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Review Articles

New Prospects for Retinal Pigment Epithelium Transplantation

Gullapalli, Vamsi K. MBBS. PhD*; Zarbin, Marco A. MD, PhD

Author Information
Asia-Pacific Journal of Ophthalmology: July/August 2022 - Volume 11 - Issue 4 - p 302-313
doi: 10.1097/APO.0000000000000521
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Abstract

In situ, retinal pigment epithelium (RPE) is a monolayer of polarized cells resting on Bruch membrane and situated between the overlying neurosensory retina and the subjacent vascular choroid. RPE performs essential functions that maintain normal outer retinal physiology.1 During ocular development, RPE is necessary for photoreceptor differentiation and outer segment disc formation2 and plays a role in melanocyte differentiation and vascular development of the choroid. In a fully developed eye, the tight junctions between the RPE cells comprise the outer blood-retinal barrier. The cyclically shed photoreceptor outer segments are phagocytosed by the RPE.3 The all-trans retinol generated during the visual cycle is converted to 11-cis retinal in the RPE and transported back to the photoreceptors.4 RPE also secretes numerous growth factors [eg, pigment epithelium-derived growth factor, vascular endothelial growth factor (VEGF), transforming growth factor–β (TGF–β), ciliary neurotrophic factor] that play an important role in maintaining the physiology of the photoreceptor–RPE–choriocapillaris complex. Active transport of fluid by RPE is the major force eliminating water from subretinal space thereby contributing to the adhesiveness of neural retina.6 In addition to creating a blood-retinal barrier, RPE actively contributes to the immune privilege of the subretinal space, creating an immune suppressive microenvironment by secreting immunomodulatory factors such as TGF–β, suppressing T-cell activation, and inducing T-cell apoptosis.7,8

RPE loss or dysfunction is associated with photoreceptor and choriocapillaris degeneration, which occurs in late-stage age-related macular degeneration (AMD) and Stargardt disease (STGD1). Late complications of AMD, that is, geographic atrophy (GA) and macular neovascularization (MNV), are the major causes of irreversible loss of central vision among the elderly.911 The prevalence of AMD-associated MNV or GA is approximately 1.47%, increasing to 10% in people older than 80 years.10 Currently, intravitreal injections of anti-VEGF antibodies at frequent intervals are the best available treatment for MNVs, but only 30% to 40% of the patients experience moderate visual improvement.1214 This treatment does not eliminate the MNV in most cases. Instead, it reduces leakage of fluid and blood from the MNV, with stabilization or improvement in vision. Rarely, patients receiving anti-VEGF for fibrovascular RPE detachments secondary to MNV may develop a tear in the RPE layer and subsequent contracture of the RPE resulting in an area devoid of RPE and, in most cases, profoundly decreased vision.15,16 Currently, there is no treatment for GA approved by regulatory authorities, although some promising results have been reported using complement inhibitors. Stargardt disease is the most common cause of macular degeneration in younger adults and children, although a subset of patients develops the disease after the age of 45.17 Sequence variants in ABCA4 gene result in RPE loss.18 ABCA4 gene encodes a glycoprotein called Rim protein (also termed ABCA4 or ABCR) that is present on membranes of the discs stacked in the photore-ceptor outer segment.19 Pathogenic ABCA4 mutations result in the accumulation of di-retinoid-pyridinium-ethanolamine in the photoreceptors, and these by-products are phagocytized by the RPE during outer segment disc phagocytosis.20 The intracellular accumulation of these photosensitive compounds is believed to result in progressive RPE death and secondary degeneration of adjacent photoreceptors.21 Replacing with healthy RPE in these patients could potentially slow the atrophy and potentially stabilize or improve vision.

The RPE monolayer would seem to be easy to replace, as the RPE apical villous processes spontaneously envelop and phagocytize adjacent photoreceptor outer segments. Thus, integration between host retina and transplanted RPE occurs spontaneously in contrast to the lack of robust synaptic integration that typically is observed after transplantation of donor photoreceptors into host retina. The goal of RPE transplantation is to replace RPE cells that are lost during the course of disease due to atrophy (eg, in GA or STGD1) or due to complications of MNV (ie, RPE tears).22 Because RPE cells produce numerous factors, RPE transplants may help maintain normal retinal and choroidal anatomy and physiology even in an abnormal microenvironment.12,2326 Cell replacement or cell therapeutics in the context of degenerative retinal diseases refers to replacing degenerating retinal cells with healthy ones or placement of nonretinal cells to help generate trophic factors to rescue dying retinal cells. Numerous preclinical studies have paved the path, and there are excellent reviews on various aspects of this field.2730 This concise review will focus on some of the early studies leading up to the published and ongoing human clinical trials, donor cells and scaffolds used for RPE replacement, immune suppression to improve survival, and finally, some challenges that RPE replacement faces. New developments will be discussed under each of these sections.

