Original Article Cell therapy with hiPSC-derived RPE cells and RPCs prevents visual function loss in a rat model of retinal degeneration Anna Salas,1 Anna Duarri,1,2,3 Laura Fontrodona,1 Diana Mora Ramírez,1 Anna Badia,1 Helena Isla-Magrané,1 Barbara Ferreira-de-Souza,1 Miguel Ángel Zapata,1 Ángel Raya,2,4,5 Anna Veiga,2,3 and José García-Arumí1,6,7 1Ophthalmology Research Group, Vall d’Hebron Institut de Recerca (VHIR), Vall d’Hebron Hospital Universitari, 08035 Barcelona, Spain; 2Barcelona Stem Cell Bank, Regenerative Medicine Program, IDIBELL, 08908 Hospitalet de Llobregat, Spain; 3National Stem Cell Bank-Barcelona Node, Biomolecular and Bioinformatics Resources Platform PRB2, ISCIII, 08908 Hospitalet de Llobregat, Spain; 4Catalan Institute for Research and Advanced Studies (ICREA), 08010 Barcelona, Spain; 5Networking Center of Biomedical Research in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), 28029 Madrid, Spain; 6Department of Ophthalmology, Vall d’Hebron Hospital Universitari, Vall d’Hebron Barcelona Hospital Campus, 08036 Barcelona, Spain; 7Department of Ophthalmology, Autonomous University of Barcelona, 08193 Bellaterra, Spain Photoreceptor loss is the principal cause of blindness in retinal all-trans retinol to 11-cis retinal in the visual cycle, phagocytosis of degenerative diseases (RDDs). Whereas some therapies exist photoreceptor outer segments (POS), or light absorption, among for early stages of RDDs, no effective treatment is currently others.3 Photoreceptor loss in RDDs may be the result of photore- available for later stages, and once photoreceptors are lost, ceptor dysfunction or may result from RPE loss or dysfunction. the only option to rescue vision is cell transplantation. With Nowadays, treatment strategies are mainly focused on the early stages the use of the Royal College of Surgeons (RCS) rat model of of the diseases, preventing retinal degeneration through neuroprotec- retinal degeneration, we sought to determine whether com- tion and gene therapies. To date, there is no effective treatment bined transplantation of human-induced pluripotent stem capable to revert cell death and restore visual function in later stages; cell (hiPSC)-derived retinal precursor cells (RPCs) and retinal thus, research in cell-based therapies for retina regeneration has been pigment epithelial (RPE) cells was superior to RPE or RPC the subject of intense investigation. transplantation alone in preserving retinal from degeneration. hiPSC-derived RPCs and RPE cells expressing (GFP) were Mammalian retinas have limited regenerative capacity,4 and cell- transplanted into the subretinal space of rats. In vivo moni- based therapies aiming to restore light sensitivity are highly desirable. toring showed that grafted cells survived 12 weeks in the subre- Human pluripotent stem cells (hPSCs), both embryonic (hESC) or tinal space, and rats treated with RPE + RPC therapy exhibited induced (hiPSC), can differentiate into any retinal cell type and can better conservation of the outer nuclear layer (ONL) and visual thus serve as a renewable cell source to repair degenerated retinas.5,6 response than RPE-treated or RPC-treated rats. Transplanted In this context, hESC-derived RPE cell transplantation has been suc- RPE cells integrated in the host RPE layer, whereas RPC mostly cessful in several preclinical models,6 although long-term efficacy has remained in the subretinal space, although a limited number of yet to be defined. Likewise, several clinical trials in age-related macu- cells integrated in the ONL. In conclusion, the combined trans- lar degeneration (AMD), retinitis pigmentosa, and Stargardt’s disease plantation of hiPSC-derived RPE and RPCs is a potentially have been proven safe but with only limited improvements in superior therapeutic approach to protect retina from degener- vision.7–10 These results likely suggest that at later disease stages, ation in RDDs. when photoreceptors have degenerated, transplanted RPE cells are unable to rescue vision, since they are not light sensing cells. Thus, several photoreceptor-replacement therapies have been explored to INTRODUCTION overcome this limitation, including full-thickness retinal patches,11– 14 Retinal degenerative diseases (RDDs) are a heterogeneous group of retinal progenitor cells,15–17 neural progenitor cells,18,19 and stem pathologies characterized by the progressive degeneration of photore- ceptors (PRs), ultimately leading to blindness,1 and affect 285 million people worldwide. Received 1 July 2020; accepted 4 February 2021; https://doi.org/10.1016/j.omtm.2021.02.006. Cone and rod photoreceptors are the light-sensing cells in the retina Correspondence: Anna Duarri, PhD, Ophthalmology Research Group, Vall d’Hebron Institut de Recerca, Vall d’Hebron Hospital Universitari, Passeig de la and are responsible for visual input.2 In close association with the Vall d’Hebron 119-129, Collserola Building, Laboratory 148, 08035 Barcelona, photoreceptors, the retinal pigment epithelium (RPE) provides Spain. them several crucial supporting functions, including: recycling of E-mail: anna.duarri@vhir.org 688 Molecular Therapy: Methods & Clinical Development Vol. 20 March 2021 ª 2021 The Author(s). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). www.moleculartherapy.org Figure 1. Characterization of human-induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) cells (A) RPE-like cells exhibited typical pigmented hexagonal cell morphology under bright-field and expressed green fluorescent protein (GFP). Representative immunostaining images show expression of RPE markers bestrophin-1 (BEST1), ZO-1 (zonula occludens 1), MITF (microphthalmia-associated transcription factor), and a human antigen (Ku80). Nuclei are stained with DAPI. Scale bars, 75 mm in left panels and 50 mm in middle and right panels. (B) Gene-expression levels in RPE-like cells by quantitative real- time PCR. Values are normalized to GAPDH and relative to undifferentiated hiPSC, expressed as 2DDCt (log scale). Data presented as mean ± standard deviation (SD; n = 3), five independent biological replicates. (C) Quantitative analysis by flow cytometry of RPE65, MITF, and BEST1 in RPE cell culture. Histograms show both the undifferentiated hiPSC (blue) and RPE cells (red). Results are representative of two independent experiments performed with cells from different differentiations. (D) Representative trans- mission electron microscopy (TEM; ac) and scanning electron microscopy (SEM; df) images of RPE cells in culture. RPE monolayers show polarization with apical microvilli, melanosomes, mitochondria, tight junction (red arrow), and adherent junction (red arrowhead) at the apical border and basal nuclei (N). Scale bars, 2 mm (a and b) and 0.2 mm (c) for TME images. Scale bars, 10 mm (d), 5 mm (e), and 0.5 mm (f) for SEM images. (E) In vitro phagocytosis of tetramethylrhodamine (TRITC)-labeled photoreceptor (PR) outer segments (POS; red) by RPE-like cells (GFP in green). Nuclei are stained with DAPI. Scale bar, 25 mm. cell-derived retinal progenitors,20–34 which have been proven to The Royal College of Surgeons (RCS) rat model of autosomal-reces- restore visual function in animal models of photoreceptor dysfunc- sive retinitis pigmentosa has been extensively used for cell therapeutic tion. The majority of cell replacement strategies are currently based approaches, since it is a good model for developing cell therapies on transplantation of a single cell-type rather than multiple cell types; focused on severe retinopathies. RCS rats carry a defective MERTK however, in the setting of defective RPE, transplantation of photore- gene,35 a receptor tyrosine kinase expressed in RPE cells, which leads ceptors only might not be sufficient without a healthy RPE support. In to RPE malfunction of phagocytosis of POS that in turn trigger severe that case, selective replacement of both RPE and photoreceptor cell photoreceptor cell death, beginning at 3 weeks after birth and types might be a better strategy to regenerate the retinas. continuing rapidly over 4 weeks.36,37 We previously demonstrated Molecular Therapy: Methods & Clinical Development Vol. 20 March 2021 689 Molecular Therapy: Methods & Clinical Development (legend on next page) 690 Molecular Therapy: Methods & Clinical Development Vol. 20 March 2021 www.moleculartherapy.org the potential of hESC and hiPSC to generate transplantable RPE cells, Differentiation and characterization of RPCs derived from but these cells failed to exert significant long-term improvement of vi- hiPSCs sual function.38 In the current study, we developed a protocol