Thus far, the grafted iPSC-derived RPE sheet has survived for 4 years and seems to support photoreceptors and choroidal vessels. The morphologic characteristics of the RPE are observed at the transplant site.

At the 4-year follow-up, the transplanted autologous iPSC-derived RPE sheet had survived beneath the retina with slight expansion of the pigmented area and no adverse events. The outer nuclear layer above and adjacent to the graft showed acceptable thickness and an organized structure. Fluorescein angiography and SD OCT suggested the presence of vessel-like structures confined to the grafted area associated with the remaining trunk vessel of preoperative polypoidal choroidal vasculopathy but with no exudative changes. Visual acuity has been stable with no additional injections of anti–vascular endothelial growth factor agent. The choroidal volume at the graft site is relatively preserved when compared with the volume outside this site without RPE after removal of the CNV. Indocyanine green angiography revealed a preserved choriocapillaris around the iPSC-derived RPE sheet. Dark cell-like structures with a predominantly hexagonal arrangement were observed by AO imaging in an area located near the margin of the graft sheet. The average intercell distance was found to be stable over time.

The function of the graft was assessed 4 years after surgery by color fundus photography, spectral-domain (SD) OCT, fluorescein angiography, indocyanine green angiography, and an adaptive optics (AO) retinal camera.

To report the results after 4 years of follow-up in a previously presented first case of induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) sheet autologous transplantation using multimodal imaging.

The retinal pigment epithelium (RPE) is a monolayer of pigmented cells lying between the retinal photoreceptor and choroidal layers.The RPE has important physiologic functions, including regeneration of opsins,phagocytosis of the outer segments of photoreceptors,and polarized secretion of factors such as pigment epithelium-derived factor (PEDF) apicallyand vascular endothelial growth factor (VEGF) basallyto maintain the neural retinal and choroidal vessels, respectively. Impairment of the RPE has been implicated in many retinal diseases, including age-related macular degeneration (AMD).Transplantation of healthy RPE is a potential therapeutic approach in such diseases. Unlike retinal neural cell transplants, which need to become integrated into the host neural network to function properly, RPE cells can be expected to function if they survive after transplantation. Surgical translocation of the peripheral RPE–choroid complex to the impaired macular area has been reported to be effective for a lengthy period in patients with AMD.Therefore, RPE transplantation is a feasible and promising therapy.

Age-related macular degeneration is one of the leading causes of vision loss in developed countries.It is classified as dry or wet according to the absence or presence of choroidal neovascularization (CNV).Physical disruption and functional impairment of the RPE have been observed throughout the pathologic course of both types of AMD. Eyes with AMD show retinal impairment in the macular region, which causes deterioration of central vision and impacts quality of life.Intravitreal anti-VEGF injection to suppress CNV activity is the first-line therapy for wet AMD, and its efficacy and safety have been demonstrated in several large clinical trials.However, this strategy does not target the underlying pathogenesis of AMD, and many patients require repeated injections of an anti-VEGF agent over a long period because of recurrences of CNV.Moreover, in several large clinical studies, for example, Comparison of AMD Treatments Trials (CATT)and VEGF Trap-Eye: Investigation of Efficacy and Safety in Wet AMD (VIEW)1 and VIEW2,a significant proportion of patients with AMD showed a poor or suboptimal response to an anti-VEGF therapeutic approach.

Advances in stem cell technology in the past decade have generated widespread interest in transplantation of embryonic stem cell (ESC)-derived RPEand induced pluripotent stem cell (iPSC)-derived RPEfor degenerative diseases of the RPE. Clinical trials of ESC-derived RPE transplantation for dry AMD and 2 independent clinical investigations of ESC-derived or iPSC-derived RPE sheet transplantation for wet AMD have now been reported, including 1 by our group. All of these studies have demonstrated that stem cell-derived RPE therapy is safe, feasible, and potentially effective in the short term to midterm.In September 2014, we performed an autologous iPSC-derived RPE sheet transplantation in a patient with wet AMD after removal of CNV.Herein, we report the 4-year follow-up of this first patient to have undergone iPSC-derived RPE transplantation.

