Publications by authors named "Masayo Takahashi"

152 Publications

Trends of Stem Cell Therapies in Age-Related Macular Degeneration.

J Clin Med 2021 Apr 20;10(8). Epub 2021 Apr 20.

Department of Ophthalmology, Kobe City Eye Hospital, Kobe 650-0047, Japan.

Age-related macular degeneration (AMD) is a highly prevalent irreversible impairment in the elderly population worldwide. Stem cell therapies have been considered potentially viable for treating AMD through the direct replacement of degenerated cells or secretion of trophic factors that facilitate the survival of existing cells. Among them, the safety of pluripotent stem cell-derived retinal pigment epithelial (RPE) cell transplantation against AMD, and some hereditary retinal degenerative diseases, has been discussed to a certain extent in clinical studies of RPE cell transplantation. Preparations are in progress for its clinical application. On the other hand, clinical trials using somatic stem cells are also being conducted, though these had controversial outcomes. Retinal regenerative medicine using stem cells is expected to make steady progress toward practical use while new technologies are incorporated from various fields, thereby making the role of ophthalmologists in this field increasingly important.
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http://dx.doi.org/10.3390/jcm10081785DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8074076PMC
April 2021

A ROCK Inhibitor Promotes Graft Survival during Transplantation of iPS-Cell-Derived Retinal Cells.

Int J Mol Sci 2021 Mar 22;22(6). Epub 2021 Mar 22.

RIKEN Center for Biosystems Dynamics Research, Laboratory for Retinal Regeneration, Kobe 650-0047, Japan.

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.
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http://dx.doi.org/10.3390/ijms22063237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8004718PMC
March 2021

Dynamics of Cyclooxygenase-1 Positive Microglia/Macrophage in the Retina of Pathological Model Mice as a Biomarker of the Retinal Inflammatory Diseases.

Int J Mol Sci 2021 Mar 25;22(7). Epub 2021 Mar 25.

Center for Biosystems Dynamics Research, Laboratory for Retinal Regeneration, RIKEN, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan.

In an intraocular inflammatory state, microglia residing in the retina become active and migrate inside the retina. In this study, we investigated whether cyclooxygenase-1 (COX-1) expressed by retinal microglia/macrophage can be a biomarker for the diagnosis of retinal diseases. COX-1 was immunopositive in microglia/macrophage and neutrophils, while COX-2 was immunopositive in astrocytes and neurons in the inner layer of normal retina. The number of COX-1 positive cells per section of the retinal tissue was 14 ± 2.8 (mean ± standard deviation) in normal mice, which showed significant increase in the lipopolysaccharide (LPS)-administrated model (62 ± 5.0, = 8.7 × 10). In addition to microglia, we found neutrophils that were positive for COX-1. In the early stage of inflammation in the experimental autoimmune uveoretinitis (EAU), COX-1 positive cells, infiltrating from the ciliary body into the retinal outer nuclear layer, were observed. The number of infiltrating COX-1 positive cells correlated with the severity of EAU. Taken together, the increased number of COX-1 positive microglia/macrophage with morphological changes were observed in the retinas of retinal inflammatory disease models. This suggests that COX-1 can be a marker of disease-related activities of microglia/macrophage, which should be useful for the diagnosis of retinal diseases.
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http://dx.doi.org/10.3390/ijms22073396DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8036698PMC
March 2021

Low Immunogenicity and Immunosuppressive Properties of Human ESC- and iPSC-Derived Retinas.

Stem Cell Reports 2021 Apr 25;16(4):851-867. Epub 2021 Mar 25.