to To guide hiPSCs to the retinal lineage, we used a differentiation pro- generate both retinal precursor cells (RPCs), consisting mainly in tocol based on a previous study (Figure 2A).39 Upon neuroretina in- photoreceptor precursor cells, and RPE cells from hiPSC. Our main duction, embryoid bodies (EBs) were seeded onto Matrigel to develop objective was to assess whether a cell-transplantation strategy using an anterior neuroepithelium, followed by neural rosette formation a combination of RPE cells and RPC would be superior to RPE or observed by day 7. Early neural retina and optic cups were apparent RPC therapies alone using the RCS rat model and to explore the po- by day 25 of culture and showed signs of internal lamination tential of our RPC to integrate and repopulate the degenerative surrounded by a few pigmented cells at day 60 of differentiation endogenous photoreceptor layer. (Figure 2A). We characterized retinal cells at different time points by immunocytochemistry, qRT-PCRquantitative real-time PCR, and flow cytometry. At day 21 of differentiation, cell culture comprised RESULTS retinal progenitor cells that formed neural rosettes and neuroepithe- Differentiation and characterization of RPE-like cells derived lium (Figure 2B). Retinal progenitors expressed the early-stage-spe- from hiPSCs cific markers PAX6, RAX, CHX10, and OTX2 and the photoreceptor Previously, we successfully differentiated hESCs and hiPSCs to RPE- progenitor markers CRX, recoverin, and NLR and showed a decrease like cells.38 With the use of the same protocol here, we obtained a ho- in the expression of the pluripotency genes OCT4, NANOG, and Tra- mogeneous culture of RPE cells recapitulating the native cellular 1-60 (Figures 2B and 2C; Figure S1A). At this stage, retinal progenitors morphology, including a characteristic pigmented polygonal cell were proliferative (Ki67+), as it is shown by flow cytometry (Fig- shape (Figure 1A). To facilitate in vivo detection of transplanted cells, ure S1A). At days 4550, cell culture was composed of a homogeneous we transduced RPE cells with lentiviral particles carrying a GFP re- population of more mature retinal cells, hereinafter RPCs, which along porter gene. RPE cells in culture constitutively expressed GFP and with GFP (cells transduced with lentivirus before transplantation), the human nuclear antigen Ku80 (Figure 1A). RPE cells also ex- RAX, CHX10, and PAX6, together with CRX and NRL expression, pressed specific early ocular markers, including PAX6, OTX2, we detected cell clusters containing recoverin + cells (Figures MITF, and CHX10, and the mature RPE markers bestrophin-1, 2D2F). Within RPC cultures, recoverin + photoreceptor precursors RPE65, ZO-1, SIL, PEDF, MERTK, and TYR and showed downregu- had extended axonal projections (arrows in magnification; Figure 2D). lation of OCT4 and Tra-1-60 (Figures 1A1C). Along maturation, Furthermore, we could detect expression of OPSIN and RHODOPSIN the RPE cell monolayer polarized and acquired apical-basolateral transcripts by quantitative real-time PCR (Figure 2E), similar to specialization, columnar-shape morphology with apical melanin- another study.34 Flow cytometry analysis revealed that the RPC mostly containing melanosomes and tight junctions, as observed by trans- expressed recoverin, red/green coneopsin (RG-OPSIN), and PAX6, mission electron microscopy (TEM) (Figure 1D, ac). Microvilli whereas a smaller proportion (1.1%) expressed rhodopsin (Figure 2F). were also observed in the apical side by scanning electron microscopy The manifestation of pigmented foci in our cultures, corresponding to (SEM) (Figure 1D, df). As RCS rats carry a defective mertk gene, RPE cells, was observed from day 30, although the percentage of pig- which is essential for RPE to phagocytize POS, we ensured that the mented RPE was relatively low, as demonstrated by flow cytometry at hiPSC-derived RPE cells exhibited native phagocytic function before day 45 with only 0.4% of total cells. Moreover, RPC at day 45 was not transplantation, as demonstrated by their internalization of tetrame- mitotically active, as it is shown by the low Ki67 levels (1.5%) (Fig- thylrhodamine (TRITC)-labeled POS in vitro (Figure 1E). Overall, ure 2F). At day 90 of differentiation, retinal cell cultures showed these results show that the RPE cell-differentiation protocol is robust, mature photoreceptor cells expressing CRX, recoverin rhodopsin, reproducible, and generates functional RPE-like cells. and RG-opsin, and these cells also presented longer axonal projections Figure 2. Generation and characterization of retinal precursor cells (RPCs) from hiPSCs (A) Schematic diagram of the three-step protocol at different stages of differentiation. Bright-field images of cell morphology of undifferentiated hiPSC colonies at day 5, embryoid bodies at day 1, early retinal rosettes containing RPCs at day 10, and optic cup-like structures at day 60. Scale bars, 500 mm and 100 mm. (B) At day 21, confocal images of retinal progenitor cells forming neural rosettes and expressing human antigen Ku80; eye-field primordial and neural retina markers SOX1, CHX10, OTX2, and PAX6; and early PR markers CRX and NRL. Scale bars, 50 mm. (C) Quantitative real-time PCR of gene expression at day 21 relative to undifferentiated hiPSC shows downregulation of pluripotent genes OCT4 and NANOG and upregulation of neural (PAX6, OTX2, and CHX10) and photoreceptor (CRX, RAX, and recoverin [Recov]) genes. Values are normalized to GAPDH and relative to undifferentiated hiPSC, expressed as 2DDCt (log scale). Data presented as mean ± SD (n = 3), three independent biological replicates. (D) Representative bright-field and immunocytochemistry images of RPC cultures transduced with the lentivirus SparQ-GFP at day 45 (before transplantation). The expression of GFP, neural retina marker (CHX10, PAX6, and RAX), and PR progenitor markers (NRL, RECOV, and CRX) is shown. Magnification of Recov + cells with axonal projections (white arrows). Scale bars, 75 mm (bright field); 50 mm and 10 mm in the magnifications. Nuclei are stained with DAPI. (E) uantitative real-time PCR of gene expression in RPCs at day 45 relative to undifferentiated hiPSC shows no expression of pluripotent genes (NANOG and OCT4) but upregulation of retinal markers, including mature PR markers opsin and rhodopsin. Values are normalized to GAPDH and relative to undifferentiated hiPSC, expressed as 2DDCt (log scale). Data presented as mean ± SD (n = 3), three independent biological replicates. (F) Quantitative flow cytometry analysis of RPC at day 45 with surface marker Tra-1-60 and intracellular markers RECOV, RHO, RG-OPSIN, and PAX6 (specific for RPC), MITF (for RPE cells), and Ki67 (proliferation) and the appropriate controls (undifferentiated hiPSC, retinal progenitors at day 21, and secondary antibody are shown in Figure S1). The numbers in the corner show the percentage of stained cells in this gate. Secondary antibody was used as control. Results are representative of two biological replicates. Molecular Therapy: Methods & Clinical Development Vol. 20 March 2021 691 Molecular Therapy: Methods & Clinical Development Table 1. Groups of transplanted RCS rats at postnatal days 21–24 Experiment Treatment (cell suspension injection) No. of injected cells No. of rats No. of successfully injected eyes Successful injection rates RPE 105 13 20 77% RPE + RP d21 5  104 + 5  104 20 34 85% #1 RPE + RPC d45–50 5  104 + 5  104 20 13 64% RPE + RPC d75 5  10 + 5  10 4 4 17 28 82% Sham  15 21 70% RPE 105 20 30 75% RPE + RPC d4550 5  104 + 5  104 23 32 70% #2 5 RPC d4550 10 8 11 68% Sham  10 15 75% RPE, retinal pigment epithelium; RP, retinal progenitor; RPC, retinal precursor cell; d21, day 21 of differentiation; d45–50, days between 45 and 50 of differentiation; d75, day 75 of differentiation. as compared with day 45 (Figure S1B) This indicated that our protocol model (Table 1; experiment 2). Four groups of RCS rats were subreti- enriches for a population of RG cone photoreceptors. nally injected with cell suspensions of RPE cells, RPC cells at days 45–50 of differentiation, a combination of RPE cells and RPC, or me- Establishing the optimal RPC developmental time window for dium. All transplanted cells expressed GFP, and the injected eyes were transplantation periodically monitored by fluorescence fundus imaging (FFI) and op- Studies have shown that the maturity of stem cell-derived photore- tical coherence tomography (OCT) for 12 weeks. We first followed ceptors is key for successful engraftment, maturation, and function the presence and evolution of cell grafts in the host retinas (Figure 3). in the correct host retinal layer.24,28 Thus, we first determined the In all groups, retinas appeared detached and formed a subretinal bleb best retinal cells’ differentiation time window to improve retinal con- (Figures 3A3C, a and b) containing the injected cell suspension. servation and visual function recovery in the RCS model. In a pilot 1 week after injection, the bleb was absorbed, and cell grafts were de- study (Table 1, experiment 1), we cotransplanted hiPSC-derived tected as fluorescent dots randomly distributed around the injection RPE cells together with retinal progenitors at day 21, RPC at days area (Figures 3A3C, c and d). 