Lessons from Early Clinical Studies of RPE Transplants

Peyman and coworkers31 first reported human RPE transplants in AMD patients in 1991. Subsequently, numerous reports have described the results of allogeneic and autologous RPE transplantation.3243 These surgeries have involved MNV excision and placement of suspended allogeneic RPE cells,31 autologous RPE cells,36,42 patches of RPE,33,43 or autografts of RPE-choroid.36,39,40,42,44,45 RPE transplantation has not been successful in the majority of patients undergoing surgery in these studies, but a few patients demonstrated moderate visual improvement [eg, ≥2 lines on an Early Treatment Diabetic Retinopathy Study (ETDRS) visual acuity chart] following autografts with some even showing vision of 20/80 or better.36,38,39,41,42,44 It is important to note that most patients undergoing RPE transplants in these studies had advanced photoreceptor degeneration, so marked visual improvement after surgery was not expected.

Allogeneic RPE transplants failed when placed in conjunction with MNV excision. Immune rejection of RPE transplants resulted in subretinal fibrosis and chronic fluid leakage in the dissection bed.32–34 In autologous transplants, graft failure was due to intraoperative and postoperative subretinal hemorrhage, failure of graft revascularization, fibrous encapsulation of the graft, and development of epiretinal membrane, proliferative vitreoretinopathy, and retinal detachment.38,39,41

Preclinical models of RPE transplantation may provide some insight into the basis of transplant failure. RPE cells transplanted as a suspension, for example, need to adhere to their basement membrane within 24 hours, otherwise, they will undergo apoptosis (anoikis).46 RPE survival improves when implanted as a monolayer vs as a cell suspension.47,48 Also, RPE suspensions typically show only sparse areas of monolayer formation with many areas exhibiting multilayering, which typically does not support photoreceptor survival well. Successful RPE transplants in laboratory animals have involved transplantation onto normal Bruch membrane or onto native RPE.4955 Bruch membrane is abnormal in AMD,56–59 which may hinder proper attachment and resurfacing.6063

In summary, the early studies indicated that for RPE transplants to succeed, healthy, phenotypically, and genotypically stable, readily and ethically available donor RPE cells need to be implanted as a monolayer with minimal surgical trauma to the host tissue. The degree of retinal atrophy in the host should not be extensive, and, finally, immune reaction to the graft should be modulated if the transplant is allogeneic.

Donor Cells for RPE Transplantation

Human adult donor eyes can be a source, either as isolated RPE sheets or after cell culture. However, access to donor tissue within a brief time frame after donor death, variability in the viability of the harvested cells due to donor age and comorbidities, lack of consistent and uniform growth of polarized, fully functional cells in culture, and inconsistent survival on aged human Bruch membrane precludes their use.60 Cultures of RPE isolated from donor adult human eyes typically yield low numbers of viable RPE in cell culture. Fortunately, RPE isolated from human donor eyes has been shown to harbor a subpopulation of cells that can exhibit stem celllike properties of self-renewal and multipotency.6466 Monolayers of these cells on polyester scaffolds survived in the subretinal space of rabbit eyes67 and cynomolgus monkeys.68 Injection of these cells into Royal College of Surgeons rats, which exhibit photoreceptor loss due to impaired phagocytosis of outer segments by RPE, results in photoreceptor rescue.69 No human trials have been conducted using these cells yet. RPE from fetal eyes can grow robustly in culture, but ethical issues limit their use.

Other potential sources afford stable and readily available RPE cells for transplantation. Stem cells are undifferentiated cells that have the capacity for self-renewal (ie, can divide indefinitely to produce more karyotypically stable stem cells) and pluripotency (ie, produce cells that are destined to differentiate into more than 1 type of cell).70,71 During the course of differentiation, stem cells form intermediate populations of increasingly committed progenitor cells that have a decreasing proliferative capacity. For example, pluripotent cells can differentiate into tissues that derive from any of the 3 germ layers-ectoderm, mesoderm, and endoderm. Multipotent cells, on the other hand, can develop into cell types from a single germ layer. Embryonic stem cells (ESCs) are derived from the inner cell mass of the 5-day old blastocyst and are capable of differentiating into cells of all 3 germ layers.72,73 Initial cultures of RPE derived from ESCs (ESC-RPE) were described by Kilmanskaya and Chung.7476 These cells exhibited the molecular characteristics of RPE including expression of RPE65, pigment epithelium-derived growth factor, cellular retinaldehyde binding protein, and phagocytosis of rod outer segments.74