Although the presence or survival of pigmented cells or cell sheets was confirmed by examination of the fundus in the above-mentioned studies, evaluation of the functional status of the transplanted RPE in vivo was difficult to achieve. We conducted the transplantation in only 1 patient with a large fibrotic scar and poor vision for this first safety test, and it was difficult to obtain reliable data by microperimetry or multifocal electroretinography because of unstable fixation. Therefore, we attempted to assess the function of the transplanted iPSC-derived RPE using multimodal imaging. Given that functioning RPE is expected to support the choroidal vasculature by secreting VEGF, we assessed the function of the transplanted iPSC-derived RPE by evaluating the temporal changes in choroidal thickness and vessel volumes in the area of the transplant. Using spectral-domain (SD) OCT, fluorescein angiography (FA), and indocyanine green angiography (ICGA), we compared our findings in the area containing the iPSC-derived RPE transplant with those in the surrounding area of defective RPE after removal of the CNV. We also attempted to analyze the morphologic features of the iPSC-derived RPE cells in the graft sheet in vivo using images obtained by an adaptive optics (AO) retinal camera.

High-resolution en face fundus images were obtained to observe the patient’s iPSC-derived RPE cells using an AO retinal camera(rtx1; Imagine Eyes, Orsay, France). The AO camera illuminates a 4° square field with 850-nm infrared flashes to acquire en face images of the retina with a transverse optical resolution of 250 line pairs/mm. The patient was instructed to fixate on an internal target that was positioned by the operator to select areas of interest in the retina. After the acquisitions, images captured outside of the transplant region and out-of-focus images were discarded. A subset of AO images acquired 2, 16, 20, and 48 months after surgery was then selected for further analysis. These AO images were aligned using coregistration software (i2k; DualAlign, Clifton Park, NY). Each image was colocalized with the images obtained by color fundus photography, infrared scanning laser ophthalmoscopy, and SD OCT. The positions of the RPE cells in the images were marked manually by 5 experienced retinal imaging specialists (ST, MM, KG, MD, and NC) working independently. The RPE cells were defined as “bright at the border and dark at their center” in accordance with a previous report.Based on the cell positions determined in each AO image, the intercell distance and neighboring cell counts were computed using Voronoi statistical software (AOdetect, Imagine Eyes, Orsay, France). The reproducibility of the intercell distance assessment between operators was estimated by computing the intraclass correlation coefficient, and changes in the intercell distance over time were analyzed. We also observed the natural RPE cells in a patient with retinal detachment as a result of central serous chorioretinopathy as a control.

Fluorescein angiography and ICGA were performed using a standard digital imaging system (Heidelberg Engineering). Fluorescein 500 mg (Alcon Pharma, Tokyo, Japan) or indocyanine green 25 mg (Daiichi Sankyo Company, Tokyo, Japan) was injected via a peripheral venous catheter over 10 seconds and followed by a 10-ml flush of physiologic saline. The patient was instructed to direct her gaze toward the center of the screen. The vessel area on ICGA images was measured in a 6 × 6-mm square at the center of the fovea. The ICGA fundus photographs were converted to 8-bit and binary images using the Niblack method with ImageJ software to quantify the vessel area. We used 5 points in the black area and averaged the reflectivity of these points to set the minimum value.

The choroidal volume was measured using the method described by Chhablani et al.In brief, each of the 31 images in a volume scan was outlined manually by 2 experienced ophthalmologists (ST, YH), and choroidal volume maps were computed using the built-in SD OCT tool. The choroidal thickness was defined as the distance between Bruch’s membrane and the outer border–chorioscleral interface. The fovea was defined according to the infrared and OCT images, thinning of the inner retina, and foveal depression. The choroidal thickness and volume in the 1-mm, 3-mm, and 6-mm areas from the foveal center were then calculated according to the Early Treatment Diabetic Retinopathy Study grid. The choroid at the iPSC-derived RPE transplant site was defined representatively as the sum of the macular, inner superior, and inner inferior areas in the Early Treatment Diabetic Retinopathy Study grid; the rest of the area after removal of the CNV was measured outside the transplant site (defective RPE area).

