July 2005
Volume 46, Issue 7
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Retina  |   July 2005
Grafting of ARPE-19 and Schwann Cells to the Subretinal Space in RCS Rats
Author Affiliations
  • Shaomei Wang
    From the John A. Moran Eye Center, University of Utah Health Science Center, Salt Lake City, Utah; and
  • Bin Lu
    From the John A. Moran Eye Center, University of Utah Health Science Center, Salt Lake City, Utah; and
  • Patrick Wood
    The Miami Project to Cure Paralysis, University of Miami School of Medicine, Miami, Florida.
  • Raymond D. Lund
    From the John A. Moran Eye Center, University of Utah Health Science Center, Salt Lake City, Utah; and
Investigative Ophthalmology & Visual Science July 2005, Vol.46, 2552-2560. doi:10.1167/iovs.05-0279
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      Shaomei Wang, Bin Lu, Patrick Wood, Raymond D. Lund; Grafting of ARPE-19 and Schwann Cells to the Subretinal Space in RCS Rats. Invest. Ophthalmol. Vis. Sci. 2005;46(7):2552-2560. doi: 10.1167/iovs.05-0279.

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      © 2017 Association for Research in Vision and Ophthalmology.

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Abstract

purpose. To study the distribution of the human retinal pigment epithelium (hRPE) cell line ARPE-19 and human Schwann (hSC) cells grafted to the subretinal space of the Royal College of Surgeon (RCS) rat and the relation of graft cell distribution to photoreceptor rescue.

methods. Cell suspensions of both donor types were injected into the subretinal space of 3-week-old dystrophic RCS rats through a transscleral approach, human fibroblast and medium were used as control grafts. All animals were maintained on oral cyclosporine. At 1, 2, 4, 6, 15, 28, and 36 weeks after grafting, animals were killed. Human cell-specific markers were used to localize donor cells.

results. Both donor cell types, as revealed by antibodies survived for a substantial time. Their distribution was very different: hRPE cells formed a large clump early on and, with time, spread along the host RPE in a layer one to two cells deep, whereas hSCs formed many smaller clumps, mainly in the subretinal space. Both cells rescued photoreceptors beyond the area of donor cell distribution. The number of surviving cells declined with time.

conclusions. Both hRPE and hSC grafts can survive and rescue photoreceptors for a substantial time after grafting. The number of both donor cell types declined with time, which could be an immune-related problem and/or due to other factors intrinsic to the host RCS retina. The fact that rescue occurred beyond the area of donor cell distribution suggests that diffusible factors are involved, raising the possibility that the two cell types function in a similar manner to rescue photoreceptors.