ESCs can be generated by somatic nuclear transfer from an adult, neonatal, or fetal somatic cell into an unfertilized oocyte, whose nucleus has been removed.7779 The oocyte reprograms the donor nuclear DNA, develops in a normal embryonic pattern, and forms embryonic stem cells (NT-ESCs). Genetically matched cell lines for autologous transplants might be created with this approach.80 Efforts to improve the efficiency of production and stability of the pluripotent cells are ongoing.81,82 Unfortunately, immune response to mitochondrial DNA primarily derived from the oocyte can elicit an immune response.83

Adult skin fibroblasts can be induced to become pluripotent stem cells (iPSCs) by transfer of transcription factors such as Oct4, Sox2, Klf4, and c-Myc that can reactivate developmentally regulated genes.84,85 Somatic mitochondrial point mutations can arise spontaneously in individual iPSC cells derived from skin fibroblasts. These mutations increase with the age of the donor.86 Aged adult skin in AMD patients thus may not be the ideal source even though autologous cells would avoid immune rejection. In contrast to somatic nuclear transfer, factor-based reprogramming can leave an epigenetic memory of the tissue of origin within the iPSC line.87,88

iPSCs can be generated from peripheral blood cells that are CD34+ by retroviral transduction89 or via oriP/EBNA1-based transfection. CD34+ hematopoietic stem cells isolated from bone marrow are another source. Using this technique, Sharma et al48 created iPSC RPE from AMD patients. These cells, when placed as a sheet on a biodegradable scaffold in the subretinal space of RCS rats or in a laser-induced RPE injury pig model, rescued degenerating retina.

Additional cell preparations under consideration for subretinal transplantation are bone marrow mesenchymal stem cells (BMSCs) and umbilical cord mesenchymal stem cells. Both cell preparations offer the possibility of autologous cell transplantation and have been shown to rescue photoreceptors in RCS rats.9193 Intravenously injected BMSCs can home into areas of chemically induced RPE loss in mice.94 Intravitreal injection also results in similar migration.95 RPE derived from mouse BMSCs that have been transplanted into subretinal space of RCS rats rescue photoreceptors.96,97 Autologous BMSCs can be enriched for cells with RPE differentiation potential by cell sorting based on surface markers (C35+C38). These cells can then be induced to differentiate into RPE by coculturing with mitomycin C-inactivated RPE cells96,98 or by using a lentiviral vector to insert a stable RPE65 transgene.97,99 Huang et al100 reported the use of ciliary neurotrophic factor to promote more efficient differentiation of BMSCs to RPE. Pigmented spheres isolated from BMSC-derived neurospheres have been used to generate cells that exhibit RPE-markers and phagocytose photoreceptor outer segments.101

Stem Cell–Derived RPE Transplantation in AMD

Schwartz et al102104 reported the use of ESC-RPE suspensions to treat 9 AMD patients with GA. A human embryonic stem cell (hESC) line, MA09, generated a master cell bank, and the RPE grown from this line were isolated, purified, expanded, and cryopreserved at passage-2 for clinical use. RPE suspensions were delivered to the subretinal space via a 38-gauge retinotomy and injected near the area of GA. Low dose tacrolimus and mycophenolyate mofetil were used for immune suppression before surgery and for 6 weeks after surgery after which only the latter was continued for another 6 weeks. Complications were associated with this regimen in the AMD patients indicating that many older patients probably will not tolerate sustained systemic immune suppression.102 ESC-RPE (50,000–150,000 cells/eye) were transplanted as a bolus injection with the goal of delivering cells in the area of transition between atrophic and clinically intact-appearing RPE. After surgery, the transplants seemed to expand with increased pigmentation in the subretinal space but showed limited growth in the area of GA. ESC-RPE showed no signs of tumor formation. Among the 8 eyes that did not develop cataract after surgery, median improvement in best-corrected visual acuity was 14 ETDRS letters by month-12 (interquartile range 3.0–19.0 letters). Fellow eyes lost a median of 1 letter by month-12 (interquartile range –5.0 to +6.1 letters). There was no control group, and the study was not masked.

Using the same MA09 ESC-RPE preparation and immuno-suppression protocol, Song et al105 reported similar findings at 1-year follow-up in 4 Asian patients, 2 with atrophic AMD, and 2 with STGD1.

