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1.
J Clin Med ; 13(13)2024 Jun 27.
Article in English | MEDLINE | ID: mdl-38999321

ABSTRACT

Background/Objectives: To approach the clinical properties of pachydrusen that differ from conventional drusen, we investigated the incidence of macular neovascularization (MNV) in fellow eyes and the treatment outcomes of intravitreal aflibercept (IVA) in MNV eyes of unilateral MNV patients with pachydrusen in the fellow eye. Methods: We retrospectively studied 261 consecutive patients with treatment-naïve unilateral MNV. Patients were classified into four groups according to the type of drusen in the fellow eye: the pachydrusen group (n = 49), the soft drusen group (n = 63), the subretinal drusenoid deposit (SDD) group (n = 24), and the no drusen group (n = 125). The development of the MNV in the fellow eye was evaluated for five years, and the retreatment proportion after three monthly aflibercept injections was evaluated for one year. Results: The choroidal thickness in the fellow eyes and MNV eyes was the greatest in the pachydrusen group (all p < 0.001). The 5-year incidence of MNV in the pachydrusen group was similar to that in the soft drusen group and no drusen group. The pachydrusen group had a lower retreatment rate than the other groups did (pachydrusen group: 46.4%; soft drusen group: 78.1%; SDDs: 87.5%; no drusen group: 83.3%). Conclusions: Unilateral MNV patients with pachydrusen in the fellow eye had a lower retreatment rate (46.4%/1 year); therefore, aflibercept monotherapy using the PRN regimen is one of the preferred treatment methods for MNV patients with pachydrusen in the fellow eye.

2.
J Ophthalmol ; 2024: 3673930, 2024.
Article in English | MEDLINE | ID: mdl-38322501

ABSTRACT

Purpose: To evaluate the one-year outcomes of switching to brolucizumab with and without a loading dose regimen (three monthly injections) in eyes with aflibercept-resistant neovascular age-related macular degeneration (nAMD). Methods: We retrospectively studied nAMD patients who had retinal exudate under bimonthly injections of aflibercept and were switched to brolucizumab from aflibercept. Patients were grouped into intravitreal brolucizumab injection (IVBr) with a loading dose regimen (loading group) and without a loading dose regimen (nonloading group). We assessed the best-corrected visual acuity (BCVA), central retinal thickness (CRT) at the fovea, subfoveal choroidal thickness (SFCT), IVBr status (number of injections and last injection interval), and retinal exudate status on optical coherence tomography. Results: Overall, 52 eyes received ≥1 IVBr; 26 eyes received ≥3 IVBr with 12-month follow-up. A total of 13 eyes in the loading group and 13 eyes in the nonloading group were reviewed. One year after switching, BCVA changed from 0.28 ± 0.25 to 0.19 ± 0.28 in the loading group (P=0.28) and from 0.25 ± 0.20 to 0.23 ± 0.25 in the nonloading group (P=0.92). The mean CRT decreased from 263.6 ± 40.7 µm to 221.7 ± 54.6 µm in the loading group (P=0.03), while it only changed from 244.9 ± 77.2 µm to 221.0 ± 78.7 µm in the nonloading group (P=0.26). Both the loading and nonloading groups achieved 69% dry macula. The number of injections received was significantly higher in the loading group (7.6 ± 0.6 vs. 6.8 ± 0.4, P < 0.001). Two patients (4.2%) developed intraocular inflammation. Conclusion: Switching to brolucizumab from aflibercept for eyes with nAMD with resistance to bimonthly injections of aflibercept is a valuable treatment option with and without the loading regimen. This trial is registered with UMIN000023676.