Spectral-domain OCT (Spectralis; Heidelberg Engineering, Heidelberg, Germany) images acquired before and 1 week and 1, 3, 6, 12, 24, 36, and 48 months after surgery were used to analyze the retinal and choroidal thicknesses. Single OCT images measuring 9.0 mm in length were acquired using a raster scan protocol of 512 (horizontal) × 128 (vertical) A-scans per data set (total, 65 536 axial scans/volume) in 0.8 seconds. One hundred B-scan images were averaged to reduce speckle noise. The volume scan was performed with raster imaging consisting of 31 high-resolution B-scans for each eye. All 31 OCT macular B-scans were acquired in a continuous automated sequence that covered an area of 30° × 25° centered on the fovea. The total retinal thickness at the fovea was measured manually using the built-in software.

Color photographs were obtained using a digital fundus camera (IMAGEnet; Topcon Corp, Tokyo, Japan). The graft area was calculated after manual delimitation of the dark-pigmented zone that was visible in the photographic images using ImageJ (National Institutes of Health, Bethesda, MD).

We have observed the patient periodically with basic ophthalmic examinations, including best-corrected visual acuity, fundus examination, SD OCT, and FA and ICGA as necessary. Adaptive optics imaging was performed at several visits when the patient’s general health status permitted.

The treatment strategy used in this patient was approved under the Regenerative Medicine Law in November 2014, and the transplantation surgery was performed in September 2014. All the details of the technique used, production of the sheet of cells, assessment of its quality and safety, surgical procedure, and postoperative course for up to 24 months have been described previously.In brief, the patient was a 77-year-old Japanese woman who had been diagnosed with a subtype of neovascular AMD and polypoidal choroidal vasculopathy and had experienced a steady decrease in vision despite receiving 13 anti-VEGF injections over 4 years. After entry into our clinical study, iPSC clones were obtained from the skin tissue using GLIS1, L-MYC, SOX2, KLF4, and OCT3/4. The iPSCs were subsequently differentiated into RPE sheets without an synthetic scafold. A 1.3 × 3-mm iPSC-derived RPE sheet was then inserted beneath the fovea immediately after removal of the neovascular membrane.

This clinical study was approved by the institutional review boards and ethics committees at the collaborating sites and by the Minister of Health, Labor, and Welfare (Japan). Written informed consent was obtained from the patient, and the study adhered to the tenets of the Declaration of Helsinki. All experiments were reviewed and approved by the institutional review boards of the Foundation for Biomedical Research and Innovation and Riken Center for Developmental Biology.

The RPE cells on the transplanted iPSC-derived RPE sheet were not visualized easily with the AO camera because the sheet was not completely flat. However, we repeatedly obtained images of dark hexagonal cell-like structures at the upper margin of the graft sheet in an area where the marginal RPE cells were a natural brown color on the fundus photographs, and the SD OCT images showed a continuous solid line at the RPE level ( Fig 4 A–C). Using the retinal vessels as landmarks in the AO images, the 5 observers independently identified the RPE cells as structures that were defined by their round shape with a grayish center and relatively bright surrounding margin ( Fig 4 B, D). The average number of the closest neighboring cells obtained by Voronoi analysis at 2, 16, 20, and 48 months was 5.67. The morphologic features of the iPSC-derived RPE cells were stable at the different time points after transplantation. At 48 months, the average distance between the iPSC-derived RPE cells measured by the 5 observers was 13.46±1.03 μm ( Fig 4 F), with an intraclass correlation coefficient of 0.76 (range, 0.62–0.80). An image from the control patient with central serous chorioretinopathy showed regular hexagonal cells with the same definition, which were observed only in the area of serous retinal detachment ( Fig 4 E).

Images showing the retinal pigment epithelium (RPE) cell-like structure at the upper margin of the graft sheet acquired using an adaptive optics (AO) camera and by fundus photography and spectral-domain (SD) OCT. A , Fundus photograph obtained 1 month after the surgery. The marginal area has a more natural orange color (similar to that of a normal fundus) than the surrounding area with RPE defect. A′ , Magnified image of the white-dotted box in ( A ). B , Adaptive optics image of the area in the yellow box in ( A′ ). The white boxed area was magnified further for counting RPE cells. Hexagonal cells were observed repeatedly at the upper graft margin by AO. C , Identification of the graft iPSC-derived RPE cells by SD OCT. The yellow box indicates the area examined by AO ( A′ , B ). C′ , Magnified SD OCT image showing the continuous hyperreflective line (between the blue arrows) suggesting the presence of RPE cells. The red line shows the area in which the cells were counted. D , Example of analysis using a Voronoi image in ( B ). The cells were counted by several specialists in the area close to the landmark retinal vessel. E , Magnified view of an AO image of the RPE layer in a 42-year-old patient with central serous chorioretinopathy. The RPE cells were described as bright at the border and dark at their center on AO images. F , Graph showing the average intercell distance at 2, 16, 20, and 48 months after transplantation. Scale bars: ( B ) 500 μm, and ( E ) 500 μm.