Kashani et al106 transplanted 3.5 × 6.25 mm2 sheets of ESC-derived RPE monolayers into areas of GA in 5 AMD patients with the goal of resurfacing Bruch membrane. The RPE had spontaneously differentiated from an ESC line, NIH-H9, through removal of soluble growth factors. Second-passage hESC-RPE were cryopreserved as an intermediate cell bank.107,108 A nano-engineered parylene-C scaffold was used for delivery.109 The scaffolds were 6 microns thick to provide mechanical support with 0.3-micron thick, 40-micron diameter diffusion zones to facilitate nutrient transfer from the choriocapillaris. The diffusion zones occupied around 60% of the scaffold surface area. Passage-3 RPE was seeded onto these vitronectin-coated parylene-C membranes and grown to confluence for around 4 weeks, achieving a density of 105 cells/scaffold. These cells demonstrated RPE markers (eg, RPE65) and phagocytosis of photoreceptor outer segments.108 The scaffold could not be implanted in 1 patient due to the presence of fibrinoid debris in the subretinal space. For the other 4 patients, the RPE scaffold was delivered into the area of GA. Low-dose tacrolimus was used for immune suppression. The appearance, location, and size of the implants did not change during follow-up, which ranged from 120 to 365 days (mean 260 days) after surgery. Four of the 5 subjects showed no substantial change in vision from baseline, but 1 transplant recipient improved by 17 ETDRS letters. Two subjects developed stable fixation over the implant. This degree of visual improvement is observed infrequently in untreated GA patients.110 Contralateral fellow eyes showed no significant change in vision or worsened during follow-up. A total of 16 patients have undergone transplantation thus far, and the study is ongoing.111

Mandai et al112 transplanted a sheet of autologous iPSC-derived RPE into the subretinal space of an AMD patient in conjunction with subfoveal MNV. iPSCs were generated from skin fibroblasts using nonintegrating episomal vectors carrying GLIS1, L-MYC, SOX2, KLF4, OCT3/4 and differentiated into RPE as described previously.113,114 Pigmented colonies of RPE were picked manually and cultured to confluence. The pigmented cells were verified as RPE based on their ultrastructural appearance, presence of retinoid cycle enzymes (RPE65, cellular retinaldehyde binding protein), phagocytosis proteins (MERTK), chloride channels (BEST1), and tight junction proteins (ZO-1). In addition, iPSC-derived RPE transepithelial resistance was measured as was the ability of the RPE to phagocytose porcine rod photoreceptor outer segments. The autologous iPSC-derived RPE cells were assessed for quality and safety before transplantation, and whole-genome sequencing, whole-genome methylation profiling, and expression analyses also were done. Confluent cultures of iPSC-RPE were prepared as sheets without any scaffold and were cut using laser microdissection. A 1.3 × 3 mm2 RPE sheet was delivered to the subretinal space using a modified 20-gauge cannula. One year after surgery, the sheet seemed to be intact, however, there was no improvement in the patient’s vision (stable at 20/200), which is not surprising given the degree of foveal atrophy evident before surgery. There was no clinical or angiographic evidence of graft rejection in this patient, who was not immune suppressed. Transplantation surgery for a second patient was halted after the discovery of 6 gene mutations that were not present in the patient’s fibroblasts from which the iPSCs were derived. Three mutations were gene deletions. Three were nucleotide changes (single nucleotide polymorphism, SNP). One SNP was in a low-risk oncogene.115 The mutations are thought to have been the result of the reprogramming method.

Da Cruz et al116 reported the use of hESC-derived RPE transplants to treat 2 AMD patients with subfoveal MNV associated with significant subretinal hemorrhage. A 6 × 3 mm2 patch of a well-differentiated RPE monolayer resting on a vitronectin-coated polyethylene terephthalate (PET) membrane was transplanted into the subretinal space and positioned under the macula. Patients were immune-suppressed with perioperative oral prednisone and intravitreal implants providing sustained delivery of fluocinolone acetonide. One patient developed a severe retinal detachment after the transplant procedure and underwent successful retinal reattachment surgery. In the patient with the least foveal atrophy before surgery, vision improved 29 ETDRS letters, from 20/640 to 20/160, and reading speed improved from 0 words/minute to around 50 words/ minute by 12 months after surgery. In the patient with the postoperative retinal detachment, who had more profound foveal atrophy before the transplant procedure, vision improved 21 ETDRS letters, from 20/800 to 20/150, and reading speed improved from 0 words/minute to around 50 words/minute by 12 months after surgery. Because vision can improve after subretinal surgery alone in this setting, with approximately 25% of eyes improving 10 or more ETDRS letters, and because there were no control surgeries in this series, one cannot attribute these improvements to the transplants with complete certainty.36,117119 There was, however, anatomic evidence of integration of the RPE transplant with host retina and focal improvement in photoreceptor anatomy over the transplant in both patients.