3.
J Ophthalmol ; 2021: 6683532, 2021.
Article in English | MEDLINE | ID: mdl-33996151

ABSTRACT

PURPOSE: To evaluate the clinical characteristics of neovascular age-related macular degeneration (nAMD) patients without typical drusen. METHODS: We retrospectively studied 165 eyes in 165 patients with treatment-naïve nAMD, including typical AMD and polypoidal choroidal vasculopathy (PCV). According to the fellow eye condition, the patients were divided into nAMD with and without typical drusen groups. Eyes with soft drusen or subretinal drusenoid deposits were classified into the nAMD with the typical drusen group. Smoking status and diagnoses of hypertension and diabetes were identified from hospital records and patient recall. We assessed best-corrected visual acuity (BCVA), central retinal thickness (CRT) at the fovea, subfoveal choroidal thickness (SFCT), and the number of injections received. RESULTS: The nAMD without typical drusen group was significantly younger (77.9 ± 7.6 vs. 71.8 ± 8.3, P < 0.001) and had thicker SFCT at baseline (207.9 ± 99.5 vs. 260.1 ± 113.2 µm, P=0.007) and a higher proportion of PCV (30.6 vs. 63.1%, P < 0.001). The proportion of ever-smokers was significantly higher in the nAMD without typical drusen group (54.8 vs. 70.9%, P=0.036). There were no statistically significant differences in the proportion of patients with hypertension or diabetes; BCVA, CRT, or SFCT changes; or the number of injections between the nAMD with and without typical drusen groups. CONCLUSION: The clinical features of patients in the nAMD without typical drusen group were almost identical to those of pachychoroid-driven choroidal neovascularization (CNV) patients. The nAMD without typical drusen group had a significantly higher proportion of ever-smokers than the nAMD with typical drusen group. Smoking could be a risk factor for the development of pachychoroid-driven CNV.

4.
Int J Mol Sci ; 22(6)2021 Mar 22.
Article in English | MEDLINE | ID: mdl-33810153

ABSTRACT

Currently, retinal pigment epithelium (RPE) transplantation includes sheet and single-cell transplantation, the latter of which includes cell death and may be highly immunogenic, and there are some issues to be improved in single-cell transplantation. Y-27632 is an inhibitor of Rho-associated protein kinase (ROCK), the downstream kinase of Rho. We herein investigated the effect of Y-27632 in vitro on retinal pigment epithelium derived from induced pluripotent stem cells (iPS-RPE cells), and also its effects in vivo on the transplantation of iPS-RPE cell suspensions. As a result, the addition of Y-27632 in vitro showed suppression of apoptosis, promotion of cell adhesion, and higher proliferation and pigmentation of iPS-RPE cells. Y-27632 also increased the viability of the transplant without showing obvious retinal toxicity in human iPS-RPE transplantation into monkey subretinal space in vivo. Therefore, it is possible that ROCK inhibitors can improve the engraftment of iPS-RPE cell suspensions after transplantation.


Subject(s)
Graft Survival/drug effects , Induced Pluripotent Stem Cells/cytology , Protein Kinase Inhibitors/pharmacology , Stem Cell Transplantation , rho-Associated Kinases/antagonists & inhibitors , Amides/pharmacology , Animals , Apoptosis/drug effects , Biomarkers , Cell Adhesion/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Cytokines/metabolism , Humans , Immunohistochemistry , Inflammation Mediators/metabolism , Macaca fascicularis , Pyridines/pharmacology , Retinal Pigment Epithelium/drug effects , Retinal Pigment Epithelium/metabolism
5.
Prog Retin Eye Res ; 84: 100950, 2021 09.
Article in English | MEDLINE | ID: mdl-33482342

ABSTRACT

Retinal pigment epithelial (RPE) cells have several functions, including support of the neural retina and choroid in the eye and immunosuppression. Cultured human RPE cells directly suppress inflammatory immune cells. For instance, they directly suppress the activation of T cells in vitro. In contrast, transplanted allogeneic human RPE cells are rejected by bystander immune cells such as T cells in vivo. Recently, human embryonic stem cell-derived RPE cells have been used in several clinical trials, and human induced pluripotent stem cell (iPSC)-RPE cells have also been tested in our clinical study in patients with retinal degeneration. Major safety concerns after stem cell-based transplantation surgery include hyper-proliferation, tumorigenicity, or ectopic tissue formation, but these events have currently not been seen in any of these patients. However, if RPE cells are allogeneic, there are concerns about immune rejection issues that have been raised in previous clinical trials. We therefore performed a preclinical study of allogeneic iPSC-RPE cell transplantation in animal rejection models. We then conducted autogenic or allogeneic iPSC-RPE cell transplantation in clinical studies of patients with age-related macular degeneration. In this review, we focus on immunological studies of RPE cells, including iPSC-derived cells. iPSC-RPE cells have unique inflammatory (immunosuppressive and immunogenic) characteristics like primary cultured RPE cells. The purpose of this review is to summarize the current findings obtained from preclinical (basic research) and clinical studies in iPSC-RPE cell transplantation, especially the immunological aspects.


