International Journal of Molecular Sciences Article Transplantation of Human Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium in a Swine Model of Geographic Atrophy Anna Duarri 1,2,3,† , Eduardo Rodríguez-Bocanegra 4,5,† , Gema Martínez-Navarrete 6,7 , Marc Biarnés 4,5 , Miriam García 4,5 , Lucía Lee Ferraro 4,5 , Bernd Kuebler 1 , Begoña Aran 1,2 , Elisabeth Izquierdo 8 , Eli Aguilera-Xiol 8 , Ricardo P. Casaroli-Marano 9,10 , Esteve Trias 11,12 , Eduardo Fernandez 6,7 , Ángel Raya 1,6,13 , Anna Veiga 1,2, * and Jordi Monés 4,5, * 1 Program for Clinical Translation of Regenerative Medicine in Catalonia–P-CMR[C], Bellvitge Biomedical Research Institute (IDIBELL), Hospitalet de Llobregat, 08908 Barcelona, Spain; anna.duarri@vhir.org (A.D.); bkuebler@idibell.cat (B.K.); baran@idibell.cat (B.A.); araya@idibell.cat (Á.R.) 2 National Stem Cell Bank-Barcelona Node, Biomolecular and Bioinformatics Resources Platform PRB2, ISCIII, IDIBELL, Hospitalet de Llobregat, 08908 Barcelona, Spain  3  Ophthalmology Research Group, Vall d’Hebron Institut de Recerca (VHIR), 08036 Barcelona, Spain 4 Barcelona Macula Foundation: Research for Vision, 08022 Barcelona, Spain; Citation: Duarri, A.; eduardo.rodriguez-bocanegra@med.uni-tuebingen.de (E.R.-B.); mbiarnes@barcelonamaculafound.org (M.B.); Rodríguez-Bocanegra, E.; mgarcia@barcelonamaculafound.org (M.G.); lferraro@barcelonamaculafound.org (L.L.F.) Martínez-Navarrete, G.; Biarnés, M.; 5 Institut de la Màcula, Centro Médico Teknon, 08022 Barcelona, Spain 6 Networking Research Center of Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), García, M.; Ferraro, L.L.; Kuebler, B.; Aran, B.; Izquierdo, E.; Aguilera-Xiol, 28029 Madrid, Spain; gemamartineznavarrete@gmail.com (G.M.-N.); e.fernandez@umh.es (E.F.) 7 Institute of Bioengineering, Universidad Miguel Hernández, 03202 Alicante, Spain E.; et al. Transplantation of Human 8 Specific Pig S.L, 08820 Barcelona, Spain; elisabeth.izquierdo@specipig.com (E.I.); Induced Pluripotent Stem eli.aguilera@specipig.com (E.A.-X.) Cell-Derived Retinal Pigment 9 Banc de Sang i Teixits (BST), Institute of Biomedical Research (IIB-Sant Pau), 08025 Barcelona, Spain; Epithelium in a Swine Model of rcasaroli@ub.edu Geographic Atrophy. Int. J. Mol. Sci. 10 Department of Surgery, School of Medicine and Health Science, Hospital Clinic de Barcelona, 2021, 22, 10497. https://doi.org/ University of Barcelona, 08036 Barcelona, Spain 10.3390/ijms221910497 11 LEITAT Technological Center, 08005 Barcelona, Spain; etrias@leitat.org 12 Advanced Therapies Unit, Hospital Clínic de Barcelona, 08005 Barcelona, Spain 13 Institució Catalana de Recerca i Estudis Avançats (ICREA), 08010 Barcelona, Spain Academic Editors: Sandra Petrus-Reurer, * Correspondence: aveiga@idibell.cat (A.V.); jmones@institutmacula.com (J.M.) † These authors contributed equally to this work. Arturo Ortín-Martínez and Luis Alarcon-Martinez Abstract: Background: The aim of this study was to test the feasibility and safety of subretinal Received: 17 September 2021 transplantation of human induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium Accepted: 24 September 2021 (RPE) cells into the healthy margins and within areas of degenerative retina in a swine model of Published: 28 September 2021 geographic atrophy (GA). Methods: Well-delimited selective outer retinal damage was induced by subretinal injection of NaIO3 into one eye in minipigs (n = 10). Thirty days later, a suspension Publisher’s Note: MDPI stays neutral of hiPSC-derived RPE cells expressing green fluorescent protein was injected into the subretinal with regard to jurisdictional claims in space, into the healthy margins, and within areas of degenerative retina. In vivo follow-up was published maps and institutional affil- performed by multimodal imaging. Post-mortem retinas were analyzed by immunohistochemistry iations. and histology. Results: In vitro differentiated hiPSC-RPE cells showed a typical epithelial morphology, expressed RPE-related genes, and had phagocytic ability. Engrafted hiPSC-RPE cells were detected in 60% of the eyes, forming mature epithelium in healthy retina extending towards the border of the atrophy. Histological analysis revealed RPE interaction with host photoreceptors in the healthy Copyright: © 2021 by the authors. retina. Engrafted cells in the atrophic zone were found in a patchy distribution but failed to form an Licensee MDPI, Basel, Switzerland. epithelial-like layer. Conclusions: These results might support the use of hiPSC-RPE cells to treat This article is an open access article atrophic GA by providing a housekeeping function to aid the overwhelmed remnant RPE, which distributed under the terms and might improve its survival and therefore slow down the progression of GA. conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Int. J. Mol. Sci. 2021, 22, 10497. https://doi.org/10.3390/ijms221910497 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 10497 2 of 21 Keywords: age-related macular degeneration (AMD); geographic atrophy; pig; animal model; stem cells; iPSC; RPE; retina; regenerative medicine; advanced cell therapy 1. Introduction Age-related macular degeneration (AMD) is the most common cause of blindness in people older than 50 years in industrialized countries, and affects between 14.9 and 21.5 million people in Europe alone [1]. AMD is a degenerative, chronic, and progressive disease involving the retinal pigment epithelium (RPE) and choroid, which trigger the degeneration of the outer retina in the macula region of the eye [2]. The etiology of AMD is multifactorial and includes multiple genetic and environmental factors [3]. There are two forms of AMD—exudative (wet, neovascular) or atrophic (dry). The first one is related to the development of choroidal neovascularization and accounts for about 20% of all cases of AMD. Late-stage dry AMD can manifest as geographic atrophy (GA), which affects the macular region of the retina, causing progressive and irreversible loss of central vision due to the degeneration of retinal pigment epithelium (RPE) and photoreceptor cells, and accounts for 80% of all cases. At present, while there are the intravitreal injections of anti- VEGF factors used in the management of wet AMD, there is no effective curative treatment or preventive therapy for the dry form of this disease. Once RPE and photoreceptor cells are lost, cell replacement or prosthetic devices are the only potential therapeutic options [4,5]. Despite the evident degeneration of RPE and photoreceptors in AMD, the inner retina and neural connections seem to be preserved, to some extent, for a limited time, supporting the realistic potential of cell-based therapy to restore vision [6]. Human pluripotent stem cells—both embryonic (hESC) and induced (hiPSC)—have the ability to self-renew and to differentiate into all retinal cell types needed for retinal regeneration [7]. Indeed, hESC- derived RPE cells are currently being investigated in 20 early-phase human clinical trials to treat retinopathies (reviewed in [8–11]) (https://clinicaltrials.gov/ queried on 1 March 2019). Follow-up studies have shown that transplantation of RPE sheets is tolerable, but requires very invasive surgical procedures with associated safety concerns. By contrast, transplantation of RPE cell suspensions requires minimal surgery, making this procedure more appealing for clinical applications and more suitable for wider and easier adoption by the ophthalmological community [12–17]. Nevertheless, more pre-clinical research is needed to better understand how RPE cell suspensions survive and integrate, mature, and interact with host photoreceptors, particularly those derived from hiPSC. Due to their similar dimensions and anatomy, large animals can play important roles in the development and adaptation of human surgical techniques and are good pre-clinical models for cell replacement treatments [18]. In particular, the pig eye and retina resemble their human counterparts in terms of size and shape, number, and distribution of photore- ceptors, vasculature, and function [19]. Accordingly, several transgenic pig models have been developed to study retinitis pigmentosa [20–24]; however, the disease progression is slow, which hinders the evaluation of cell therapy benefits, and the models are expensive to maintain. To overcome these limitations and to bypass the genetic component, and also the aging and environmental factors that underlie retinal diseases, we have developed a pig model of well-delimited outer retinal degeneration through localized subretinal injection of sodium iodate (NaIO3 ), which resembles the selective degeneration of RPE or outer retina in humans, with special interest in two major aspects of human GA associated with the dry form of AMD, damage of outer retinal layers with preservation of the inner layers, and lesions will well-defined distinct margins between the atrophic area and the healthy area [25]. Other, less well-delimited, models have been established, such as intravenous in- jection of iodoacetic acid [26], light injury [27–29], scraping injury [29,30], or high-pressure injury [30], and these models have been used in studies of allogenic and xenogenic cell transplantation in healthy or degenerated porcine retinas using either porcine or human fe- tal neuroretina cells or sheets [27,29–32], mouse retinal progenitor cells [28], hESC-derived Int. J. Mol. Sci. 2021, 22, 10497 3 of 21 RPE monolayer on biocompatible scaffolds [33,34], or porcine iPSC-derived RPE and retinal progenitor cells [26,35]. The lack of pre-clinical studies in swine models involving injection of subretinal cell suspensions of human iPSC-derived retinal cells prompted us to test whether hiPSC-derived RPE cell transplantation in a model mimicking end-stage AMD might be a potential approach to treat this disease. Here, we investigated the feasibility and safety, survival, and integration of subretinal xenotransplantation of hiPSC-RPE cell suspensions in a NaIO3 -induced GA model in miniature pigs. 2. Results 2.1. Generation and Characterization of hiPSC-RPE Cells To facilitate the identification of implanted hiPSC-RPE cells, we generated an hiPSC line (CBiPS30-4F-5) constitutively expressing GFP while maintaining pluripotency and normal karyotype (Supplementary Figure S1A,B). Differentiated and purified hiPSC-RPE cells were pigmented, maintained GFP expression, and had a hexagonal-shaped morphol- ogy (Figure 1A). Additionally, hiPSC-RPE cells expressed molecular markers of RPE such as PAX6 and OTX2, and the RPE cell-specific markers bestrophin-1 (basal membrane), MITF (optic vesicle), ZO-1 (tight junctions), SIL, RPE65 and CRALBP (visual cycle), PEDF (growth factors), and TYR (pigmentation), as detected by immunocytochemistry and qPCR (Figure 1A,C). Reassuringly, vertical confocal sections showed correct apical localization of ZO-1 and basolateral localization of bestrophin-1 in cells (Figure 1A). To assess the purity of hRPE-GFP cells, the hiPSC line together with hRPE-GFP derived from it were tested in parallel for Tra-1-60, a pluripotency marker, and for CD140b and CD59, RPE-specific mark- ers, by flow cytometry (Figure 1B; Supplementary Figure S2). hRPE-GFP cells expressed CD140b and CD59 but not Tra-1-60, indicating the absence of undifferentiated cells in our culture. TEM analysis of the ultrastructure of hiPSC-RPE cells cultured in Transwell inserts showed a monolayer of pigmented cuboidal epithelial cells with cytoplasmic polarization, with nuclei located to the basolateral side, and microvilli, well-formed tight junctions, and melanin-containing melanosomes on the apical side (Figure 1C, top). Likewise, SEM analy- sis showed microvilli on the apical surface and a polygonal-shaped cellular morphology at days 30 and 60 in culture (Figure 1C, bottom). We also assessed the integrity of tight junctions’ dynamics in hiPSC-RPE cells by measuring transepithelial electrical resistance (TEER). TEER values were first detected at day 3 of Transwell culture and increased until day 11, when they reached a plateau at ~200 Ω·cm2 , in contrast to hiPSC culture (Figure 1E). We then tested the functionality of hiPSC-RPE cells in vitro by analyzing their phagocytosis ability, finding that the cells phagocytosed bovine-purified TRITC-labeled POS (Figure 1F). 