Degeneration of photoreceptors as a result of genetic defects affecting either the photoreceptors themselves or associated cells, such as the retinal pigment epithelium (RPE), represents the leading cause of blindness in humans for which no suitable treatment exists. Animal models, particularly rodents with diseases homologous or analogous to human disorders, provide an opportunity to explore potential therapeutic approaches that may eventually be applied in the clinic. The potential of cell-based therapy to rescue photoreceptors from death has attracted considerable interest, and methods used in studies have included transplanting healthy RPE into the subretinal space, with the object of replacing the defective host RPE cells and so rescuing photoreceptors 1 2 3 4 5 6 7 8 9 ; grafting iris pigment epithelium 10 11 ; injecting cells that release trophic factors to improve the environment in diseased eyes 12 13 ; and introducing stem cells. 14 These studies have, for the most part, achieved successful morphologic rescue, and several groups have examined the functional consequences as well. 7 9 13 15  
Very little attention has been given, however, to the disposition of donor cells over time and their relation to the rescue process, in part due to the difficulty of labeling donor cells effectively. 
In this study, we used two donor cell types: the human RPE (hRPE) cell line ARPE-19 and human Schwann (hSC) cells. Both have been shown to rescue photoreceptors and visual function when injected into the subretinal space of the Royal College of Surgeon (RCS) rat, an animal in which photoreceptors degenerate due to an RPE cell defect. 16 17 In both cases, the donor cells were human-derived, allowing use of human-specific markers to label the cells noninvasively, track donor cells at various stages after grafting, and relate their distribution to photoreceptor cell survival patterns. 
This is part of a series of studies conducted to show that both hRPE cells and hSCs can rescue photoreceptors and maintain visual function for a substantial time after grafting into RCS rats. 7 9 15 However, the rescue effort tends to deteriorate over time. There are many explanations, but one likely reason is graft rejection, since these were xenografts, and the immunosuppression used in this study may have been not sufficient. 18 Therefore, the main purpose of this study was to address a series of questions: (1) How do donor cells distribute in the subretinal space? (2) How is donor cell location related to photoreceptor rescue? (3) How does the number of donor cells change with time? (4) In hRPE cell transplantation, what is the mechanism of photoreceptor rescue? Is the rescue effect beyond donor cell location? (5) How do rescue and cell distribution compare with hRPE and hSCs, given that hRPE may function by replacing dysfunctional RPE, whereas Schwann cells may release growth factors? 
Methods
Animals
In the main part of this study, we used pigmented dystrophic RCS rats (rdy +, p +), which were either left without surgery or underwent transplantation of either the hRPE cell line ARPE-19 or hSCs into the subretinal space. A total of 60 rats were examined at various time points from 1 week to 36 weeks after surgery. Twenty-four received an ARPE19 graft in one eye. Three were killed at each of the following postgrafting time points: 1, 2, 4, 6, 15, 22, 28, or 36 weeks. A further 24 received hSC grafts. Three were killed at each of the following postgrafting time points: 1, 2, 4, 6, 12, 15, 22, or 28 weeks. Sham-surgery animals that received injections of human fibroblasts (n = 6) or medium alone (n = 6) were control subjects. Two from each sham control were killed at 2, 15, or 22 weeks after surgery. All rats were bred and reared in our animal colony and kept on a 12-hour light–dark cycle. They were maintained on cyclosporine (Cy; Novartis, Basel, Switzerland), administered in the drinking water (210 mg/L; blood concentration, ∼300 μg/L 9 ) from 1 day before transplantation until they were killed. In addition, an intraperitoneal injection of dexamethasone was given for 2 weeks (2.5 mg/kg per day, starting on the day of surgery). These studies were conducted with the approval and under the supervision of the Institutional Animal Care Committee at the University of Utah. All animals were treated in accordance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research. 
Transplantation
ARPE-19 donor cells 19 were obtained from ATCC (American Type Culture Collection, Manassas, VA). Purified hSCs and human fibroblasts freshly isolated from peripheral nerves of adult human organ donors 20 were prepared at The Miami Project to Cure Paralysis (University of Miami, FL). For the transplantation procedure, rats were anesthetized with 2,2,2 tribromoethanol (230 mg/kg) and the eyes received topical ophthaine anesthesia. The pupils were dilated with tropicamide, and the eyes were proptosed slightly with a thread. Cell cultures were trypsinized, washed, and delivered into the subretinal space through a small scleral incision, as a suspension in 2 μL of DMEM/F12 medium (Invitrogen, Carlsbad, CA) using a fine glass pipette (internal diameter, 75–150 μm) attached by tubing to a 10-μL syringe (Hamilton, Reno, NV). The cell suspension contained ∼2 × 105 cells. The cornea was punctured to reduce intraocular pressure and to limit the efflux of cells. A sham-operation group was treated the same, except the transplant contained medium alone or human fibroblasts. Immediately after injection, the fundus was examined to check for retinal damage or signs of vascular distress. Any animals showing such problems were removed from the study and are not included in the results. Donor cells were checked for viability at the beginning and end of a transplantation session using dye exclusion. A typical amount of greater than 95% intact cells was obtained at both time points. 
Histology
Rats were overdosed with pentobarbital sodium (Sigma-Aldrich, St. Louis, MO) and perfused with phosphate-buffered saline (PBS). The superior pole of each eye was marked with a suture to maintain orientation, and the eyes were removed and immersed in 4% paraformaldehyde for one hour. Eyes were infiltrated with sucrose and embedded in OCT, and coronal sections (10 μm) were cut on a cryostat. Four sections (50 μm apart)/slide were collected in five slides per series. One slide per series was stained with cresyl violet (CV) for assessing the injection site and the retinal lamination. Further slides were stained with the human-specific nuclear marker MAB1281 (Chemicon, Temecula, CA) for ARPE-19 and the hSC marker-p75 (University of Miami, FL) for hSCs. The protocol for processing the human-specific nuclear marker was conducted according to the manufacturer’s data sheet. The sections were visualized with a peroxidase substrate (Nova Red; Vector Laboratories, Burlingame, CA). For hSC marker-p75 (dilution 1:100), a standard immunochemical technique was applied. Sections were finally visualized by the peroxidase substrate (Nova Red; Vector Laboratories). All sections were lightly counterstained with CV before being mounted (Vector mount; Vector Laboratories). Photographs were obtained (Image-pro-Plus; Media Cybernetics, Silver Spring, MD), and montage pictures were produced (Photoshop; Adobe Systems, Inc., Mountain View, CA). 
Quantitation