Subject(s)
Induced Pluripotent Stem Cells , Macular Degeneration , Animals , Cell Differentiation , Humans , Retina , Retinal Pigment Epithelium , Stem Cell Transplantation
6.
Int J Mol Sci ; 21(9)2020 Apr 27.
Article in English | MEDLINE | ID: mdl-32349277

ABSTRACT

Recently, we successfully transplanted an autograft, or major histocompatibility complex (MHC)-matched allografts, from induced-pluripotent-stem-cell-derived retinal pigment epithelial (iPSC-RPE) cells in patients with age-related macular degeneration. However, there was an issue regarding immune rejection after transplantation. In this study, we established a preoperational in vitro "drug-lymphocytes-grafts immune reaction (Drug-LGIR)" test to determine the medication for immune rejection using host immunocompetent cells (lymphocytes) and transplant cells (target iPSC-RPE cells) together with different medications. The adequacy of the test was assessed by in vivo transplantation in monkey models together with medication based on in vitro data. In the results of Drug-LGIR tests, some drugs exhibited significant suppression of RPE cell-related allogeneic reactions, while other drugs did not, and the efficacy of each drug differed among the recipient monkeys. Based on the results of Drug-LGIR, we applied cyclosporine A or local steroid (triamcinolone) therapy to two monkeys, and successfully suppressed RPE-related immune rejections with RPE grafts, which survived without any signs of rejection under drug administration. We propose that our new preoperational in vitro Drug-LGIR test, which specifies the most efficacious medication for each recipient, is useful for controlling immune attacks with personalized treatment for each patient after retinal transplantation.


Subject(s)
Epithelial Cells , Graft Rejection/immunology , Graft Rejection/therapy , Induced Pluripotent Stem Cells , Precision Medicine , Retinal Pigment Epithelium/cytology , Stem Cell Transplantation , Animals , Biomarkers , Cell Differentiation/drug effects , Cells, Cultured , Cyclosporine/administration & dosage , Disease Models, Animal , Epithelial Cells/cytology , Epithelial Cells/drug effects , Heterografts , Humans , Immunohistochemistry , Immunophenotyping , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Lymphocytes/immunology , Lymphocytes/metabolism , Macaca fascicularis , Postoperative Complications , Precision Medicine/methods , Retinal Pigment Epithelium/metabolism , Stem Cell Transplantation/adverse effects , Stem Cell Transplantation/methods , Steroids/administration & dosage , Transplantation, Heterologous , Treatment Outcome
7.
J Ophthalmol ; 2019: 9428738, 2019.
Article in English | MEDLINE | ID: mdl-31316826

ABSTRACT

PURPOSE: No standard therapy for RPE tear, a complication of neovascular age-related macular degeneration, exists even though RPE tears cause severe vision loss, and promotion of cell proliferation and/or migration could be a candidate RPE tear therapy. The aim of this study is to evaluate the effect of Rho-associated coiled-coil containing kinase (ROCK) inhibitor Y27632 on retinal pigment epithelial (RPE) cell motility during wound healing. METHODS: Human RPE cells were cultured in media with and without 10 µM Y27632. A luminescent cell viability assay and vinculin immunocytochemistry were used to test the Y27632 effect on RPE cell adhesion. The mean size of vinculin puncta was quantified from immunofluorescence images. RPE cell motility during wound healing was evaluated using time-lapse imaging and measuring cell migration distances and cell coverage rate in wound fields. RESULTS: The number of adhered RPE and mean size of vinculin puncta were, respectively, 20519 cells and 3.65 µm2 under nontreatment and 23569 cells and 0.66 µm2 under Y27632 treatment. Cell migration distance and cell coverage percentage for untreated and Y27632-treated cells were 98.9 and 59.4% and 203.4 and 92.5%, respectively. CONCLUSIONS: Inhibition of ROCK signaling by using 10 µM Y27632 promoted RPE cell motility during wound healing by reducing RPE cell adhesion strength.