Figure 4 Images showing the retinal pigment epithelium (RPE) cell-like structure at the upper margin of the graft sheet acquired using an adaptive optics (AO) camera and by fundus photography and spectral-domain (SD) OCT. A , Fundus photograph obtained 1 month after the surgery. The marginal area has a more natural orange color (similar to that of a normal fundus) than the surrounding area with RPE defect. A′ , Magnified image of the white-dotted box in ( A ). B , Adaptive optics image of the area in the yellow box in ( A′ ). The white boxed area was magnified further for counting RPE cells. Hexagonal cells were observed repeatedly at the upper graft margin by AO. C , Identification of the graft iPSC-derived RPE cells by SD OCT. The yellow box indicates the area examined by AO ( A′ , B ). C′ , Magnified SD OCT image showing the continuous hyperreflective line (between the blue arrows) suggesting the presence of RPE cells. The red line shows the area in which the cells were counted. D , Example of analysis using a Voronoi image in ( B ). The cells were counted by several specialists in the area close to the landmark retinal vessel. E , Magnified view of an AO image of the RPE layer in a 42-year-old patient with central serous chorioretinopathy. The RPE cells were described as bright at the border and dark at their center on AO images. F , Graph showing the average intercell distance at 2, 16, 20, and 48 months after transplantation. Scale bars: ( B ) 500 μm, and ( E ) 500 μm.

The choroid comprises larger vessels and the choriocapillaris. In the late ICGA images (obtained at 7–10 minutes), the presence of diffuse hyperfluorescence with a punctate appearance adjacent to the graft suggested that the choriocapillaris around the iPSC-derived RPE sheet was relatively preserved (indicated by the yellow arrowheads in Fig 3 F). Using the Niblack method, we then measured the area of the vessels in the 6 × 6-mm boxed area that included the graft. There was no appreciable change in the vessel area measured as total white pixels (considered to represent the density of medium to large choroidal vessels) over 48 months.

Given that the RPE is reported to have an important role in maintaining the choriocapillaris by basal secretion of VEGF, we evaluated the choroidal thickness and volume on an Early Treatment Diabetic Retinopathy Study chart before and after surgery ( Fig 3 A–E ). The overall choroidal thickness decreased over time but is relatively preserved around the graft ( Fig 3 C). The choroidal volume decreased at the graft site for up to 12 months after surgery but stabilized thereafter. In contrast, there was a steady decrease in the choroidal volume outside the transplant site (from 0.56 mmto 0.43 mm, i.e., a decrease of 27%, and from 2.31 mmto 1.38 mm, i.e., a decrease of 43%, respectively, at 48 months; Fig 3 E). There was no change in central choroidal thickness in the fellow eye during the 4-year follow-up period (114 μm before surgery and 115 μm 48 months later; Fig S1 C, D).

Choroidal thickness and volume before and after removal of choroidal neovascularization and induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) sheet transplant surgery. A , Example of choroidal segmentation on a spectral-domain OCT image. The choroidal thickness was defined as the distance between Bruch’s membrane and the outer border and chorioscleral interface (red dot). B , Application of an Early Treatment of Diabetic Retinopathy Study (ETDRS) grid on the fundus photograph. The transplant site was defined as the sum of the macular, inner superior, and inner inferior areas in the ETDRS grid. C , Choroidal thickness map within the ETDRS grid before and 1, 3, 12, 24, and 48 months after surgery. D , Graph showing the change in the choroidal volume in the macular area (gray line), inner circle (dotted black line), and outer circle (solid black line) on the ETDRS grid. E , Graph showing the rate of reduction in choroidal volume at the transplant site (solid black line) and adjacent to the transplant site (dotted black line). F , Late-phase indocyanine green angiography (ICGA) images obtained 3, 12, 33, and 48 months after surgery. An area of filling shadow was preserved around the iPSC-derived RPE graft (yellow arrowheads). G , Change in medium and large choroidal vessels in the choroid after transplantation. A 6-mm-square ICGA image around the graft (left image, yellow box) was converted to a binary Image using ImageJ software (middle), and the vessel area was measured (right graph). IA = indocyanine green angiography.

