomy and implantation injector. The hRPE-laden scaffolds demonstrated relatively good incorporation into the host retina over an eight-week observation period, with some indication of a gliotic scar formation, and a likely neuroinflammatory response in the transplanted area despite the use of immunosuppression.

Center for Eye Research Department of Ophthalmology Oslo University Hospital and Institute for Clinical Medicine Faculty of Medicine University of Oslo 0450 Oslo Norway

Department of Cell Biology Faculty of Science Charles University 11000 Prague Czech Republic

Department of Ophthalmology Justus Liebig University Giessen University Hospital Giessen and Marburg GmbH 35392 Giessen Germany

Department of Ophthalmology University Hospital Kralovske Vinohrady and 3rd Faculty of Medicine Charles University 10034 Prague Czech Republic

Institute for Clinical and Experimental Medicine 14021 Prague Czech Republic

Institute of Animal Physiology and Genetics Academy of Sciences of the Czech Republic 27721 Libechov Czech Republic

Institute of Experimental Medicine Academy of Sciences of the Czech Republic 11720 Prague Czech Republic

Institute of Macromolecular Chemistry Academy of Sciences of the Czech Republic 16206 Prague Czech Republic

Karl Landsteiner Institute for Retinal Research and Imaging 1030 Vienna Austria

Moorfields Eye Hospital Abu Dhabi 62807 United Arab Emirates

Research Center Principe Felipe Stem Cell Therapies in Neurodegenerative Diseases Laboratory 46012 Valencia Spain

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Electrospun poly(l-lactide-co-dl-lactide) nanofibrous scaffold as substrate for ex vivo limbal epithelial cell cultivation

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Progress in Stem Cells-Based Replacement Therapy for Retinal Pigment Epithelium: In Vitro Differentiation to In Vivo Delivery

Retinal Pigment Epithelium Cell Development: Extrapolating Basic Biology to Stem Cell Research

Mitochondrial Dysfunction in a High Intraocular Pressure-Induced Retinal Ischemia Minipig Model