2 weeks postinjection, retinas had 4550, and more mature RPC at day 75; RPE cells alone; or medium almost recovered their natural position, and the fluorescent clumps (sham) into the subretinal space of RCS rats at postnatal days between seemed to be partially reduced in size (Figures 3A3C, e and f). 21 and 24, corresponding to the initial stages of photoreceptor degen- The level of fluorescence remained very similar from 4 to 12 weeks eration. We then analyzed visual function by electroretinography in the RPE + RPC group, indicating the survival of most of the cells (ERG) and retina conservation by histology at 8 and 12 weeks post- present at week 2 (Figure 3C, gl). However, the fluorescent cell transplantation (Figure S2). Cotransplantation of RPE + RPC at grafts in the RPE and the RPC groups were reduced drastically days 45–50 and day 75 of differentiation led to a significant preserva- from 4 weeks after transplantation (Figures 3A and 3B, g and h), tion of visual function at 12 weeks compared with RPE-transplanted and very few green cells were observed at 12 weeks (Figures 3A and rats and the sham group (p < 0.05) and was superior to that in retinas B, k and l) even though rats were under immunosuppression, stating transplanted with RPE + retinal progenitors at day 21 (Figures the difficulty of RPE or RPC survival when transferred alone in the S2AS2C). We also observed a better preservation of the outer nu- retina of the RCS model. clear layer (ONL) from 8 to 12 weeks with RPE + RPC differentiated for 45–50 days or for 75 days when compared with RPE + retinal pro- Combined RPE + RPC therapy induces a better survival of genitors differentiated for 21 days, RPE cells alone, or sham (Fig- endogenous photoreceptors than RPC or RPE cell therapies ure S2D). These data suggest that day 21 differentiated retinal progen- The RCS rat model is primarily characterized by severe degeneration itor cultures consist of immature progenitors that fail to preserve or of photoreceptors as a consequence of the RPE cell dysfunction, improve retinal function and that RPC days 45–75 cultures consist which can be easily observed by OCT at postnatal day 60.40 To eval- of precursor cells at an equivalent stage of development to those of uate whether RPE + RPC, RPC, or RPE cell therapies could rescue RCS rats. Based on these results, we established our transplantable photoreceptor degeneration, we examined the total retina thickness developmental time window at days 45–50. and the photoreceptor layer thickness (consisting of ONL + outer and inner photoreceptor segments) in all groups. Quantifications Transplanted RPE + RPCs survive in the subretinal space of RCS were performed at 4 weeks (Figures 4A4F), 8 weeks (Figures rats and can be monitored in vivo over 12 weeks 4G4L), and 12 weeks (Figures 4M4R) postinjection, and retinal In a second experiment, we performed a comprehensive study of the preservation of the grafted area was normalized by the thickness of progression, survival, and integration of transplanted cells in the RCS the contralateral area of the same eye and compared between groups. 692 Molecular Therapy: Methods & Clinical Development Vol. 20 March 2021 www.moleculartherapy.org Figure 3. In vivo time course of GFP-fluorescent cell survival in the subretinal space of the RCS rat (A) Images from the same eye injected with RPE cells alone at 1, 2, 4, 8, and 12 weeks postinjection (PI). (B) Images from the same eye injected with RPC alone. (C) Images from the same eye injected with the combination of RPE + RPC alone. Images show fundus retinographies (a, c, e, g, i, and k) and fluorescent retinographies under blue filter (b, d, f, h, j, and l) in the same area, where grafted GFP+ cells are observed as green spots. At 4 weeks after transplantation, retinal micrographs showed no dif- Combined RPE + RPC therapy preserves visual function better ferences in terms of retina preservation among the four groups (Fig- than RPC or RPE cell therapies alone ure 4E). Conversely, at 8 and 12 weeks postinjection, we observed a We previously showed that RPE transplantation in the subretinal significant rescue of the total retina (Figures 4K and 4Q) and photo- space of RCS rats partially preserved retinal function up to 8 weeks receptor layer thickness (Figures 4L and 4R) in the group treated with postinjection.38 To assess whether combined RPE + RPC therapy combined RPE + RPC compared with RPC, RPE, and sham groups. was superior to RPC or RPE cell transplantation at preserving re- Moreover, at 12 weeks, the RPC group exhibited a marked reduction sponses to light stimuli, we compared the electrical response of the of both total retina and ONL thickness compared to 8 weeks (Figures different treatment groups by scotopic ERG at 4, 8, and 12 weeks post- 4Q and 4R). Of note, we did not observe any nuclei in the ONL of injection (Figures 5A5C). Animals were stimulated with green light sham-injected eyes36 at 12 weeks postinjection, whereas we detected from 1.1 to 1.9 log cd $ s $ m2 to acquire the best retinal response, some nuclear layers in RPE-, RPC-, and RPE + RPC-transplanted and ERG waves were analyzed by amplitude and implicit time quan- eyes (Figures 4M4P). In summary, combined RPE + RPC cell ther- tification (Figures 5D5G). Measurements shown in Figure 4 are apy is better at preserving retinas from degeneration than RPE and those corresponding to 1.9 log cd $ s $ m2. At 4 weeks postinjection, RPC cell therapies. ERG responses obtained from RPE and RPE + RPC cell-engrafted Molecular Therapy: Methods & Clinical Development Vol. 20 March 2021 693 Molecular Therapy: Methods & Clinical Development Figure 4. Structural analysis of transplanted retinas by optical coherence tomography (OCT) Eyes injected with medium (sham; A, G, and M; n = 6 eyes), RPE cells (B, H, and N; n = 8 eyes), RPC (C, I, and O; n = 6 eyes), or the combination of RPE and RPC (RPE + RPCs; D, J, and P; n = 10 eyes) were analyzed at 4, 8, and 12 weeks PI by OCT, obtaining retina micrographs of the grafted area and a contralateral area of the same eye (not shown). Cross-sections of the retinas were analyzed, quantifying total retina thickness (TOTAL RETINA; area between blue and orange lines) and PR layer thickness (PR LAYER; area between green and orange lines). The preservation of the thickness of total retina and PR layer was compared among groups at 4 (E and F), 8 (K and L), and 12 weeks PI (Q and R), calculating the difference between the grafted area and the contralateral area of the same eye. Data presented as mean ± SD. Statistical significance was calculated by one-way ANOVA, followed by Tukey’s multiple comparison tests. *p < 0.05; **p < 0.005 between RPE + RPC and sham groups; #p < 0.05; ##p < 0.005 between RPE + RPC and RPE groups. 