Figure 3 Choroidal thickness and volume before and after removal of choroidal neovascularization and induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) sheet transplant surgery. A , Example of choroidal segmentation on a spectral-domain OCT image. The choroidal thickness was defined as the distance between Bruch’s membrane and the outer border and chorioscleral interface (red dot). B , Application of an Early Treatment of Diabetic Retinopathy Study (ETDRS) grid on the fundus photograph. The transplant site was defined as the sum of the macular, inner superior, and inner inferior areas in the ETDRS grid. C , Choroidal thickness map within the ETDRS grid before and 1, 3, 12, 24, and 48 months after surgery. D , Graph showing the change in the choroidal volume in the macular area (gray line), inner circle (dotted black line), and outer circle (solid black line) on the ETDRS grid. E , Graph showing the rate of reduction in choroidal volume at the transplant site (solid black line) and adjacent to the transplant site (dotted black line). F , Late-phase indocyanine green angiography (ICGA) images obtained 3, 12, 33, and 48 months after surgery. An area of filling shadow was preserved around the iPSC-derived RPE graft (yellow arrowheads). G , Change in medium and large choroidal vessels in the choroid after transplantation. A 6-mm-square ICGA image around the graft (left image, yellow box) was converted to a binary Image using ImageJ software (middle), and the vessel area was measured (right graph). IA = indocyanine green angiography.

During the same 4-year period, the fellow eye received 11 intravitreal aflibercept injections and underwent 1 session of photodynamic therapy for an exudative change and 1 vitrectomy procedure for vitreous hemorrhage. The findings on fundus photography, SD OCT, FA, and ICGA before surgery and 4 years later are shown in Figure S1 (available at www.ophthalmologyretina.org ). Decimal visual acuity in the fellow eye was 0.3 initially and 4 years later was 0.5, despite the expansion of the lesion to the temporal side and a progressive atrophic change in the macular area of the outer retina seen on SD OCT.

There was no systemic deterioration in the patient’s general health nor any adverse events in the transplanted eye according to the Common Terminology Criteria for Adverse Events version 4.0 criteria (whereby adverse events of grade ≥2 are considered serious). The patient’s clinical course in the 4 years since removal of the CNV and the iPSC-derived RPE transplant surgery is summarized in Figures 1 and 2 . During this time, there have been no hemorrhagic events or exudative changes. Furthermore, no leak suggestive of recurrence of active CNV has been detected on FA, except for a possible slight increase in the pigmented area of the graft on fundus photographs ( Fig 1 A–C). The SD OCT images acquired at each FA examination show intermittent degenerative cystic edema but without leakage ( Figs 1 C and 2 A). As described in our previous report,the trunk structure of the polypoidal choroidal vasculopathy vascular network appears to be present after removal of the CNV on FA, ICGA, and SD OCT. Moreover, a vessel-like structure observed to be extending from the trunk after surgery was confined to the grafted area ( Figs 1 C and 2 A). Although retinal thinning has progressed over the RPE defect after removal of the CNV, the structures of the outer nuclear layer on and adjacent to the graft area have remained ( Fig 2 A). The foveal thickness, which is an indicator of exudative change, has remained essentially stable for 4 years, except for morphologic fluctuation of nonleaking cystic edema. There are no features of marked stratified proliferation of the graft on SD OCT. The patient’s best-corrected visual acuity, which had continually decreased before surgery to 0.09 in decimal visual acuity (lower than 20/200 on a Snellen chart), despite a total of 13 anti-VEGF injections, has remained stable in the 4 years since surgery ( Fig 2 C).