Laboratory for Retinal Regeneration, RIKEN Center for Biosystems Dynamics Research, Kobe 650-0047, Japan; RIKEN Program for Drug Discovery and Medical Technology Platforms (DMP), RIKEN Cluster for Science, Technology and Innovation Hub, Saitama 351-0198, Japan. Electronic address:

ESC- and iPSC-derived retinal transplantation is a promising therapeutic approach for disease with end-stage retinal degeneration, such as retinitis pigmentosa and age-related macular degeneration. We previously showed medium- to long-term survival, maturation, and light response of transplanted human ESC- and iPSC-retina in mouse, rat, and monkey models of end-stage retinal degeneration. Because the use of patient hiPSC-derived retina with a disease-causing gene mutation is not appropriate for therapeutic use, allogeneic transplantation using retinal tissue/cells differentiated from a stocked hESC and iPSC line would be most practical. Here, we characterize the immunological properties of hESC- and iPSC-retina and present their three major advantages: (1) hESC- and iPSC-retina expressed low levels of human leukocyte antigen (HLA) class I and little HLA class II in vitro, (2) hESC- and iPSC-retina greatly suppressed immune activation of lymphocytes in co-culture, and (3) hESC- and iPSC-retina suppressed activated immune cells partially via transforming growth factor β signaling. These results support the use of allogeneic hESC- and iPSC-retina in future clinical application.
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http://dx.doi.org/10.1016/j.stemcr.2021.02.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8072071PMC
April 2021

Immunological aspects of RPE cell transplantation.

Prog Retin Eye Res 2021 Jan 19:100950. Epub 2021 Jan 19.

Laboratory for Retinal Regeneration, Laboratory for Retinal Regeneration, RIKEN Center for Biosystems Dynamics Research Kobe, Japan; Department of Ophthalmology, Kobe City Eye Hospital, Kobe, Japan.

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.
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http://dx.doi.org/10.1016/j.preteyeres.2021.100950DOI Listing
January 2021

Stem-cell-based therapies for retinal degenerative diseases: Current challenges in the establishment of new treatment strategies.

Dev Growth Differ 2021 Jan 21;63(1):59-71. Epub 2021 Jan 21.

Laboratory for Retinal Regeneration, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan.

Various advances have been made in the treatment of retinal diseases, including new treatment strategies and innovations in surgical devices. However, the treatment of degenerative retinal diseases, such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD), continues to pose a significant challenge. In this review, we focus on the use of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to treat retinal diseases by harnessing the ability of stem cells to differentiate into different body tissues. The retina is a tissue specialized for light sensing, and its degradation leads to vision loss. As part of the central nervous system, the retina has very low regenerative capability, and therefore, treatment options are limited once it degenerates. Nevertheless, innovations in methods to induce the generation of retinal cells and tissues from ESCs/iPSCs enable the development of novel approaches for these irreversible diseases. Here we review some historical background and current clinical trials involving the use of stem-cell-derived retinal pigment epithelial cells for AMD treatment and stem cell-derived retinal cells/tissues for RP therapy. Finally, we discuss our future vision of regenerative treatment for retinal diseases with a partial focus on our studies and introduce other interesting approaches for restoring vision.
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http://dx.doi.org/10.1111/dgd.12704DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7986097PMC
January 2021

A Variable Scheduling Maintenance Culture Platform for Mammalian Cells.

SLAS Technol 2021 Apr 3;26(2):209-217. Epub 2020 Dec 3.

Laboratory for Biologically Inspired Computing, RIKEN Center for Biosystems Dynamics Research, Suita, Osaka, Japan.

Cell culturing is a basic experimental technique in cell biology and medical science. However, culturing high-quality cells with a high degree of reproducibility relies heavily on expert skills and tacit knowledge, and it is not straightforward to scale the production process due to the education bottleneck. Although many automated culture systems have been developed and a few have succeeded in mass production environments, very few robots are permissive of frequent protocol changes, which are often required in basic research environments. LabDroid is a general-purpose humanoid robot with two arms that performs experiments using the same tools as humans. Combining our newly developed AI software with LabDroid, we developed a variable scheduling system that continuously produces subcultures of cell lines without human intervention. The system periodically observes the cells on plates with a microscope, predicts the cell growth curve by processing cell images, and decides the best times for passage. We have succeeded in developing a system that maintains the cultures of two HEK293A cell plates with no human intervention for 192 h.
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http://dx.doi.org/10.1177/2472630320972109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7985857PMC
April 2021

Melanin concentration and depolarization metrics measurement by polarization-sensitive optical coherence tomography.