694 Molecular Therapy: Methods & Clinical Development Vol. 20 March 2021 www.moleculartherapy.org Figure 5. Visual function analysis by electroretinogram (AC) Representative electroretinogram recordings at 4, 8, and 12 weeks PI. Scotopic electroretinogram responses were recorded from eyes injected with medium (sham; blue lines, n = 7 eyes), RPE-differentiated cells (RPE; red lines, n = 8 eyes), RPC-differentiated cells (RPC; green lines, n = 8 eyes), or the combination of RPE and RPCs (RPE + RPC; black lines, n = 9 eyes). Measurements were recorded under a light stimulus of 1.9 log cd $ s $ m2 and 1 ms of duration. (DG) Electroretinographic waves were quantified and compared among treatment groups at the three time points by the measurement of the b-wave amplitude (D) and its implicit time (F) and the a-wave amplitude (E) and its implicit time (G). Data presented as mean ± SD. Statistical significance was calculated by two-way ANOVA, followed by Tukey’s multiple comparison tests comparing sham with RPE, RPC, and RPE + RPC data (*), comparing RPE with RPE + RPC data (#), or comparing RPC with RPE + RPC data (+). *p < 0.05; **p < 0.005; ***p < 0.0005; ****p < 0.0001; #p < 0.05; ##p < 0.005; ###p < 0.0005; +p < 0.05; ++p < 0.005; +++p < 0.0005. eyes were clearly higher than those obtained from sham-injected eyes, in the RPE + RPC cell-injected group than in the sham group, and this exhibiting a significantly higher b-wave and a significantly shorter im- significance was also observed at 12 weeks postinjection (Figure 5E). plicit time (Figures 5D5F). However, RPC-engrafted eyes did not In addition, the implicit time of the a-wave was significantly shorter in produce significantly better ERG signals at 4 weeks. Furthermore, all of the cell-injected groups compared to the sham group at all three eyes injected with the RPE + RPC combination showed a better time points assessed, indicating a faster response with the cell thera- b-wave response than eyes injected with RPE and PRC alone (Fig- pies (Figure 5G). Overall, the functional results obtained by ERG ure 5D). This significant improvement in the visual function by demonstrate the superiority of the combined RPE + RPC therapy combined therapy was consistent until 12 weeks postinjection. Never- over the RPE and the RPC cell therapies alone in preserving visual theless, the retinal signal of all animals inevitably decreased along this function over 12 weeks after transplantation. time period, likely due to the progressive retinal neurodegeneration of the model and the effect of immunosuppression with cyclosporin RPC and RPE cells integrate in the degenerating rat retina A, which has been shown to reduce visual response in RCS rats.40 Consistent with the OCT data, postmortem histological analyses of A-wave amplitudes at 4 weeks postinjection were significantly better eyes transplanted with RPE + RPC at 8 and 12 weeks revealed Molecular Therapy: Methods & Clinical Development Vol. 20 March 2021 695 Molecular Therapy: Methods & Clinical Development Figure 6. Postmortem analysis of RPE and RPC integration in the RCS rat retina (A) Hematoxylin and eosin of rat eye cryosections injected with sham (control), RPE cells, RPC, and RPE + RPC at 8 weeks (a) and 12 weeks (b) PI (n = 3 eyes/group). Scale bar, 50 mm. (B) Immunofluorescence analysis of Rho, blue opsin, RPE65, and Recov in sections of RCS rat retinas at 8 weeks PI with sham (control), RPE cells, RPC, and RPE + RPC cell suspensions. Scale bars, 25 mm. (C) Immunofluorescence staining with Rho, blue opsin, RPE65, and Recov in sections of RCS rat retinas at 12 weeks PI with sham (control), RPE, RPC, and RPE + RPC cell suspensions. Brackets indicate outer nuclear layer (ONL). Scale bars, 25 mm. Nuclei were stained with DAPI. (D) Quantification of ONL nuclei rows (top graph) and ONL thickness (bottom graph) in retinal paraffin sections (n = 35 eyes/group). Data are expressed as mean ± SD. *p < 0.01; ***p < 0.0005; **p < 0.001 versus 8 weeks; #p < 0.01; ##p < 0.001 versus control at 8 weeks; yp < 0.05; yyp < 0.00001 versus control at 12 weeks, calculated using Student’s t test. GCL, ganglion cell layer; INL, inner nuclear layer. extensive conservation of the photoreceptor layer that extended that engrafted RPE cells were distributed in GFP+ cell patches, similar around the injection site, and this was better compared to that of to what we observed in vivo, and exhibited a regular hexagonal the eyes transplanted with RPC or RPE cells only (Figure S3A). At morphology and tight junctions, shown by ZO-1 staining 8 weeks postinjection, we observed well-defined engrafted cell clusters (Figure S3B). containing pigmented cells in both RPE + RPC and RPE cell groups but not in the RPC group (Figure 6A, a). The rescue of the photore- We next assessed the integration of transplanted cells in the host ceptor layer was more manifest at 12 weeks postinjection (Figure 6A, retina. Analysis of the combined RPE + RPC therapy at 1 week post- b). Flat-mounted RPE layers of eyes injected with RPE + RPC showed injection showed that transplanted cells remained in the subretinal 696 Molecular Therapy: Methods & Clinical Development Vol. 20 March 2021 www.moleculartherapy.org Figure 7. Identification of RPE and RPC engrafted in the RCS rat retina (A) Immunostaining of retinal sections at 8 weeks PI with RPE showing engraftment of GFP+ RPE cells in the host RPE layer (n = 4 eyes). (a) Bright-field image corresponding to Figure 6B showing pigmented GFP+ RPE cells. (b) GFP+ RPE cells expressed RPE65. (c and c’) Costaining with Rho and opsin showing interaction of engrafted GFP+ RPE with endogenous photoreceptors. (B) Immunostaining of retinal sections at 8 weeks PI with RPC showing integration of RPC into the ONL (n = 4 eyes). (d and e) Staining with Recov. White dashed square in (d) indicates the area enlarged in (d’). (f) Magnification of Figure 6B showing that GFP+ RPC expressed Rho. (C) Immunostaining of retinal sections at 8 weeks PI with PRE + RPC (n = 5 eyes). (gj) Staining with Recov, RPE65, and human antigen Ku80. White arrow- heads indicate GFP+ RPC cells integrated to ONL in (d), (e), (f), (g), and (h). GFP+ RPE cells integrate into the host RPE layer as a discrete monolayer (i) or as clusters (g) and (j). Scale bars, 25 mm. GFP in green. Nuclei were stained with DAPI. space (Figure S4A), evidenced by GFP+ and Ku80+ cells (Figure S4B). of the ONL was considerably better in the RPE + RPC-treated group, Because of the assortment of RPE cells and RPC, some transplanted as shown by the expression of rhodopsin, opsin, and recoverin by cells expressed RPE65, and others expressed CRX and opsin, and endogenous photoreceptors (Figure 6C). only a few cells were proliferative (Ki67+), similar to what we observed at 8 weeks (Figure S3C). It has been described that retinas transplanted with photoreceptor cells expressing fluorescent reporters can exchange cellular material Notably, at 8 weeks postinjection, the conservation of the ONL was with host cells.41 In the RPC and combined RPE + RPC groups, we evident in all cell treatments in the cryosection, as shown by the also found some GFP signals in the inner nuclear layer and the gan- expression of recoverin, rhodopsin, and opsin (photoreceptor glion cell layer, probably due to material transfer to neighboring rat markers) in contrast to sham-injected retinas (Figure 6B). Most of cells (Figure S3D), a phenomenon that was not observed in the the transplanted RPE cells localized in the RPE layer, expressed RPE cell therapy group. RPE65, acquired pigmentation, and interacted with endogenous POS (Figures 6B, 6C, and 7, a, b, c, c’, and i). We also observed Although RCS rats were immunosuppressed during cell treatments, that GFP+ RPE cells formed clusters in the subretinal space and we observed activated microglia and Müller glial cells in the subretinal coexpressed RPE65 and Ku80, with no evidence of nuclei fragmen- space surrounding GFP+ cells, which was more evident in the com- tation (Figure 7, i and j). Most transplanted RPCs remained in the bined RPE + RPC therapy, as evidenced by Iba1 and glutamine syn- subretinal space (Figures 6B and 6C), but we observed some RPC thetase staining (Figure S5). It is worthy of mention that the retinal integrated into the ONL, coexpressing recoverin and extending their degeneration itself also triggered the activation of microglia and projections through the layers (Figure 7, dh). We also observed Müller glia cell in the subretinal space of sham-injected retinas that in sham-injected eyes, ONL thickness was reduced and con- (Figure S5). tained less rows of nuclei in the ONL, whereas RPE- and RPC- transplanted retinas maintained similar rows of nuclei and ONL Taken together, these results indicate that although most of the trans- thickness. In contrast, in RPE + RPC-transplanted retinas, ONL planted RPC remained in the subretinal space, the combined RPE + was thicker and contained more rows of nuclei (Figure 6D) (p < RPC grafts survived longer and better conserved the ONL than 0.001). RPE- or RPC-only engrafted cells in the 12-week study period. The number of GFP+ cells at 12 weeks postinjection was significantly DISCUSSION lower in all cell-transplanted eyes than in equivalent eyes studied at As part of our preclinical cell therapy studies, we successfully differ- 8 weeks (Figure 6C), which resulted in a progressive reduction of entiated hiPSC into RPE cells with phagocytic activity and developed ONL and retinal thickness (Figure 6D). However, the conservation a new differentiation protocol to obtain RPC expressing precursor Molecular Therapy: Methods & Clinical Development Vol. 20 March 2021 697 Molecular Therapy: Methods & Clinical Development photoreceptor markers. We established that the best RPC develop- (2) it allows the transplantation