Spectral-domain (SD) OCT images and best-corrected visual acuity in the 4 years after removal of choroidal neovascularization and induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) sheet transplant surgery. A , A horizontal SD OCT image including the fovea before and 3, 12, 33, and 48 months after surgery (corresponding to the fluorescein angiography assessments shown in Fig 2) with a magnified view at the fovea. Preoperative SD OCT revealed a hyperreflective mass (yellow asterisk) accompanying a trunk vessel (orange arrow), network vessels (orange arrowheads), retinal edema, and outer retinal tubulation (red arrowheads). The location of the transplanted iPSC-derived RPE sheet on the SD OCT images corresponding to each fundus image is marked by the yellow line. Part of the outer nuclear layer structure was retained on and adjacent to the graft sheet (yellow arrowheads). B , Graph showing change in total foveal thickness measured by OCT images. C , Graph showing change in best-corrected visual acuity after surgery. The yellow line in the SD OCT infra-red (IR) images corresponds to the yellow line on the graft area on the IR fundus photography.

Figure 2 Spectral-domain (SD) OCT images and best-corrected visual acuity in the 4 years after removal of choroidal neovascularization and induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) sheet transplant surgery. A , A horizontal SD OCT image including the fovea before and 3, 12, 33, and 48 months after surgery (corresponding to the fluorescein angiography assessments shown in Fig 2) with a magnified view at the fovea. Preoperative SD OCT revealed a hyperreflective mass (yellow asterisk) accompanying a trunk vessel (orange arrow), network vessels (orange arrowheads), retinal edema, and outer retinal tubulation (red arrowheads). The location of the transplanted iPSC-derived RPE sheet on the SD OCT images corresponding to each fundus image is marked by the yellow line. Part of the outer nuclear layer structure was retained on and adjacent to the graft sheet (yellow arrowheads). B , Graph showing change in total foveal thickness measured by OCT images. C , Graph showing change in best-corrected visual acuity after surgery. The yellow line in the SD OCT infra-red (IR) images corresponds to the yellow line on the graft area on the IR fundus photography.

Fundus photographs, fluorescein angiographic images, and indocyanine green angiographic images of the eye before and after the transplantation of induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) sheet. A , Photographic images of the fundus before and after removal of choroidal neovascularization (CNV) and fibrotic tissue (blue dots) and iPSC-derived RPE sheet transplant surgery before and 3, 12, and 48 months after surgery. The red arrows point to the main pigmented graft. Each magnified image in the white box shows island graft cells indicating an increase in pigmentation over time (green arrows), and the border line of atrophy indicated a slight expansion of the area of the RPE defect (black dot). B , The graft area identified by the presence of a pigmented area on the color fundus photographs was calculated using ImageJ (red demarcation line for the main graft and green demarcation line for the fragments). The graph shows the area of the main graft (red), fragments (green), and total area (blue) over 4 years. C , Preoperative indocyanine green angiography (IA) and fluorescein angiography (FA) images before and 3, 12, 33, and 48 months after surgery. The preoperative IA images show typical polyps (yellow arrow) and a sea fan-shaped network of vessels and trunk vessels of CNV (yellow asterisk). There was no evident leakage on the FA images suggestive of a newly formed CNV network at any of the follow-up examinations, except that no-expanding hyperfluorescein spots were observed continuously at the preoperative trunk of the polypoidal choroidal vasculopathy network throughout the follow-up period (white asterisk).

Figure 1 Fundus photographs, fluorescein angiographic images, and indocyanine green angiographic images of the eye before and after the transplantation of induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) sheet. A , Photographic images of the fundus before and after removal of choroidal neovascularization (CNV) and fibrotic tissue (blue dots) and iPSC-derived RPE sheet transplant surgery before and 3, 12, and 48 months after surgery. The red arrows point to the main pigmented graft. Each magnified image in the white box shows island graft cells indicating an increase in pigmentation over time (green arrows), and the border line of atrophy indicated a slight expansion of the area of the RPE defect (black dot). B , The graft area identified by the presence of a pigmented area on the color fundus photographs was calculated using ImageJ (red demarcation line for the main graft and green demarcation line for the fragments). The graph shows the area of the main graft (red), fragments (green), and total area (blue) over 4 years. C , Preoperative indocyanine green angiography (IA) and fluorescein angiography (FA) images before and 3, 12, 33, and 48 months after surgery. The preoperative IA images show typical polyps (yellow arrow) and a sea fan-shaped network of vessels and trunk vessels of CNV (yellow asterisk). There was no evident leakage on the FA images suggestive of a newly formed CNV network at any of the follow-up examinations, except that no-expanding hyperfluorescein spots were observed continuously at the preoperative trunk of the polypoidal choroidal vasculopathy network throughout the follow-up period (white asterisk).