Sci Rep 2020 11 11;10(1):19513. Epub 2020 Nov 11.

Department of Ophthalmology, Kobe City Eye Hospital, Kobe, Hyogo, Japan.

Imaging of melanin in the eye is important as the melanin is structurally associated with some ocular diseases, such as age-related macular degeneration. Although optical coherence tomography (OCT) cannot distinguish tissues containing the melanin from other tissues intrinsically, polarization-sensitive OCT (PS-OCT) can detect the melanin through spatial depolarization of the backscattered light from the melanin granules. Entropy is one of the depolarization metrics that can be used to detect malanin granules in PS-OCT and valuable quantitative information on ocular tissue abnormalities can be retrived by correlating entropy with the melanin concentration. In this study, we investigate a relationship between the melanin concentration and some depolarization metrics including the entropy, and show that the entropy is linearly proportional to the melanin concentration in double logarithmic scale when noise bias is corrected for the entropy. In addition, we also confirm that the entropy does not depend on the incident state of polarization using the experimental data, which is one of important attributes that depolarization metrics should have. The dependence on the incident state of polarization is also analyzed for other depolarization metrics.
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http://dx.doi.org/10.1038/s41598-020-76397-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7658243PMC
November 2020

Capacity of Retinal Ganglion Cells Derived from Human Induced Pluripotent Stem Cells to Suppress T-Cells.

Int J Mol Sci 2020 Oct 22;21(21). Epub 2020 Oct 22.

Laboratory for Retinal Regeneration, RIKEN Center for Biosystems Dynamics Research, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan.

Retinal ganglion cells (RGCs) are impaired in patients such as those with glaucoma and optic neuritis, resulting in permanent vision loss. To restore visual function, development of RGC transplantation therapy is now underway. Induced pluripotent stem cells (iPSCs) are an important source of RGCs for human allogeneic transplantation. We therefore analyzed the immunological characteristics of iPSC-derived RGCs (iPSC-RGCs) to evaluate the possibility of rejection after RGC transplantation. We first assessed the expression of human leukocyte antigen (HLA) molecules on iPSC-RGCs using immunostaining, and then evaluated the effects of iPSC-RGCs to activate lymphocytes using the mixed lymphocyte reaction (MLR) and iPSC-RGC co-cultures. We observed low expression of HLA class I and no expression of HLA class II molecules on iPSC-RGCs. We also found that iPSC-RGCs strongly suppressed various inflammatory immune cells including activated T-cells in the MLR assay and that transforming growth factor-β2 produced by iPSC-RGCs played a critical role in suppression of inflammatory cells in vitro. Our data suggest that iPSC-RGCs have low immunogenicity, and immunosuppressive capacity on lymphocytes. Our study will contribute to predicting immune attacks after RGC transplantation.
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http://dx.doi.org/10.3390/ijms21217831DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7660053PMC
October 2020

Truncating Variants Contribute to Hearing Loss and Severe Retinopathy in -Associated Retinitis Pigmentosa in Japanese Patients.

Int J Mol Sci 2020 Oct 22;21(21). Epub 2020 Oct 22.

Department of Ophthalmology, Kobe City Eye Hospital, Kobe, Hyogo 650-0047, Japan.