8.
Invest Ophthalmol Vis Sci ; 60(5): 1298-1308, 2019 04 01.
Article in English | MEDLINE | ID: mdl-30933257

ABSTRACT

Purpose: To report occurrence of acute severe inflammation after surgical implantation of mycoplasma-infected induced pluripotent stem cell-derived RPE (iPS-RPE) cells into the eyes of healthy primates, and determine the immunopathological mechanisms of the inflammation. Methods: Ophthalmic allogeneic transplantation of iPS-RPE cells was performed in the subretina of major histocompatibility complex (MHC)-matched (two eyes) and MHC-mismatched (one eye) healthy cynomolgus monkeys. The clinical course after transplantation was observed using color fundus photography, fluorescence angiography, and optical coherence tomography. After the animals were killed at 1 month after surgery, eyeballs were removed and pathologically examined. Microorganisms were analyzed by PCR methods and BLAST analysis using preserved graft iPS-RPE cells and the recipients' vitreous humor. Mixed lymphocyte-RPE assay was performed on the mycoplasma-infected and noninfected iPS-RPE cells in vitro. Results: In tested eyes, abnormal findings were observed in the grafted retina 2 weeks after surgery. Here, we observed retinal vasculitis and hemorrhage, retinal detachment, and infiltration of inflammatory cells into the retina of the eyes. One month after surgery, animals were killed due to the severe immune responses observed. Using PCR methods, sequence analysis detected mycoplasma-DNA (Mycoplasma arginini species) in both the grafted RPE cells and the collected vitreous fluids of the monkeys. Mixed lymphocyte-RPE assay revealed that the infected iPS-RPE cells enhanced the proliferation of inflammatory cells in vitro. Conclusions: Transplantation of graft iPS-RPE cells contaminated with mycoplasma into the subretina caused severe ocular inflammation. Mycoplasma possesses the ability to cause immune responses in the host.


Subject(s)
Cell Transplantation/adverse effects , Eye Infections/microbiology , Induced Pluripotent Stem Cells/cytology , Mycoplasma Infections/pathology , Mycoplasma/isolation & purification , Retinal Pigment Epithelium/transplantation , Animals , Cell Transplantation/methods , DNA, C-Form/analysis , Disease Models, Animal , Eye Infections/etiology , Inflammation/pathology , Macaca fascicularis , Mycoplasma Infections/etiology , Postoperative Complications/microbiology , Retinal Detachment/pathology , Retinal Pigment Epithelium/cytology , Retinal Pigment Epithelium/pathology , Retinal Vasculitis/pathology
9.
J Ophthalmol ; 2019: 7189241, 2019.
Article in English | MEDLINE | ID: mdl-31016039

ABSTRACT

PURPOSE: The evaluation of drug-induced cytotoxicity is of great importance for the clinical application of pharmaceutical products, and human-induced pluripotent stem cells (hiPSCs) have received considerable scrutiny as a cell source for in vitro cytotoxicity testing. The aim of this study is to validate the concept of cytotoxicity testing using hiPSC-derived retinal pigment epithelium (hiPSC-RPE) by comparing the responsiveness of human fetal RPE (hfRPE) and human RPE cell line (ARPE19) to recombinant tissue plasminogen activator (rtPA). METHODS: HfRPE, two types of hiPSC-RPE, and ARPE19 were cultured in media with or without rtPA. A lactate dehydrogenase release assay was performed to investigate the dose- and time-dependent effects of rtPA on cell death. RPE function was evaluated by measuring the secretion of pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) and RPE-specific gene expression. RESULTS: Rates of cell damage in hfRPE and both hiPS-RPE were increased by rtPA supplementation (2000 and 4000 µg/ml) for 1 hour, whereas ARPE19 cell damage was increased by supplementation with rtPA at concentrations higher than 50 µg/ml. Although 100 µg/ml rtPA for 24 hours did not affect RPE cell function, sustained rtPA exposure induced prolonged cytotoxic effects in hfRPE and two hiPSC-RPE, but not ARPE19. CONCLUSION: The responsiveness of hiPSC-RPE to rtPA is similar to that of hfRPE in terms of cell death and cell function. Thus, hiPSC-RPE is a valuable cell source for in vitro cytotoxicity testing.