of an accurate cell number in a local- mental time window for transplantation in RCS rats at postnatal ized area; and (3) it enables a better isolation, characterization, and day 21 is days 45–75, as immature progenitors from an earlier stage cryopreservation, as well as quality control of the transplantable cell (day 21) were unable to preserve retinal degeneration or visual func- type. For therapeutic applications, the capacity of grafted cells trans- tion. Collectively, our data demonstrate for the first time that RPE planted subretinally to functionally integrate into the host retina relies cells and RPC in a combined therapy can preserve both endogenous on their survival and migratory capacity.48 In the majority of cases, photoreceptors and visual function in a manner superior to that using however, this type of transplantation leads to a low number of cells only RPE cells or RPC. The rationale for choosing a transplantation integrating into the correct layer, thus limiting the effectiveness of scheme combining RPE cells with RPC, both derived from hiPSC, the therapy. We show that although RPE cells and RPC survived up was based on our previous study,38 where we found that transplanta- to 12 weeks in the subretinal space and mainly exerted protective ef- tion of RPE cells into the RCS rat model of severe photoreceptor fects on endogenous photoreceptors, only a low number RPC and degeneration, caused by RPE phagocytosis dysfunction, was unable RPE cells integrated into the correct layer. There are several possible to halt photoreceptor-degenerative processes and preserve visual explanations for this. First, RCS rats exhibit severe photoreceptor loss, function. but the RPE cell layer remains mostly intact. Accordingly, trans- planted RPE cells may not have sufficient space to accommodate Several retinal cell types have been successfully transplanted into into the layer close to Bruch’s membrane. Second, transplantation wild-type or degenerative animal models, and most were cells ob- of RPC in retinas retaining endogenous photoreceptors may lead to tained from the neuroretinas of young animals.28,42 These studies low integration rates because of the presence of limiting membranes, show that neuroretinal cells can survive for long periods in the host as we showed at 1 week post-transplantation (Figure S4). Recent retina, integrate into the ONL, and mature as photoreceptors, form- studies indicate that transplantation of cell suspensions into retinas ing synaptic connections with bipolar cells,24 suggesting that the mul- lacking photoreceptors might be a better model to study photore- tipotent capacity of retinal progenitor cells is key to ensure the proper ceptor engraftment and synaptic connectivity with host bipolar formation of retinal structures, making them valuable for retinal cells.20,45 Other studies point toward the use of chemical modulators regeneration therapy. In terms of translating this approach to treat of glial response, such as matrix metalloproteinase 2 (MMP2) or ami- patients, fetal retinal tissue may present limitations, including tissue noadipic acid, to reduce physical impairment of limiting membranes availability and ethical acceptability. Accordingly, derivation of and to enhance donor retinal integration into the ONL49–53. Finally, retinal cells from hESC and hiPSC represents the most promising transplantation of RPC into a neurodegenerative environment has cell source for cell therapy. Several protocols have been used to several implications that can reduce transplanted cell survival and generate photoreceptors in 2D cultures and 3D retinal organoids integration, such as gliosis, inflammation, and activated immune from both hESC and hiPSC,23,28–30,33,43–46 yielding different popula- response. tions of postmitotic cone and rod photoreceptor precursors. Our dif- ferentiation protocol in 2D yielded a population of RPC consisting of Histological analyses of eyes transplanted with combined RPE + RPC an assortment of postmitotic precursors and more mature photore- revealed the preservation of retinal anatomy adjoining the engrafted ceptor cells at day 45 of differentiation. The expression profile of area and the rescue of photoreceptors, which correlates well with the photoreceptor genes in our retinal RPC cultures is very similar to functional data. Of note, the fact that the area of photoreceptor rescue that recently reported by Mellough et al.34 and reviewed by Llonch extended beyond the limits of the injection site suggests a non-cell- et al.,47 who described the highest peak of hESC-derived photorecep- autonomous trophic effect of transplanted cells on the survival of tors expressing OPSINSW and RHODOPSIN between days 40 and 60 host photoreceptors rather than their replacement. This was previ- of differentiation. The failure of transplanted immature RPCs to sur- ously observed by others,18,19 but the neuroprotective mechanism is vive in the host eye indicates that only postmitotic precursors may still unknown. Previous work by Klassen et al.54 found that mouse ret- survive and integrate.24 Indeed, we found that our retinal cells at inas transplanted with RPC showed preservation of host photorecep- day 21 of differentiation failed to exert a protective effect on host pho- tors by a rescue mechanism that was not previously observed after in- toreceptors or visual function. In this line, the absence of mitotically travitreal injections,55 indicating that for cell rescue purposes, active or undifferentiated cells is particularly important to prevent subretinal injections are more effective. In addition, material transfer teratoma formations. Although the retinal progenitors at day 21 did between cells has been proposed as a new alternative mechanism in not have any effect on host retinas, no neoplastic structures were which retinal cells can benefit.5 This novel concept is based on the ex- evident, suggesting a postmitotic state of differentiated RPC from change of cytoplasmic material between the engrafted retinal cells and that stage onward in differentiation. the host photoreceptors. Previous reports observed that the nonpho- toreceptor fraction of retinal progenitor cells presented limited capac- We optimized the trans-scleral route of administration, in the form of ity to integrate into the ONL upon transplantation and suggested that cell suspension, into the subretinal space of rat eyes to achieve high the material transfer observed from donor to host cells could be survival rates. This offers major advantages over other routes or cell photoreceptor-photoreceptor specific.24,46,56–58 This is in accordance sources: (1) it is technically easier and less traumatic than the trans- with our results using the combined RPE + RPC therapy, where we corneal route, the transplantation of RPE sheets, or retinal patches; observed some degree of material exchange with the rat retina,31 likely 698 Molecular Therapy: Methods & Clinical Development Vol. 20 March 2021 www.moleculartherapy.org due to RPCs rather than the RPE, since this phenomenon was not small clumps, and EBs were formed in conical-bottom 96-well plates evident in eyes injected with RPE cells alone. Thus, the fact that the in mTeSR1 medium (STEMCELL Technologies). After 48 h, EBs were combined therapy induced conservation of host ONL despite the transferred to low-attachment plates with retinal induction medium minimal integration of transplanted RPC, in combination with (RIM) containing DMEM: nutrient mixture F-12 (DMEM/F12; some cytoplasmic transfer, suggests that the efficacy of the combined Gibco), 10% KSR (Gibco), 0.1 mM nonessential amino acids (Gibco), therapy is partially due to a neuroprotective effect rather than func- 1% N2 (Gibco), 2% B27 (Gibco), supplemented with 10 ng/mL tional integration. Noggin (STEMCELL Technologies), 10 ng/mL DKK-1 (STEMCELL Technologies), 10 ng/mL IGF-1 (R&D Systems), and 5 ng/mL basic Conclusions fibroblast growth factor (bFGF; Millipore). At day 5, EBs were plated We show here the potential benefit of combined RPC and RPE cell onto Matrigel-coated plates (5–10 EBs per cm2) to facilitate rapid therapy in the RCS rat model of retinitis pigmentosa. Cells not only conversion into retinal progenitors43 and were maintained in RIM survived in the host retina but also were able to delay disease progres- without KSR, which was changed every other day. To enrich for sion and preserve visual function better than RPC or RPE therapies. retinal progenitors, neural rosettes were manually picked and In late-stage retinal degeneration, when the irreversible loss of photo- expanded on Matrigel-coated