A series of retinal sections were chosen and stained with human-specific antibodies, with the CV-stained sections used as a reference. Human nuclear-marker–positive cells were counted manually on four sections per slide per series. The total number per series was achieved by multiplying by five (five slides per series); the total number for each animal was the sum of the series, after correction by the Abercrombie method. 21 Total number N = (n · D)/(D + T), where n is nuclear profile counts; D is mean nuclear diameter; and T is section thickness. Three eyes were counted in each age group. For hSCs, it was not possible to count individual cells, because they tended to clump, and, with the cytoplasmic stain used, individual cells could not be resolved. The number of individual clumps was counted in each section, but a total number of clumps per eye was not attempted because of their irregular size and the high chance of sectioning through some clumps several times. The image-analysis program (Image Pro-Plus; Media Cybernetics) was used to make the following measurements: the length per retinal section containing either hRPE or hSCs; the length of retina in which the outer nuclear layer was more than four cells thick (this was measured in all animal groups at ≥6 weeks after grafting); and the length of the whole retinal section. The ratios between length of donor cell distribution and the length of the whole retinal section, as well as the length of rescued photoreceptors and the length of the whole retinal section, were calculated. A comparison between the ratios of length of donor cell distribution and the length of rescued cells was made by t-test. 
Results
CV-Stained Sections
The CV-stained sections allowed examination of the general lamination of the retina and the injection site. It was easy to identify the injection site (Fig. 1A) , because of the characteristic damage to the choroid. One week after injection, hRPE grafts were easily seen in CV-stained sections as a single clump in the subretinal space (Fig. 1A) ; there was usually some disruption of inner retina immediately adjacent to the graft site (Figs. 1A 1B , arrows). As early as 2 weeks, the donor cells were spread along the host RPE layer (Fig. 1C) . By 6 weeks, the donor cells formed a sheet up to two to four cells deep on the host RPE and a monolayer at 15 weeks. The hSCs were harder to identify in CV-stained sections because they formed smaller clumps, usually spreading in both directions along the subretinal space from the injection site (see Fig. 5D ). At later time points, it was almost impossible to identify individual cells. 
Distribution of Donor Cells and Relation to Photoreceptor Rescue
The human-specific nuclear marker strongly labeled donor hRPE cells (Figs. 1B 1D 1E 1F 1G 1H 1I) . The hRPE cell nuclei were mostly oval or round with a long diameter 14.08 ± 3.87 μm (the mean ± SD was achieved by measuring 100 antibody-stained cells). At early time points, the clump of cells in CV sections was composed largely of antibody-positive cells (Fig. 1B) . Clumps ranged from ∼341 × 80 to 1289 × 392 μm in area and were 872 ± 407 μm (mean ± SE) in length. By 2 weeks after injection, the donor cells spread from the injection site centrally in the retina (Fig. 1D) , covering maximum areas of ∼1208 × 16 to 1285 × 132 μm, ∼1066 ± 357 μm in length. By 4 to 6 weeks, donor cells formed a continuous layer two to six cells deep over the host RPE layer (Figs. 1E 1F 2A 2B) , and covered an area ∼989 ± 397 μm in length, ∼14% ± 0.1% (mean ± SD) of the length of a retinal section. In sham-surgery RCS retinas, there were two to three layers of photoreceptors remaining at 2 months of age. The rescued photoreceptors (four or more cells thick) covered ∼2610 ± 1047 μm or 38% ± 0.03% of length of the section. The difference between the lengths of donor cell distribution and the photoreceptor rescue was significant (P < 0.001). By 15 weeks after injection, the donor cells formed a layer one to two cells deep, covering ∼2289 ± 194.43 μm in length, ∼29% of the retinal section along the host RPE layer (Figs. 1G 2C 2D) , compared with nonsurgical, dystrophic retinas in which the ONL contained only an intermittent layer of cells. The area of photoreceptor rescue was 4274 ± 130 μm in length, ∼56% ± 0.03% of the retina section. Again, the difference between the graft length and length of photoreceptor rescue was significant (P < 0.001). With time, the number of donor cells declined dramatically, but even at 28 to 36 weeks after surgery (Figs. 1H 1I) , there were still rescued photoreceptors extending beyond the distribution of donor cells. Detailed quantitation was not made at these time points. In sham-injected retinas (Fig. 1J , human fibroblasts), there was local photoreceptor rescue around the injection site, but the effect disappeared by 22 weeks after injection. 
Changes in Number of Donor Cells with Time after Grafting
Approximately 200,000 hRPE cells were injected into the subretinal space at the age of 23 days. One week later, 53,505 ± 5,766 cells (∼27% of the total injected cells) were counted. By 2 weeks after grafting, the number of donor cells was reduced to 42,105.01 ± 10,347.77 (∼20.05% survival). By 4 weeks after grafting, the donor cells were reduced dramatically to 22,368 ± 6,933 (∼11.18% ± 3.5%), almost half of the amount surviving at 2 weeks. The number of donor cells declined further with age. By 6 weeks, the surviving cells amounted to 10,412.54 ± 3,994.57 (∼5.20% of the total injected cells), and by 15 weeks after grafting, the number was reduced to 3,293.90 ± 1,493.02 (∼1.64% survival). There were still 403.01 ± 189.57 cells (0.2% survival) at 28 weeks after grafting (Fig. 3) , and even at 36 weeks after injection, a few labeled donor cells were observed. 
Distribution of Donor hSCs Revealed by the hSC Antibody
The distribution of hSCs in the subretinal space was rather different from that of hRPE cells. hSCs formed many small clumps of various sizes. They did not have close contact with host RPE layer, apart from at the injection site (Figs. 4B 5B 5C 6A) . The size and shape of the clumps were irregular. Some were ball-like (20 μm in diameter) or oval (20 × 45 μm), others were irregularly shaped or sheetlike (850 μm in length; Figs. 4 5 6 ), and some single cells were visible (Fig. 4D , arrows). They were distributed on both sides of the injection site within the subretinal space. Sometimes, labeled cells were spread over 25% to 75% of the retinal length in a single section, extending as far as the retinal margin on one side. The hSC-specific antibody stained the whole cell body (Fig. 4) , and as a result, it was impossible to count individual cells, because they were closely apposed within the clumps. At early time points, there were ∼4 to 13 clumps per section (Figs. 5A 5B) , distributed over an average of 31.6% of the retinal section. This number was reduced over time (Figs. 5C 5D 6A)to one to two clumps at the late time points (Figs. 4H 4G) . There was evidence of a few hSCs migrating into inner retina (Fig. 4D)at both early and late time points. The control staining was negative when primary or secondary antibodies were omitted. 
Donor Cell Distribution in Relation to Photoreceptor Rescue
From the early time point (1 week after grafting), the donor hSCs spread in the subretinal space in both directions (Fig. 5A) . Counting 15 sections per eye, three eyes per age group revealed that there were ∼4 to 13 clumps per section, extending over an average of 31.6% ± 6.58% of the length of a retinal section. By 2 weeks after grafting, there were ∼6 to 12 clumps per section, covering 37.7% ± 3.5% of the length of a retinal section. By 4 weeks, there were approximately three to nine clumps per section, covering 30.4% ± 7.0% of the length of the retinal section (Fig. 5B) . Photoreceptor rescue in the region of the donor cells covered 43.4% ± 9.7% of the length of a retinal section. The difference between the lengths of graft distribution and rescued photoreceptors was highly significant (P < 0.0001). By 6 weeks after injection, there were three to eight clumps per section, covering an average of 42.23% ± 6.9% of the length of the retinal section (Figs. 5C 6A) . The rescued photoreceptors (four or more cells deep) occupied ∼51.35% ± 5.8% of the length of the retinal section. The difference between the lengths of graft distribution and the rescued photoreceptors was again significant (P < 0.005). By 15 weeks, the number of antibody-stained hSCs was dramatically reduced. There were one to four clumps per section (Fig. 4G) , covering ∼22.95% ± 9.5% of the length of a retinal section. Rescued photoreceptors occupied 35.12% ± 9.7% of the length of the retinal section. The difference between the lengths of hSC distribution and the rescued photoreceptors was significant (P < 0.0001). By 22 weeks (Fig. 4H) , antibody-stained hSCs were reduced, with usually only one to two clumps per section observed, often around the injection site. There was still photoreceptor rescue beyond the region of donor cell distribution. By 28 weeks after grafting, only a few hSCs stained, and sometimes the cells were seen along blood vessels and on the vitreous surface of the retina, but photoreceptor rescue was still evident. 
