Survival of Allogeneic Porcine Retinal Pigment Epithelial Sheets after Subretinal Transplantation

Abstract

purpose. To examine morphology after transplantation of organized primary porcine RPE sheets into the porcine subretinal space.

methods. Primary RPE sheets were harvested from freshly enucleated female porcine eyes and embedded in a thin slice of 50% gelatin and 300 mM sucrose before subretinal transplantation into male pigs by using vitrectomy techniques. Thirty-eight animals that underwent surgery were observed for up to 3 months without immune suppression.

results. Four days after surgery, the subretinal space contained a multilayer of heavily pigmented RPE that was predominantly Barr body positive. One month after transplantation, there was marked shortening of the outer segments with an intact external limiting membrane. The transplant bed contained a pigmented monolayer in some regions, whereas in other regions the graft was folded into multilayers with degenerated inner layers of transplanted cells despite synthesis of basement membrane. The choroidal vessels and choriocapillaris remained patent in the transplant bed. Barr body positive cells were still present 3 months after surgery. There was no infiltration of the graft site with inflammatory cells.

conclusions. Allogeneic RPE grafts survive in the subretinal space up to 3 months after surgery, and the choriocapillaris remains patent in the transplant bed, although there are many heavily pigmented cells within the transplant bed that are Barr body negative by 3 months. Further work is needed to produce uniform repopulation of a sizable portion of Bruch’s membrane with a monolayer of transplanted RPE.

The retinal pigment epithelium (RPE) is a hexagonally packed monolayer of cells present on the inner aspects of Bruch’s membrane in the human eye. The RPE performs many functions that are important in the maintenance of the integrity of the outer retina, including phagocytosis of the distal tips of photoreceptor outer segments, transport and reisomerization of bleached visual pigments, and maintaining the integrity of the blood–outer retinal barrier. In addition, the RPE is necessary to maintain the integrity of the subjacent choriocapillaris. Pharmacologic damage to the RPE by intravitreous injection of ornithine, iodate, or iodoacetic acid leads to secondary atrophy of the subjacent choriocapillaris, ^{1 2 3 4 5} and surgical RPE removal in pigs, cats, and the primates leads to choriocapillaris atrophy. ^{6 7 8 9} Progressive choriocapillaris atrophy has been documented in patients undergoing surgical excision of choroidal neovascular lesions in age-related macular degeneration (AMD) and presumed ocular histoplasmosis syndrome, probably due to removal of the native RPE with the choroidal neovascular complex. ^{10 11 12} Thus, it is expected that RPE dysfunction would lead to widespread dysfunction of the outer retina and choriocapillaris. 

An interest in RPE transplantation has been fueled by the realization that numerous disorders that affect the outer retina–RPE–choriocapillaris interface may be treatable with this technique. For example, two genes that are expressed in the RPE, RPE65 and CRALBP, have been determined to cause some cases of retinitis pigmentosa. ^{13 14 15} In animal models, a defect in RPE phagocytosis is responsible for the severe changes seen in the Royal College of Surgeons rat. Transplantation of normal RPE rescues the outer segments in this animal model. ¹⁶ Thus, it is hoped that RPE cell transplantation will be of benefit in select cases of human RP and possibly other tapetoretinal degenerations. 

A more immediate need for successful RPE transplantation also exists in the management of AMD. Loss of the RPE precedes loss of the choriocapillaris in patients with nonexudative AMD, and RPE transplantation may prevent or reverse choriocapillaris atrophy in these patients. ^{10 11 12} Surgical removal of choroidal neovascular lesions has been performed in exudative AMD, but visual recovery is limited in these eyes, because native RPE is removed with the choroidal neovascular membrane. ²⁰ RPE transplantation, autologous RPE translocation, and iris pigment epithelial transplantation have been performed in a limited number of patients with exudative and nonexudative AMD, but to date central visual acuity has not improved significantly in most patients studied. ^{21 22 23 24 25 26 27 28 29 30}  