10.
J Ophthalmol ; 2018: 2353428, 2018.
Article in English | MEDLINE | ID: mdl-30538852

ABSTRACT

PURPOSE: To evaluate the effect of smoking on the outcome of antivascular endothelial growth factor (VEGF) therapy in patients with neovascular age-related macular degeneration (nAMD). METHODS: This retrospective case-control study included 64 eyes in 59 patients with treatment-naïve nAMD. Smoking habits were obtained from hospital records and patient recall. The patients were divided into ever-smokers and never-smokers. The patients were treated with ranibizumab or aflibercept for at least 1 year. Outcome measures were best-corrected visual acuity (BCVA), central retinal thickness (CRT) at the fovea, subfoveal choroidal thickness (SCT), and number of injections received. RESULTS: There were no statistically significant differences in BCVA, CRT, or SCT changes between ever-smokers and never-smokers. The number of injections received was significantly higher in ever-smokers with a history of heavy smokers (never-smokers vs. heavy smokers: 5.3 ± 2.6/year vs. 7.3 ± 2.5/year; P=0.048 and mild smokers vs. heavy smokers: 5.2 ± 2.5/year vs. 7.3 ± 2.5/year; P=0.043). There was no significant difference in the baseline CRT or presence of atrophic retinal pigment epithelium in the fellow eyes of patients with nAMD according to smoking status; however, the baseline CRT in eyes with nAMD was significantly thinner in ever-smokers than in never-smokers (P=0.02). CONCLUSION: The anti-VEGF therapy was frequently required in nAMD patients with a history of heavy smoking. Heavy smoking could cause poor therapeutic response in nAMD patients.

11.
Stem Cell Reports ; 9(5): 1501-1515, 2017 11 14.
Article in English | MEDLINE | ID: mdl-29103970

ABSTRACT

Antibody-mediated rejection is characterized by donor-specific antibody produced by B cells. However, to our knowledge, B cell invasion and antibody in the inflamed retina after transplantation of retinal pigment epithelial (RPE) cells has not been reported. To determine if RPE transplantation could be performed using allografts, we established in vivo immune rejection models with induced pluripotent stem cell (iPSC)-RPE allografts and determined whether RPE-specific antibody could be detected in these models. We detected alloantibodies in the serum from recipient monkeys that had immune attacks in the retina in an immunofluorescent assay using the transplanted iPSC-RPE cells as the antigen. In addition to T cell and antigen-presenting cell immunity, peripheral blood cells and lymph nodes in animal models with allogeneic iPSC-RPE cells also had activated B cells, which were probably secreting alloantibodies. Using serum and transplanted cells, alloreactive antibody can be detected for the diagnosis of immune rejection after transplantation.


Subject(s)
Graft Rejection/immunology , Induced Pluripotent Stem Cells/transplantation , Isoantibodies/immunology , Retinal Pigment Epithelium/transplantation , Stem Cell Transplantation/methods , Animals , Antigen Presentation , B-Lymphocytes/immunology , Cell Differentiation , Cells, Cultured , Graft Rejection/prevention & control , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/immunology , Isoantibodies/therapeutic use , Macaca fascicularis , Retinal Pigment Epithelium/cytology , Retinal Pigment Epithelium/immunology , Stem Cell Transplantation/adverse effects , T-Lymphocytes/immunology , Transplantation, Homologous
12.
N Engl J Med ; 376(11): 1038-1046, 2017 03 16.
Article in English | MEDLINE | ID: mdl-28296613

ABSTRACT

We assessed the feasibility of transplanting a sheet of retinal pigment epithelial (RPE) cells differentiated from induced pluripotent stem cells (iPSCs) in a patient with neovascular age-related macular degeneration. The iPSCs were generated from skin fibroblasts obtained from two patients with advanced neovascular age-related macular degeneration and were differentiated into RPE cells. The RPE cells and the iPSCs from which they were derived were subject to extensive testing. A surgery that included the removal of the neovascular membrane and transplantation of the autologous iPSC-derived RPE cell sheet under the retina was performed in one of the patients. At 1 year after surgery, the transplanted sheet remained intact, best corrected visual acuity had not improved or worsened, and cystoid macular edema was present. (Funded by Highway Program for Realization of Regenerative Medicine and others; University Hospital Medical Information Network Clinical Trials Registry [UMIN-CTR] number, UMIN000011929 .).