is a common causal gene of retinitis pigmentosa (RP), a progressive blinding disease due to retinal degeneration. Genetic alterations in can lead to two types of RP, non-syndromic and syndromic RP, which is called Usher syndrome, with impairments of vision and hearing. The complexity of the genotype-phenotype correlation in -associated RP (-RP) has been reported. Genetic and clinical characterization of -RP has not been performed in Japanese patients. In this study, genetic analyses were performed using targeted panel sequencing in 525 Japanese RP patients. Pathogenic variants of were identified in 36 of 525 (6.9%) patients and genetic features of -RP were characterized. Among 36 patients with -RP, 11 patients had syndromic RP with congenital hearing problems. Amino acid changes due to alterations were similarly located throughout entire regions of the protein structure in non-syndromic and syndromic RP cases. Notably, truncating variants were detected in all syndromic patients with a more severe retinal phenotype as compared to non-syndromic RP cases. Taken together, truncating variants could contribute to more serious functional and tissue damages in Japanese patients, suggesting important roles for truncating mutations in the pathogenesis of syndromic -RP.
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http://dx.doi.org/10.3390/ijms21217817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7659936PMC
October 2020

Retinal Pigment Epithelial Cells Derived from Induced Pluripotent Stem (iPS) Cells Suppress or Activate T Cells via Costimulatory Signals.

Int J Mol Sci 2020 Sep 5;21(18). Epub 2020 Sep 5.

Laboratory for Retinal Regeneration, RIKEN Center for Biosystems Dynamics Research, 2-2-3 Minatojima-minamimachi, Chuo-ku Kobe 650-0047, Japan.

Human retinal pigment epithelial (RPE) cells derived from induced pluripotent stem (iPS) cells have immunosuppressive properties. However, RPE cells are also known as immunogenic cells, and they have major histocompatibility complex expression and produce inflammatory proteins, and thus experience immune rejection after transplantation. In this study, to confirm the immunological properties of IPS-RPE cells, we examined whether human RPE cells derived from iPS cells could suppress or stimulate inflammatory T cells from uveitis patients via costimulatory signals. We established T cells from patients with active uveitis as target cells and used iPS-RPE cells as effector cells. As a result, cultured iPS-RPE cells inhibited cell proliferation and the production of IFN-γ by activated uveitis CD4 T cells, especially Th1-type T cells. In contrast, iPS-RPE cells stimulated T cells of uveitis patients. The iPS-RPE cells constitutively expressed B7-H1/CD274 and B7-DC/CD273, and suppressed the activation of T cells via the PD-1 receptor. iPS-RPE expressed these negative costimulatory molecules, especially when RPE cells were pretreated with recombinant IFN-γ. In addition, iPS-RPE cells also expressed B7-H3/CD276 costimulatory molecules and activated uveitis T cells through the B7-H3-TLT-2 receptor. Thus, cultured iPS-derived retinal cells can suppress or activate inflammatory T cells in vitro through costimulatory interactions.
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http://dx.doi.org/10.3390/ijms21186507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7554762PMC
September 2020

Microglia dynamics in retinitis pigmentosa model: formation of fundus whitening and autofluorescence as an indicator of activity of retinal degeneration.

Sci Rep 2020 09 7;10(1):14700. Epub 2020 Sep 7.

Laboratory for Retinal Regeneration, Center for Biosystems Dynamics Research, RIKEN, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, 650-0047, Japan.

In patients with retinitis pigmentosa (RP), color fundus photography and fundus autofluorescence (FAF) have been used to estimate the disease progression. To understand the origin and the diagnostic interpretation of the fundus color and FAF, we performed in vivo imaging of fundus color and FAF together with histological analyses of the retinal degeneration process using the RP model mice, rd10. FAF partly represented the accumulation of microglia in the photoreceptor outer segments. Fundus whitening suggested the presence of apoptotic cells, which spatiotemporally preceded increase in FAF. We observed two patterns of FAF localization, arcuate and diffuse, each indicating different pattern of apoptosis, wavy and diffuse, respectively. Diffuse pattern of apoptosis was suppressed in dark-raised rd10 mice, in which outer nuclear layer (ONL) loss was significantly suppressed. The occupancy of FAF correlated with the thinning rate of the ONL. Fractalkine, a microglia chemotactic factor, was detected in apoptotic photoreceptors, suggesting chemokine-induced recruitment of microglia into the ONL, which paralleled with accelerated ONL loss and increased FAF occupancy. Thus, we propose that the degree of photoreceptor apoptosis and the rate of ONL thinning in RP patients might be read from the fundus color and the FAF.
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http://dx.doi.org/10.1038/s41598-020-71626-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7477572PMC
September 2020

Reproducible production and image-based quality evaluation of retinal pigment epithelium sheets from human induced pluripotent stem cells.