2.2. Generation of a Swine Model with Geographic Atrophy-like Features Based on our previous work [25], we used the subretinal injection of NaIO3 to reca- pitulate the main features of GA in minipigs. Specifically, the right eye of 7 pigs (pigs 1–7; Table 1) was injected with the optimal concentration of NaIO3 (0.01 mg/mL). One month later, in vivo spectral domain optical coherence tomography (SD-OCT) analysis of these animals revealed selective atrophy of outer retinal layers (Figure 2A, a; Table 1; Sup- plementary Figure S3). We also explored whether more severe atrophy could affect the outcome of the RPE therapy. Thus, three pigs (pigs 8–10) were injected with a higher dose of NaIO3 (0.1 mg/mL), which resulted in severe atrophy with dramatic thinning of all retinal layers (Figure 2A, b; Supplementary Figure S3). In all pigs analyzed, outer retinal atrophy was maintained over time and a well-delimited transition zone between healthy and atrophic retinas was observed, thus mimicking the alterations seen in patients with advanced age-related macular degeneration and GA. Int. J. Mol. Sci. 2021, 22, 10497 4 of 21 Int. J. Mol. Sci. 2021, 221, 497 4 of 21 Figure 1. Characterization of hiPSC-derived RPE cells expressing GFP. (A) Representative bright-field and immunostain- Figure 1. Characterization of hiPSC-derived RPE cells expressing GFP. (A) Representative bright-field and immunostaining ing images showing pigmented hiPSC-RPE at 30 days in culture and the expression of endogenous GFP and RPE markers: images showing pigmented hiPSC-RPE at 30 days in culture and the expression of endogenous GFP and RPE markers: BEST-1 (bestrophin-1), ZO-1 (zonula occludens 1), PAX6 (paired box 6), MITF (microphthalmia-associated transcription BEST-1 (bestrophin-1), factor), ZO-1 (zonula OTX2 (orthodenticle homeobox occludens 2), and1), PAX6 RPE65 (paired box (anti-retinal 6), MITF pigment (microphthalmia-associated epithelium-specific 65 kDa protein).transcription hiPSC- factor), RPE OTX2 (orthodenticle cells are negative for homeobox OCT4 (data2),notand RPE65Scale shown). (anti-retinal bars: 25pigment epithelium-specific μm. Representative x–z scans65ofkDa protein). polarized hiPSC-RPE hiPSC-RPE cells cells are(green), negativeapical ZO-1 (red), for OCT4 (dataand notbasolateral shown). Scale bestrophin bars: 25(white). Cell nuclei werex–z µm. Representative stained scanswith DAPI (blue). of polarized (B) Quanti- hiPSC-RPE cells tative flow cytometry analysis of hiPSC and hPRE-GFP cells with surface markers Tra-1-60 (undifferentiated (green), apical ZO-1 (red), and basolateral bestrophin (white). Cell nuclei were stained with DAPI (blue). (B) Quantitative cells), CD140b, and CD59 (RPE-specific), and the appropriate isotype controls. The numbers in the corners show the percentage flow cytometry analysis of hiPSC and hPRE-GFP cells with surface markers Tra-1-60 (undifferentiated cells), CD140b, and of stained cells in this gate. (C) Gene expression levels of OCT4, OTX2, SIL (silver), CRLBP (cellular retinal aldehyde- CD59 (RPE-specific), and the appropriate isotype controls. The numbers in the corners show the percentage of stained cells binding protein), PEDF (pigment epithelium-derived factor), and TYR (tyrosinase) in hiPSC-RPE cells by qPCR. Values in this gate. (C) Gene are normalized expression to GAPDH and levels relativeoftoOCT4, hiPSC,OTX2, and are SIL (silver), as expressed CRLBP 2^-ΔΔCt (cellular retinal (log scale). (D)aldehyde-binding protein), Representative electron PEDF (pigmentimages microscopy epithelium-derived of hiPSC-RPE cells factor), and TYR in Transwell (tyrosinase) insert indays cultures at hiPSC-RPE 30 and 60 cells by qPCR. in culture. TEM Values images are normalized (top) showing to pigmented GAPDH cuboidaltoepithelial and relative monolayer, hiPSC, and are expressed as 2-∆∆Ct with apical microvilli, melanosomes (log scale). (asterisk), basal (D) Representative nuclei electron (N), mitochondria microscopy images of (m), tight junctions (arrow), and adherent hiPSC-RPE cells in Transwell insert cultures at days 30junctions (arrowheads). Scale bars: and 60 in culture. TEM5 images μm (left), 1 μm (top) (right). pigmented showing SEM imagescuboidal (bot- tom) showing the apical microvilli and polygonal cell morphology. Scale bars: 5 μm (middle left); 2 μm (bottom left); 1 epithelial monolayer, with apical microvilli, melanosomes (asterisk), basal nuclei (N), mitochondria (m), tight junctions μm (right). (E) Graph showing transepithelial electrical resistance (TEER) for hiPSC and hiPSC-RPE cells at indicated days (arrow), and adherent in culture. junctions (F) Confocal images(arrowheads). Scale bars: showing phagocytosis 5 µm (left), of isolated 1 µm (right). TRITC-labeled SEM bovine images (bottom) photoreceptor outershowing segmentsthe apical microvilli (POS, and polygonal red) by hiPSC-RPE cellinmorphology. (green) Scale bars: x–y culture. Representative 5 µm (middle and left);show x–z images 2 µmtotal (bottom left); 1 µm POS TRITC-labeled (right). (E) (red) Graph taken showing up by transepithelial the cells. Nucleielectrical resistance are stained with DAPI(TEER) forScale (blue). hiPSC and bar: 25hiPSC-RPE μm. cells at indicated days in culture. (F) Confocal images showing phagocytosis of isolated TRITC-labeled bovine photoreceptor outer segments (POS, red) by hiPSC-RPE (green) in culture. Representative x–y and x–z images show total TRITC-labeled POS (red) taken up by the cells. Nuclei are stained with DAPI (blue). Scale bar: 25 µm. Int. J. Mol. Sci. 2021, 22, 10497 5 of 21 Table 1. List and details of procedures and findings of each animal in this study. hiPSC-RPE NaIO3 Follow- Evidence of hiPSC-RPE Animal In vivo Cells Post-Mortem Analysis Dose Up In Vivo Injected Affected GFP+ Cells (Area (mg/mL) Months FAF SD-OCT Layers in and Layers) the Atrophy Selective atrophy of Healthy retina, 1 0.01 3.3 × 105 6 Yes Yes ONL, RPE ONL atrophy (RPE) Healthy retina, Selective atrophy of 2 0.01 3.3 × 105 0.5 * ND ND ONL, RPE atrophy and border ONL (RPE) Selective atrophy of 3 0.01 3.3 × 105 3 No No ONL, RPE No cells ONL Selective atrophy of 4 0.01 3.3 × 105 3 No No ONL, RPE No cells ONL Selective atrophy of Healthy retina and 5 0.01 2.5 × 105 3 Yes Yes ONL, RPE ONL vitreous Selective atrophy of 6 0.01 2.5 × 105 3 Yes Yes ONL, RPE Healthy retina (RPE) ONL Selective atrophy of Atrophy (INL, RPE), 7 0.01 2.5 × 105 3 Yes Yes ONL, RPE ONL border and vitreous Marked atrophy and INL, ONL, 8 0.1 thinning of all retinal 3.3 × 105 3 No No No cells RPE layers Marked atrophy and INL, ONL, Healthy retina and 9 0.1 thinning of all retinal 3.3 × 105 3 Yes Yes RPE border (RPE) layers 10 0.1 Retinal detachment ND ND ND ND ND ND *: Exitus for reasons unrelated to the study before the first follow-up after the cell injection. ND: Not done; FAF: fundus autofluorescence; ONL: outer nuclear layer; INL; inner nuclear layer; SD-OCT: spectral-domain optic coherence tomography; RPE: retinal pigmented epithelium; GFP; green fluorescent protein. Three animals, two with selective atrophy (animal 3 and 4) and one with severe atrophy (animal 8), were histologically analyzed because they showed no presence of hiPSC-RPE cells. Animals 3 and 4 showed selective atrophy, mostly limited to the integrity of RPE and the outer nuclear layer (ONL) (Figure 2B,C). In the selective atrophies, photore- ceptors were absent from the border of the atrophy (Figure 2C, a–c), RPE cells were focally lost (Figure 2C, e), and the remaining RPE layer in the atrophic zone showed alterations in both cell morphology and organization compared with the healthy RPE layer (insets in Figure 2C, d,f), consistent with previous observations [25,36]. By contrast, the severe atro- phy in pig 8 affected RPE, the ONL, and the inner nuclear layer (INL), with a well-delimited border (Figure 2D,E). Additionally, the bipolar cells (PKC-positive) in the INL and pho- toreceptors (recoverin-positive) were missing in the transition zone (Figure 2E, a–c). RPE cells were lost (as judged by the absence of the RPE65 signal), and this was accompanied by Bruch’s membrane (BM) atrophy (Figure 2D,E, d,f). We also observed that activated astrocytes (GFAP-positive) in the atrophic zone were densely packed (Figure 2E, d–f) and formed a membrane-like structure posterior to the photoreceptor segments in the subretinal space (Supplementary Figure S4A), likely due to the accumulation of junctions within glial processes, as has been described in patients with GA [37]. Int. J. Mol. Sci. 2021, 221, 497 *: Exitus for reasons unrelated to the study before the first follow-up after the cell injection. ND: Not done; Int. J. Mol. Sci. 2021, 22, 10497 FAF: fund 6 of 21 autofluorescence; ONL: outer nuclear layer; INL; inner nuclear layer; SD-OCT: spectral-domain optic coherence tomogr phy; RPE: retinal pigmented epithelium; GFP; green fluorescent protein. Figure 2. Selective and severe atrophies induced by subretinal injection of NaIO3 in porcine retinas. (A) Fundus porci Figure 2. Selective and severe atrophies induced by subretinal injection of NaIO in porcine retinas. retinas at 1 month after NaIO3 subretinal injection (0.2 mL) at a concentration of 0.01 mg/mL3 (a). A well-demarcated roun (A) area is shown byFundus porcine IR fundus retinasafter imaging at 1 month NaIO3 after NaIO injection subretinal in3 pig injection 4. The 30° fundus (0.2IRmL) image at awith concentration overlying ofposition of 0.01 mg/mL (a). A well-demarcated round area is shown by IR fundus Scan (middle panel; green line) and cross-sectional SD-OCT B-scan demonstrate selective loss of the imaging after NaIO 3 injection outer retinal laye in pig 4. The ◦ fundus IR image with overlying position of B-Scan (middle panel; green line) The outer plexiform layer30and inner nuclear layer are directly on the damaged RPE, as occurs in GA in humans. (b) contrast, a and cross-sectional severe round atrophy SD-OCT is shown B-scan by IRdemonstrate and SD-OCTselectiveimaginglossafterofNaIO the outer retinalinlayers. 