Discussion
The present results tracked both homologous- and heterologous-type cell transplants from early time points after transplantation to long-term survival by using donor cell–specific antibodies. Both hRPE and hSCs survived for a substantial time after grafting into the subretinal space of RCS rats, but the number of surviving cells declined with time. Both hRPE and hSCs preserved photoreceptors from degeneration for a substantial time. For both cell types, the rescued photoreceptors extended beyond the region of donor cell distribution, suggesting that the rescue effects may depend on diffusible factors and not necessarily on cell-contact–mediated events. 
Identification of Transplanted Cells
In many earlier studies exploring the effects of cell transplantation, emphasis was placed on photoreceptor rescue, and donor cell survival was not explored, in part, because of inadequate methods for localizing donor cells. As a result, correlation of rescue and donor cell survival over time has not been studied. Among the methods of identifying donor cells in the past, some have relied on donor cell markers such as pigment granules (when transplanting to albino hosts); but, with that method, there is always concern that host cells (even in the RCS rats where phagocytosis is compromised but not completely inhibited) may take up inclusion bodies of cells that have died and give false positives. 22 23 24 Use of extrinsic markers, again giving positive results, is limited by the lack of stability of the marker in the cell, possible additional challenges to the immune system, and the risk that the marker may disturb cell function and, although not necessarily affecting donor cell survival, may affect the ability of the cell to rescue photoreceptors. 25 26 27 The Y chromosome has also been used in identifying donor cells, but the small dotlike signals in the in situ hybridization technique are sometimes difficult to detect. 28 29  
Del Priore et al. 18 used a porcine-specific molecule to track survival of porcine RPE cells transplanted to the subretinal space of normal albino rabbits under triple immune suppression conditions. Their results show a remarkable similarity to the present data from ARPE-19 cells. Four weeks after grafting, Del Priore et al. counted 10.5% remaining of the 40,000 cells originally injected. This number declined to 0.25% to 0.5% after 12 weeks. In our study, we recorded 11.18% of donor cells at 4 weeks of survival and 1.64% after 15 weeks. It should be noted that we started with more donor cells (200,000) and a less rigorous immune-suppression regimen, together with a different donor–host xenograft pairing. Substantial donor cell loss in the early posttransplantation period has also been found in grafting of dopamine cells in animal models of Parkinson’s disease, in which ∼5% to 6% of human dopamine-producing neurons harvested from 6.5- to 8-week-old fetuses survived after grafting into Cy-immunosuppressed rats. The survival rates of dopamine-producing neurons in patients receiving grafts of human nigral tissue ranged from 1% to 4%. 30  
The decline in cell number with time is most likely due to the inadequacy of Cy in immunoprotection of the grafts. 18 It is poorly absorbed 31 and a subgroup of T cells has been shown to become resistant to it. 32 33 In culture, some of the RPE cells are activated and that may accelerate the rejection cascade after transplantation. 22 The expression of MHC class II antigens on RPE cells of rabbits can be upregulated by treatment with IFN-γ, 22 and such activated RPE cells induce a rejection cascade after transplantation. 22 34 In long-term experiments, graft failure was associated with invasion of macrophages and glial cells but not lymphocytes. 35 36 A pilot study revealed that under the same immunosuppression protocol, macrophage and microglial activation were reduced somewhat, but not completely. 37  
Another confounding factor is that in RCS rats, as in some forms of age-related macular degeneration, leaky vessels appear 38 that may abrogate the privileged site, compromising Cy’s efficacy. The number of 5-bromo-2-deoxyuridine (BrdU)-labeled human fetal RPE cells injected subretinally into rabbit eyes declines starting from day 14 after transplantation, also suggesting rejection. 39  
It should be noted that in a behavioral study examining acuity over time after ARPE-19 or hSC grafts, 15 some individual animals showed severe decline, whereas others sustained high performance levels for as much as 4 months after grafting, raising the possibility that absorption of CyA or activational states of intrinsic cells, such as microglia, may vary among animals. The response to xenografts, especially delivered through a transscleral approach may give little insight into immune considerations relevant to clinical management. It would be much better to explore the survival capability of allogeneic and syngeneic grafts, to explore possible requirements for sustained immunosuppressive therapy over time. This exploration is being undertaken in ongoing studies. 
Mechanism of the Rescue Effect
The present results, in conjunction with those in previous studies, raise interesting questions as to the mode of action of the transplanted cells in rescuing photoreceptors. Previous functional studies have shown similar rescue of function with hRPE cells and hSCs, by using ERG analysis, 40 41 threshold response recording from the superior colliculus, 42 43 optomotor responses, and acuity measures. 15 It might be presumed that the ARPE-19 cells would assume the full range of roles of RPE cells, particularly in sustaining rod function. However, a recent adaptation study provides evidence that in the RCS rat, rods fail to function at low luminance levels and that whereas ARPE-19 cells sustain rods anatomically, 7 8 9 there is no evidence of restoration of low-luminance vision. 43 Similarly, SC cells failed to restore low-luminance vision in this animal. The mechanism of SC efficacy may relate to the ability of these cells to produce a number of growth factors known to sustain photoreceptors when injected into the vitreous or introduced by vectors into the vitreous or the subretinal space. 44 45 46 The finding in the current study that rescue extended beyond the area of distribution of donor hSCs is consistent with the idea that these cells function by release of diffusible factors. The similar finding with ARPE-19 cell grafts suggests that these cells, too, may function by release of diffusible factors. It is known that RPE cells produce a rang of factors, such as brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor (bFGF), and pigment epithelium-derived factor (PEDF), 47 48 49 all of which have been shown to rescue photoreceptors. 50 51 52 Together, these results suggest that ARPE-19 cells, like Schwann cells, function as a local cell delivery system for growth factors rather than necessarily as a homologous cell replacement. 
Summary
The present study revealed that both hRPE and hSC grafts can survive and rescue photoreceptors for a substantial time after grafting into the subretinal space. The number of both donor cell types, as identified by human-specific antibodies declined with time, which could be an immune-related problem, and/or due to other factors intrinsic to the host RCS retina. The fact that rescue after grafting of both hRPE cells and hSCs produced a larger area of photoreceptor rescue than that covered by the donor cells suggests that diffusible factors are involved, raising the possibility that the two cell types function in a manner similar to rescue photoreceptors. 
 