Subject(s)
Induced Pluripotent Stem Cells/cytology , Macular Degeneration/therapy , Retinal Pigment Epithelium/cytology , Aged , Cell Culture Techniques , Cell Differentiation , Feasibility Studies , Female , Fibroblasts , Humans , Male , Retinal Pigment Epithelium/transplantation , Transplantation, Autologous
13.
Invest Ophthalmol Vis Sci ; 58(1): 211-220, 2017 01 01.
Article in English | MEDLINE | ID: mdl-28114582

ABSTRACT

Purpose: To develop a clinically applicable transplantation device and surgical procedure for extracellular matrix-scaffold-supported human-induced pluripotent stem cell-derived retinal pigment epithelium (hiPSC-RPE) cell sheet transplantation for clinical use. Methods: The developed surgical device consisted of a custom-designed hand piece and a cannula. The subretinal transplantation of hiPSC-RPE cell sheets was performed in 12 rabbits. The results evaluated were the graft condition (damage or fold), side (front or back), position (center, near, far), and direction (anterior, posterior, right, left) immediately after surgery and the graft condition (shrinking or fold) 2 weeks after surgery. These results were evaluated by fundus photography and optical coherence tomography, followed by immersion-fixed histology. Results: All grafts could be transplanted without obvious damage. The transplanted grafts included 2 of 12 folded grafts, 12 of 12 front side, 12 of 12 center position, 10 of 12 anterior direction, and 2 of 12 right direction immediately after surgery, whereas transplantation with a distance between an inlet and an outlet greater than graft and the coaxial direction of the flow paths and the insertion device posed the correct condition and direction. Two weeks after the surgery, the transplanted grafts included two folded grafts and four shrunken grafts; however, complete drainage of subretinal fluid for adhesion between the graft and the host prevented shrunken grafts. Conclusions: A developed surgical device and procedure allow grafts to be transplanted into the targeted transplantation site safely and reproducibly. This surgical method will provide additional information on the advancement of future RPE transplantation therapies.


Subject(s)
Cell Transplantation/instrumentation , Extracellular Matrix , Macular Degeneration/surgery , Retinal Diseases/surgery , Retinal Pigment Epithelium/cytology , Tissue Scaffolds , Animals , Cell Line , Disease Models, Animal , Equipment Design , Humans , Induced Pluripotent Stem Cells , Macular Degeneration/pathology , Rabbits , Retinal Diseases/diagnosis , Retinal Pigment Epithelium/transplantation , Tomography, Optical Coherence , Transplantation, Autologous
14.
Stem Cell Reports ; 7(4): 635-648, 2016 10 11.
Article in English | MEDLINE | ID: mdl-27641649

ABSTRACT

There is an ongoing controversy as to whether major histocompatibility complex (MHC) matching is a solution for allogeneic stem cell transplantation. In the present study, we established retinal pigment epithelial (RPE) cells from induced pluripotent stem cells (iPSCs) in MHC homozygote donors. We observed no rejection signs in iPSC-derived RPE allografts of MHC-matched animal models without immunosuppression, whereas there were immune attacks around the graft and retinal tissue damage in MHC-mismatched models. In an immunohistochemical examination of MHC-mismatched allografts, the transplanted RPE sheets/cells were located in the subretinal space, but the RPE exhibited inflammatory and hypertrophic changes, and many inflammatory cells, e.g., Iba1+ cells, MHC class II+ cells, and CD3+ T cells, invaded the graft area. Conversely, these inflammatory cells poorly infiltrated the area around the transplanted retina if MHC-matched allografts were used. Thus, cells derived from MHC homozygous donors could be used to treat retinal diseases in histocompatible recipients.


Subject(s)
Epithelial Cells/metabolism , Epithelial Cells/transplantation , Homozygote , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Major Histocompatibility Complex/genetics , Retinal Pigment Epithelium/cytology , Animals , Biomarkers , Epithelial Cells/cytology , Epithelial Cells/immunology , Heterozygote , Histocompatibility Testing , Immunohistochemistry , Macaca fascicularis , Major Histocompatibility Complex/immunology , Tissue Donors , Transplantation, Homologous
16.
Nippon Ganka Gakkai Zasshi ; 120(11): 754-63, 2016 Nov.
Article in Japanese | MEDLINE | ID: mdl-30074740

ABSTRACT

Age-related macular degeneration is one of the leading causes of blindness characterized by progressive dysfunction of retinal pigment epithelium (RPE) and RPE transplantation. The replacement of pathological RPE with healthy RPE, is being investigated for AMD treatment. In recent years increasing attention has been given to human induced pluripotent stem cells (hiPSCs) as a useful cell source for RPE transplantation. We generated hiPSC-derived RPE (hiPSC-RPE) cell sheets optimized to meet clinical use requiring efficacy, consistency, and safety. These grafts consist of a monolayer of cells without any artificial scaffolds, and express typical RPE markers, form tight junctions that exhibit polarized secretion of growth factors, and show phagocytotic ability and gene expression patterns similar to those of native RPE. Additionally, autologous non-human primate iPSC-RPE cell sheets showed no immune rejection or tumor formation. These results suggest that autologous hiPSC-RPE cell sheets may serve as a useful cell source for RPE transplantation.