3 injection The pig 9. A outer zone (r transition arrows) is plexiform observed layer between and the inneratrophic nuclearandlayerhealthy retina are directly oninthe SD-OCT damaged in RPE, both as(a)occurs and (b). in GARedinsquares humans.indicate t enlargement (b)of Bythe atrophic contrast, border. a severe (B) Photomerge round composition atrophy is shown by IR and of SD-OCT hematoxylin imagingand after eosinNaIO (H&E) staining of the lesi 3 injection in induced withpig 0.01 9. A mg/mL transition NaIO zone3 in pig 4. Arrows indicate the border of the atrophy. Scale bar: 200 μm. (C) (Top pane (red arrows) is observed between the atrophic and healthy retina in SD-OCT Images of H&E staining showing different parts of porcine healthy and atrophic retina of the lesion induced with 0. in both (a) and (b). Red squares indicate the enlargement of the atrophic border. (B) Photomerge mg/mL NaIO3. Scale bars: 50 μm. (Bottom panels) Immunofluorescence images of different areas (healthy (a,d), bord composition of hematoxylin and eosin (H&E) staining of the lesion induced with 0.01 mg/mL NaIO3 (b,e), and mild atrophy (c,f)) labeled with glial fibrillary acidic protein (GFAP), retinal pigment epithelium-specific 65 kD in pigand protein (RPE65) 4. Arrows indicate rhodopsin. Insetstheinborder (C (d) of andthe atrophy. (f)) show an Scale bar: 200 µm. enlargement of the(C)RPE (Top panels) layer Images stained withofRPE65. Sca bars: 50 μm.H&E(D)staining Photomergeshowing differentofparts composition H&Eofstaining porcineof healthy and induced the lesion atrophic with retina 0.1ofmg/mL the lesionNaIOinduced 3 in pig 8. Arrow indicate thewith 0.01 of border mg/mL NaIO3 .Scale the atrophy. Scalebar: bars:200 50 μm. (Bottom µm. (E) (Top panels) panels) Immunofluorescence Images of H&E staining images of different showing different parts areas and porcine healthy (healthy (a,d),retina atrophic borderof (b,e), and mild the lesion atrophy induced with(c,f)) labeledNaIO 0.1 mg/mL with3glial . Scalefibrillary bars: 50acidic protein panels) Im μm. (Bottom munofluorescence (GFAP), images of the different retinal pigment zones of the65 epithelium-specific eyes kDa (healthy protein(a,d), (RPE65) border and (b,e), and severe rhodopsin. Insetsatrophy in (C (d)(c,f)) label and (f)) show an enlargement of the RPE layer stained with RPE65. Scale bars: 50 µm. (D) Photomerge Int. J. Mol. Sci. 2021, 22, 10497 7 of 21 composition of H&E staining of the lesion induced with 0.1 mg/mL NaIO3 in pig 8. Arrows indicate the border of the atrophy. Scale bar: 200 µm. (E) (Top panels) Images of H&E staining showing different parts of porcine healthy and atrophic retina of the lesion induced with 0.1 mg/mL NaIO3 . Scale bars: 50 µm. (Bottom panels) Immunofluorescence images of the different zones of the eyes (healthy (a,d), border (b,e), and severe atrophy (c,f)) labeled with recoverin, protein kinase C alpha (PKC), GFAP, and RPE65. Scale bars: 50 µm. Nuclei are stained with DAPI (blue). NaIO3 , sodium iodate; IR, infrared image; SD-OCT, spectral-domain optic coherence tomography; RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; GFP, green fluorescent protein; Chor, choroid. 2.3. In Vivo Imaging of Transplanted hiPSC-RPE Cells in the Porcine Eyes One month following NaIO3 injection, hiPSC-RPE cells were injected into the subreti- nal space of animals 1–7 with selective atrophy and animals 8 and 9 with severe atrophy (Table 1). Animal 10 was not transplanted because of the development of retinal detach- ment during the follow-up after atrophy induction, and animal 2 died two weeks after transplantation from causes unrelated to the procedure. To assess the traceability of the injected cells, in vivo post-operative follow-up was performed using SD-OCT, and fundus autofluorescence (FAF) and infrared (IR) imaging. SD-OCT images were acquired with simultaneous visualization of the FAF in the IR channel. Constitutive GFP expression of hiPSC-RPE cells was clearly detectable through FAF imaging as hyper-autofluorescence signals in pigs 1, 5, 6, 7, and 9 (60% of the animals) (Figures 3 and 4; Table 1). In pigs 1 and 5, hyper-autofluorescence signals were only visible in the first ophthalmological exam after hiPSC-RPE cell injection, whereas in pigs 6, 7, and 9, the signals were maintained up to three months (latest time-point analyzed). Two weeks after cell injection, FAF imaging revealed a hyper-autofluorescence speckle in the atrophied area and its border (animal 6, compare Figure 3A (before cell injection) and Figure 3B (after cell injection)). Additionally, SD-OCT analysis showed a thickening of the outer retinal layer due to the new appear- ance of subretinal hyper-reflective material that might correspond to the transplanted hiPSC-RPE cells, both in the healthy retina area and within the atrophic zone (Figure 3B), and this was also observed in animals 1, 5, 6, 7, and 9. At three months, FAF imaging detected homogeneous signals of hyper-autofluorescence in healthy areas of the retina (animals 7 and 9, Figure 4A). Cells appeared well-organized in a monolayer, localized between the RPE and photoreceptors as a new hyper-reflective layer, as visualized by SD-OCT (Figure 4A, e). Conversely, the engraftment of hiPSC-RPE cells in atrophic areas occurred in a much more punctual manner, and was predominantly at the atrophy border (Figure 4B). 2.4. Post-Mortem Analysis of Retinas Transplanted with hiPSC-RPE Cells Animal 1 was euthanized at 6 months, animal 2 died and was analyzed at 2 weeks, and the remaining animals were euthanized at 3 months (Table 1). Surviving hiPSC-RPE cells were found engrafted in areas of healthy retina in 5 animals (1, 2, 5, 6, and 9), and at the margins of the atrophy and/or within the atrophied retina in 4 animals (1, 2, 7, and 9) (Table 1). Post-mortem analysis of animal 2 at 2 weeks following transplantation revealed hyper- autofluorescence corresponding to engrafted hiPSC-RPE cells in an area covering the healthy retina, the limiting border, and a small part of the atrophy area (Figure 5A, circle denotes the atrophic zone, and Figure 5B). Within the healthy retina, GFP-positive cells exhibited an elongated cell morphology, interacted with POS (recoverin and rhodopsin), and expressed the human nuclear antigens Ku80 and bestrophin-1. We also found isolated GFP+ cells within the atrophied area (Figure 5D). RPE cells, both in the healthy retina area and within the atrophic zone (Figure 3B), and this was also observed in animals 1, 5, 6, 7, and 9. At three months, FAF imaging detected homogeneous signals of hyper-autofluorescence in healthy areas of the retina (animals 7 and 9, Figure 4A). Cells appeared well-organized in a monolayer, localized between the Int. J. Mol. Sci. 2021, 22, 10497 RPE and photoreceptors as a new hyper-reflective layer, as visualized by SD-OCT (Figure 8 of 21 4A, e). Conversely, the engraftment of hiPSC-RPE cells in atrophic areas occurred in a much more punctual manner, and was predominantly at the atrophy border (Figure 4B). Figure 3. Representative image of FAF and SD-OCT of (A) prior to and (B) two weeks after hiPSC-RPE cell subretinal Figure 3. Representative image of FAF and SD-OCT of (A) prior to and (B) two weeks after hiPSC- injection in pig 6. (A) The day before hiPSC-RPE cell injection, no evidence of hyper-autofluorescence due to GFP expres- sion is observed on FAF, and RPEacell subretinal marked injection atrophy in pigof6.all(A) and thinning Thelayers retinal day before hiPSC-RPE is present on SD-OCT.cell (B) injection, no evidence Fifteen days of hyper-autofluorescence due to GFP expression is observed on FAF, and a after the hiPSC-RPE cell injection, several foci of hyper-autofluorescence are observed inside the area of atrophy on FAF, marked atrophy and but also an increase in retinal thickness as compared with (A). Green squares delineate scan area and green horizontal line thinning of all retinal layers is present on SD-OCT. (B) Fifteen days after the hiPSC-RPE cell injection, show scan directions andseveral level offoci rightofimage FAF, fundus autofluorescence; hyper-autofluorescence are observedGFP, green inside fluorescence the protein; area of atrophy on SD-OCT, FAF, but also an spectral-domain optic coherence tomography; RPE, retinal pigment epithelium. increase in retinal thickness as compared with (A). Green squares delineate scan area and green horizontal line show scan directions and level of right image FAF, fundus autofluorescence; GFP, Int. J. Mol. Sci. 2021, 221, 497 green fluorescence protein; SD-OCT, spectral-domain optic coherence tomography; 8 of 21 RPE, retinal pigment epithelium. Figure 4. Multimodal fundus imaging showing the presence of hiPSC-RPE cells in healthy (A) and atrophic (B) retina in Figure 4. Multimodal fundus imaging showing the presence of hiPSC-RPE cells in healthy (A) and pigs 9 and 7 respectively, at 3 months post-injection. Well-demarcated round areas by IR fundus (a) four months after NaIO3 subretinal injection.atrophic FAF (b)(B) retinahyper-autofluorescence showing in pigs 9 and 7 respectively, generatedat 3bymonths the GFPpost-injection. Well-demarcated expression in subretinal in- round areas jected hiPSC-RPE cells . Pseudo-colored images (c) from increased autofluorescence in FAF imaging using Fiji show wide- spread (A, c,d) and localized (B, c,d) hiPSC-RPE cells (in green) outside the atrophic area, but also inside the atrophy region (B, d). On FAF-guided SD-OCT, a well-organized RPE monolayer appears between native RPE and photoreceptors (A, e) as a new hyper-reflective layer of hiPSC-RPE cells in healthy retina. A granular distribution of the hiPSC-RPE cells (hyper-reflective dots) is shown in the outer retinal layers of the atrophic retina (B, e). Green squares delineate scan area Int. J. Mol. Sci. 2021, 22, 10497 9 of 21 by IR fundus (a) four months after NaIO3 subretinal injection. FAF (b) showing hyper- autofluorescence generated by the GFP expression in subretinal injected hiPSC-RPE cells. Pseudo- colored images (c) from increased autofluorescence in FAF imaging using Fiji show widespread (A, c,d) and localized (B, c,d) hiPSC-RPE cells (in green) outside the atrophic area, but also inside the atrophy region (B, d). On FAF-guided SD-OCT, a well-organized RPE monolayer appears between native RPE and photoreceptors (A, e) as a new hyper-reflective layer of hiPSC-RPE cells in healthy retina. A granular distribution of the hiPSC-RPE cells (hyper-reflective dots) is shown in the outer retinal layers of the atrophic retina (B, e). Green squares delineate scan area and green horizontal line show scan directions and level. FAF, fundus autofluorescence; GFP, green fluorescence pro- tein; NaIO3 , sodium iodate; SD-OCT, spectral-domain optic coherence tomography; RPE, retinal Int. J. Mol. Sci. 2021, 221, 497 9 of 21 pigment epithelium. Figure 5. Analysis of engrafted hiPSC-RPE cells in the host porcine retinas at two weeks post-transplantation in pig 4. (A) Figure 5. Analysis of engrafted hiPSC-RPE cells in the host porcine retinas at two weeks post-transplantation in pig 4. FAF shows hyper-fluorescence corresponding to hiPSC-RPE cells located in an area covering the healthy retina, the limit- (A) FAF shows hyper-fluorescence corresponding to hiPSC-RPE cells located in an area covering the healthy retina, the ing margin, and a small part of the atrophy (circle denotes the atrophic zone). (B) Immunohistochemical analysis shows limiting hiPSC-RPEmargin, and a small in cell integration part of the retinas healthy atrophyclose (circletodenotes the atrophic the margin zone). (B) of the atrophy Immunohistochemical stained with RPE65 and analysis recoverinshows (RE- hiPSC-RPE cell integration in healthy retinas close to the margin of the atrophy stained with RPE65 and recoverin COV) (white arrow indicates the atrophy border). Scale bar: 250 μm. (C) Confocal images show engrafted hiPSC-RPE cells (RECOV) (white (GFP inarrow indicates green) the atrophy in healthy border). with retina, stained Scalerecoverin bar: 250 µm.and(C) Confocal(RECOV rhodopsin images show and engrafted hiPSC-RPE RHO), expressing cells (GFP bestrophin-1 (BEST-1) in and green) in Ku80 (human healthy nuclear retina, stained marker). with Astrocytes recoverin show normal and rhodopsin (RECOV morphology and RHO),(GFAP). Scale expressing bars: 25 μm. bestrophin-1 (D) Immu- (BEST-1) and nofluorescence Ku80 imagesmarker). (human nuclear of hiPSC-RPE integration Astrocytes in atrophic show normal areas stained morphology withScale (GFAP). RPE65. Scale bars: bars:(D) 25 µm. 25 Immunofluorescence μm. Cell nuclei were stained of images with DAPI (blue). hiPSC-RPE FAF, fundus integration autofluorescence; in atrophic areas stained RPE, retinal with pigment RPE65. Scaleepithelium; bars: 25 µm.OS, outer Cell segments; nuclei ONL, outer were stained with nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; Chor, choroid; GFP, green fluorescent protein. DAPI (blue). FAF, fundus autofluorescence; RPE, retinal pigment epithelium; OS, outer segments; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; Chor, choroid; GFP, green fluorescent protein. Surviving hiPSC-RPE cells were found engrafted in areas of the healthy retina at 3 months in 3 animals Surviving (5, 6, and hiPSC-RPE cells9), andfound were at theengrafted margins of in the atrophy, areas or in the of the healthy atrophic retina at 3 retina, monthsinin23animals animals (9(5,and 7; Figures 6, and 9), and at6 the andmargins 7, respectively; Table 1). of the atrophy, Moreover, or in we retina, the atrophic could detect GFP-positive in 2 animals (9 and 7;cells in the6vitreous Figures of two pigs (pigs and 7, respectively; Table51). and 7; Supplementary Moreover, we could Figure detect S4B). hiPSC-RPE cells formed a well-organized epithelium-like layer GFP-positive cells in the vitreous of two pigs (pigs 5 and 7; Supplementary Figure S4B).