The ultimate success of RPE cell transplantation depends on the development of surgical techniques that allow for repopulation of denuded Bruch’s membrane by transplanted cells, with simultaneous control of cell–surface interactions between transplanted RPE and damaged or diseased underlying tissue. We have demonstrated that intact sheets of adult human RPE can be harvested from donor eyes. ³¹ The RPE cell membrane, intracellular organelles, and intercellular adhesions are intact and cell viability is more than 85% 24 hours after harvesting. ³¹ The purpose of the current manuscript is to describe the anatomic results obtained after transplanting RPE sheets into the subretinal space in a healthy porcine eye, using modern pars plana vitrectomy techniques. 

Methods

Preparation of Gelatin Sheets

Gelatin was used as a matrix for harvested RPE because of the in vivo compatibility and safety of the gelatin. Gelatin blocks (50% wt/vol) were firm enough to manipulate during the harvesting procedure. Porcine skin gelatin powder with a rigidity of 300 blooms (Sigma-Aldrich, St. Louis, MO) was sterilized with gamma irradiation (2.7 megarads) and dissolved in minimum essential medium (MEM; Invitrogen-Gibco, Grand Island, NY) to increase the rigidity of the gelatin. ³¹ The addition of 300 mM sucrose (Sigma-Aldrich) to the gelatin solution maintained the gelatin sheets in a solid phase at temperatures below 37°C, but allowed the sheets to melt within minutes at 37°C. ³¹ Once the gelatin dissolved, the solution was poured into 35 mm tissue culture dishes (Falcon 3001; BD Biosciences, Lincoln Park, NJ) and allowed to cool for 15 minutes and solidify at room temperature. Solid gelatin blocks were stored at 4°C. Gelatin blocks that were less than 24 hours old were used throughout the experiments because of the time-dependent variations in the rigidity and the melting point of the gelatin. Gelatin blocks were cut into 15 × 30-mm triangular pieces and mounted on a vibratome (Series 1000; Technical Products International, St Louis, MO) with the basal side facing a 102 μm thick steel blade (Personna; American Safety Razor Company, Staunton, VA). The vibratome was cleansed with 70% alcohol, and the moving platform that holds the blade was sterilized by exposing it to 100% ethylene oxide gas for 4 hours followed by 8 hours of aeration. Smooth gelatin sheets were cut from the blocks at a thickness of 100 μm and stored in CO₂-free medium (CFM; Invitrogen-Gibco) at 4°C. The entire procedure was performed in tissue culture hoods in a class-100 clean room. 

Harvesting of Retinal Pigment Epithelial Sheets

RPE sheets were harvested as we have described elsewhere, ³¹ by a modification of the technique described by Pfeffer. ³² Briefly, freshly enucleated porcine eyes were cleaned of extraocular tissue. The suprachoroidal space of the posterior pole was sealed with cyanoacrylate glue, and a small scleral incision was made 3 mm posterior to the limbus until the choroidal vessels were exposed. Tenotomy scissors were introduced through this incision into the suprachoroidal space, and the incision was extended circumferentially. Four radial relaxing incisions were made in the sclera, and the sclera was peeled away from the periphery to the optic nerve with care taken to avoid tearing the choroid. The eyecup was then incubated with 25 U/mL dispase (Invitrogen-Gibco) for 30 minutes and rinsed with CFM, and a circumferential incision was made into the subretinal space along the ora serrata. The loosened RPE sheets were separated from the remainder of the ocular tissue and placed on a slice of 50% gelatin with the apical RPE surface facing upward. Contamination with choroidal cells was avoided by visualizing the RPE sheets under a dissecting microscope during harvest. The gelatin film containing the RPE sheet was then incubated in a humidified atmosphere of 5% CO₂ and 95% air at 37°C for 5 minutes, to allow the gelatin to melt and encase the RPE sheet. The specimen was kept at 4°C for 5 minutes to solidify the liquid gelatin and then stored in CFM at 4°C. 