Sci Rep 2020 09 1;10(1):14387. Epub 2020 Sep 1.

Laboratory of Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya University, Nagoya, 464-8601, Japan.

Transplantation of retinal pigment epithelial (RPE) sheets derived from human induced pluripotent cells (hiPSC) is a promising cell therapy for RPE degeneration, such as in age-related macular degeneration. Current RPE replacement therapies, however, face major challenges. They require a tedious manual process of selecting differentiated RPE from hiPSC-derived cells, and despite wide variation in quality of RPE sheets, there exists no efficient process for distinguishing functional RPE sheets from those unsuitable for transplantation. To overcome these issues, we developed methods for the generation of RPE sheets from hiPSC, and image-based evaluation. We found that stepwise treatment with six signaling pathway inhibitors along with nicotinamide increased RPE differentiation efficiency (RPE6iN), enabling the RPE sheet generation at high purity without manual selection. Machine learning models were developed based on cellular morphological features of F-actin-labeled RPE images for predicting transepithelial electrical resistance values, an indicator of RPE sheet function. Our model was effective at identifying low-quality RPE sheets for elimination, even when using label-free images. The RPE6iN-based RPE sheet generation combined with the non-destructive image-based prediction offers a comprehensive new solution for the large-scale production of pure RPE sheets with lot-to-lot variations and should facilitate the further development of RPE replacement therapies.
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http://dx.doi.org/10.1038/s41598-020-70979-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7462996PMC
September 2020

HLA-Matched Allogeneic iPS Cells-Derived RPE Transplantation for Macular Degeneration.

J Clin Med 2020 Jul 13;9(7). Epub 2020 Jul 13.

Tomey Corporation, Nagoya 451-0051, Japan.

Immune attacks are key issues for cell transplantation. To assess the safety and the immune reactions after iPS cells-derived retinal pigment epithelium (iPS-RPE) transplantation, we transplanted HLA homozygote iPS-RPE cells established at an iPS bank in HLA-matched patients with exudative age-related macular degeneration. In addition, local steroids without immunosuppressive medications were administered. We monitored immune rejections by routine ocular examinations as well as by lymphocytes-graft cells immune reaction (LGIR) tests using graft RPE and the patient's blood cells. In all five of the cases that underwent iPS-RPE transplantation, the presence of graft cells was indicated by clumps or an area of increased pigmentation at 6 months, which became stable with no further abnormal growth in the graft during the 1-year observation period. Adverse events observed included corneal erosion, epiretinal membrane, retinal edema due to epiretinal membrane, elevated intraocular pressure, endophthalmitis, and mild immune rejection in the eye. In the one case exhibiting positive LGIR tests along with a slight fluid recurrence, we administrated local steroid therapy that subsequently resolved the suspected immune attacks. Although the cell delivery strategy must be further optimized, the present results suggest that it is possible to achieve stable survival and safety of iPS-RPE cell transplantation for a year.
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http://dx.doi.org/10.3390/jcm9072217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7408794PMC
July 2020

A discrete neuronal circuit induces a hibernation-like state in rodents.

Nature 2020 07 11;583(7814):109-114. Epub 2020 Jun 11.

Faculty of Medicine, University of Tsukuba, Tsukuba, Japan.