(Figure 6B,C). His- tological hiPSC-RPE analysis confirmed cells formed normal gross a well-organized retinal morphology epithelium-like in areas layer (Figure containing 6B,C). Histologicalen- grafted analysiscells, with no confirmed neoplastic normal grossstructures (Figure 6B). retinal morphology in Surviving hiPSC-RPE areas containing cells were engrafted cells, pigmented with at levelsstructures no neoplastic similar to(Figure those of host 6B). RPE cells Surviving (asterisk in hiPSC-RPE Figure cells were6B; Figure 6E). pigmented at Transplanted levels similar tocells extended those of host close to the RPE cells limiting (asterisk in border of theTransplanted Figure 6B,E. atrophy (Figurecells6C, arrow extended indicates close to thethelimiting atrophyborder border),ofexpressed the atrophy bestrophin-1 (Figure 6C, and RPE65 arrow (Figurethe indicates 6D,atrophy a,b; Supple- bor- der), expressed bestrophin-1 and RPE65 (Figure 6D, a,b; Supplementary mentary Figure S4C), and formed both mononuclear and multinuclear layers with a Figure S4C), and formednormal nearly both mononuclear pattern (insetsandinmultinuclear layersMoreover, Figure 6C, c,d). with a nearly normal engrafted pattern (insets hiPSC-RPE cells in Figurenumerous showed 6C, c,d). Moreover, immunolabeled engrafted hiPSC-RPE phagosomes cells showed numerous (recoverin-positive immunola- or rhodopsin-posi- tive) in cross-sections, indicating a potential interaction with host photoreceptors (Figure 6D, e,e’,e’’; Figure 6E) [38]. Int. J. Mol. Sci. 2021, 22, 10497 10 of 21 Int. J. Mol. Sci. 2021, 221, 497 10 of 21 beled phagosomes (recoverin-positive or rhodopsin-positive) in cross-sections, indicating a 0 00 potential interaction with host photoreceptors (Figure 6D, e,e ,e Figure 6E) [38]. Figure 6. Analysis of engrafted hiPSC-RPE cells in healthy porcine retinas at three months post-transplantation in pigs 6 Figure 6. Analysis of engrafted hiPSC-RPE cells in healthy porcine retinas at three months post-transplantation in pigs 6 and and 9. (A) Post-fixed FAF and IR of transplanted hiPSC-RPE cells in healthy retinas near the atrophy border. Atrophy is 9. (A) Post-fixed marked FAF and with a dashed IR (B) line. of transplanted Compositions hiPSC-RPE cells inand of hematoxylin healthy eosinretinas (H&E)near the atrophy staining showing border. AtrophyhiPSC-RPE transplanted is marked with a dashed line. (B) Compositions of hematoxylin and eosin (H&E) staining showing transplanted cells (asterisk) in healthy retina. Arrow indicates the atrophy border. Scale bar: 400 μm. White dashed square indicates hiPSC-RPE cellsa (asterisk) in healthy retina. higher magnification image Arrow (rightindicates the atrophy panel) showing border. Scale hiPSC-RPE the transplanted bar: 400 µm.cell White layerdashed square (asterisk). Noteindicates a higher that green fluo- rescence couldimage magnification not be(right seen after panel) H&E staining. showing (C) Confocal composition the transplanted hiPSC-RPE cell of integrated hiPSC-RPE layer (asterisk). cell layer Note that greenin the healthy fluorescence retinanot could extending be seentowards after H&E thestaining. transition(C) zone of the atrophic Confocal composition area of in integrated pig 9 immunolabeled hiPSC-RPEwith RPE65 cell layer in and recoverin the healthy (RE- retina COV). Scale extending bars: 1000 towards μm. Inset shows the transition zone ofmagnification the atrophic areaof theinmargin of the atrophy indicated pig 9 immunolabeled with RPE65by aanddashed square. recoverin Arrows (RECOV). indicate Scale bars:atrophy border. 1000 µm. Scale bars: Inset shows 50 μm. (D) magnification of Immunohistochemical the margin of the atrophy staining of engrafted indicated hiPSC-RPE by a dashed cells inindicate square. Arrows healthy retina showing the expression of RPE-specific markers bestrophin-1 (BEST-1) and RPE65 (a,b). Retinal glia was stained atrophy border. Scale bars: 50 µm. (D) Immunohistochemical staining of engrafted hiPSC-RPE cells in healthy retina with GFAP. The human nuclear antigen Ku80 was used to detect human cells corresponding to transplanted hiPSC-RPE showing the expression of RPE-specific markers bestrophin-1 (BEST-1) and RPE65 (a,b). Retinal glia was stained with GFAP. cells (c,d, insets show magnification of Ku80+ nuclei). Photoreceptor cells were detected by rhodopsin (RHO) marker (e). The human nuclear Enlargement antigenimmunofluorescence RHO (e’,e’’) Ku80 was used to detect human images cells corresponding showing contact between tohiPSC-RPE transplanted hiPSC-RPE cells cells (c,d, insets and photoreceptor outer show magnification of Ku80+ nuclei). Photoreceptor cells were detected by rhodopsin segments revealing numerous immunolabeled phagosomes (white arrowheads). Dashed lines indicate the GFP+ hiPSC- (RHO) marker (e). Enlargement RHO (e 0 ,e00 ) immunofluorescence images showing contact between hiPSC-RPE cells and photoreceptor outer segments revealing RPE layer. Scale bars: 25 μm. (E) Bright-field images of control and transplanted retinas (left panels). Immunohistochem- ical staining numerous of control retina immunolabeled and transplanted phagosomes retina with Dashed (white arrowheads). engrafted hiPSC-RPE lines indicate the cells showing GFP+ the expression hiPSC-RPE of bars: layer. Scale RPE- specific 25 µm. (E) marker RPE65images Bright-field and photoreceptor of control andmarker recoverin transplanted (RECOV) retinas (right panels). (left panels). Scale bars: 25 μm. Immunohistochemical Cell nuclei staining were of control stained with DAPI (blue). FAF, fundus autofluorescence; IR, infrared image; RPE, retinal retina and transplanted retina with engrafted hiPSC-RPE cells showing the expression of RPE-specific marker RPE65 andpigment epithelium; ONL, outer nuclear layer; marker photoreceptor INL, inner nuclear recoverin layer; GCL, (RECOV) ganglion (right panels).cell layer; Scale Chor, bars: 25 µm.choroid; GFP, were Cell nuclei greenstained fluorescent with protein. DAPI (blue). FAF, fundus autofluorescence; IR, infrared image; RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear FAF imaging of pig 7 revealed several hiPSC-RPE cells engrafted in a punctuated layer; GCL, ganglion cell layer; Chor, choroid; GFP, green fluorescent protein. manner within the atrophic zone, accumulating in the vicinity of the border with healthy retina (Figure 7A, arrowheads). Immunohistochemical analysis of cryosections revealed the discrete integration of transplanted cells in the RPE layer along the atrophy and in the margins (Figure 7B,C, a). We also observed hiPSC-RPE cells engrafted within the remain- ing INL (Figure 7C, b–d), although in some areas, hiPSC-RPE cells formed epithelial-like structures (Figure 7C, b). Int. J. Mol. Sci. 2021, 22, 10497 11 of 21 Int. J. Mol. Sci. 2021, 221, 497 11 of 21 Figure7.7. Analysis Figure AnalysisofofhiPSC-RPE hiPSC-RPEcell cellengraftment engraftmentininatrophic atrophicporcine porcineretinas retinasininpigs pigs77and and1.1.(A) (A)In In vivo vivo FAF FAF corresponding corresponding to the atrophic area transplanted with hiPSC-RPE cells in pig 7 at three months post-transplantation shows punctual inte- to the atrophic area transplanted with hiPSC-RPE cells in pig 7 at three months post-transplantation shows punctual gration of cells that are more prominent in the atrophic border (white arrowheads). Scale bar: 600 μm. (Green horizontal integration of cells that are more prominent in the atrophic border (white arrowheads). Scale bar: 600 µm. (Green horizontal line show OCT scan level, not shown)(B) Immunostaining composition of the atrophic area stained with RPE65 and re- line show coverin OCT scan (RECOV) level, not punctual corroborates shown)(B) Immunostaining engraftment composition of hiPSC-RPE of the atrophic cells. Arrows area stained indicate atrophy withScale borders. RPE65 bar:and 250 recoverin (RECOV) μm. (C) Detailed corroborates confocal imagespunctual engraftment of hiPSC-RPE of hiPSC-RPE cells engrafted cells. Arrows in the atrophy marginindicate atrophy (a) located in theborders. RPE layer Scale bar: (RPE65) 250 µm. (C)with interacting Detailed confocal images photoreceptors of hiPSC-RPE (RECOV), and in the cells engrafted atrophy in thethe (b–e) within atrophy margin RPE layer (a) located (RPE65) and theinremaining the RPE layerINL. (RPE65) interacting Transplanted with photoreceptors hiPSC-RPE expressed human (RECOV), and nuclei in the atrophy marked with Ku80(b–e)(d).within the RPE Astrocyte layer (RPE65) is disorganization and the remaining shown by GFAP (e,f) and INL. the lack ofhiPSC-RPE Transplanted photoreceptor by RECOV expressed (c) nuclei human and rhodopsin marked (RHO) with Ku80 (e). Scale bars: 25 μm. (d). Astrocyte At six months, disorganization is few hiPSC- shown by RPE cells integrated in the RPE layer (RPE65) within the atrophic area (f). Scale bars: 50 μm. Cell GFAP (e,f) and the lack of photoreceptor by RECOV (c) and rhodopsin (RHO) (e). Scale bars: 25 µm. At six months, fewnuclei were stained with DAPI (blue). hiPSC-RPE FAF, cells fundus autofluorescence; integrated RPE, retinal in the RPE layer (RPE65) withinpigment epithelium; the atrophic area (f).ONL, Scaleouter bars:nuclear 50 µm. layer; INL, inner Cell nuclei nuclear were stained layer; GCL, ganglion cell layer; Chor, choroid; GFP, green fluorescent protein. with DAPI (blue). FAF, fundus autofluorescence; RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; Chor, choroid; GFP, green fluorescent protein. 3. Discussion FAF Here,imaging we usedofa pig 7 revealed miniature severalofhiPSC-RPE pig model cells engrafted GA that resembles in a punctuated some advanced atrophic manner form of within humanthe AMD atrophic zone, features to accumulating in the vicinity study the subretinal of the border hiPSC-derived with RPE cell healthy transplan- retina tation (Figure 7A, arrowheads). as a potential therapy to treatImmunohistochemical GA. Our main objective analysis wasoftocryosections revealed assess the approach’s the discrete and feasibility, integration of transplanted the safety, survival, and cells in the RPE potential layertoalong of cells the atrophy integrate within and in the the tissue, margins (Figure 7B,C, a). We also observed hiPSC-RPE cells engrafted particularly at the margins of the localized well-defined GA lesions. Much progress has within the remaining INL been(Figure achieved 7C, in b–d), although the treatment ofin thesome areas,form exudative hiPSC-RPE of AMD,cells which formed was onceepithelial-like considered structures (Figure 7C, to be a devastating b). condition. However, there is no current treatment for GA, the atrophic form of AMD, and several recent trials have failed to demonstrate efficacy against this 3. Discussion progressing blinding condition [39]. RPE cell degeneration and death is prevalent in GA and,Here, within wea used few ayears, miniature leads pig model to very of GAimpairment severe that resembles some advanced of visual function and atrophic legal form of human AMD features to study the subretinal hiPSC-derived blindness [40]. Attempts to regenerate or rescue already dead tissue appear to be a chal- RPE cell transplanta- tion lengeasbecause a potential therapy of the to treat extremely GA. Our main sophisticated objective was tridimensional to assess network