Cytokeratin Staining

Harvested RPE sheets were rinsed in phosphate-buffered saline (PBS), fixed with 10% phosphate-buffered formalin for 5 minutes, and washed again with PBS. The cells were treated with 3% hydrogen peroxide for 5 minutes to quench endogenous peroxidase activity and 1% bovine serum albumin (Sigma-Aldrich) to block nonspecific binding sites. Cells were incubated at 37°C for 1 hour with a mixture of monoclonal anti-pan cytokeratin antibodies to cytokeratin-1, -4, -5, -6, -8, -10, -13, -18, and -19 (Sigma-Aldrich). The cells were washed twice with PBS, incubated for 30 minutes with biotinylated goat anti-mouse IgG, and incubated with an avidin-biotin peroxidase complex (Extravidin; Sigma-Aldrich). Visualization was achieved by using 3-amino-9-ethyl-carbazole chromogen counterstained with Mayer’s hematoxylin. The sites of antibody deposition were visible as brownish-red granular spots. An irrelevant IgG primary antibody (anti-human von Willebrand antibody; Sigma-Aldrich) was also used and showed no background staining. All the harvested cells were positive for pancytokeratin, indicating that the cells were of epithelial origin. 

Cell-Viability Analysis

RPE sheets were transferred to a Petri dish containing MEM and incubated at 37°C for 5 minutes to melt the gelatin. Cell viability was assessed by a commercial assay (Live/Dead Viability/Cytotoxicity Kit; Molecular Probes, Eugene, OR). ³³ This kit contains two probes: calcein and ethidium homodimer. It relies on the intracellular esterase activity to identify the living cells, which cleaves the calcein to form a green fluorescent membrane-impermeable product. In dead cells, ethidium can easily pass through the compromised membranes to attach to the DNA, yielding a red fluorescence. At least three different areas each containing approximately 250 cells were counted under 100× magnification. The viability of the RPE sheet was expressed as the average ratio of live cells to the total number of cells in these three different areas. 

Delivery of RPE Sheets into the Porcine Subretinal Space

All surgical procedures were performed in one eye of 4- to 6-month-old (30–45 kg) domestic pigs. All animals were cared for in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Animals were sedated, underwent endotracheal intubation, and were maintained on 1.5% to 2.0% isoflurane, as previously described. ⁶ A sterile field was established around one eye and a three-port pars plana vitrectomy was performed with instruments and surgical techniques currently used for vitreous surgery in the human. A bent 33-gauge cannula (Synergetics, Chesterfield, MO) was introduced into the vitreous cavity and into the subretinal space under direct visualization. A bleb neurosensory retinal detachment was created by injecting approximately 50 mL of Hanks’ balanced salt solution (HBSS; Invitrogen-Gibco) into the subretinal space. Intraocular diathermy was applied as necessary to control intraocular bleeding. The RPE transplant had been preloaded into the broad end of a tapered pipette for injection into the subretinal space. The pipette was placed through the right-hand sclerotomy, and the tip was introduced through the retinotomy into the subretinal space under direct visualization. The transplant was injected into the subretinal space, and we waited approximately 10 minutes to allow the transplant to unfold spontaneously. A fluid–air exchange was then performed; vitreous and subretinal fluid was aspirated under passive suction with a soft-tip silicone cannula, and the retina was reattached under air. The instruments were withdrawn, and the sclerotomies closed with 7-0 coated Vicryl sutures (Ethicon, Somerville, NJ). The infusion cannula was removed, the infusion port was sutured closed with 7-0 Vicryl, and additional air was injected through the pars plana through a 30-gauge needle to reform the globe to a tactile pressure of approximately 20 mm Hg. Two milligrams dexamethasone (Anpro Pharmaceuticals, Arcadia, CA) and 20 mg gentamicin sulfate (Elkins-Sinn, Cherry Hill, NJ) was injected subconjunctivally. Subcutaneous buprenorphine 0.005 mg/kg (Reckitt and Colman Pharmaceuticals, Inc., Richmond, VA) was administered for postoperative pain control. 