Discussion

24 Thomas M.A.

Kaplan H.J. Surgical removal of subfoveal neovascularization in the presumed ocular histoplasmosis syndrome. 25 Thomas M.A.

Dickinson J.D.

Melberg N.S.

et al. Visual results after surgical removal of subfoveal choroidal neovascular membranes. 26 Cekic O.

Ohji M.

Keserc I.B.

et al. Evaluation of choroidal perfusion of the new central macular area by dilution analysis of indocyanine green angiography after macular translocation. 7 van Zeeburg E.J.

Maaijwee K.J.

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et al. A free retinal pigment epithelium-choroid graft in patients with exudative age-related macular degeneration: results up to 7 years. 14 Klimanskaya I.

Hipp J.

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et al. Derivation and comparative assessment of retinal pigment epithelium from human embryonic stem cells using transcriptomics. , 15 Hirami Y.

Osakada F.

Takahashi K.

et al. Generation of retinal cells from mouse and human induced pluripotent stem cells. Surgical excision of CNV was reported to be a beneficial treatment for wet AMD first in the 1990s.However, despite its anatomic success, the overall visual outcome is relatively poor as a result of loss of the RPE, which is often removed as a part of the CNV and fibrovascular tissuebut is critical for preservation of photoreceptors and the choriocapillaris.Reconstruction of the RPE after removal of the CNV and transplantation of an RPE sheet with choroid harvested from beneath the peripheral portion of the retina has shown impressive results in some patients, providing proof-of-concept for this therapeutic approach.However, the surgery required for harvesting RPE and reintroducing it in the submacular area is invasive and the availability of RPE cells is limited. Hence, human stem cellswould provide an unlimited supply of RPE for transplantation.

27 Schwartz S.

Tan G.

Nagiel A.

et al. Subretinal transplantation of embryonic stem cell-derived retinal pigment epithelium for the treatment of macular degeneration: an assessment at 4 years. 17 Schwartz S.D.

Regillo C.D.

Lam B.L.

et al. Human embryonic stem cell-derived retinal pigment epithelium in patients with age-related macular degeneration and Stargardt’s macular dystrophy: follow-up of two open-label phase 1/2 studies. , 28 da Cruz L.

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et al. Phase 1 clinical study of an embryonic stem cell-derived retinal pigment epithelium patch in age-related macular degeneration. In this research, we have confirmed that a subretinally transplanted autologous iPSC-derived RPE sheet can survive for at least 4 years. Fundus photographs showed an acute increase in the pigmented area in the first 3 months, followed by a smaller increase. The graft sheet was initially curled on the margin, but gradually flattened in the ensuing 8 weeks; during this time, there may have been some migration of RPE cells outward from the sheet. Although the subsequent pigmented area did not show the same shape as the original rectangular sheet, it was approximately 1.5 times the size of the original sheet and covered the target area well. The mild increase in the pigmented area may also represent increased pigmentation. We found small patches of pigmentation in the area adjacent to the main graft sheet, which were likely to have resulted from small fragments being dispersed from the main sheet during the surgical procedure when the sheet was guided to the fovea via mild injection flow from a 1-mm syringe. An increase in the size of the transplanted area was also reported in a previous study of embryonic stem cell-derived RPE transplantation; however, this increase, as in our study, did not manifest as stratified protrusion or abnormal growth on SD OCT. We found no sign of graft rejection by SD OCT, FA, or ICGA, either, which added further evidence of safety of the sustainable use of iPSC-derived RPE compared with that of ESC-derived RPE in dry and wet AMD.

We have not seen improvement in vision or retinal sensitivity in our patient, probably because the photoreceptor layer in the treated eye had been damaged severely before surgery by a long course of exudative AMD and contained fibrotic scarring, leaving limited potential for visual recovery. Nevertheless, the patient has been satisfied with the subjective improvement in brightness of vision during follow-up, which is attributable primarily to resolution of exudative changes after removal of CNV and fibrotic scar tissue.

16 Kamao H.

Mandai M.