the approach’s of synapses of each feasibility, and the safety, survival, and potential of cells to integrate retinal cell to numerous different types of surrounding cells. Results of cell transplantationwithin the tissue, particularly studies showathighthe margins variabilityof of thecell localized survival well-defined and a lack of GA lesions. Much integration progress in atrophic has retinas, been achieved in the treatment of the exudative form of AMD, which which might be caused by factors that trigger graft rejection, either by autoimmune re- was once considered to be a devastating sponse [41], acquired condition. immuneHowever, responsethere is no [35], or by current treatment environmental for GA, factors the atrophic associated with form of AMD, and several recent trials have failed to demonstrate the degenerative retinal state. Moreover, damage to RPE and BM layers implies efficacy against this a disrup- progressing blinding condition [39]. RPE cell degeneration and death tion of the retinal-blood barrier, which is potentially more susceptible to inflammatory is prevalent in GA and, within a few years, leads to very severe impairment of visual function response [42]. More importantly, perhaps, photoreceptor loss initiates a cascade of nega- and legal blind- ness [40]. Attempts to regenerate or rescue already dead tissue appear to be a challenge tive neuronal remodeling of the retina and cell death, in addition to reactive gliosis of the because of the extremely sophisticated tridimensional network of synapses of each retinal astrocytes and Müller cells and the formation of scar tissue [43]. Accordingly, one of the cell to numerous different types of surrounding cells. Results of cell transplantation studies theoretical aims of stem cell-based therapy with pluripotent stem cell-derived retinal show high variability of cell survival and a lack of integration in atrophic retinas, which cells—integrating into and rewiring host cells—might be a challenging endeavor. Another might be caused by factors that trigger graft rejection, either by autoimmune response [41], potential beneficial mechanism would rely on neurotrophic factor secretion without ac- acquired immune response [35], or by environmental factors associated with the degener- tual stem-cell integration into the host tissue. A third potential mechanism of benefit ative retinal state. Moreover, damage to RPE and BM layers implies a disruption of the would be stem cell survival and limited integration into the host tissue without true Int. J. Mol. Sci. 2021, 22, 10497 12 of 21 retinal-blood barrier, which is potentially more susceptible to inflammatory response [42]. More importantly, perhaps, photoreceptor loss initiates a cascade of negative neuronal remodeling of the retina and cell death, in addition to reactive gliosis of the astrocytes and Müller cells and the formation of scar tissue [43]. Accordingly, one of the theoretical aims of stem cell-based therapy with pluripotent stem cell-derived retinal cells—integrating into and rewiring host cells—might be a challenging endeavor. Another potential beneficial mechanism would rely on neurotrophic factor secretion without actual stem-cell integration into the host tissue. A third potential mechanism of benefit would be stem cell survival and limited integration into the host tissue without true rewiring, which could assist the overwhelmed host cells with their housekeeping functions. In any case, regenerative effects appear to be highly unlikely within areas of long-standing well-established atrophy due to the aforementioned reasons, especially due to neuronal remodeling. Therefore, the interest is focused on exploring if regenerative processes may still occur at the margins of the atrophy, where the retinal network and microenvironment are partially preserved. AMD is characterized by extracellular deposits, and a hallmark of the disease is the presence of different types of drusen [44,45], which have been demonstrated to be toxic for RPE cells [46]. In addition, it is known that cell-to-cell transmission of pathogenic proteins plays a role in neurodegenerative diseases [47]. In a similar line, a decline in the endo-lysosomal function within the RPE may lead to functional deficits and an increase of intracellular debris such as lipofuscin [48,49], which might overwhelm the capacity of RPE to remove metabolic debris from the visual cycle, ultimately leading to RPE cell death and atrophy [50,51]. Therefore, we hypothesize that RPE cells injected around and at the boundaries of the atrophy, where there are still viable cells, might play a role in assisting with housekeeping functions of the overwhelmed host RPE cells. This would not need the complex integration with photoreceptors that true regeneration would require, but only survival and a certain degree of integration to aid host RPE cells in removing the debris and deposits occurring in the setting of AMD. This might potentially help the host RPE cells to survive longer. The present study was designed with this rationale in mind; specifically, to evaluate whether hiPSC-RPE cells could survive and (partially) integrate into the retina of the margins of the atrophic areas. The lack of pre-clinical studies on hiPSC-based therapies in swine models of end- stage AMD raised the question of whether hiPSC-RPE cells’ suspension could integrate into porcine eyes, either in the normal retina or in the induced GA. There are various techniques to induce GA models, such as laser-high-pressure or light-induced injuries, retinal scraping, or iodoacetic acid toxicity [25–28,32]. NaIO3 -induced localized selective outer retinal damage in the porcine retina mimics that feature of human GA and represents a reproducible model that can be maintained over time [25], and also has utility to test the behavior of injected stem cell-derived RPE cells in the boundaries of the atrophic retina. FAF imaging was found to be a useful tool to detect the hiPSC-RPE cells in retinas. Although lipofuscin accumulates with age [52], the animals used in this study were too young to have an accumulation of this pigment, and FAF imaging was able to capture the hyper-autofluorescence emitted by GFP from the injected hiPSC-RPE cells, surrounded by the dark fundus of the retina. Several studies have reported attempts at pre-clinical RPE cell therapy in the pig model, with experimental designs varying with regards to the cell delivery method em- ployed (single cells [35] or sheets [33,34,53,54]), the species origin (swine [31,35,54] or human [27,33,34,53]), and the RPE cell source (pluripotent stem cells [33–35,53], adult [54], or fetal [27,31] RPE cells). To our knowledge, the present study is the first one to employ single-cell injection of human iPSC-derived RPE cells in pigs. Upon subretinal transplanta- tion, the capacity of grafted cells to functionally integrate into the host retina relies on their survival and migratory capacity [55]. In line with this, our results show that transplanted RPE cells achieved high survival rates and organized forming an RPE monolayer in the presence of a healthy environment, such as in regions of the healthy retina and atrophy border. Compared to RPE sheets on scaffolds, we observed that the administration of cell Int. J. Mol. Sci. 2021, 22, 10497 13 of 21 suspension into the subretinal space of the eye offers advantages: (i) it is technically easier and less traumatic than the transplantation of RPE sheets or scaffolds, (ii) it reduces the chance to trigger an immune response or inflammation due to the presence of scaffolds’ products, (iii) it allows the injection of an accurate cell number in a localized area, and (iv) it enables better isolation, characterization, and cryopreservation, as well as quality control of the transplantable cell type. However, in atrophic areas where the environment is hostile and the rate of transplanted cell survival decreased, the transplantation of RPE sheets may increase their survival. As expected, hiPSC-RPE cells failed to engraft properly as an epithelial-like structure within atrophic areas, and only three animals showed some isolated surviving cells. Al- though our animals were under immunosuppression, we cannot exclude the possibility that the combination of severe disruption of the RPE/BM layers together with the presence of xenogenic cells caused an immune response and graft rejection observed in some eyes, which is similar to the rejection observed by Sohn et al. [35]. Another possibility is the implication of choriocapillaris and the vascular endothelial growth factor (VEGF) in this model of GA, similar to the neovascular form of human AMD. Future studies on the VEGF levels in the healthy retina and in the GA before and after iPSC in our mode would be interesting to elucidate its role, if any, in the advanced stage of the dry form of AMD. The RPE cells generated from hiPSC in this study have previously been shown to have specific RPE functions in vitro, such as phagocytosis of photoreceptor outer segments, ion transport, polarized factor secretion, membrane potential, and gene expression, similar to their native counterparts [56,57]. Extending our previous work on differentiation of hESC and hiPSC to RPE cells [58–60], here, we have further characterized differentiated RPE cells from human cord blood-derived iPSC, and shown that they exhibit cell properties, in vitro functions, and gene expression similar to native RPE cells. Our RPE confluent cultures expressed apical tight junctions and reached a TEER of >200 Ω·cm2 , which is similar to that seen in vivo [56], suggesting a strong polarization and good tight junction integrity, important for their barrier function. In the eye, RPE cells interact continuously with photoreceptor light-sensitive outer segments and are responsible for the phagocytosis of shed distal outer segments—a daily process that is essential for the renewal and survival of photoreceptors [61]. We showed here that hiPSC-RPE cells have the capacity to phagocytize POS in vitro. This is important to consider for the optimization of protocol conditions and the establishment of specific characteristics to ensure the transplantation of functional cells. We found that hiPSC-RPE cells indeed engrafted to some extent in the host healthy retina near the border of the atrophy in five animals and were able to form a mature quiescent long-term extensive epithelial-like layer in the healthy porcine retina in some animals. More importantly, several engrafted hiPSC-RPE cells showed numerous immunolabeled phagosomes, suggesting a potential interaction with host photoreceptors. This would support the rationale of using hiPSC-RPE injection as a regenerative therapy with the intention of assisting native RPE in decreasing the rate of atrophy progression. Prior studies of transplanted hESC-RPE cells in a rabbit model of GA showed reduced pigmentation that was recovered over time [62,63]. In line with this, we confirmed that although the hiPSC-RPE cells used here acquired pigmentation in vitro, in the short-term integration period in porcine retinas, the cells lost their morphology and pigmentation according to their proliferative state, resembling the in vitro re-maturation process after cell expansion. However, at three months post-transplantation, RPE cell pigmentation and functional layer formation were re-established. From a safety perspective, iPSC-based therapies must overcome the clinical hurdle of tumorigenicity, associated with the transplantation of undifferentiated cells or progenitors with increased genomic instability. In this regard, we first assessed the cell integrity and purity of our hiPSC-RPE cultures to exclude undifferentiated cells using a protocol of cell sorting and purification by extensive culture and passaging. Upon transplantation, there was no evidence of neoplastic or teratoma structures