Figure 1.
 
Retinal sections with hRPE graft. (A) CV-stained section 1 week after grafting, showing a clump of donor cells (quad arrow). Triangle: injection site. (B) Section adjacent to (A), stained with human-specific nuclear marker. Quad arrow: antibody-positive donor cells. (C, D) Retinal sections 2 weeks after grafting stained with CV (C) and human nuclear marker (D). Arrows: donor cells; triangle: injection site. (E, F) Human nuclear marker-positive donor cells (arrows) at 4 and 6 weeks after grafting. (G) Five weeks after grafting. Arrows: donor cells. The ONL was approximately three to five cells deep. (H) The ONL was three to four cells deep, even at 28 weeks after grafting. (I) Donor cells (arrows) surviving at 36 weeks after transplantation. (J) Human fibroblasts injected retina 15 weeks after surgery. Arrows: localized photoreceptor rescue. INL, inner nuclear layer; ONL, outer nuclear layer; RGC, retinal ganglion cell layer; RPE, retinal pigment epithelium. Scale bars, 50 μm
Figure 1.
 
Retinal sections with hRPE graft. (A) CV-stained section 1 week after grafting, showing a clump of donor cells (quad arrow). Triangle: injection site. (B) Section adjacent to (A), stained with human-specific nuclear marker. Quad arrow: antibody-positive donor cells. (C, D) Retinal sections 2 weeks after grafting stained with CV (C) and human nuclear marker (D). Arrows: donor cells; triangle: injection site. (E, F) Human nuclear marker-positive donor cells (arrows) at 4 and 6 weeks after grafting. (G) Five weeks after grafting. Arrows: donor cells. The ONL was approximately three to five cells deep. (H) The ONL was three to four cells deep, even at 28 weeks after grafting. (I) Donor cells (arrows) surviving at 36 weeks after transplantation. (J) Human fibroblasts injected retina 15 weeks after surgery. Arrows: localized photoreceptor rescue. INL, inner nuclear layer; ONL, outer nuclear layer; RGC, retinal ganglion cell layer; RPE, retinal pigment epithelium. Scale bars, 50 μm
Figure 2.
 