Hibernating mammals actively lower their body temperature to reduce energy expenditure when facing food scarcity. This ability to induce a hypometabolic state has evoked great interest owing to its potential medical benefits. Here we show that a hypothalamic neuronal circuit in rodents induces a long-lasting hypothermic and hypometabolic state similar to hibernation. In this state, although body temperature and levels of oxygen consumption are kept very low, the ability to regulate metabolism still remains functional, as in hibernation. There was no obvious damage to tissues and organs or abnormalities in behaviour after recovery from this state. Our findings could enable the development of a method to induce a hibernation-like state, which would have potential applications in non-hibernating mammalian species including humans.
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http://dx.doi.org/10.1038/s41586-020-2163-6DOI Listing
July 2020

Polarization-sensitive optical coherence tomography for estimating relative melanin content of autologous induced stem-cell derived retinal pigment epithelium.

Sci Rep 2020 05 6;10(1):7656. Epub 2020 May 6.

Department of Ophthalmology, Kobe City Eye Hospital, Kobe, Hyogo, Japan.

Transplantation of autologous human induced pluripotent stem cell-derived retinal pigment epithelial (hiPSC-RPE) sheets is a promising therapy for age-related macular degeneration (AMD). As melanin content is a representative feature of healthy RPE, we used polarization-sensitive optical coherence tomography (PS-OCT) to estimate the relative melanin content of RPE in diseased and non-diseased area, and in human iPSC-RPE sheets in vitro and in vivo by evaluating the randomness of polarization (entropy). Two aged Japanese women, one with neovascular AMD that underwent transplantation of an autologous hiPSC-RPE cell sheet and another with binocular dry AMD, were selected for this study. Entropy value was minimal in cells containing no melanin, whereas that of human RPE and hiPSC-RPE sheets was high. En face entropy of the cultured hiPSC-RPE sheet was compared with its grey-scale photo and its values were found to be inversely correlated with the extent of absence of pigmentation in vitro. En face entropy maps were compared to colour fundus photographs, fundus autofluorescence images, and fluorescein angiography images from patients. Entropy values of intact and defective RPEs and of iPSC-RPE transplant areas were determined in vivo using PS-OCT B-scan images. PS-OCT was found to be applicable in the estimation of relative melanin content of cultured and transplanted RPEs in regenerative medicine.
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http://dx.doi.org/10.1038/s41598-020-64601-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7203130PMC
May 2020

A Strategy for Personalized Treatment of iPS-Retinal Immune Rejections Assessed in Cynomolgus Monkey Models.

Int J Mol Sci 2020 Apr 27;21(9). Epub 2020 Apr 27.

Laboratory for Retinal Regeneration, RIKEN Center for Biosystems Dynamics Research, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan.

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.
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http://dx.doi.org/10.3390/ijms21093077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7247695PMC
April 2020

[Toward establishment of regenerative cell therapy for retinitis pigmentosa using iPS cell derived retinal sheet].

Nihon Yakurigaku Zasshi 2020 ;155(2):93-98

Laboratory for Retinal Regeneration, Center for Biosystems Dynamics Research, Riken.

Retinitis pigmentosa (RP) is a group of hereditary diseases that involve loss of photoreceptors. There has been no established treatment for RP, and it is now the 2 leading cause of blindness in Japan. Previous clinical researches using human fetal retina transplantation suggested some functional recovery in vision, but it did not become a standard therapy because of ethical concerns for using fetus tissues. Invention of induced pluripotent stem cells (iPSC) in 2006 and the establishment of retinal organoids induction protocol from ES/iPS cells have paved a way of cell therapy for RP without ethical concerns. Our team has shown that mouse iPSC derived retinas can survive and mature after subretinal transplantation to the end-stage retinal degeneration model mice. Further, human ESC derived retinas survived and matured in retinal degeneration monkey models. Recently, we have established a qualitative and quantitative evaluation tool for photoreceptor synapses, QUANTOS, and showed that photoreceptors in mouse iPSC derived retina can form photoreceptor synapses in a time dependent manner after transplantation. We are now moving toward 1 in human clinical trial using iPSC derived retina for RP.
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http://dx.doi.org/10.1254/fpj.19124DOI Listing
March 2020

Ethical and professional challenges encountered by Japanese healthcare professionals who provide genetic counseling services.