RPE transplantation was performed on 38 eyes. Thirty-three eyes were processed for histologic examination, with five eyes excluded from further analysis because of retinal detachment or extensive subretinal and preretinal hemorrhage. Animals were killed by intravascular injection of pentobarbital sodium between 4 days and 3 months after surgery. 

Tissue Fixation and Processing

The transplant-recipient eye was enucleated immediately after death and immersed in 2% glutaraldehyde and 10% formalin in PBS. Globes were opened with a full-thickness circumferential incision posterior to the ora serrata and fixed for at least 48 hours before storage in 0.2 M sodium cacodylate buffer (pH 7.4). The posterior eyecup was inspected with a dissecting microscope and a 6-mm corneoscleral trephine was used to trephine the transplant area from the eyecups. The trephined specimens were processed for light microscopy by washing in 0.2 M sodium cacodylate buffer and placing in 1% osmium tetroxide at room temperature for 1 hour. Samples were then dehydrated in a graded alcohol series and embedded in resin (EM-Bed 812; Electron Microscopy Sciences, Fort Washington, PA). One-micrometer plastic sections were cut and stained with Richardson stain before viewing with light microscopy. Stained sections were photographed with a 35-mm camera attached to an upright microscope (Olympus Instruments, Overland Park, KS). Thin sections were cut for transmission electron microscopy, placed on copper grids, and stained with saturated uranyl acetate and 0.3% lead citrate. Sections were then viewed with an electron microscope (100B; JEOL, Tokyo, Japan). 

Identification of Transplanted Cells

We used a modified Barr-body staining technique to identify the transplant after surgery. Unstained sections were deplasticized and stained by using a modification of Guard’s sex chromatin staining method. ³⁴ Briefly, tissue sections were placed sequentially in 95% alcohol, then 70% alcohol, and then Biebrich Scarlet for 2 minutes each. Sections were then placed in 50% alcohol for 5 minutes and rinsed with 50% alcohol, stained in fast green solution for 1 to 2 hours, and rinsed in 50% alcohol for 5 minutes before dehydration in 70%, 95%, and absolute ethyl alcohol for 2 minutes each. Sections were then placed in two changes of xylene for 2 minutes each and mounted (Permount; Fisher Scientific, Pittsburgh, PA). Sections were viewed with an upright microscope at 100× under oil immersion, and cells were considered Barr-body positive if intranuclear red staining was observed. Histologic sections of retina, RPE, and choroid from female and male pigs served as positive and negative controls, respectively. 

Results

We were able to deliver the RPE sheet subretinally in 33 (88%) of 38 eyes without significant complications. Five eyes were excluded from further analysis because of retinal detachment (one eye), cataract formation (one eye), bleeding precluding further tissue processing (two eyes), and endophthalmitis (one eye). 

Four days after surgery a hyperpigmented patch were readily visible in the transplant bed (Fig. 1A) . The transplant sheets were multilayered with extensive outer segment shortening over the transplant (Fig. 1B) . The inner retina and outer nuclear layers were intact and the choriocapillaris was patent. Numerous Barr-body–positive cells (Fig. 1C) were visible within the pigmented multilayer. Bruch’s membrane appears normal ultrastructurally in the transplant bed (Fig. 1D) . 