or any other adverse event in any Int. J. Mol. Sci. 2021, 22, 10497 14 of 21 of the transplanted porcine eyes studied, in accordance with other studies in different animal models and in humans [12,58,59,64]. Furthermore, there was no evidence of en- dophthalmitis, retinal edema, uveitis, epiretinal membrane, proliferative vitreoretinopathy, encapsulation, or retinal rupture in the immunosuppressed animals. One animal devel- oped a retinal detachment due to persistence and enlargement of the bleb. Therefore, the current implantation subretinal transplant surgical technique appears to be safe and reproducible. Despite not performing vitrectomy and posterior hyaloid removal, epiretinal membrane formation was not an issue, which is usually the main complication of this type of subretinal transplantation. Several human phase I/II clinical trials using RPE derived from hESC are in progress in patients under immunosuppression (e.g., NCT01344993). However, one important advantage of using hiPSC-derived cells would be the possibility to create HLA-haplotype banking of hiPSC (i.e., from umbilical cord banks) to cover most of the HLA types of the population [65], which would circumvent the need for immunosuppression in the elderly patients and would favor long-term transplanted cell survival. This might be a key advantage of using iPSC over hESC for the clinical translation of this therapy to a large population of patients. However, several strengths and limitations of the present study warrant further discussion. The major limitations of this study were the lack of a true animal model of AMD and the impossibility of quantitatively measuring the magnitude of the lesion induced with the NaIO3 . Despite these obstacles, this model offers a quite selective outer retinal damage within a lesion with well-demarcated distinct boundaries, representing an optimal model to evaluate the feasibility of stem cell engrafting at the margins of the lesion. Our present study was also limited by the low number of animals used and the relatively short follow-up time. Testing more animals and over longer time periods will be necessary to evaluate the efficacy of the procedure, as will be including a control group that does not receive a RPE cell injection. These limitations notwithstanding, we believe that the results of our observational study clearly support the feasibility of hiPSC-derived RPE cell transplantation in pigs and set the foundation for future preclinical trials addressing efficacy. In summary, we showed that subretinal NaIO3 is a robust model mimicking some of the features of human GA, and seems to be appropriate to test stem cell-based transplanta- tion. Subretinal injection of hiPSC-RPE cell suspensions is a feasible approach with a high rate of long-term cell survival in areas of a healthy retina, at the borders of the atrophy. Additionally, we provided findings suggestive of iPSC-derived RPE integration, including phagosomes from retinal phagocytosis, which might underscore the use of these RPE cells to treat atrophic AMD in a supportive housekeeping role by improving the survival of overwhelmed RPE cells and photoreceptors at the boundaries of the atrophy, decreasing the rate of progression of the GA, and consequently delaying the onset of loss of visual function. Human phase I/II studies are needed as a next step to assess the safety and efficacy of these cells on visual function deterioration or lesion size progression in patients with GA secondary to AMD. 4. Materials and Methods 4.1. Cell Culture Ethics review board and competent authority approval were obtained for this study (472 380 1; 12 July 2019). An hiPSC line derived from cord blood CD133+ cells, CBiPS30-4F- 5 [66], was obtained from the Spanish Stem Cell Bank. hiPSC were maintained and expanded in mTeSR1 medium (Stem Cell Technologies, Vancouver, BC, Canada) on MatrigelTM (Corning, NY, USA)-coated plates at 37 ◦ C with 5% (v/v) CO2 . 4.2. Generation of hiPSC Expressing GFP and Their Differentiation to RPE Cells To facilitate the detection of RPE cells in the host retina, we first generated an hiPSC line constitutively expressing green fluorescent protein (GFP) by lentiviral transduction (Supplementary Materials and Methods). The hiPSC-GFP line was differentiated to RPE Int. J. Mol. Sci. 2021, 22, 10497 15 of 21 cells as previously described [58–60]. The hiPSC-GFP line was expanded on Matrigel- coated 10 cm plates in mTeSR1 medium until the colonies reached 60% confluence. Then, the medium was replaced with RPE medium (DMEM/F12 (Gibco, supplemented with 5% Knockout Serum Replacement (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 1% N2 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 2% B27 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 0.1 µM dexamethasone (Sigma Aldrich, St. Louis, MO, USA), 10 mM β-glycerolphosphate (Sigma Aldrich, St. Louis, MO, USA), 20 ng/mL human IGF-1 (R&D Systems, Minneapolis, MN, USA), and 10 mM nicotinamide (Sigma Aldrich, St. Louis, MO, USA) and changed every 2–3 days for 6–8 weeks. Pigmented areas were manually picked, dissected with 0.25% trypsin, and re-plated on Matrigel-coated plates. After 3–4 weeks of differentiation, when cells reached confluence, they were purified by incubation with 0.05% trypsin for 2 min to remove fibroblast-like cells. The remaining cells were disaggregated in TrypLE Select (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), re-seeded on Matrigel, and maintained on RPE medium. Single hiPSC-derived RPE (hiPSC-RPE) cells were sorted on a Moflo XDP cell sorter (Beckman Coulter, Brea, CA, USA), and only GFP+ cells were expanded for injection (Supplementary Figure S1C,D). hiPSC-RPE cells were frozen and stored for later use at passages 2–4. 4.3. Immunocytochemistry hiPSC-RPE cells were fixed in 4% paraformaldehyde for 20 min at room temperature, permeabilized, and blocked with tris-buffered saline (TBS) containing 0.5% (v/v) Triton X-100 and 6% (v/v) normal donkey serum (Sigma-Aldrich; St. Louis, MO, USA) for 1 h at room temperature. Primary antibodies were incubated at 4 ◦ C overnight in phosphate- buffered saline (PBS), 0.1% (v/v) Triton X-100, and 6% (v/v) normal donkey serum, and secondary antibodies (1:500; Jackson Immunoresearch Laboratories, West Grove, PA, USA) were incubated in the same buffer at 37 ◦ C for 2 h. Nuclei were stained with 40 ,6-diamidino- 2-phenylindole (DAPI) (Sigma-Aldrich; St. Louis, MO, USA) and cells were mounted in an anti-fading mounting medium. Confocal images were obtained on a TCS SP5 confocal microscopy (Leica Microsystems, Wetzlar, Germany) using a 40× or 63× oil immersion objective, and z-stacks were acquired at 1024 × 1024 pixels. Antibodies are listed in Supplementary Table S1. 4.4. Messenger RNA Expression by Quantitative Reverse-Transcriptase Polymerase Chain Reaction Total RNA from hiPSC-RPE cells was isolated using TRIzol reagent (Sigma Aldrich, St. Louis, MO, USA), and cDNA was synthesized using the Cloned AMV First-Strand Synthesis Kit (both from Invitrogen, Carlsbad, CA, USA) [67]. Gene expression levels were analyzed by qRT-PCR (qPCR) reaction using SYBR green chemistry (Life Technologies, Carlsbad, CA, USA) and run on the ABI PRISM 7900HT platform (Applied Biosystems, Foster City, CA, USA) by a standard two-step amplification program: hot start activation (95 ◦ C for 5 min), denaturing step (95 ◦ C for 15 s), and annealing/extension step (60 ◦ C for 30 s), repeated for 40 cycles. Primer sequences are listed in Supplementary Table S2. Threshold cycle (Ct) values were normalized to those of the housekeeping gene GAPDH and relative to hiPSC genes, and were expressed as 2-∆∆Ct (log scale). 4.5. Transepithelial Electrical Resistance Measurements hiPSC-RPE cells were cultured on Matrigel-coated Transwell filter inserts (Millipore, Burlington, MA, USA) in RPE medium until the complete formation of the epithelial monolayer. Electrical measurements were performed every 3 days for 3 weeks in biological triplicates using an epithelial voltohmmeter (EVOM; World Precision Instruments Inc., Sarasota, FL, USA). Transepithelial electrical resistance (TEER) values were corrected by subtracting values of a blank and multiplied by the cell growth area. Values were presented in Ω·cm2 . Int. J. Mol. Sci. 2021, 22, 10497 16 of 21 4.6. Electron Microscopy For transmission electron microscopy (TEM), hiPSC-RPE cells were cultured on Matrigel-coated Transwell inserts and fixed with 2.5% (w/v) glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2–7.4, for 2 h at 4 ◦ C. Post-fixation was carried out in 1% (w/v) osmium tetroxide for 2 h at 4 ◦ C, and samples were then dehydrated with an ascending ethanol series and embedded in epoxy resin. Ultrathin sections were examined in a Jeol 1011 transmission electron microscope (Jeol, Tokyo, Japan). For scanning electron microscopy (SEM), RPE cells were cultured and fixed as for TEM. Fixed cells were dehydrated with an ascending ethanol series, dried in a critical point dryer, and metalized, and then examined in a Jeol JSM-6390LV scanning electron microscope (Jeol, Tokyo, Japan). 4.7. Photoreceptor Outer Segment Phagocytosis Assays hiPSC-RPE phagocytosis activity was assessed by analyzing the binding and internal- ization of photoreceptor outer segments (POS) labeled with tetramethylrhodamine isoth- iocyanate (TRITC; Sigma-Aldrich Corp.St Louis, MO, USA), as described previously [68]. Unlabeled bovine POS were obtained from Fondation Voir et Entendre (Paris, France). POS were incubated with TRITC in labeling buffer containing 10% sucrose, 20 mM disodium phosphate (Sigma Aldrich, St. Louis, MO, USA), pH 7.2, and 5 mM taurine (Sigma Aldrich, St. Louis, MO, USA), and rotated for 1 h in the dark. Subsequently, POS-TRITC were washed twice with labeling buffer and once with DMEM/F12 medium, and were then resuspended in DMEM/F12. hiPSC-RPE cells were incubated with POS-TRITC for 4 h at 37 ◦ C with 5% (v/v) CO2 in RPE medium, washed four times in PBS with Ca2+ and Mg2+, and then fixed in 4% (w/v) paraformaldehyde for 20 min at room temperature. Confocal images were taken with a TCS SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany). 4.8. Animals, Preoperative Preparation, and Study Design Experiments in pigs were approved by the Institutional Ethics Committee for Animal Experimentation and were carried out in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Ten healthy female miniature (mini) pigs (Sus scrofa domesticus) (provided by Specific Pig S.L., Barcelona, Spain) aged 7–8 months and 20–30 kg body weight were used in the study (preoperative preparation and medication are described in Supplementary Materials and Methods). The present study is a dose- escalating exploratory pre-clinical study to assess the feasibility of RPE cell injection. The nature of this study is observational and, to maximize the variables explored, we used 2 doses of NaIO3 and 2 concentrations of cells. Histology and immunostaining were performed and analyzed by researchers blinded to the sample experimental condition. 