Subject(s)
Induced Pluripotent Stem Cells , Retinal Pigment Epithelium/transplantation , Stem Cell Transplantation , Animals , Gene Expression Regulation , Humans , Macular Degeneration/therapy
17.
Stem Cells ; 33(5): 1543-53, 2015 May.
Article in English | MEDLINE | ID: mdl-25728228

ABSTRACT

Retinitis pigmentosa (RP) is a group of visual impairments characterized by progressive rod photoreceptor cell loss due to a genetic background. Pigment epithelium-derived factor (PEDF) predominantly secreted by the retinal pigmented epithelium (RPE) has been reported to protect photoreceptors in retinal degeneration models, including rd1. In addition, clinical trials are currently underway outside Japan using human mesenchymal stromal cells and human neural stem cells to protect photoreceptors in RP and dry age-related macular degeneration, respectively. Thus, this study aimed to investigate the rescue effects of induced pluripotent stem (iPS)-RPE cells in comparison with those types of cells used in clinical trials on photoreceptor degeneration in rd1 mice. Cells were injected into the subretinal space of immune-suppressed 2-week-old rd1 mice. The results demonstrated that human iPS-RPE cells significantly attenuated photoreceptor degeneration on postoperative days (PODs) 14 and 21 and survived longer up to at least 12 weeks after operation than the other two types of graft cells with less immune responses and apoptosis. The mean PEDF concentration in the intraocular fluid in RPE-transplanted eyes was more than 1 µg/ml at PODs 14 and 21, and this may have contributed to the protective effect of RPE transplantation. Our findings suggest that iPS-RPE cells serve as a competent source to delay photoreceptor degeneration through stable survival in degenerating ocular environment and by releasing neuroprotective factors such as PEDF.


Subject(s)
Induced Pluripotent Stem Cells/cytology , Mesenchymal Stem Cells/cytology , Neural Stem Cells/cytology , Neuroprotective Agents/metabolism , Retinal Degeneration/therapy , Retinal Pigment Epithelium/cytology , Stem Cell Transplantation , Animals , Apoptosis , Cell Proliferation , Graft Survival , Humans , Mice , Photoreceptor Cells, Vertebrate/metabolism , Retinal Degeneration/pathology
18.
Invest Ophthalmol Vis Sci ; 56(2): 1051-62, 2015 Jan 20.
Article in English | MEDLINE | ID: mdl-25604685

ABSTRACT

PURPOSE: The purpose of this study was to determine whether human retinal pigment epithelial (RPE) cells from induced pluripotent stem (iPS) cells could inhibit T-cell activation in vitro. METHODS: Cultured iPS-derived RPE (iPS-RPE) cells were established from fresh skin tissues or dental pulp cells obtained from healthy donors or a retinal patient after informed consent was obtained. To confirm expression of the specific markers on iPS and iPS-RPE cells, immunohistochemistry, quantitative RT-PCR (qRT-PCR), and flow cytometry were performed. Target T cells were obtained from peripheral blood mononuclear cells of healthy donors. Target T cells were assessed for proliferation by incorporation of bromodeoxyuridine or carboxyfluorescein succinimidyl ester for production of cytokines such as IFN-γ. Expression of TGFß and other candidate molecules by iPS-RPE cells was evaluated with flow cytometry, ELISA, multiplex cytokine array, immunohistochemistry, and qRT-PCR. RESULTS: The RPE cells we established from iPS cells had many characteristics of mature RPE cells but no characteristics of pluripotent stem cells. Cultured iPS-RPE cells inhibited cell proliferation and production of IFN-γ by activated CD4(+) T cells. In some bystander T cells, iPS-derived RPE cells induced CD25(+)Foxp3(+) regulatory T cells in vitro. Induced pluripotent stem-RPE cells constitutively expressed TGFß and suppressed activation of T cells via soluble TGFß, because TGFß-downregulated iPS-RPE cells did not inhibit this T-cell activation. CONCLUSIONS: Cultured iPS-derived retinal cells fully suppress T-cell activation. Transplantation of iPS-RPE cells into the eye might be a therapy for retinal disorders.