Stem Cell–Derived RPE Transplantation in Stargardt Macular Dystrophy

Schwartz et al103,104 transplanted MA09-derived RPE cells in 9 patients with STGD1 as described above. Vision-related quality-of-life measures increased for general and peripheral vision, and near and distance activities, 3 to 12 months after surgery.120

In a dose-escalation trial in 12 patients with advanced STGD1, Mehat et al121 evaluated the safety and potential efficacy of subretinal injection of the ESC-RPE derived from MA09 cell line as well. ESC-RPE were placed in the transition zone between atrophic and nonatrophic areas, and the same immunosuppression regimen was used. Immune suppression was achieved with low-dose tacrolimus that was started 1 week before transplantation and titrated to achieve serum trough levels of 3 to 7 ng/mL for 6 weeks after transplantation. Additionally, mycophenolate was given for 12 weeks posttransplant at an escalating dose regimen starting on the day of the surgery to the maximum dosage of 1 g twice a day. Increased pigmentation was noted within the injected area in all patients with some pigment migration into the GA. Modest improvement in best-corrected visual acuity in 4 of 12 subjects either was not sustained or was matched by a similar improvement in the untreated fellow eye. Microperimetry demonstrated no evidence of benefit at 12 months in the 12 subjects. In 1 patient receiving the highest ESC-RPE dose, localized retinal thinning and reduced sensitivity in the area of hyperpigmentation suggested the potential for harm. Patient-reported quality of life using the 25-item National Eye Institute Visual Function Questionnaire indicated no significant change.

Long-term results of these studies are awaited. However, transplantation of RPE may not succeed long-term in STGD1 because the underlying problem arises in the photoreceptors. Stem cell-derived RPE may survive but are prone to lipofuscin accumulation and related inflammation and cell death just as native RPE.122

Cell Delivery

Cell Suspensions Versus Cell Sheets

Subretinal injection of cell suspensions can be accomplished easily through a small retinotomy but may result in uneven donor cell distribution in the subretinal space, RPE multilayer formation (with variable rescue of overlying photoreceptors), and potential complications from efflux of cells into the vitreous cavity. Injection of a polarized cell sheet, with or without a support scaffold, requires a larger retinotomy, a larger retinal detachment, and specialized instruments to assure placement of the polarized RPE in the correct orientation and location. Theoretically, cell suspension delivery could cover a larger area while sheet delivery is limited to the size of a sheet that can be manipulated for successful placement. (Cell proliferation and migration of the cells off of the scaffold and into the adjacent area might eliminate this restriction.) Potential advantages to transplanting sheets of cells on a scaffold are 4-fold. First, one can transplant cells that are differentiated and organized anatomically, resembling the in situ configuration. Highly differentiated transplanted cells may be better suited for subfoveal RPE transplantation than RPE suspensions.47,123 Once the foveal photoreceptors are detached, they begin to degenerate. Using differentiated RPE may limit the time foveal photoreceptors are without RPE support. This issue is moot in an area of preexisting photoreceptor atrophy. A second potential advantage is that fewer cells and, hence, lesser antigen load might be delivered with scaffolds than with cell suspensions. Third, it may be possible to integrate growth factors, immuno-modulatory molecules, or other useful moieties into the scaffold, thus prolonging RPE graft survival and photoreceptor survival. In cell culture studies, polarized sheets were found to be less prone to oxidative stress-induced cell death than nonpolarized and subconfluent ESC-RPE.123 Finally, a monolayer of differentiated RPE transplant on scaffold might recapitulate the passive and active immune privilege features of native RPE.

Scaffolds

In addition to supporting a polarized monolayer of functional RPE, an ideal scaffold should be biocompatible (eg, should not elicit an inflammatory or fibrotic reaction in the retina) or biodegradable at a rate slow enough to allow the transplanted cells to be established (without the byproducts of biodegradation causing local inflammation or structural damage), should have transport and diffusion properties resembling normal Bruch membrane, should not migrate in the subretinal space after surgical delivery, and must have handling properties that enable tissue delivery without damaging the donor cells, the overlying host retina, or the subjacent Bruch membrane–horiocapillaris complex.