Nine days after surgery there was excellent preservation of the outer nuclear layer, but the outer segments remain shortened (Fig. 2A) . Two populations of cells were visible in the subretinal space: flatter hyperpigmented cells lining Bruch’s membrane and rounder macrophage-like cells between this layer and the outer segments (Figs. 2A 2B) . In adjacent regions elongated, columnar pigmented cells lined Bruch’s membrane with shortened outer segments (Fig. 2C) . Inner and outer nuclear layers appeared intact although there was shortening of the photoreceptor outer segments (Fig. 2D) . In another eye there was folding of the transplanted sheet with a pseudo lumen formed between the apposed apical borders of the cells (Fig. 2E) . 

Seventeen days after surgery, there was a heterogeneous distribution of intracellular pigment at the transplant site, and the outer nuclear layer and outer limiting membrane appeared normal (Fig. 3A) . Photoreceptor outer segment shortening persisted and the choriocapillaris remained perfused (Figs. 3A 3B) . Occasional inflammatory cells were visible within the lumen of the choriocapillaris (Figs. 3C 3D) without any extravasation or subretinal infiltration. The outer limiting membrane, composed of footplates of Müller cells, was intact, and photoreceptor nuclei appeared normal (Fig. 3E) . 

One month after surgery, the multilayered transplant was visible in the subretinal space (Fig. 4A) . The transplant bed contained a pigmented multilayer along Bruch’s membrane and a lightly pigmented layer, closer to the neurosensory retina, that contained degenerated cells (Fig. 4A) . Basement membrane material was present adjacent to cells that were not in contact with Bruch’s membrane (Fig. 4B) . The two outermost layers of cells appeared intact and were aligned apex to apex with visible basement membrane (Fig. 4B) . A third innermost layer of cells appeared grossly disrupted with extensive cell degeneration, despite the presence of basement membrane. 

Three months after surgery there was hyperpigmentation at the transplant site (Fig. 5A) . There was a heavily pigmented monolayer along Bruch’s membrane with occasional cells with a dome-shaped, rounder apical profile (Fig. 5B) . The outer nuclear layer was intact, and photoreceptor outer segment lengths appeared to be normal in the transplant bed (Fig. 5B) . Round, heavily pigmented macrophage-like cells were present in some regions, possibly accounting for the hyperpigmentation at this site. Barr-body–positive cells were visible (Figs. 5C 5D) within the heavily pigmented monolayer along Bruch’s membrane. 

Discussion

Short-Term Survival and Morphology of Transplanted RPE Sheets

The morphology of the RPE sheets and subretinal space at different time points after transplantation is shown schematically in Fig. 6 . Several days after transplantation, a large sheet of donor RPE was present in the subretinal space. The graft itself was multilayered, possibly due to contraction or folding of the sheet. There was marked shortening of the photoreceptor outer segments over the transplant site, but the choriocapillaris and the large vessels of the choroid were patent in the transplant bed. Shortening of photoreceptor outer segments was probably due to the creation of a serous separation between the RPE and neurosensory retina, because similar changes are seen after subretinal fluid injection. ^{6 7 8 9 35 36 37 38} One month after transplantation, some RPE from the original sheet had reattached to the host Bruch’s membrane and survived in the subretinal space. Cellular debris was present at the apical border of the sheet, possibly representing degeneration of cells not in contact with Bruch’s membrane. Photoreceptor outer segments remained shortened, and the choriocapillaris was patent. Three months after surgery, Barr-body–positive cells were identified adjacent to Bruch’s membrane within the transplant bed, indicating survival of some transplanted RPE, despite the absence of systemic immune suppression. Outer segment length returned to normal, although we could not determine whether this was due to transplanted RPE or host RPE at the transplant site. 