Okamoto S.

et al. Characterization of human induced pluripotent stem cell-derived retinal pigment epithelium cell sheets aiming for clinical application. 29 Le Y.Z.

Bai Y.

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Zheng L. Temporal requirement of RPE-derived VEGF in the development of choroidal vasculature. , 30 Saint-Geniez M.

Kurihara T.

Sekiyama E.

et al. An essential role for RPE-derived soluble VEGF in the maintenance of the choriocapillaris. 4 Dawson D.W.

Volpert O.V.

Gillis P.

et al. Pigment epithelium-derived factor: a potent inhibitor of angiogenesis. We attempted to evaluate the postoperative changes in the choroid as a potential indicator of RPE function and the morphologic features of the iPSC-derived RPE as a way of assessing the possible functionality of the transplanted iPSC-derived RPE as an alternative to tests of visual function. Our iPSC-derived RPE in culture showed many physiologic and functional properties of RPE cells, including secretion of VEGF and PEDF with specific polarity.Endothelial growth factor is an angiogenic growth factor that contributes to development of new choroidal vessels,and PEDF helps to maintain the structures of the retina as well as the choriocapillaris by stabilizing the endothelium.The choroidal volume in the grafted area decreased in the first 6 months but was subsequently preserved better than that in the surrounding area with bare RPE. A possible explanation for this finding is that secretion of VEGF also may have affected the adjacent area. Indeed, the area containing the CNV–RPE complex that was removed was much larger than the area containing the transplanted RPE, and the relatively small grafted portion also may have been affected by the surrounding environment. By 6 months after the surgery, a balance may have been achieved such that the choroidal thickness became stable. Furthermore, it would take some time for the marginal cells that had migrated out to become functional. However, these observations are based on 1 case study, and the effect of RPE grafting on the choroid will need to be investigated further in the future.

After transplantation, the thickness and structure of the photoreceptor layer as well as the choroidal volume and choriocapillaris were retained better at and adjacent to the transplanted site than in the area outside the transplant site, where the RPE had been removed together with the CNV and fibrovascular tissue. It was noteworthy that on SD OCT, vessel-like structures under the iPSC-derived RPE were confined to the graft area, suggesting that the iPSC-derived RPE has angiogenic properties. However, we have not observed any exudative changes or hemorrhages from these vessels so far. It is also intriguing that on ICGA, the choriocapillaris was retained only around the transplant, whereas the density of medium to large choroidal vessels did not differ between the area with or without iPSC-derived RPE within the RPE-deleted area. These observations suggested that iPSC-derived RPE cells may secrete VEGF and PEDF after transplantation, as they do in culture to support the photoreceptors and choroidal choriocapillaris.

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et al. In vivo autofluorescence imaging of the human and macaque retinal pigment epithelial cell mosaic. , 32 Scoles D.

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et al. Analysis of RPE morphometry in human eyes. Previous experiments were able to visualize individual RPE cells using AO technology in combination with various imaging methods.In particular, earlier workusing AO scanning laser ophthalmoscopy showed that RPE cells could be seen directly using an infrared reflectance method in particular conditions where the photoreceptor layer was compromised or missing. Using an infrared flood-illumination AO retinal camera, we reached similar conclusions based on images acquired in a patient affected by central serous chorioretinopathy in whom the RPE cell mosaic was visible only in areas where the retina was detached ( Fig 4 E). The ability of this device to detect cells that contain melanin has been reported in investigations of patients with dry AMD.In the present study, the arrangement of the iPSC-derived RPE cells observed on AO images in the subretinal space were mostly hexagonal, as judged by the neighboring cell counts. The average spacing between adjacent iPSC-derived RPE cells was close to the values previously found using AO scanning laser ophthalmoscopy in the natural RPE cell mosaic of patients with retinal diseases.This spacing is also close to that of the healthy RPE cell size around the macula by immune histologic analysis from a human donor eye (10.4–13.6 μm).

Although this is a preliminary report with limited evaluation based on the available in vivo imaging methods, we believe the results presented here suggest that iPSC-derived RPE has some functional properties and a healthy physiologic environment could be restored by this approach. The next stage of this research will include further evaluation of the potency of iPSC-derived RPE transplantation in a larger number of patients and disease states.