plates. From day 30, retinal progenitors receptors leads to blindness, the combined hiPSC-derived RPC and were cultured in RIM without KSR, supplemented with 10 ng/mL RPE cell therapy might represent a more feasible and complete option IGF-1, 20 mM taurine, and 500 nM all-trans retinoic acid (Sigma) un- for cell replacement than RPC or RPE therapy alone. til the end of the protocol. In addition, 10 mM g-secretase inhibitor, DAPT (Sigma), a Notch inhibitor, was added to media from days MATERIALS AND METHODS 28 to 32. Before day 45 of retinal differentiation, RPCs destined for Cell culture conditions subretinal injection were transduced with lentiviral particles carrying The hiPSC line CBiPS30-4F-5,59 which was previously used to suc- a GFP reporter gene (cell characterization is described in Supple- cessfully generate RPE cells,38 was obtained from the Spanish Stem mental materials and methods). Cell Bank upon Ethics Review Board and competent authority approval. Cells were cultured feeder free on Matrigel (Corning)- Animals coated plates and expanded in chemically defined mTeSR1 medium All procedures were approved by the Animal Care and Use Commit- (STEMCELL Technologies) at 37 C with 5% (v/v) CO2. The medium tee of the Vall d’Hebron Research Institute and were performed in was changed every other day until cells were ready for passaging. Col- accordance with the tenets of the European Community (86/609/ onies were detached using 0.5 mM EDTA (Thermo Fisher Scientific) CEE) and the Association for Research in Vision and Ophthalmology. for 2 min at room temperature. Animals used were 21- to 24-day-old dystrophic (rdy2/p+) RCS rats, which were maintained on a 12-h light/dark cycle with ad libitum hiPSC differentiation to RPE and RPCs food and water. To avoid human cell graft rejection, animals were im- Differentiation of CBiPS30-4F-5 hiPSC to RPE cells was performed as munosuppressed by oral gavage with cyclosporin A 20 mg/kg/day described.38 Briefly, hiPSC colonies on Matrigel (Corning)-coated (Novartis Pharma AG). Treatment was maintained throughout the plates were expanded in mTeSR1 medium (STEMCELL Technologies) entire experiment, starting 2 days before transplantation. until they reached 70% confluence. Retinal induction was performed by replacing mTeSR1 with RPE medium containing Dulbecco’s modified Surgery and transplantation Eagle’s medium (DMEM): nutrient mixture F-12 (DMEM/F12; Gibco) Before surgery, GFP-expressing differentiated RPE cells and RPCs with 10% knockout serum replacement (KSR; Gibco), 0.1 mM nones- were dissociated in TrypLE Select (Gibco), neutralized, and passed sential amino acids (Gibco), 1% N2 (Gibco), 2% B27 (Gibco), 0.1 mM through a 70-mm filter to remove cell clumps. Cells suspended in dexamethasone (Sigma), 10 mM b-glycerophosphate (Sigma), 20 ng/ serum-free DMEM/F12 medium at 5  104 cells/mL. Four groups mL human insulin growth factor (IGF)-1 (R&D Systems), and of RCS rats were used for a single-cell injection (2 mL in total) into 10 mM nicotinamide (Sigma), which were replenished every other the subretinal space at postnatal days 21–24 as follows (see Table 1): day. After 6 weeks of differentiation, visible pigmented foci were manu- group 1, transplantation of 100,000 RPE cells; group 2, cotransplan- ally isolated and expanded in RPE medium on Matrigel-coated plates. tation of 50,000 RPE cells and 50,000 RPC mixture (note that in For purification and expansion, RPE cultures were treated with 0.05% experiment #1, two additional animal groups were included for pilot trypsin (Gibco) for 2 min to remove fibroblast-like cells, and then re- studies of transplantation of RPE + RPC at different time points of the maining attached RPE cells were disaggregated using 0.25% trypsin differentiation process to assess the best option); group 3, transplan- and cultured in monolayers. For all experiments, hiPSC-RPE cells tation of 100,000 RPC at days 4550; and group 4, injection of me- expanded 2–4 times and were frozen in 10% DMSO in fetal bovine dium as a control (sham). Animals were anesthetized with a mixture serum medium. Lentiviral transduction and cell characterization are of 2% isoflurane (Arrane; Baxter Laboratories)/1% O2 and main- described in Supplemental materials and methods. tained with a rat nasal mask. Pupils were dilated with tropicamide (10 mg/mL Colircusi Tropicamida; Alcon Cusí Laboratories). Under Retinal differentiation was based on a published protocol39 with slight a surgical microscope, simple sutures were performed in the upper modifications. When confluent, hiPSC colonies were dissociated into eyelid and upper limbal conjunctiva with Prolene 7/0 Ethicon Molecular Therapy: Methods & Clinical Development Vol. 20 March 2021 699 Molecular Therapy: Methods & Clinical Development (Johnson & Johnson) in order to expose the superior bulbar conjunc- trode), the head (reference electrode), and the cornea, located in tiva, and a 2-mm sclerotomy was performed using a 20G blade, 4 mm the objective lens (corneal electrode). Scotopic retinal responses from the limbus. 2 mL of cell suspension was loaded into a 32G blunt were recorded using light flashes (1 ms duration, light intensities needle attached to a 10-mL Nanofil syringe (World Precision Instru- ranging from 1.1 to 1.9 log cd $ s $ m2, with 10 sweeps and ments), which was introduced tangentially into the subretinal space 10- to 60-s intervals, depending on the intensity). Waveforms through the sclerotomy. Finally, the needle and securing sutures were analyzed using LabScribe ERG software (Phoenix Research were carefully removed, and a drop of tobramycin and dexametha- Laboratories), and the a- and b-wave amplitudes and latencies sone (Tobradex 3 mg/mL + 1 mg/mL; Alcon Cusí Laboratories) were determined. was topically administered just after surgery as a local anti-inflamma- tory and antibiotic prophylaxis. Those eyes that did not present an Histology and immunochemistry evident fluorescent bleb in the subretinal space assessed by fundus im- RPE flat mounts aging and OCT were excluded from the study. At 8 and 12 weeks postinjection, eyes were enucleated and dissected in PBS to remove cornea, lens, and retina. The posterior In vivo analyses eyecups were fixed in 4% (w/v) paraformaldehyde (PFA) for 1 h, Animals were maintained over 12 weeks, and structural and func- rinsed 3 times for 10 min with phosphate-buffered saline (PBS), tional analyses were performed periodically by FFI, OCT, and Ganz- and flattened by making four radial incisions. For detection of feld ERG using the Micron III platform (Phoenix Research Labora- ZO-1, eyecups were subjected to immunochemistry, as described tories). For all procedures, animals were anesthetized with inhaled below. 2% isofluorane and placed on a heating pad at 37 C. Pupils were dilated with a mixture of tropicamide and phenylephrine (100 mg/ Histology mL Colircusí Fenilefrina; Alcon Cusí Laboratories), and 2% Methocel At 1, 8, or 12 weeks postinjection, eyes were enucleated and fixed gel (OmniVision) was administered to the cornea to favor contact in 4% PFA overnight at 4 C. After 3 rinses with PBS, eyes were with the lens. subjected to dehydration using a successive sucrose concentration as follows: 15% for 30 min, 20% for 60 min, and 30% overnight, Color fundus and fluorescent images were taken at the day of sur- all at 4 C. Eyes were embedded in optimum cutting temperature gery and 1, 2, 4, 8, and 12 weeks postinjection using the Micron III compound (Tissue-Tek), frozen, and stored at 80 C. Thin serial imaging microscope (Phoenix Research Laboratories). RPE and sections of 10 mm were cut using a Cryostat microtome (Leica) RPC grafts were identified as green fluorescent spots in the eye and collected on Superfrost glass slides. Transversal cryosections fundus under the retina. Images from different time points in the were stained with hematoxylin and eosin by standard protocols. same animal were compared to monitor the presence of the trans- Images were acquired on an FSX100 microscope (Olympus Life planted cells. Following FFI and with the animal still anesthetized, Sciences). OCT images were taken by image-guided tomography (Micron IV-OCT2; Phoenix Research Laboratories). For each eye, the scan- Immunohistochemistry ner was placed over the injected area, and three images were taken Cryosections and RPE flat mounts were washed in PBS and permea- near the injection point. One image was also taken in a noninjected bilized and blocked in PBS containing 0.5% Triton X-100 and 6% area at the opposite site of the eye. Finally, the images were analyzed normal donkey serum (NDS; Millipore) for 