J Genet Couns 2020 12 6;29(6):1004-1014. Epub 2020 Feb 6.

Laboratory for Retinal Regeneration, RIKEN, Center for Biosystems Dynamics Research, Kobe, Japan.

It is important to identify ethical and professional challenges associated with genetic counseling services and systems to improve these services. In previous studies, specific challenges in genetic counseling were categorized into 16 domains. However, these studies were limited to a few countries, and genetic counseling differs according to national cultures or systems. Thus, additional efforts should be made to collect and analyze challenges in genetic counseling to address these issues. We interviewed 48 genetic counseling professionals in Japan (including 29 clinical geneticists, 17 genetic counselors, and 2 other professionals) about anecdotes that included ethical professional challenges. Thematic analysis was used to code the interview data, and anecdotes were categorized according to the ethical and professional challenges. The anecdotes (n = 333) were classified into the 16 previously identified domains and three unique subcategories: 'lack of understanding about genetic professionals or departments of genetic counseling by other professionals and patients', 'insufficient communication skills to carry out counseling on the part of the genetic counseling professionals', and 'lack of a system for self-improvement'. Many of the anecdotes also noted the emotional responses domain. The challenges experienced by Japanese genetic counseling professionals described herein will improve the quality of the service these professionals provide. Furthermore, the results can assist development of high-quality genetic counseling systems in countries developing these systems.
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http://dx.doi.org/10.1002/jgc4.1225DOI Listing
December 2020

Publisher Correction: Preconditioning the Initial State of Feeder-free Human Pluripotent Stem Cells Promotes Self-formation of Three-dimensional Retinal Tissue.

Sci Rep 2020 Feb 5;10(1):2237. Epub 2020 Feb 5.

Regenerative & Cellular Medicine Kobe Center, Sumitomo Dainippon Pharma Co., Ltd., Chuo, Kobe, 650-0047, Japan.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-020-58892-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7002801PMC
February 2020

Bilateral retinitis after influenza virus infection in a case report.

Am J Ophthalmol Case Rep 2020 Mar 3;17:100584. Epub 2020 Jan 3.

Department of Ophthalmology, Kobe City Eye Hospital, 2-1-8 Minatojima-Minamimachi, Chuo-ku, Kobe-shi, Hyogo, 650-0047, Japan.

Purpose: To report 2 years' longitudinal retinal changes using spectral domain optical coherence tomography (SD-OCT) images in a case of retinitis after influenza virus infection.

Observations: A 48-year-old female complained of scotoma in the central visual field after influenza virus infection. Her best visual acuity was 20/16, her fundus examination was normal, and fluorescein angiography demonstrated no evident leakage in either the retina or the optic disc. However, SD-OCT images showed a disrupted, blurred inner-segment ellipsoid zone in the macula of both eyes. Two steroid pulse therapy sessions in the first 3 months showed temporary improvement of the central scotoma. However, atrophy of the photoreceptor layer at the juxta fovea gradually progressed in OCT images during the follow-up period. In contrast, the fovea itself was mostly intact and visual acuity was maintained in the 2-year period.

Conclusions And Importance: We experienced a unique case of retinitis after influenza infection, in whom progressive atrophy of the photoreceptor layer was observed in SD-OCT images.
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http://dx.doi.org/10.1016/j.ajoc.2019.100584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6962652PMC
March 2020

Deep Learning Classification Models Built with Two-step Transfer Learning for Age Related Macular Degeneration Diagnosis.