Subject(s)
Immunity, Cellular , Induced Pluripotent Stem Cells/cytology , Lymphocyte Activation/immunology , Retinal Pigment Epithelium/immunology , T-Lymphocytes/immunology , Cell Proliferation , Cells, Cultured , DNA/genetics , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Gene Expression Regulation , Humans , Immunohistochemistry , Induced Pluripotent Stem Cells/immunology , Real-Time Polymerase Chain Reaction , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/pathology , Transforming Growth Factor beta/biosynthesis , Transforming Growth Factor beta/genetics
19.
Invest Ophthalmol Vis Sci ; 55(12): 8309-18, 2014 Nov 11.
Article in English | MEDLINE | ID: mdl-25389202

ABSTRACT

PURPOSE: For the transplantation of human induced pluripotent stem cell-derived retinal pigment epithelium (hiPSC-RPE), determination of the maturation status of these cells is essential, and the degree of pigmentation (dPG) can serve as a good indicator of this status. The aim of this study was to establish a method of objectively and quantitatively evaluating the dPG of hiPSC-RPE. METHODS: Two observers determined the dPG subjectively by observing recorded images of hiPSC-RPE as follows: the dPG of a single cell was classified into three different pigmentation stages, and the overall dPG was compared between two cell groups to identify the group with the higher dPG. The κ statistic was applied to assess interobserver reproducibility. Next, the dPG of single cells and cell groups was objectively determined by the lightness of the hue, saturation, and value (HSL) color space, and the correlation between the subjective evaluation and time-dependent change in the objective dPG of hiPSC-RPE was investigated. RESULTS: The κ statistic was 0.88 and 0.81 in the single-cell and cell-group observations, respectively. The objective dPG of single cells and cell groups was highly correlated with the subjective dPG. However, the observers were occasionally unable to subjectively determine the group with the higher dPG. The objective dPG increased in a time-dependent manner. CONCLUSIONS: The lightness of the HSL color space can be used to objectively and quantitatively evaluate the dPG of hiPSC-RPE in culture. The objective evaluation was consistent and was able to better identify small differences than subjective evaluation.


Subject(s)
Embryonic Stem Cells/cytology , Epithelial Cells , Induced Pluripotent Stem Cells/cytology , Pigmentation , Retinal Pigment Epithelium/cytology , Embryonic Stem Cells/metabolism , Epithelial Cells/metabolism , Eye Proteins/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Nerve Growth Factors/metabolism , Observer Variation , Reproducibility of Results , Retinal Pigment Epithelium/metabolism , Serpins/metabolism , Vascular Endothelial Growth Factor A/metabolism
20.
Stem Cell Reports ; 2(2): 205-18, 2014 Feb 11.
Article in English | MEDLINE | ID: mdl-24527394

ABSTRACT

Age-related macular degeneration (AMD) causes severe visual impairment due in part to age-dependent impairment of retinal pigment epithelium (RPE). It has been suggested that autologous human induced pluripotent stem cells (hiPSCs) may represent a useful cell source for the generation of graft RPE. We generated hiPSC-derived RPE (hiPSC-RPE) cell sheets optimized to meet clinical use requirements, including quality, quantity, consistency, and safety. These cell sheets are generated as a monolayer of cells without any artificial scaffolds, express typical RPE markers, form tight junctions that exhibit polarized secretion of growth factors, and show phagocytotic ability and gene-expression patterns similar to those of native RPE. Additionally, upon transplantation, autologous nonhuman primate iPSC-RPE cell sheets showed no immune rejection or tumor formation. These results suggest that autologous hiPSC-RPE cell sheets may serve as a useful form of graft for use in tissue replacement therapy for AMD.


Subject(s)
Cell Culture Techniques , Epithelial Cells/cytology , Induced Pluripotent Stem Cells/cytology , Retinal Pigment Epithelium/cytology , Animals , Biomarkers/metabolism , Cell Differentiation , Cluster Analysis , Epithelial Cells/immunology , Epithelial Cells/metabolism , Epithelial Cells/transplantation , Gene Expression Profiling , Humans , Macaca fascicularis , Rats
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