4.9. NaIO3 Subretinal Injection To generate a pig model with features resembling some of those more characteristic of GA (damage of outer retinal layers with preservation of the inner layers, and lesions with well-defined distinct margins between the atrophic area and the healthy area), the proce- dure was based on a previous report that had defined the optimal NaIO3 concentration [25]. Degeneration of outer retinal layers was induced in the right eye of 10 minipigs through localized subretinal injection of NaIO3 . To observe the behavior of the cells under different conditions of atrophy, we used three different NaIO3 concentrations: 0.01 mg/mL in pigs 1–7, and 0.1 mg/mL in pigs 8–10 (Table 1). Animals were placed in lateral recumbence with the snout slightly turned. The right eyes and the periocular skin were cleaned with povidone iodine solution diluted 1:10 in saline serum. Two blepharostats were placed to maximize the operating field and to avoid canthotomy, if possible. Two 23G valve trocars were inserted 1–1.5 mm posterior to the limbus, one for the 23G-endoilluminator and the other for the 25/41G-subretinal injection cannula connected to the ACCURUS® Surgical System through an extension tube and a viscous fluid control system (all apparatus from Int. J. Mol. Sci. 2021, 22, 10497 17 of 21 Alcon Cusí S.A., Barcelona, Spain). No infusion canula was used. No vitrectomy was performed. Atrophy was induced by subretinal injection of 0.1–0.2 mL NaIO3 between the arcades using a subretinal injection cannula (25/41G DeJuan; Bausch + Lomb S.A., Madrid, Spain) near and superior to the optic nerve head. Trocars were removed without sutures of the self-sealing sclerotomies, and the eye pressure was checked. 4.10. Subretinal Injection of hiPSC-RPE Single Cell Suspension hiPSC-RPE subretinal transplantation was performed 30 days after the lesion induction with NaIO3 , when outer retinal atrophy was well-established. The cultured hiPSC-RPE cells were disaggregated with TrypLE Express ( Invitrogen, Carlsbad, CA, USA), washed 3 times in PBS, and resuspended in DMEM/F12 medium. A subretinal bleb of 0.1–0.2 mL with a suspension of hiPSC-RPE cells containing 2.5 × 105 or 3.3 × 105 cells (Table 1) was created with an injection cannula (25/41G DeJuan; Bausch + Lomb). Retinotomy and bleb induction were performed in the healthy margins and within areas of the degenerative retina, such that it was also covered with cells. Cell survival after passing through the cannula was tested in vitro by flow cytometry using Propidium Iodide Cell survival rates that were similar to those of noninjected cells (Supplementary Figure S1E,F). In addition, 7 days before the cell transplantation and until euthanasia, animals were immunosuppressed by a daily oral administration of cyclosporine (Sandimmun Neoral, 15 mg/kg; Novartis Farmacéutica S.A., Basel, Switzerland) and prednisone (Prednisone Kern Pharma, 1 mg/kg; Kern Pharma S.L., Barcelona, Spain). 4.11. Postoperative Follow-Up All animals were periodically assessed with a complete ophthalmic examination, summarized in Table 1. Prior to hiPSC-RPE cell subretinal injection, all animals were evaluated to assess the atrophy and rule out complications. After subretinal injections, animals 1 and 3–9 were evaluated at days 30, 60, and 90. Animal 2 died two weeks after cell injection for reasons unrelated to the study, but the eyes were successfully conserved. Animal 10 developed a retinal detachment due to a persistent enlargement of the bleb and was euthanized. Animal 1 was also analyzed in vivo at four, five, and six months to assess long-term cell survival. Animals 5–7 were further evaluated at day fifteen after hiPSC-RPE subretinal injection to discard inflammation or immunological response in the first two weeks. In all cases, the ophthalmological examinations were performed under sedation and the untreated contralateral eyes were considered as controls. Assessment of the retinas was carried out with infrared (IR), fundus autofluorescence (FAF; excitation filter at 488 nm; barrier >500 nm), and spectral-domain optical coherence tomography (SD-OCT) imaging using the Spectralis HRA+OCT® device (Heidelberg Engineering, Heidelberg, Germany). All SD-OCT exams were performed with tracking activated from baseline if possible. Euthanasia was carried out under sedation and by applying an intravenous sodium pentobarbital overdose at 80 mg/kg. 4.12. Histology and Immunohistochemistry Porcine eyes were enucleated, fixed in 4% (w/v) paraformaldehyde, and processed as described [25]. Tissue-Tek optimal cutting temperature compound (Sakura Finetek, Torrance, CA, USA -embedded retinal cross-sections (20 µm) were analyzed by hematoxylin and eosin (H&E) staining following a standard protocol. Images were taken with a Leica DM6000 microscope (Leica Microsystems, Wetzlar, Germany). Prior to the immunohistochemistry, porcine retinal transverse cryosections were incu- bated with 0.1 M citrate buffer (pH 6.0) for 1 h at 60 ◦ C to expose antigens. Immunohisto- chemistry of cryosections was performed as described above in the immunocytochemistry section. Int. J. Mol. Sci. 2021, 22, 10497 18 of 21 4.13. Image Processing Longitudinal analysis of fundus images was performed by aligning IR and FAF images to create overlapped images using FIJI software (ImageJ software; NIH, Bethesda, MD, USA). Images were processed using LAS AF software (Leica Microsystems, Wetzlar, Germany), FIJI software, and/or Photoshop (Adobe Systems Inc., San Jose, CA, USA). Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/ijms221910497/s1. Author Contributions: Conceptualization, J.M., E.F., Á.R. and A.V.; methodology, A.D., E.R.-B., G.M.-N., M.B., M.G., L.L.F., B.K., E.I., E.A.-X. and B.A.; validation, A.D., M.B., M.G., L.L.F., E.F., A.V. and J.M.; formal analysis, A.D., E.R.-B., M.B. and J.M.; investigation, A.D., E.R.-B., G.M.-N., M.B., M.G., L.L.F., B.K. and J.M.; writing—original draft preparation, A.D., E.R.-B., M.B. and J.M.; writing— review and editing, A.D., R.P.C.-M., E.F., Á.R., A.V., and J.M.; visualization, A.D., E.R.-B., M.B. and G.M.-N.; supervision, A.V. and J.M.; project administration, A.V. and J.M.; funding acquisition, R.P.C.-M., E.T., Á.R., A.V. and J.M. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by Spanish Ministry of Science and Innovation (MICINN, RTI2018-095377-B-100), Instituto de Salud Carlos III ISCIII/FEDER (PRB2 PT13/0001/0041; TerCel RD16/0011/0024 and Oftared-RETICS RD16/0008), ERA-NET EuroNanoMed III/ISCIII (AC19/00080), the Catalan Government, AGAUR (2017-SGR-899), CERCA Programme/Generalitat de Catalunya, and by Fundació Carmen i Mª José Godó (Fundació CMJ Godó 2017). Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, approved by the Institutional Ethics Committee for Animal Experimentation of Specific Pig S.L (P16026; 22 September 2016), and was carried out in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available in Supplementary Materials. Acknowledgments: We thank Lola Mulero for technical assistance (Histology Unit, IDIBELL). Conflicts of Interest: Jordi Monés is a Scientific consultant for Cellcure, Reneuron, Novartis, Roche, Bayer Allegro, and Kodiek. Grants from Novartis, Bayer, Roche, Thrombogenics, and Iveric. Stock options from Iveric and Notalvision. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. The authors declare no conflict of interest. References 1. Colijn, J.M.; Buitendijk, G.H.; Prokofyeva, E.; Alves, D.; Cachulo, M.L.; Khawaja, A.P.; Cougnard-Gregoire, A.; Merle, B.M.; Korb, C.; Erke, M.G.; et al. Prevalence of Age-Related Macular De-generation in Europe: The Past and the Future. Ophthalmology 2017, 124, 1753–1763. [CrossRef] 2. Chirco, K.R.; Sohn, E.; Stone, E.M.; Tucker, B.; Mullins, R.F. Structural and molecular changes in the aging choroid: Implications for age-related macular degeneration. Eye 2016, 31, 10–25. [CrossRef] [PubMed] 3. Chen, Y.; Bedell, M.; Zhang, K. Age-related Macular Degeneration: Genetic and Environmental Factors of Disease. Mol. Interv. 2010, 10, 271–281. [CrossRef] 4. Bracha, P.; Moore, N.A.; Ciulla, T. Induced pluripotent stem cell-based therapy for age-related macular degeneration. Expert Opin. Biol. Ther. 2017, 17, 1113–1126. [CrossRef] [PubMed] 5. Lewis, P.M.; Ayton, L.N.; Guymer, R.H.; Lowery, A.J.; Blamey, P.J.; Allen, P.J.; Luu, C.D.; Rosenfeld, J.V. Advances in implantable bionic devices for blindness: A review. ANZ J. Surg. 2016, 86, 654–659. [CrossRef] [PubMed] 6. Humayun, M.S.; Weiland, J.; Fujii, G.Y.; Greenberg, R.; Williamson, R.; Little, J.; Mech, B.; Cimmarusti, V.; Van Boemel, G.; Dagnelie, G.; et al. Visual perception in a blind subject with a chronic microelectronic retinal prosthesis. Vis. Res. 2003, 43, 2573–2581. [CrossRef] 7. Borooah, S.; Phillips, M.; Bilican, B.; Wright, A.; Wilmut, I.; Chandran, S.; Gamm, D.; Dhillon, B. Using human induced pluripotent stem cells to treat retinal disease. Prog. Retin. Eye Res. 2013, 37, 163–181. [CrossRef] 8. Jones, M.K.; Lu, B.; Girman, S.; Wang, S. Cell-based therapeutic strategies for replacement and preservation in retinal degenerative diseases. Prog. Retin. Eye Res. 2017, 58, 1–27. [CrossRef] Int. J. Mol. Sci. 2021, 22, 10497 19 of 21 9. Chichagova, V.; Hallam, D.; Collin, J.; Zerti, D.; Dorgau, B.; Felemban, M.; Lako, M.; Steel, D.H. Cellular regeneration strategies for macular degen-eration: Past, present and future. Eye 2018, 32, 946–971. [CrossRef] 10. Higuchi, A.; Kumar, S.S.; Benelli, G.; Alarfaj, A.A.; Munusamy, M.A.; Umezawa, A.; Murugan, K. Stem Cell Therapies for Reversing Vision Loss. Trends Biotechnol. 2017, 35, 1102–1117. [CrossRef] 11. Zarbin, M. Cell-Based Therapy for Degenerative Retinal Disease. Trends Mol. Med. 2016, 22, 115–134. [CrossRef] [PubMed] 12. Mandai, M.; Watanabe, A.; Kurimoto, Y.; Hirami, Y.; Morinaga, C.; Daimon, T.; Fujihara, M.; Akimaru, H.; Sakai, N.; Shibata, Y.; et al. Autologous Induced Stem-Cell-Derived Reti-nal Cells for Macular Degeneration. N. Engl. J. Med. 2017, 376, 1038–1046. [CrossRef] 13. Schwartz, S.D.; Regillo, C.D.; Lam, B.L.; Eliott, D.; Rosenfeld, P.J.; Gregori, N.Z.; Hubschman, J.P.; Davis, J.L.; Heilwell, G.; Spirn, M.; et al. Human embryonic stem cell-derived retinal pig-ment epithelium in patients with age-related macular degeneration and Stargardt’s macular dystrophy: Follow-up of two open-label phase 1/2 studies. Lancet 2015, 385, 509–516. [CrossRef] 14. Song, W.K.; Park, K.-M.; Kim, H.-J.; Lee, J.H.; Choi, J.; Chong, S.Y.; Shim, S.H.; Del Priore, L.V.; Lanza, R. Treatment of Macular Degeneration Using Embryonic Stem Cell-Derived Retinal Pigment Epithelium: Preliminary Results in Asian Patients. Stem Cell Rep. 2015, 4, 860–872. [CrossRef] 15. Falkner-Radler, C.I.; Krebs, I.; Glittenberg, C.; Považay, B.; Drexler, W.; Graf, A.; Binder, S. Human retinal pigment epithelium (RPE) transplantation: Outcome after autologous RPE-choroid sheet and RPE cell-suspension in a randomised clinical study. Br. J. Ophthalmol. 2011, 95, 370–375. [CrossRef] [PubMed] 16. Mehat, M.S.; Sundaram, V.; Ripamonti, C.; Robson, A.G.; Smith, A.J.; Borooah, S.; Robinson, M.; Rosenthal, A.N.; Innes, W.; Weleber, R.G.; et al. Transplantation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells in Macular Degeneration. Ophthalmology 2018, 125, 1765–1775. [CrossRef] 17. Schwartz, S.D.; Hubschman, J.-P.; Heilwell, G.; Franco-Cardenas, V.; Pan, C.; Ostrick, R.M.; Mickunas, E.; Gay, R.; Klimanskaya, I.; Lanza, R. Embryonic stem cell trials for macular degeneration: A preliminary report. Lancet 2012, 379, 713–720. [CrossRef] 18. Ribitsch, I.; Baptista, P.M.; Lange-Consiglio, A.; Melotti, L.; Patruno, M.; Jenner, F.; Schnabl-Feichter, E.; Dutton, L.C.; Connolly, D.J.; van Steenbeek, F.G.; et al. Large Animal Models in Regenerative Medicine and Tissue Engineering: To Do or Not to Do. Front. Bioeng. Biotechnol. 2020, 8, 972. [CrossRef] 19. Sanchez, I.; Martin, R.; Ussa, F.; Fernandez-Bueno, I. The parameters of the porcine eyeball. Graefe’s Arch. Clin. Exp. Ophthalmol. 2011, 249, 475–482. [CrossRef] [PubMed] 20. Kraft, T.W.; Allen, D.; Petters, R.M.; Hao, Y.; Peng, Y.-W.; Wong, F. Altered light responses of single rod photoreceptors in transgenic pigs expressing P347L or P347S rhodopsin. Mol. Vis. 2005, 11, 1246–1256. 21. Petters, R.M.; Alexander, C.A.; Wells, K.D.; Collins, E.B.; Sommer, J.R.; Blanton, M.R.; Rojas, G.; Hao, Y.; Flowers, W.L.; Banin, E.; et al. Genetically engineered large animal model for studying cone photoreceptor survival and degeneration in retinitis pigmentosa. Nat. Biotechnol. 