Broadly, the scaffolds that have been explored can be grouped into those that are naturally produced, those that are synthetically generated, and those that are a hybrid of the natural and synthetic materials. Corneal Descemet membrane, anterior lens capsule, retinal internal limiting membrane, and amniotic membrane are examples of nonbiodegradable scaffolds. Anterior lens capsule has permeability properties similar to Bruch membrane and is convenient and easily available material to use if a transplant recipient needs to undergo cataract surgery as well. The lens capsule supports growth of ARPE-19 cells in culture especially if the cells are centrifuged onto the membranes as opposed to gravity-based seeding.124 Subretinal implantation of lens capsule has been reported to be difficult surgically when attempted in pigs due to curling and adhesion of the membrane to forceps.125,126 Descemet membrane has been shown to support RPE and iris pigment epithelial growth in culture.127 Similarly, internal limiting membrane has been shown to support porcine RPE and ARPE-19 cells in culture, but human adult donor RPE do not adhere.128 Amniotic membrane is another candidate that has the benefit of having long-term data on safety and established good manufacturing practices for ocular surface pathologies.129 Clinical grade, decellularized human amniotic membrane support human ESC-RPE growth on the basement membrane side. These cells express bestrophin, secrete VEGF, and phagocytose photo-receptor outer segments,130 confirming previous studies.131,132 When RPE-amniotic membrane complex was implanted in the subretinal space of Royal College of Surgeons rats, photoreceptor rescue occurred.130 The disadvantages of natural scaffolds include: access to tissue, variability in quality and thickness of the tissue, and the risk of disease transmission. None of these materials have been used in human trials.

An alternative to natural tissues is scaffolds constructed using naturally occurring polymers such as collagen, hyaluronic acid, gelatin, or silk, which also are biodegradable. Excellent in-depth reviews of the various scaffold materials, manufacturing techniques, and modifications of these polymers have been published.28,29 A brief summary of the RPE supportive scaffolds and some of the newer techniques are presented below.

Collagen, which is present in Bruch membrane, has been used to design scaffolds that support RPE cultures,133 iPSC-derived RPE,113 and RPE transplants in animal models.134,135 Collagen scaffolds can be very thick and may need to be dissolved before subretinal implantation. Thinner nanofibrous membranes from collagen type 1 manufactured by electrospinning136 and multilaminar films using Langmuir-Schaefer deposition support human ESC-RPE.137139 These have not been translated to in vivo studies yet. Gelatin-encased RPE cells have been transplanted in porcine eyes140 and rabbit eyes.141 The gelatin usually dissolves overtime, and no significant immune reaction was reported. Two reports of RPE transplantation using gelatin scaffolds published previously did not show any improvement in posttransplant vision, but the graft in 1 patient did not seem to be attached to the graft bed.43,142 Biodegradable fibrinogen was used to encase fetal human RPE for subretinal transplantation in rabbit eyes.143 The authors reported a monolayer of cells at 1 month, but there was an inflammatory response. More recently, iPSC-derived RPE on fibrin was shown to grow into a monolayer with cobblestone morphology and express RPE markers.144 The fibrin rigidity could be modulated to allow for manipulation involved in subretinal transplantation. Scaffolds created using fibroin, the insoluble protein in silk, have been shown to promote excellent attachment and cultures of ARPE-19.145,146 Three-dimensional construction of a biohybrid retina using silk hydrogels and films as a framework to house RPE, Muller cells, and neural retina has been reported.147 Although subscleral placement in rabbits did not induce inflammation,148 viability of subretinal placement remains to be seen.

Various synthetic polymers have been shown to support RPE cultures. The advantage of synthetic membranes is that production can be controlled, thereby maintaining uniformity of the end product consistently. The production can be scaled up. Parameters such as thickness, porosity, surface topography, and surface adsorption can be modified to optimize cell attachment, survival, and function. Finally, the risk of disease transmission that might occur from human donor tissues is eliminated.

Synthetic membranes can be biodegradable and those that support RPE growth include poly (L-lactide),149 poly-lactic-co-glycolic acid,136 polyurethane,150 polycaprolactone,151 and poly lactide caprolactone.152 Toxicity of the byproducts of biodegradation is a concern.153

Nonbiodegradable membranes include parylene C, polyethylene terephthalate (PET; polyester), polydimethylsiloxane (PDMS), polyimide, and aggregates of carbon nanotubes (bucky-paper). Parylene-C membrane with properties mimicking those of Bruch membrane154 has been shown to support RPE, safe when placed in subretinal space,107,108 and phase I/IIa clinical trials of subretinal human ESC-RPE on parylene-C scaffolds in patients with GA is ongoing (NCT02590692). Polyester membranes have also been shown to support human RPE152 and nontoxic in sub-retinal space of rabbits67 and pigs116 When human ESC-RPE on polyester scaffold was implanted in 2 patients with MNV as part of a phase I clinical trial, the cells appeared to have survived up to 12 months and the patients showed improved visual fixation and increased reading speed.116 Human ARPE-10 and iPSC-derived RPE have been shown to grow as a monolayer on PDMS scaffold.155,156 In addition to surface modification of the PDMS that helps promote cell attachment, the opposite surface lends itself to further modification like coating with dexamethasone-loaded liposomes to suppress oxidative stress-induced angiogenesis.157 Ultrathin polyimide membrane is another nonbiodegradable membrane that is well tolerated in the subretinal space of rats and rabbits in addition to supporting human ESC-RPE.158 With the exception of parylene C and PET/polyester membranes, none of the other scaffolds have been used in human trials.