Identification of Transplanted RPE

Barr-body staining of deplasticized specimens is a simple, accurate, and inexpensive technique to distinguish donor RPE from host RPE after RPE transplantation. This technique is preferable to the use of cytoplasmic markers such as melanin to identify transplanted cells, because these markers may be unreliable if donor cell fragments are ingested by host RPE or macrophages. In theory, it is possible that inactivated X-chromosome from degenerated donor RPE could be ingested by native RPE or macrophages and yield a false-positive signal, but under these circumstances the signal would be cytoplasmic rather than intranuclear. Other markers that have also been used to identify transplanted cells include pretransplant labeling of donor cells with di-I, nuclear yellow, or 5-bromo-2-deoxyuridine (BrdU). ^{39 40 41 42 43} More recent studies have used a Y-chromosome probe to identify male feline RPE transplanted into the female feline subretinal space ⁴⁴ and a DNA probe against a porcine-specific repetitive chromosomal marker to identify porcine RPE placed in the rabbit subretinal space. ^{45 46} Both techniques produce a high specificity rate for donor tissue with sensitivity as high as 50% to 70%. ^{45 46} Green fluorescent protein has a high sensitivity and specificity but this technique is limited to identification of passaged cells genetically modified to express the protein. ^{47 48} Development of transgenic animals expressing green fluorescent protein should allow this marker to be used to track primary RPE harvested from mice with eventual expansion to other species. 

In this study, we demonstrated some survival of transplanted RPE 3 months after surgery despite the absence of immune suppression. It is difficult to quantify RPE survival at different time points using a Barr-body technique, but we can draw some general inferences about the morphology of the subretinal space after surgery. First, the choriocapillaris was intact and perfused under the transplant site in the sections examined. Other investigators have shown that functional RPE is necessary to maintain choriocapillaris perfusion. ^{1 2 3 4 5 6 7 8 9} Thus, the results of the present study suggest that there is intact functioning of the RPE monolayer at the transplant site over the area of perfused choriocapillaris. We recognize that we could not determine the relative contribution of the donor and host RPE to choriocapillaris preservation in this model, because the host RPE was not removed. 

RPE Survival without Immune Suppression

In the present study, we did not observe any evidence of overt graft rejection despite the fact that adult tissue was used for transplantation. However, we observed rare lymphocytic cells within the lumen of the choriocapillaris underlying the transplant site, without evidence of any lymphocytic infiltration. It is likely that local or systemic immune suppression would improve graft survival in this animal model because adult RPE is immunogenic, but we cannot exclude the possibility that other factors, including the presence of native RPE at the transplant site, may inhibit transplant survival. Immune suppression with intraocular cyclosporin A increases short-term survival of human fetal RPE xenografts in the rabbit subretinal space, ⁴⁹ although daily cyclosporine (20 mg daily, intramuscularly) does not completely prevent loss of RPE grafted from brown rabbits into albino rabbits. ⁵⁰ The number of BrdU-labeled human fetal RPE injected subretinally into rabbit eyes declines starting 14 days after transplantation, suggesting possible rejection. ⁵¹ The subretinal space is an immune-privileged site, but this immune privilege is relative, rather than absolute. ^{52 53}  

Synthesis of Basement Membrane by Transplanted Cells

We observed the presence of basement membrane adjacent to the transplanted RPE sheet within 3 weeks after surgery. Dispase cleaves the attachment between the RPE sheet and its native basement membrane, and RPE sheets harvested with this enzyme do not contain basement membrane material. ³¹ Thus, the basement membrane present several weeks after surgery represents new basement membrane synthesized by the transplanted RPE. This formation of new membrane is not surprising, because RPE can synthesize basement membrane components in vivo and in vitro. The basal lamina and inner collagen layer contain collagen IV, laminin, fibronectin, and proteoglycans, including heparan sulfate and chondroitin sulfate, and embryonic RPE are capable of depositing all these components on the basal side of the cell and of continuously turning over basal lamina components. ^{54 55} Basement membrane synthesis was seen in the present study but not in previous studies in which porcine fetal RPE microaggregates were transplanted into the rabbit subretinal space, ⁴⁶ perhaps because of longer RPE survival after sheet transplantation, with sufficient time for basement membrane synthesis. Alternatively, basement membrane synthesis might require the prolonged cell–cell contact present in RPE sheets, a suggestion that is supported by tissue culture studies showing that extracellular matrix synthesis is highest after RPE establish a confluent monolayer. ⁵⁴  