1 h at room temperature. with InSight 3D Voxeleron software for the quantitative assessment Primary and secondary antibodies (see Table S2) were diluted in 0.1% of whole retina thickness and the photoreceptor layer. The percent- Triton X-100 and 6% NDS in PBS and incubated in a humidified age of preservation of total retina thickness and photoreceptor layer chamber overnight at 4 C or 2 h at 37 C, respectively. Slides were thickness in Figure 4 was calculated by applying the following for- mounted in mounting medium containing 40 ,6-diamidino-2-phenyl- mula, using the values of a noninjected area and the injected area of indole (DAPI), and images were obtained on a DMI6000 confocal mi- the same eye: croscopy (Leica Microsystems). ðinjected area thicknessÞ  ðnon injected area thicknessÞ % preservation =  100: non injected area thickness Functional responses of the retinas were recorded at 4, 8, and Statistics 12 weeks postinjection using the Phoenix Ganzfeld ERG system All quantitative data were analyzed using GraphPad Prism software (Phoenix Research Laboratories). Dark-adapted rats (12–16 h) (GraphPad Software). The unpaired two-tailed Student’s t-test was were anesthetized under dim red light, and pupils were dilated as applied to determine statistical significance between 2 groups. For described. Three electrodes were placed on the tail (ground elec- more than 2 groups, 1- or 2-way ANOVA with a Tukey-corrected 700 Molecular Therapy: Methods & Clinical Development Vol. 20 March 2021 www.moleculartherapy.org post hoc test was used. Statistical significance was considered at <0.05 9. Mandai, M., Watanabe, A., Kurimoto, Y., Hirami, Y., Morinaga, C., Daimon, T., Fujihara, M., Akimaru, H., Sakai, N., Shibata, Y., et al. (2017). Autologous Induced with a confidence interval of 95%. Stem-Cell-Derived Retinal Cells for Macular Degeneration. N. Engl. J. Med. 376, 1038–1046. SUPPLEMENTAL INFORMATION 10. Schwartz, S.D., Hubschman, J.-P., Heilwell, G., Franco-Cardenas, V., Pan, C.K., Supplemental Information can be found online at https://doi.org/10. Ostrick, R.M., Mickunas, E., Gay, R., Klimanskaya, I., and Lanza, R. (2012). 1016/j.omtm.2021.02.006. Embryonic stem cell trials for macular degeneration: a preliminary report. Lancet 379, 713–720. 11. Arai, S., Thomas, B.B., Seiler, M.J., Aramant, R.B., Qiu, G., Mui, C., de Juan, E., and ACKNOWLEDGMENTS Sadda, S.R. (2004). Restoration of visual responses following transplantation of intact The authors thank Lola Mulero, Begoña Aran, and Bernd Kuebler retinal sheets in rd mice. Exp. Eye Res. 79, 331–341. (Core Facilities and Stem Cell Bank at P-CMR[C]-IDIBELL) for tech- 12. Ghosh, F., Wong, F., Johansson, K., Bruun, A., and Petters, R.M. (2004). nical assistance. We thank CERCA Program/Generalitat de Catalu- Transplantation of full-thickness retina in the rhodopsin transgenic pig. Retina 24, nya for institutional support. Histological processing was performed 98–109. by the ICTS-NANBIOSIS-U20/FVPR at the Vall d’Hebron Institute 13. Aramant, R.B., and Seiler, M.J. (2002). Transplanted sheets of human retina and of Research (VHIR). The data that support the findings of this study retinal pigment epithelium develop normally in nude rats. Exp. Eye Res. 75, 115–125. are available in the Supplemental information. This research project was funded by grants from La Marató de TV3 Foundation (484/C/ 14. Silverman, M.S., Hughes, S.E., Valentino, T.L., and Liu, Y. (1992). Photoreceptor transplantation: anatomic, electrophysiologic, and behavioral evidence for the 2012), PRB2-ISCIII-SGEFI- FEDER-PE I+D+i 2013-2016 (PT13/ functional reconstruction of retinas lacking photoreceptors. Exp. Neurol. 115, 0001/0041), and ISCIII-FEDER RETICS (Oftared; RD16/0008). 87–94. 15. Liu, Y., Chen, S.J., Li, S.Y., Qu, L.H., Meng, X.H., Wang, Y., Xu, H.W., Liang, Z.Q., and AUTHOR CONTRIBUTIONS Yin, Z.Q. (2017). Long-term safety of human retinal progenitor cell transplantation in retinitis pigmentosa patients. Stem Cell Res. Ther. 8, 209. Conception and design, collection and assembly of data, data analysis and interpretation, and manuscript writing, A.S., A.D., and L.F.; 16. Luo, J., Baranov, P., Patel, S., Ouyang, H., Quach, J., Wu, F., Qiu, A., Luo, H., Hicks, C., Zeng, J., et al. (2014). Human retinal progenitor cell transplantation preserves collection and assembly of data, D.M.R., A.B., B.F.-d.-S., M.A.Z., vision. J. Biol. Chem. 289, 6362–6371. and A.R.; conception and design, financial support, and final approval 17. Klassen, H., Kiilgaard, J.F., Warfvinge, K., Samuel, M.S., Prather, R.S., Wong, F., of manuscript, A.V. and J.G.-A.; final approval of manuscript, A.D., Petters, R.M., la Cour, M., and Young, M.J. (2012). Photoreceptor Differentiation A.V., and J.G.-A. following Transplantation of Allogeneic Retinal Progenitor Cells to the Dystrophic Rhodopsin Pro347Leu Transgenic Pig. Stem Cells Int. 2012, 939801. DECLARATION OF INTERESTS 18. Cuenca, N., Fernández-Sánchez, L., McGill, T.J., Lu, B., Wang, S., Lund, R., Huhn, S., The authors declare no competing interests. and Capela, A. (2013). Phagocytosis of photoreceptor outer segments by transplanted human neural stem cells as a neuroprotective mechanism in retinal degeneration. Invest. Ophthalmol. Vis. Sci. 54, 6745–6756. REFERENCES 19. Tsai, Y., Lu, B., Bakondi, B., Girman, S., Sahabian, A., Sareen, D., Svendsen, C.N., and 1. Pascolini, D., and Mariotti, S.P. (2012). Global estimates of visual impairment: 2010. Wang, S. (2015). Human iPSC-Derived Neural Progenitors Preserve Vision in an Br. J. Ophthalmol. 96, 614–618. AMD-Like Model. Stem Cells 33, 2537–2549. 2. (1995). Simple anatomy of the retina. In Webvision: The Organization of the Retina 20. Collin, J., Zerti, D., Queen, R., Santos-Ferreira, T., Bauer, R., Coxhead, J., Hussain, R., and Visual System, H. Kolb, E. Fernandez, and R. Nelson, eds. (University of Utah Steel, D., Mellough, C., Ader, M., et al. (2019). CRX Expression in Pluripotent Stem Health Sciences Center). Cell-Derived Photoreceptors Marks a Transplantable Subpopulation of Early Cones. 3. Strauss, O. (2005). The retinal pigment epithelium in visual function. Physiol. Rev. 85, Stem Cells 37, 609–622. 845–881. 21. Tucker, B.A., Park, I.-H., Qi, S.D., Klassen, H.J., Jiang, C., Yao, J., Redenti, S., Daley, 4. Hamon, A., Roger, J.E., Yang, X.-J., and Perron, M. (2016). Müller glial cell-depen- G.Q., and Young, M.J. (2011). Transplantation of adult mouse iPS cell-derived photo- dent regeneration of the neural retina: An overview across vertebrate model systems. receptor precursors restores retinal structure and function in degenerative mice. PLoS Dev. Dyn. 245, 727–738. ONE 6, e18992. 22. Santos-Ferreira, T., Postel, K., Stutzki, H., Kurth, T., Zeck, G., and Ader, M. (2015). 5. Gasparini, S.J., Llonch, S., Borsch, O., and Ader, M. (2019). Transplantation of pho- Daylight vision repair by cell transplantation. Stem Cells 33, 79–90. toreceptors into the degenerative retina: Current state and future perspectives. Prog. Retin. Eye Res. 69, 1–37. 23. Lamba, D.A., Gust, J., and Reh, T.A. (2009). Transplantation of human embryonic stem cell-derived photoreceptors restores some visual function in Crx-deficient 6. Zarbin, M., Sugino, I., and Townes-Anderson, E. (2019). Concise Review: Update on mice. Cell Stem Cell 4, 73–79. Retinal Pigment Epithelium Transplantation for Age-Related Macular Degeneration. Stem Cells Transl. Med. 8, 466–477. 24. MacLaren, R.E., Pearson, R.A., MacNeil, A., Douglas, R.H., Salt, T.E., Akimoto, M., Swaroop, A., Sowden, J.C., and Ali, R.R. (2006). Retinal repair by transplantation 7. Schwartz, S.D., Regillo, C.D., Lam, B.L., Eliott, D., Rosenfeld, P.J., Gregori, N.Z., of photoreceptor precursors. Nature 444, 203–207. Hubschman, J.-P., Davis, J.L., Heilwell, G., Spirn, M., et al. (2015). Human embryonic stem cell-derived retinal pigment epithelium in patients with age-related macular 25. Pearson, R.A., Barber, A.C., Rizzi, M., Hippert, C., Xue, T., West, E.L., Duran, Y., degeneration and Stargardt’s macular dystrophy: follow-up of two open-label phase Smith, A.J., Chuang, J.Z., Azam, S.A., et al. (2012). Restoration of vision after trans- 1/2 studies. Lancet 385, 509–516. plantation of photoreceptors. Nature 485, 99–103. 8. Song, W.K., Park, K.-M., Kim, H.-J., Lee, J.H., Choi, J., Chong, S.Y., Shim, S.H., Del 26. Singh, M.S., Charbel Issa, P., Butler, R., Martin, C., Lipinski, D.M., Sekaran, S., Priore, L.V., and Lanza, R. (2015). Treatment of macular degeneration using embry- Barnard, A.R., and MacLaren, R.E. (2013). Reversal of end-stage retinal degeneration onic stem cell-derived retinal pigment epithelium: preliminary results in Asian pa- and restoration of visual function by photoreceptor transplantation. Proc. Natl. Acad. tients. Stem Cell Reports 4, 860–872. Sci. USA 110, 1101–1106. Molecular Therapy: Methods & Clinical Development Vol. 20 March 2021 701 Molecular Therapy: Methods & Clinical Development 27. Lakowski, J., Gonzalez-Cordero, A., West, E.L., Han, Y.