Retinal sections with hRPE grafts stained with a human-specific nuclear marker. (B) Overall view of the distribution of donor cells at 6 weeks after surgery. Arrows: donor cells. Under the donor cells the ONL was six to eight cells deep, whereas distant from donor cells, the ONL was two to three cells deep. (A) High-power image of outline b in (B) showing donor cells (arrows). (D) Overall view of donor cell distribution at 15 weeks after surgery. Arrows: donor cells. (C) High-power image of outline d showing donor cells forming monolayer (arrows). Scale bars: (A, C) 200 μm; (B, D) 50 μm.
Figure 2.
 
Retinal sections with hRPE grafts stained with a human-specific nuclear marker. (B) Overall view of the distribution of donor cells at 6 weeks after surgery. Arrows: donor cells. Under the donor cells the ONL was six to eight cells deep, whereas distant from donor cells, the ONL was two to three cells deep. (A) High-power image of outline b in (B) showing donor cells (arrows). (D) Overall view of donor cell distribution at 15 weeks after surgery. Arrows: donor cells. (C) High-power image of outline d showing donor cells forming monolayer (arrows). Scale bars: (A, C) 200 μm; (B, D) 50 μm.
Figure 3.
 
Number of ARPE-19 cells revealed by the human-specific nuclear marker. Data were collected from serial sections in three animals per postgrafting time point and were corrected by the Abercrombie method. Error bars, SD.
Figure 3.
 
Number of ARPE-19 cells revealed by the human-specific nuclear marker. Data were collected from serial sections in three animals per postgrafting time point and were corrected by the Abercrombie method. Error bars, SD.
Figure 4.
 
Retinal sections with hSC graft stained with hSC antibody. (A, B) Antibody-positive hSCs formed ball-like (A, arrows) and sheetlike (B, triangles) at 1 week after transplantation. hSCs formed irregular shapes at 2 (C, triangles) and 4 (D, left- and right-pointing arrows) weeks after grafting. Filled arrows: migrating hSCs. The ONL showed rescued cells with inner segments (IS) at 6 (E), 12 (F), and 15 (G) weeks after grafting. Triangles: hSCs. (H) Labeled hSCs (triangles) at 22 weeks after surgery. (I) High-power image of hSCs at 1 week after grafting (quad arrow). Scale bars: (A–F, H) 100 μm; (G, I) 50 μm.
Figure 4.
 
Retinal sections with hSC graft stained with hSC antibody. (A, B) Antibody-positive hSCs formed ball-like (A, arrows) and sheetlike (B, triangles) at 1 week after transplantation. hSCs formed irregular shapes at 2 (C, triangles) and 4 (D, left- and right-pointing arrows) weeks after grafting. Filled arrows: migrating hSCs. The ONL showed rescued cells with inner segments (IS) at 6 (E), 12 (F), and 15 (G) weeks after grafting. Triangles: hSCs. (H) Labeled hSCs (triangles) at 22 weeks after surgery. (I) High-power image of hSCs at 1 week after grafting (quad arrow). Scale bars: (A–F, H) 100 μm; (G, I) 50 μm.
Figure 5.
 
(A) Overall view of the distribution of hSCs 1 week after grafting. Arrows: hSCs in clumps in the subretinal space; right-pointing, open arrow: injection site. (Aa) High-power of the outline in (A); triangle: ball-like hSCs. (B) Overall view of the distribution of hSCs 4 weeks after grafting. Arrows: donor hSCs; (Bb) high-power image of the outline in (B). (C) Overall view of the distribution of hSCs 6 weeks after grafting. Arrows: donor hSCs; triangle: injection site. (Cc) High-power image of the outlined area in (C). (D) Section adjacent to (A) stained with CV; (Dd) High-power image of the outlined area in (D); triangle: injection site. Scale bars: (A–D) 200 μm; (Aa–Dd) 20 μm.
Figure 5.
 