Summary

Our results are applicable to the study of adult RPE sheets transplanted into the normal porcine subretinal space and thus can be extrapolated to some but not all potential clinical applications. In patients with tapetoretinal degenerations, the subretinal space and Bruch’s membrane may be normal for many years before the clinical onset of disease, and choriocapillaris atrophy is a late manifestation of outer retina and RPE dysfunction. Thus, the results obtained here may be extrapolated to RPE sheet transplantation surgery performed early in the course of the disease in these patients, but may not be applicable to surgery at later time points when choriocapillaris atrophy and outer retina disorganization are present. Similarly, these results cannot be applied directly to RPE sheet transplantation after submacular surgery in exudative AMD, in which disruption of Bruch’s membrane by the disease process or submacular membranectomy may have a profound effect on the behavior of the transplanted cells by two distinct mechanisms. First, disease within Bruch’s membrane or damage to it, including surgical removal of the inner layers, adversely affects graft reattachment, survival, and proliferation in the subretinal space. ^{56 57} Second, violation of the integrity of Bruch’s membrane may increase the chance of graft rejection by disrupting the partial immune privilege of the subretinal space. 

The ultimate success of RPE transplantation depends on understanding the many factors that control the initial reattachment of the transplant to Bruch’s membrane and its subsequent survival in the subretinal space. Basic questions that should be addressed include determining the number of cells that survive after subretinal transplantation and whether the transplanted cells will proliferate in an abnormal subretinal environment. Systematic attention to these issues is ultimately necessary to ensure survival of the RPE after subretinal transplantation. 

 Supported by Grant EY10311 from the National Eye Institute, the Foundation Fighting Blindness, The Eye Surgery Fund, the Robert L. Burch III Fund, The Macula Foundation, and Research to Prevent Blindness, Inc. LVDP is a Robert L. Burch III Scholar.

 Submitted for publication June 27, 2003; revised October 6 and November 10, 2003; accepted November 19, 2003.

 Disclosure: L.V. Del Priore, None; T.H. Tezel, None; H.J. Kaplan, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

RPE transplant 4 days after surgery showing (A) hyperpigmentation in the region of the transplanted tissue (arrow). (B) The transplant sheet was multilayered (arrow), with extensive outer segment shortening over the transplant. The inner retina and outer nuclear layer (ONL) were intact, and the choriocapillaris (CC) was patent. No inflammatory cells were observed. (C) Numerous Barr bodies were present (red arrows) within the nucleus of pigment-containing cells. (D) Bruch’s membrane appeared normal ultrastructurally in the transplant bed. The retinal pigment epithelial basal lamina (BL), inner collagen layer (ICL), elastin layer (EL), outer collagen layer (OCL), and basal lamina of the choriocapillaris (BL, cc) appeared normal, and the choriocapillaris lumen (cc) appeared patent.

Figure 2.

 

RPE transplant 9 days after surgery. (A) Hyperpigmented cells were visible in the transplant bed. Two populations of heavily pigmented cells were visible in the subretinal space: flatter hyperpigmented cells closer to Bruch’s membrane (arrows) and rounder macrophage-like cells (MP) closer to the outer segments. The choriocapillaris (CC) was patent under the transplant bed. There was excellent preservation of the outer nuclear layer (ONL) and outer limiting membrane (OLM), but the outer segments were shortened. (B) Round, heavily pigmented cells that appear to be macrophages (MP) were visible between a more basal RPE layer and the outer retina. Note bleb (bl) between Bruch’s membrane and pigment layer. There was irregular chromatin condensation in the overlying outer nuclear layer (arrowheads) and marked shortening of the photoreceptor outer segments ( ). The lumen of choriocapillaris vessels (cc) was visible. (C) In an adjacent region, elongated, columnar pigmented cells lined Bruch’s membrane (BM) with shortened outer segments. The lumen of the choriocapillaris (cc) was intact. (D) In the second eye there was variable pigmentation within cells after subretinal transplantation. (E) A third eye demonstrating folding of transplanted sheet with a space formed between the apical borders of the cells (arrows). Inner (INL) and outer (ONL) nuclear layers appeared intact, although there was severe shortening of the photoreceptor outer segments.