1997, 15, 965–970. [CrossRef] 22. Ross, J.W.; De Castro, J.P.F.; Zhao, J.; Samuel, M.; Walters, E.; Rios, C.; Bray-Ward, P.; Jones, B.W.; Marc, R.E.; Wang, W.; et al. Generation of an Inbred Miniature Pig Model of Retinitis Pigmentosa. Investig. Ophthalmol. Vis. Sci. 2012, 53, 501–507. [CrossRef] 23. Li, Z.Y.; Wong, F.; Chang, J.H.; Possin, D.E.; Hao, Y.; Petters, R.M.; Milam, A.H. Rhodopsin transgenic pigs as a model for human retinitis pigmentosa. Investig. Ophthalmol. Vis. Sci. 1998, 39, 808–819. 24. Shaw, L.C.; Skold, A.; Wong, F.; Petters, R.; Hauswirth, W.W.; Lewin, A. An allele-specific hammerhead ribozyme gene therapy for a porcine model of autosomal dominant retinitis pigmentosa. Mol. Vis. 2001, 7, 6–13. [PubMed] 25. Monés, J.; Leiva, M.; Peña, T.; Martínez, G.; Biarnés, M.; Garcia, M.; Serrano, A.; Fernandez, E. A Swine Model of Selective Geographic Atrophy of Outer Retinal Layers Mimicking Atrophic AMD: A Phase I Escalating Dose of Subretinal Sodium Iodate. Investig. Ophthalmol. Vis. Sci. 2016, 57, 3974–3983. [CrossRef] [PubMed] 26. Zhou, L.; Wang, W.; Liu, Y.; De Castro, J.F.; Ezashi, T.; Telugu, B.P.V.; Roberts, R.M.; Kaplan, H.J.; Dean, U.C. Differentiation of induced pluripotent stem cells of swine into rod photoreceptors and their integration into the retina. Stem Cells 2011, 29, 972–980. [CrossRef] [PubMed] 27. Li, S.Y.; Yin, Z.Q.; Chen, S.J.; Chen, L.F.; Liu, Y. Rescue from light-induced retinal degeneration by human fetal retinal transplanta- tion in minipigs. Curr. Eye Res. 2009, 34, 523–535. [CrossRef] [PubMed] 28. Warfvinge, K.; Kiilgaard, J.F.; Lavik, E.B.; Scherfig, E.; Langer, R.; Klassen, H.J.; Young, M.J. Retinal progenitor cell xenografts to the pig retina: Morphologic integration and cytochemical differentiation. Arch. Ophthalmol. 2005, 123, 1385–1393. [CrossRef] 29. Klassen, H.; Warfvinge, K.; Schwartz, P.H.; Kiilgaard, J.F.; Shamie, N.; Jiang, C.; Samuel, M.; Scherfig, E.; Prather, R.S.; Young, M.J. Isolation of Progenitor Cells from GFP-Transgenic Pigs and Transplantation to the Retina of Allorecipients. Cloning Stem Cells 2008, 10, 391–402. [CrossRef] 30. Klassen, H.; Kiilgaard, J.F.; Zahir, T.; Ziaeian, B.; Kirov, I.; Scherfig, E.; Warfvinge, K.; Young, M.J. Progenitor Cells from the Porcine Neural Retina Express Photoreceptor Markers after Transplantation to the Subretinal Space of Allorecipients. Stem Cells 2007, 25, 1222–1230. [CrossRef] 31. Ghosh, F.; Engelsberg, K.; English, R.V.; Petters, R.M. Long-term neuroretinal full-thickness transplants in a large animal model of severe retinitis pigmentosa. Graefe’s Arch. Clin. Exp. Ophthalmol. 2007, 245, 835–846. [CrossRef] Int. J. Mol. Sci. 2021, 22, 10497 20 of 21 32. Klassen, H.; Kiilgaard, J.F.; Warfvinge, K.; Samuel, M.S.; Prather, R.S.; Wong, F.; Petters, R.M.; La Cour, M.; Young, M.J. Photoreceptor Differentiation following Trans-plantation of Allogeneic Retinal Progenitor Cells to the Dystrophic Rhodopsin Pro347Leu Transgenic Pig. Stem Cells Int. 2012, 2012, 939801. [CrossRef] 33. Brant Fernandes, R.A.; Koss, M.J.; Falabella, P.; Stefanini, F.R.; Maia, M.; Diniz, B.; Ribeiro, R.; Hu, Y.; Hinton, D.; Clegg, D.O.; et al. An Innovative Surgical Technique for Sub-retinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Pigmented Epithelium in Yucatan Mini Pigs: Preliminary Results. Ophthalmic Surg. Lasers Imaging Retina 2016, 47, 342–351. [CrossRef] 34. Koss, M.J.; Falabella, P.; Stefanini, F.R.; Pfister, M.; Thomas, B.B.; Kashani, A.H.; Brant, R.; Zhu, D.; Clegg, D.O.; Hinton, D.R.; et al. Subretinal implantation of a monolayer of hu-man embryonic stem cell-derived retinal pigment epithelium: A feasibility and safety study in Yucatán minipigs. Graefe’s Arch. Clin. Exp. Ophthalmol. 2016, 254, 1553–1565. [CrossRef] 35. Sohn, E.H.; Jiao, C.; Kaalberg, E.; Cranston, C.; Mullins, R.F.; Stone, E.M.; Tucker, B.A. Allogenic iPSC-derived RPE cell transplants induce immune response in pigs: A pilot study. Sci. Rep. 2015, 5, 11791. [CrossRef] 36. Bhutto, I.A.; Ogura, S.; Baldeosingh, R.; McLeod, D.S.; Lutty, G.A.; Edwards, M.M. An Acute Injury Model for the Phenotypic Char-acteristics of Geographic Atrophy. Investig. Ophthalmol. Vis. Sci. 2018, 59, AMD143–AMD151. [CrossRef] [PubMed] 37. Edwards, M.M.; McLeod, D.S.; Bhutto, I.A.; Grebe, R.; Duffy, M.; Lutty, G.A. Subretinal Glial Membranes in Eyes with Geographic Atrophy. Investig. Ophthalmol. Vis. Sci. 2017, 58, 1352–1367. [CrossRef] [PubMed] 38. Nandrot, E.F.; Anand, M.; Almeida, D.; Atabai, K.; Sheppard, D.; Finnemann, S.C. Essential role for MFG-E8 as ligand for αvβ5 integrin in diurnal retinal phagocytosis. Proc. Natl. Acad. Sci. USA 2007, 104, 12005–12010. [CrossRef] 39. Holz, F.G.; Sadda, S.R.; Busbee, B.; Chew, E.Y.; Mitchell, P.; Tufail, A.; Brittain, C.; Ferrara, D.; Gray, S.; Honigberg, L.; et al. Efficacy and Safety of Lampalizumab for Geographic At-rophy Due to Age-Related Macular Degeneration: Chroma and Spectri Phase 3 Randomized Clinical Trials. JAMA Ophthalmol. 2018, 136, 666–677. [CrossRef] [PubMed] 40. Sunness, J.S.; Gonzalez-Baron, J.; Applegate, C.A.; Bressler, N.M.; Tian, Y.; Hawkins, B.; Barron, Y.; Bergman, A. Enlargement of atrophy and visual acuity loss in the geographic atrophy form of age-related macular degeneration. Ophthalmology 1999, 106, 1768–1779. [CrossRef] 41. Kyger, M.; Worley, A.; Adamus, G. Autoimmune Responses against Photoreceptor Antigens during Retinal Degeneration and Their Role in Macrophage Recruitment into Retinas of RCS Rats. J. Neuroimmunol. 2013, 254, 91–100. [CrossRef] 42. Romero-Vazquez, S.; Llorens, V.; Soler-Boronat, A.; Figueras-Roca, M.; Adan, A.; Molins, B. Interlink between Inflammation and Oxidative Stress in Age-Related Macular Degeneration: Role of Complement Factor H. Biomedicines 2021, 9, 763. [CrossRef] 43. Jones, B.W.; Watt, C.B.; Frederick, J.M.; Baehr, W.; Chen, C.-K.; Levine, E.M.; Milam, A.H.; Lavail, M.M.; Marc, R.E. Retinal Remodeling Triggered by Photoreceptor Degenerations. J. Comp. Neurol. 2003, 464, 1–16. [CrossRef] 44. Sarks, S.H. Drusen Patterns Predisposing to Geographic Atrophy of the Retinal Pigment Epithelium. Aust. J. Ophthalmol. 1982, 10, 91–97. [CrossRef] 45. Bressler, S.B.; Maguire, M.G.; Bressler, N.M.; Fine, S.L. Relationship of Drusen and Abnormalities of the Retinal Pigment Epithelium to the Prognosis of Neovascular Macular Degeneration. The Macular Photocoagulation Study Group. Arch. Ophthalmol. 1990, 108, 1442–1447. [CrossRef] 46. Usui, H.; Nishiwaki, A.; Landiev, L.; Kacza, J.; Eichler, W.; Wako, R.; Kato, A.; Takase, N.; Kuwayama, S.; Ohashi, K.; et al. In Vitro Drusen Model - Three-Dimensional Spheroid Culture of Retinal Pigment Epithelial Cells. J. Cell Sci. 2018, 132, jcs215798. [CrossRef] 47. Guo, J.L.; Lee, V.M.Y. Cell-to-Cell Transmission of Pathogenic Proteins in Neurodegenerative Diseases. Nat. Med. 2014, 20, 130–138. [CrossRef] [PubMed] 48. Toops, K.A.; Tan, L.X.; Lakkaraju, A. Apolipoprotein E Isoforms and AMD. Adv. Exp. Med. Biol. 2016, 854, 3–9. [CrossRef] [PubMed] 49. Toops, K.A.; Tan, L.X.; Jiang, Z.; Radu, R.A.; Lakkaraju, A. Cholesterol-Mediated Activation of Acid Sphingomyelinase Disrupts Autophagy in the Retinal Pigment Epithelium. Mol. Biol. Cell 2015, 26, 1–14. [CrossRef] [PubMed] 50. Sparrow, J.R.; Parish, C.A.; Hashimoto, M.; Nakanishi, K. A2E, a Lipofuscin Fluorophore, in Human Retinal Pigmented Epithelial Cells in Culture. Investig. Ophthalmol. Vis. Sci. 1999, 40, 2988–2995. 51. Sparrow, J.R.; Cai, B.; Fishkin, N.; Jang, Y.P.; Krane, S.; Vollmer, H.R.; Zhou, J.; Nakanishi, K. A2E, a Fluorophore of RPE Lipofuscin: Can It Cause RPE Degeneration? Adv. Exp. Med. Biol. 2003, 533, 205–211. 52. Feeney-Burns, L.; Eldred, G.E. The Fate of the Phagosome: Conversion to “age Pigment” and Impact in Human Retinal Pigment Epithelium. Trans. Ophthalmol. Soc. UK 1983, 103 Pt 4, 416–421. [PubMed] 53. Sharma, R.; Khristov, V.; Rising, A.; Jha, B.S.; Dejene, R.; Hotaling, N.; Li, Y.; Stoddard, J.; Stankewicz, C.; Wan, Q.; et al. Clinical-Grade Stem Cell-Derived Retinal Pigment Epithelium Patch Rescues Retinal Degeneration in Rodents and Pigs. Sci. Transl. Med. 2019, 11, eaat5580. [CrossRef] [PubMed] 54. Del Priore, L.V.; Tezel, T.H.; Kaplan, H.J. Survival of Allogeneic Porcine Retinal Pigment Epithelial Sheets after Subretinal Transplantation. Investig. Ophthalmol. Vis. Sci. 2004, 45, 985–992. [CrossRef] [PubMed] 55. Warre-Cornish, K.; Barber, A.C.; Sowden, J.C.; Ali, R.R.; Pearson, R.A. Migration, Integration and Maturation of Photoreceptor Precursors Following Transplantation in the Mouse Retina. Stem Cells Dev. 2014, 23, 941–954. [CrossRef] 56. Hazim, R.A.; Karumbayaram, S.; Jiang, M.; Dimashkie, A.; Lopes, V.S.; Li, D.; Burgess, B.L.; Vijayaraj, P.; Alva-Ornelas, J.A.; Zack, J.A.; et al. Differentiation of RPE Cells from Integration-Free IPS Cells and Their Cell Biological Characterization. Stem Cell Res. Ther. 2017, 8, 1–17. [CrossRef] Int. J. Mol. Sci. 2021, 22, 10497 21 of 21 57. Rizzolo, L.J. Barrier Properties of Cultured Retinal Pigment Epithelium. Exp. Eye Res. 2014, 126, 16–26. [CrossRef] 58. Riera, M.; Fontrodona, L.; Albert, S.; Ramirez, D.M.; Seriola, A.; Salas, A.; Muñoz, Y.; Ramos, D.; Villegas-Perez, M.P.; Zapata, M.A.; et al. Comparative Study of Human Embryonic Stem Cells (HESC) and Human Induced Pluripotent Stem Cells (HiPSC) as a Treatment for Retinal Dystrophies. Mol. Ther. Methods Clin. Dev. 2016, 3, 16010. [CrossRef] 59. Salas, A.; Duarri, A.; Fontrodona, L.; Ramírez, D.M.; Badia, A.; Isla-Magrané, H.; Ferreira-de-Souza, B.; Zapata, M.Á.; Raya, Á.; Veiga, A.; et al. Cell Therapy with Human Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium and Retinal Precursor Cells Prevents Visual Function Loss in a Rat Model of Retinal Degeneration. Mol. Ther.-Methods Clin. Dev. 2021, 20, 688–702. [CrossRef] 60. Maqueda, M.; Mosquera, J.L.; García-Arumí, J.; Veiga, A.; Duarri, A. Repopulation of Decellularized Retinas with HiPSC-Derived Retinal Pigment Epithelial and Ocular Progenitor Cells Shows Cell Engraftment, Organization and Differentiation. Biomaterials 2021, 276, 121049. [CrossRef] 61. Strauss, O. The Retinal Pigment Epithelium in Visual Function. Physiol. Rev. 2005, 85, 845–881. [CrossRef] 62. Petrus-Reurer, S.; Bartuma, H.; Aronsson, M.; Westman, S.; Lanner, F.; André, H.; Kvanta, A. Integration of Subretinal Suspension Transplants of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells in a Large-Eyed Model of Geographic Atrophy. Investig. Ophthalmol. Vis. Sci. 2017, 58, 1314–1322. [CrossRef] 63. Plaza Reyes, A.; Petrus-Reurer, S.; Antonsson, L.; Stenfelt, S.; Bartuma, H.; Panula, S.; Mader, T.; Douagi, I.; André, H.; Hovatta, O.; et al. Xeno-Free and Defined Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells Functionally Integrate in a Large-Eyed Preclinical Model. Stem Cell Rep. 2015, 6, 9–17. [CrossRef] [PubMed] 64. Lin, T.-C.; Seiler, M.J.; Zhu, D.; Falabella, P.; Hinton, D.R.; Clegg, D.O.; Humayun, M.S.; Thomas, B.B. Assessment of Safety and Functional Efficacy of Stem Cell-Based Therapeutic Approaches Using Retinal Degenerative Animal Models. Stem Cells Int. 2017, 2017, 9428176. [CrossRef] 65. Nakatsuji, N.; Nakajima, F.; Tokunaga, K. HLA-Haplotype Banking and IPS Cells. Nat. Biotechnol. 2008, 26, 739–740. [CrossRef] [PubMed] 66. Giorgetti, A.; Montserrat, N.; Aasen, T.; Gonzalez, F.; Rodríguez-Pizà, I.; Vassena, R.; Raya, A.; Boué, S.; Barrero, M.J.; Corbella, B.A.; et al. Generation of Induced Pluripotent Stem Cells from Human Cord Blood Using OCT4 and SOX2. Cell Stem Cell 2009, 5, 353–357. [CrossRef] 67. Badia, A.; Salas, A.; Duarri, A.; Ferreira-de-Souza, B.; Zapata, M.Á.; Fontrodona, L.; García-Arumí, J. Transcriptomics Analysis of Ccl2/Cx3cr1/Crb1rd8 Deficient Mice Provides New Insights into the Pathophysiology of Progressive Retinal Degeneration. Exp. Eye Res. 2021, 203, 108424. [CrossRef] [PubMed] 68. Nandrot, E.F.; Kim, Y.; Brodie, S.E.; Huang, X.; Sheppard, D.; Finnemann, S.C. Loss of Synchronized Retinal Phagocytosis and Age-Related Blindness in Mice Lacking Alphavbeta5 Integrin. J. Exp. Med. 2004, 200, 1539–1545. [CrossRef] [PubMed]