(A) Overall view of the distribution of hSCs 1 week after grafting. Arrows: hSCs in clumps in the subretinal space; right-pointing, open arrow: injection site. (Aa) High-power of the outline in (A); triangle: ball-like hSCs. (B) Overall view of the distribution of hSCs 4 weeks after grafting. Arrows: donor hSCs; (Bb) high-power image of the outline in (B). (C) Overall view of the distribution of hSCs 6 weeks after grafting. Arrows: donor hSCs; triangle: injection site. (Cc) High-power image of the outlined area in (C). (D) Section adjacent to (A) stained with CV; (Dd) High-power image of the outlined area in (D); triangle: injection site. Scale bars: (A–D) 200 μm; (Aa–Dd) 20 μm.
Figure 6.
 
(A) Overall view of the distribution of hSCs at 6 weeks after grafting. Arrows: donor hSCs; triangle: injection site. (B) High-power image of area b showing donor hSCs (triangles); arrows: distortion of host ONL by donor cells. (C) High-power image of area c showing sheet-like distribution of hSCs (arrows). (D) Section adjacent to (A) stained with CV, showing area in c; triangles: donor cells mixed with host RPE cells. Scale bars: (A) 400 μm; (B–D) 100 μm.
Figure 6.
 
(A) Overall view of the distribution of hSCs at 6 weeks after grafting. Arrows: donor hSCs; triangle: injection site. (B) High-power image of area b showing donor hSCs (triangles); arrows: distortion of host ONL by donor cells. (C) High-power image of area c showing sheet-like distribution of hSCs (arrows). (D) Section adjacent to (A) stained with CV, showing area in c; triangles: donor cells mixed with host RPE cells. Scale bars: (A) 400 μm; (B–D) 100 μm.
Authors thank Linda White for isolation, preparation, and shipping of human Schwann cells and human fibroblasts and Xiaoying Cheng for histologic assistance. 
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Figure 1.
 
Retinal sections with hRPE graft. (A) CV-stained section 1 week after grafting, showing a clump of donor cells (quad arrow). Triangle: injection site. (B) Section adjacent to (A), stained with human-specific nuclear marker. Quad arrow: antibody-positive donor cells. (C, D) Retinal sections 2 weeks after grafting stained with CV (C) and human nuclear marker (D). Arrows: donor cells; triangle: injection site. (E, F) Human nuclear marker-positive donor cells (arrows) at 4 and 6 weeks after grafting. (G) Five weeks after grafting. Arrows: donor cells. The ONL was approximately three to five cells deep. (H) The ONL was three to four cells deep, even at 28 weeks after grafting. (I) Donor cells (arrows) surviving at 36 weeks after transplantation. (J) Human fibroblasts injected retina 15 weeks after surgery. Arrows: localized photoreceptor rescue. INL, inner nuclear layer; ONL, outer nuclear layer; RGC, retinal ganglion cell layer; RPE, retinal pigment epithelium. Scale bars, 50 μm
Figure 1.
 
Retinal sections with hRPE graft. (A) CV-stained section 1 week after grafting, showing a clump of donor cells (quad arrow). Triangle: injection site. (B) Section adjacent to (A), stained with human-specific nuclear marker. Quad arrow: antibody-positive donor cells. (C, D) Retinal sections 2 weeks after grafting stained with CV (C) and human nuclear marker (D). Arrows: donor cells; triangle: injection site. (E, F) Human nuclear marker-positive donor cells (arrows) at 4 and 6 weeks after grafting. (G) Five weeks after grafting. Arrows: donor cells. The ONL was approximately three to five cells deep. (H) The ONL was three to four cells deep, even at 28 weeks after grafting. (I) Donor cells (arrows) surviving at 36 weeks after transplantation. (J) Human fibroblasts injected retina 15 weeks after surgery. Arrows: localized photoreceptor rescue. INL, inner nuclear layer; ONL, outer nuclear layer; RGC, retinal ganglion cell layer; RPE, retinal pigment epithelium. Scale bars, 50 μm
Figure 2.
 
Retinal sections with hRPE grafts stained with a human-specific nuclear marker. (B) Overall view of the distribution of donor cells at 6 weeks after surgery. Arrows: donor cells. Under the donor cells the ONL was six to eight cells deep, whereas distant from donor cells, the ONL was two to three cells deep. (A) High-power image of outline b in (B) showing donor cells (arrows). (D) Overall view of donor cell distribution at 15 weeks after surgery. Arrows: donor cells. (C) High-power image of outline d showing donor cells forming monolayer (arrows). Scale bars: (A, C) 200 μm; (B, D) 50 μm.
Figure 2.
 