Efforts continue to optimize scaffold manufacture by modifying parameters of the scaffold to optimize cell function and surgical handling. Liu et al152 determined that fiber diameter of 350 microns was optimal for RPE growth. Using a photocrosslinked poly(ethyelene glycol) diemehtacrylate, Wendland et al159 determined that shear modulus of at least 35 kPa was required for surgical handling as determined by tendency to tear, ease of handling with the surgical tool, and durability; the handling characteristics started to vary with modulus of 800 kPa and higher. Rim et al160 combined different materials in the scaffold to enhance cell-cell and cellmatrix interaction, and Phelan et al161 sought a more benign soy protein-based fibrous scaffold to improve maturation and differentiation of RPE. Kim et al162 developed a “bioink” out of porcine Bruch membrane through sodium dodecyl sulfate treatment, lyophilization, and pepsin digestion, which consisted of collagen and glycoasminoglycans. The ink had a thermal-sensitive crosslinking behavior with liquid state at 4°C changing to gel state at 37°C. Using a 3D printer, a Bruch membrane mimetic was created that supported RPE cultures better than extracellular matrix proteins. These cells expressed RPE65, polarity, and transepithelial resistance. After placement of this Bruch membrane mimetic in the rat subretinal space, no retinal toxicity was identified.

Scaffolds may have limitations. RPE on the scaffolds can dedifferentiate or transdifferentiate via the SMAD3 pathway.163 Exposure to scaffold can result in increased interleukin 6 and monocyte chemo-attractant protein-1 in the retina, triggering microglia scarring.164 Scaffold use generally mandates instrument design for RPE-scaffold delivery into the subretinal space with minimal trauma165,166 Kashani et al111 and da Cruz et al116 used custom-designed forceps to transplant the RPE-scaffold assembly to the subretinal space.

Immune Suppression

Independent of its efficacy, systemic immune suppression can be complicated by side effects such as nephrotoxicity, hypertension, risk of malignancy, susceptibility to infection, hepatotoxicity, seizures, and even anaphylaxis, depending on the medications used. Local immune suppression should minimize the systemic complications. High-dose dexamethasone therapy is effective in preventing acute allograft rejection.167,168 Repeated intravitreous injections of dexamethasone can be well tolerated by the retina, which suggests that sustained intravitreal dexamethasone delivery may not be complicated by retinal damage. Several intravitreal sustained-release steroid formulations currently available might be considered for human RPE transplants to prevent graft rejection. Sustained-release fluocinolone acetonide implants with a therapeutic window of as long as 3 years seem desirable.169 Dexamethasone implants also have been used for immune suppression in minipigs receiving ESC-RPE monolayers seeded on a parylene-C scaffold.107 Innovative drug delivery approaches such as port delivery systems, hydrogels, and nanoparticles are being developed and may be useful for achieving adequate suppression without adverse effects.170

Donor iPSC banks are being created and are targeted for individuals homozygous at some of the major histocompatibility (MHC) loci.171175 Disparities at minor histocompatibility loci could potentially provoke immune rejection.176 Using MHC-matched donor and host tissue may be most effective in a genetically homogenous population within a geographic region, as human leukocyte antigen (HLA) alleles and haplotypes are conserved within populations. For example, one estimate is that an iPSC bank from 150 homozygous HLA-typed individuals could match 93% of the UK population, and, due to limited diversity of the Japanese population, as few as 50 such lines could potentially match 90% of the population.177 This approach does not address minor HLA matching, and minor HLA antigens can stimulate rejection.