Figure 3.

 

Seventeen days after surgery (A), there were heavily pigmented cells at the transplant site (arrow). The outer nuclear layer (ONL) appeared intact, and the outer limiting membrane (OLM) was visible. (B) Photoreceptor outer segments were shortened. The choriocapillaris (cc) was perfused under Bruch’s membrane (BM). (C) A solitary inflammatory cell (arrow) visible within the lumen of choriocapillaris beneath a round heavily pigmented cell ( ) that may be a macrophage. (D) Choriocapillaris lumen (cc) was visible with occasional inflammatory cells (arrow). Bruch’s membrane (BM) appeared normal. (E) Outer limiting membrane (OLM), composed of footplates of Müller cells, appeared intact, and photoreceptor nuclei (arrows) were visible.

Figure 4.

 

Twenty-eight days after transplantation. (A) A multilayered transplant was visible in the subretinal space. The outer nuclear layer (ONL) was identifiable, and the outer limiting membrane (OLM) was intact, with persistent outer segment shortening. The transplant site contained an outer layer of more heavily pigmented cells (arrows) and an inner layer of flatter cells (arrowheads) without pigment. The large vessels of the choroid (Ch) and choriocapillaris (cc) appeared patent. (B) Transmission electron microscopy of the transplant region. Bruch’s membrane (BM) was intact, and the choriocapillaris (cc) was perfused under the transplant bed. The transplant sheet was folded onto itself. Two inner layers of RPE along Bruch’s membrane (1) and immediately above (2), appeared relatively intact, with cells aligned microvilli to microvilli (mv). Basement membrane material was adjacent to layer 2 (solid arrows). A third layer of cells (3) appeared grossly disrupted, with extensive cell death. Basement membrane material was adjacent to these cells as well (dotted arrows).

Figure 5.

 

RPE transplant 3 months after surgery showing (A) hyperpigmentation in region of the transplant (arrows); the retinotomy was visible (arrowhead). (B) A heavily pigmented monolayer with round apical profile (arrows) was present along Bruch’s membrane (BM). The outer nuclear layer (ONL) was intact and photoreceptor outer segment lengths (OS) appeared to be normal, with a patent choriocapillaris in the transplant bed. No inflammatory cells were seen. (C) Round, heavily pigmented macrophage-like cells were present (arrowhead) in some regions. A Barr-body–positive cell (arrow) was visible within the heavily pigmented monolayer. The choriocapillaris was patent, with numerous erythrocytes ( ). (D) Region of different eye showing Barr-body–positive cells (arrows).

Figure 6.

 

Schematic of changes occurring within transplant and surrounding tissue after surgery. (A) Normal anatomy of outer segments (OS), retinal pigment epithelium (RPE), and choriocapillaris (CC). (B) Four days after surgery. RPE sheet transplant (gray cells) is delivered into subretinal space in nondebrided eye. Grafted area appears hyperpigmented due to undulation of the basal surface of the sheet. In some regions there is folding of the graft edge ( ). (C) Two to 4 weeks after surgery. Transplanted cells on the inner portion of the graft degenerate ( ). (D) One month after surgery. Portions of the sheet survive. Scattered transplanted cells fill in regions of Bruch’s membrane between host RPE. (E) Three months after surgery. Some graft cells repopulate Bruch’s membrane. Outer segment length increases, some macrophages remain in the subretinal space. Macrophages (open circles) migrate into the subretinal space, engulf pigment, and are visible at time points in (C)–(E).