Retinal sections with hRPE grafts stained with a human-specific nuclear marker. (B) Overall view of the distribution of donor cells at 6 weeks after surgery. Arrows: donor cells. Under the donor cells the ONL was six to eight cells deep, whereas distant from donor cells, the ONL was two to three cells deep. (A) High-power image of outline b in (B) showing donor cells (arrows). (D) Overall view of donor cell distribution at 15 weeks after surgery. Arrows: donor cells. (C) High-power image of outline d showing donor cells forming monolayer (arrows). Scale bars: (A, C) 200 μm; (B, D) 50 μm.
Figure 3.
 
Number of ARPE-19 cells revealed by the human-specific nuclear marker. Data were collected from serial sections in three animals per postgrafting time point and were corrected by the Abercrombie method. Error bars, SD.
Figure 3.
 
Number of ARPE-19 cells revealed by the human-specific nuclear marker. Data were collected from serial sections in three animals per postgrafting time point and were corrected by the Abercrombie method. Error bars, SD.
Figure 4.
 
Retinal sections with hSC graft stained with hSC antibody. (A, B) Antibody-positive hSCs formed ball-like (A, arrows) and sheetlike (B, triangles) at 1 week after transplantation. hSCs formed irregular shapes at 2 (C, triangles) and 4 (D, left- and right-pointing arrows) weeks after grafting. Filled arrows: migrating hSCs. The ONL showed rescued cells with inner segments (IS) at 6 (E), 12 (F), and 15 (G) weeks after grafting. Triangles: hSCs. (H) Labeled hSCs (triangles) at 22 weeks after surgery. (I) High-power image of hSCs at 1 week after grafting (quad arrow). Scale bars: (A–F, H) 100 μm; (G, I) 50 μm.
Figure 4.
 
Retinal sections with hSC graft stained with hSC antibody. (A, B) Antibody-positive hSCs formed ball-like (A, arrows) and sheetlike (B, triangles) at 1 week after transplantation. hSCs formed irregular shapes at 2 (C, triangles) and 4 (D, left- and right-pointing arrows) weeks after grafting. Filled arrows: migrating hSCs. The ONL showed rescued cells with inner segments (IS) at 6 (E), 12 (F), and 15 (G) weeks after grafting. Triangles: hSCs. (H) Labeled hSCs (triangles) at 22 weeks after surgery. (I) High-power image of hSCs at 1 week after grafting (quad arrow). Scale bars: (A–F, H) 100 μm; (G, I) 50 μm.
Figure 5.
 
(A) Overall view of the distribution of hSCs 1 week after grafting. Arrows: hSCs in clumps in the subretinal space; right-pointing, open arrow: injection site. (Aa) High-power of the outline in (A); triangle: ball-like hSCs. (B) Overall view of the distribution of hSCs 4 weeks after grafting. Arrows: donor hSCs; (Bb) high-power image of the outline in (B). (C) Overall view of the distribution of hSCs 6 weeks after grafting. Arrows: donor hSCs; triangle: injection site. (Cc) High-power image of the outlined area in (C). (D) Section adjacent to (A) stained with CV; (Dd) High-power image of the outlined area in (D); triangle: injection site. Scale bars: (A–D) 200 μm; (Aa–Dd) 20 μm.
Figure 5.
 
(A) Overall view of the distribution of hSCs 1 week after grafting. Arrows: hSCs in clumps in the subretinal space; right-pointing, open arrow: injection site. (Aa) High-power of the outline in (A); triangle: ball-like hSCs. (B) Overall view of the distribution of hSCs 4 weeks after grafting. Arrows: donor hSCs; (Bb) high-power image of the outline in (B). (C) Overall view of the distribution of hSCs 6 weeks after grafting. Arrows: donor hSCs; triangle: injection site. (Cc) High-power image of the outlined area in (C). (D) Section adjacent to (A) stained with CV; (Dd) High-power image of the outlined area in (D); triangle: injection site. Scale bars: (A–D) 200 μm; (Aa–Dd) 20 μm.
Figure 6.
 
(A) Overall view of the distribution of hSCs at 6 weeks after grafting. Arrows: donor hSCs; triangle: injection site. (B) High-power image of area b showing donor hSCs (triangles); arrows: distortion of host ONL by donor cells. (C) High-power image of area c showing sheet-like distribution of hSCs (arrows). (D) Section adjacent to (A) stained with CV, showing area in c; triangles: donor cells mixed with host RPE cells. Scale bars: (A) 400 μm; (B–D) 100 μm.
Figure 6.
 
(A) Overall view of the distribution of hSCs at 6 weeks after grafting. Arrows: donor hSCs; triangle: injection site. (B) High-power image of area b showing donor hSCs (triangles); arrows: distortion of host ONL by donor cells. (C) High-power image of area c showing sheet-like distribution of hSCs (arrows). (D) Section adjacent to (A) stained with CV, showing area in c; triangles: donor cells mixed with host RPE cells. Scale bars: (A) 400 μm; (B–D) 100 μm.
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