Experiments in macaque monkeys showed that when MHC-matched, iPSC RPE could survive in the host subretinal space without immune rejection for up to 6 months.178 Subsequently a human study was conducted in which Sugita et al179 injected allografts of iPSC-RPE cell suspensions fromanHLAhomozygous donor into the subretinal space of 5 AMD patients with MNV to study the safety and feasibility of HLA-matched transplants without the use of systemic immune suppression. Patients were treated with local steroids. To detect transplant rejection, lymphocyte-graft cells immune reaction test and detection of RPE-specific antibody were done. At the 52-week endpoint, no tumorigenicity was noted. All patients developed epiretinal membranes, which would be expected because transvitreal injection of cell suspensions into the subretinal space can lead to reflux of some cells into the vitreous. One of the patients with macular edema secondary to epiretinal membrane underwent membrane peeling that led to resolution of the edema. One patient developed aseptic endophthalmitis from triamcinolone injection. One patient showed signs of rejection that improved with ocular steroid injection. Vision did not show any significant improvement or worsening. The authors estimate that 70 donors of the HLA-3 locus homozygote can cover 80% of Japanese patients.

Another strategy is to induce tolerance, that is, absence of a destructive immune response to transplanted tissue without immune suppression.180 Tolerance can be achieved via mixed chimerism or by inducing anergy through blockade of costimulatory signals that activate T cells (eg, with belatacept). The combination of the immune-privileged site of the transplant (ie, the subretinal space) and the immune-privileged tissue that is transplanted might result in the induction of ignorance (ie, failure of the immune system to recognize transplanted tissue). An alternate way to suppress the immune reaction would be to prevent antigen presentation by knocking out MHC class II antigens. When RPE cells generated from MHC class II knock-out human ESC cells were placed in the subretinal space of rabbit eyes, immune rejection was reduced and delayed.181 In a similar approach, elimination of HLA molecules by multiplex genome editing using a CRISPR/Cas9 system and induction of CD47 expression muted the adaptive and innate immune responses to human pluripotent stem cells.182 Other studies have confirmed the effectiveness of this approach.183,184

Challenges and Developments

Cell-based therapy for relentlessly progressing blinding conditions has been a goal for several decades. With the rapid explosion of research in several areas such as stem cell-derived RPE cells, 3D printing, nanotechnology for developing supporting scaffolds, and drug delivery systems, and the successful completion of several safety trials in humans, that goal seems to be closer. However, many hurdles and questions remain.

Regardless of which cells are used and whether the cells are placed as a sheet or injected as a suspension, preparation of the cells will have to be consistent with Good Manufacturing Practice. In the initial stages of establishing the cell line from ESCs, identification of colonies of RPE traditionally is based on pigmentation. This method can be inefficient and lends itself to contamination with non-RPE cells. Plaza Reyes et al185 have identified CD140b among others as a cell surface marker and propose that this can help automate the process of identifying and isolating the correct cells for efficient enrichment during and after differentiation. Currently, it can take several months to establish an ESC-RPE cell line or iPSC-RPE.30 Generating cells from adult RPE stem cells may require only weeks. The lengthy multistep process opens itself to potential contamination and failures. Germline mutations in the cell source and mutations induced during differentiation can be a concern.186,187 Using whole-genome sequencing, single-cell RNA sequencing, and copy number variation analysis between cells at different stage of differentiation, Petrus-Reurer et al188 showed that there is a low number of variations and that the protocols can produce highly pure ESC-RPE cells. None of the mutations noted involved cancer-driver genes based on currently available databases Standardization of criteria for RPE cell selection so that variability induced from donors with different alleles, clonal variability, and variations in extent of RPE features exhibited will help. A reference dataset has been established towards that end.189 Once the cells are transplanted, is there migration of the cells to other parts of the eye or the body (biodistribution)? Will there be teratoma formation? These questions need to be answered before use in humans.47,116

Summary

RPE transplantation has progressed from successful preclinical experiments in which transplants have been documented to survive long-term, rescue photoreceptors, and restore visual function to early-stage human clinical trials in which long-term RPE transplant survival and safety have been documented. Visual outcome in these early trials has been variable with most subjects experiencing little or no improvement. Some issues that might affect the clinical results include appropriate disease selection, appropriate timing of intervention during the course of the selected disease, and manufacturing issues. Regarding cell manufacture, there is no single well-established standard. The effect of batch to batch and donor to donor variability on RPE transplant behavior may be important. Sharma et al,48 for example, demonstrated such variability, especially donor to donor, and attempted to define criteria for selection. Other concerns include contamination with infectious agents and inclusion of cells that might differentiate into a non-RPE cells or a malignant tumor. Genomic stability has been demonstrated in cells cultured for extended periods.112,186 Other areas of active investigation include modulation of immune surveillance (through sustained drug delivery, HLA/MHC matching, autologous transplants, HLA knockdown) and management of the abnormal extracellular milieu that often is present in late-stage degenerative disease.

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Keywords:

age-related macular degeneration; cell-based therapy; RPE transplant; Stargardt disease; stem cells

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