-T., Welby, E., Naeem, A., 43. Boucherie, C., Mukherjee, S., Henckaerts, E., Thrasher, A.J., Sowden, J.C., and Ali, Blackford, S.J.I., Bainbridge, J.W.B., Pearson, R.A., Ali, R.R., and Sowden, J.C. R.R. (2013). Brief report: self-organizing neuroepithelium from human pluripotent (2015). Transplantation of Photoreceptor Precursors Isolated via a Cell Surface stem cells facilitates derivation of photoreceptors. Stem Cells 31, 408–414. Biomarker Panel From Embryonic Stem Cell-Derived Self-Forming Retina. Stem 44. Eiraku, M., Takata, N., Ishibashi, H., Kawada, M., Sakakura, E., Okuda, S., Sekiguchi, Cells 33, 2469–2482. K., Adachi, T., and Sasai, Y. (2011). Self-organizing optic-cup morphogenesis in 28. Lakowski, J., Baron, M., Bainbridge, J., Barber, A.C., Pearson, R.A., Ali, R.R., and three-dimensional culture. Nature 472, 51–56. Sowden, J.C. (2010). Cone and rod photoreceptor transplantation in models of the 45. Mandai, M., Fujii, M., Hashiguchi, T., Sunagawa, G.A., Ito, S.-I., Sun, J., Kaneko, J., childhood retinopathy Leber congenital amaurosis using flow-sorted Crx-positive Sho, J., Yamada, C., and Takahashi, M. (2017). iPSC-Derived Retina Transplants donor cells. Hum. Mol. Genet. 19, 4545–4559. Improve Vision in rd1 End-Stage Retinal-Degeneration Mice. Stem Cell Reports 8, 29. Banin, E., Obolensky, A., Idelson, M., Hemo, I., Reinhardtz, E., Pikarsky, E., Ben-Hur, 1112–1113. T., and Reubinoff, B. (2006). Retinal incorporation and differentiation of neural pre- 46. Santos-Ferreira, T., Llonch, S., Borsch, O., Postel, K., Haas, J., and Ader, M. (2016). cursors derived from human embryonic stem cells. Stem Cells 24, 246–257. Retinal transplantation of photoreceptors results in donor-host cytoplasmic ex- 30. Barnea-Cramer, A.O., Wang, W., Lu, S.-J., Singh, M.S., Luo, C., Huo, H., change. Nat. Commun. 7, 13028. McClements, M.E., Barnard, A.R., MacLaren, R.E., and Lanza, R. (2016). Function 47. Llonch, S., Carido, M., and Ader, M. (2018). Organoid technology for retinal repair. of human pluripotent stem cell-derived photoreceptor progenitors in blind mice. Dev. Biol. 433, 132–143. Sci. Rep. 6, 29784. 48. Warre-Cornish, K., Barber, A.C., Sowden, J.C., Ali, R.R., and Pearson, R.A. (2014). 31. Gonzalez-Cordero, A., Kruczek, K., Naeem, A., Fernando, M., Kloc, M., Ribeiro, J., Migration, integration and maturation of photoreceptor precursors following trans- Goh, D., Duran, Y., Blackford, S.J.I., Abelleira-Hervas, L., et al. (2017). plantation in the mouse retina. Stem Cells Dev. 23, 941–954. Recapitulation of Human Retinal Development from Human Pluripotent Stem Cells Generates Transplantable Populations of Cone Photoreceptors. Stem Cell 49. Ma, J., Kabiel, M., Tucker, B.A., Ge, J., and Young, M.J. (2011). Combining chondroi- Reports 9, 820–837. tinase ABC and growth factors promotes the integration of murine retinal progenitor cells transplanted into Rho(-/-) mice. Mol. Vis. 17, 1759–1770. 32. Hambright, D., Park, K.-Y., Brooks, M., McKay, R., Swaroop, A., and Nasonkin, I.O. (2012). Long-term survival and differentiation of retinal neurons derived from hu- 50. Yao, J., Tucker, B.A., Zhang, X., Checa-Casalengua, P., Herrero-Vanrell, R., and man embryonic stem cell lines in un-immunosuppressed mouse retina. Mol. Vis. Young, M.J. (2011). Robust cell integration from co-transplantation of biodegradable 18, 920–936. MMP2-PLGA microspheres with retinal progenitor cells. Biomaterials 32, 1041– 33. Lamba, D.A., McUsic, A., Hirata, R.K., Wang, P.-R., Russell, D., and Reh, T.A. (2010). 1050. Generation, purification and transplantation of photoreceptors derived from human 51. Suzuki, T., Mandai, M., Akimoto, M., Yoshimura, N., and Takahashi, M. (2006). The induced pluripotent stem cells. PLoS ONE 5, e8763. simultaneous treatment of MMP-2 stimulants in retinal transplantation enhances 34. Mellough, C.B., Sernagor, E., Moreno-Gimeno, I., Steel, D.H.W., and Lako, M. (2012). grafted cell migration into the host retina. Stem Cells 24, 2406–2411. Efficient stage-specific differentiation of human pluripotent stem cells toward retinal 52. Yu, W.-Q., Eom, Y.S., Shin, J.-A., Nair, D., Grzywacz, S.X.Z., Grzywacz, N.M., Craft, photoreceptor cells. Stem Cells 30, 673–686. C.M., and Lee, E.-J. (2016). Reshaping the Cone-Mosaic in a Rat Model of Retinitis 35. Gal, A., Li, Y., Thompson, D.A., Weir, J., Orth, U., Jacobson, S.G., Apfelstedt-Sylla, E., Pigmentosa: Modulatory Role of ZO-1 Expression in DL-Alpha-Aminoadipic Acid and Vollrath, D. (2000). Mutations in MERTK, the human orthologue of the RCS rat Reshaping. PLoS ONE 11, e0151668. retinal dystrophy gene, cause retinitis pigmentosa. Nat. Genet. 26, 270–271. 53. Johnson, T.V., Bull, N.D., and Martin, K.R. (2010). Identification of barriers to 36. Ryals, R.C., Andrews, M.D., Datta, S., Coyner, A.S., Fischer, C.M., Wen, Y., Pennesi, retinal engraftment of transplanted stem cells. Invest. Ophthalmol. Vis. Sci. 51, M.E., and McGill, T.J. (2017). Long-term Characterization of Retinal Degeneration in 960–970. Royal College of Surgeons Rats Using Spectral-Domain Optical Coherence 54. Klassen, H.J., Ng, T.F., Kurimoto, Y., Kirov, I., Shatos, M., Coffey, P., and Young, M.J. Tomography. Invest. Ophthalmol. Vis. Sci. 58, 1378–1386. (2004). Multipotent retinal progenitors express developmental markers, differentiate 37. Perlman, I. (1978). Dark-adaptation in abnormal (RCS) rats studied electroretino- into retinal neurons, and preserve light-mediated behavior. Invest. Ophthalmol. Vis. graphically. J. Physiol. 278, 161–175. Sci. 45, 4167–4173. 38. Riera, M., Fontrodona, L., Albert, S., Ramirez, D.M., Seriola, A., Salas, A., Muñoz, Y., 55. Eberle, D., Kurth, T., Santos-Ferreira, T., Wilson, J., Corbeil, D., and Ader, M. (2012). Ramos, D., Villegas-Perez, M.P., Zapata, M.A., et al. (2016). Comparative study of hu- Outer segment formation of transplanted photoreceptor precursor cells. PLoS ONE 7, man embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) e46305. as a treatment for retinal dystrophies. Mol. Ther. Methods Clin. Dev. 3, 16010. 56. Pearson, R.A., Gonzalez-Cordero, A., West, E.L., Ribeiro, J.R., Aghaizu, N., Goh, D., 39. Lamba, D.A., Karl, M.O., Ware, C.B., and Reh, T.A. (2006). Efficient generation of Sampson, R.D., Georgiadis, A., Waldron, P.V., Duran, Y., et al. (2016). Donor and retinal progenitor cells from human embryonic stem cells. Proc. Natl. Acad. Sci. host photoreceptors engage in material transfer following transplantation of post- USA 103, 12769–12774. mitotic photoreceptor precursors. Nat. Commun. 7, 13029. 40. Adachi, K., Takahashi, S., Yamauchi, K., Mounai, N., Tanabu, R., and Nakazawa, M. 57. Decembrini, S., Martin, C., Sennlaub, F., Chemtob, S., Biel, M., Samardzija, M., (2016). Optical Coherence Tomography of Retinal Degeneration in Royal College of Moulin, A., Behar-Cohen, F., and Arsenijevic, Y. (2017). Cone Genesis Tracing by Surgeons Rats and Its Correlation with Morphology and Electroretinography. PLoS the Chrnb4-EGFP Mouse Line: Evidences of Cellular Material Fusion after Cone ONE 11, e0162835. Precursor Transplantation. Mol. Ther. 25, 634–653. 41. Ortin-Martinez, A., Tsai, E.L.S., Nickerson, P.E., Bergeret, M., Lu, Y., Smiley, S., 58. Singh, M.S., Balmer, J., Barnard, A.R., Aslam, S.A., Moralli, D., Green, C.M., Barnea- Comanita, L., and Wallace, V.A. (2017). A Reinterpretation of Cell Cramer, A., Duncan, I., and MacLaren, R.E. (2016). Transplanted photoreceptor Transplantation: GFP Transfer From Donor to Host Photoreceptors. Stem Cells precursors transfer proteins to host photoreceptors by a mechanism of cytoplasmic 35, 932–939. fusion. Nat. Commun. 7, 13537. 42. Bartsch, U., Oriyakhel, W., Kenna, P.F., Linke, S., Richard, G., Petrowitz, B., 59. Giorgetti, A., Montserrat, N., Aasen, T., Gonzalez, F., Rodríguez-Pizà, I., Vassena, R., Humphries, P., Farrar, G.J., and Ader, M. (2008). Retinal cells integrate into the outer Raya, A., Boué, S., Barrero, M.J., Corbella, B.A., et al. (2009). Generation of induced nuclear layer and differentiate into mature photoreceptors after subretinal transplan- pluripotent stem cells from human cord blood using OCT4 and SOX2. Cell Stem Cell tation into adult mice. Exp. Eye Res. 86, 691–700. 5, 353–357. 702 Molecular Therapy: Methods & Clinical Development Vol. 20 March 2021