Annu Int Conf IEEE Eng Med Biol Soc 2019 Jul;2019:2049-2052

The objective of this study was to build deep learning models with optical coherence tomography (OCT) images to classify normal and age related macular degeneration (AMD), AMD with fluid, and AMD without any fluid. In this study, 185 normal OCT images from 49 normal subjects, 535 OCT images of AMD with fluid, and 514 OCT mages of AMD without fluid from 120 AMD eyes as training data, while 49 normal images from 25 normal eyes, 188 AMD OCT images with fluid and 154 AMD images without any fluid from 77 AMD eyes as test data, were enrolled. Data augmentation was applied to increase the number of images to build deep learning models. Totally, two classification models were built in two steps. In the first step, a VGG16 model pre-trained on ImageNet dataset was transfer learned to classify normal and AMD, including AMD with fluid and/or without any fluid. Then, in the second step, the fine-tuned model in the first step was transfer learned again to distinguish the images of AMD with fluid from the ones without any fluid. With the first model, normal and AMD OCT images were classified with 0.999 area under receiver operating characteristic curve (AUC), and 99.2% accuracy. With the second model, AMD with the presence of any fluid, and AMD without fluid were classified with 0.992 AUC, and 95.1% accuracy. Compared with a transfer learned VGG16 model pre-trained on ImageNet dataset, to classify the three categories directly, higher classification performance was achieved with our notable approach. Conclusively, two classification models for AMD clinical practice were built with high classification performance, and these models should help improve the early diagnosis and treatment for AMD.
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http://dx.doi.org/10.1109/EMBC.2019.8857468DOI Listing
July 2019

Preconditioning the Initial State of Feeder-free Human Pluripotent Stem Cells Promotes Self-formation of Three-dimensional Retinal Tissue.

Sci Rep 2019 12 12;9(1):18936. Epub 2019 Dec 12.

Regenerative & Cellular Medicine Kobe Center, Sumitomo Dainippon Pharma Co., Ltd., Chuo, Kobe, 650-0047, Japan.

A three-dimensional retinal tissue (3D-retina) is a promising graft source for retinal transplantation therapy. We previously demonstrated that embryonic stem cells (ESCs) can generate 3D-retina in vitro using a self-organizing stem cell culture technique known as SFEBq. Here we show an optimized culture method for 3D-retina generation from feeder-free human pluripotent stem cells (hPSCs). Although feeder-free hPSC-maintenance culture was suitable for cell therapy, feeder-free hPSC-derived aggregates tended to collapse during 3D-xdifferentiation culture. We found that the initial hPSC state was a key factor and that preconditioning of the hPSC state by modulating TGF-beta and Shh signaling improved self-formation of 3D-neuroepithelium. Using the preconditioning method, several feeder-free hPSC lines robustly differentiated into 3D-retina. In addition, changing preconditioning stimuli in undifferentiated hPSCs altered the proportions of neural retina and retinal pigment epithelium, important quality factors for 3D-retina. We demonstrated that the feeder-free hiPSC-derived 3D-retina differentiated into rod and cone photoreceptors in vitro and in vivo. Thus, preconditioning is a useful culture methodology for cell therapy to direct the initial hPSC state toward self-organizing 3D-neuroepithelium.
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http://dx.doi.org/10.1038/s41598-019-55130-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6908610PMC
December 2019

Base-Resolution Methylome of Retinal Pigment Epithelial Cells Used in the First Trial of Human Induced Pluripotent Stem Cell-Based Autologous Transplantation.

Stem Cell Reports 2019 10 26;13(4):761-774. Epub 2019 Sep 26.

Department of Biochemistry, Kyushu University Graduate School of Medical Sciences, Fukuoka 812-8582, Japan. Electronic address:

The first-in-human trial of induced pluripotent stem cell (iPSC)-based autologous transplantation was successfully performed on a female patient with age-related macular degeneration. Here we delineated the base-resolution methylome of the iPSC-derived retinal pigment epithelium (iRPE) used in this trial. The methylome of iRPE closely resembled that of native RPE (nRPE), although partially methylated domains (PMDs) emerged in iRPE but not nRPE. Most differentially methylated regions between iRPE and nRPE appeared to originate from (de)methylation errors during differentiation, whereas errors at reprogramming resulted in aberrant genomic imprinting and X chromosome reactivation. Moreover, non-CpG methylation was prominent in nRPE but not iRPE. Intriguingly, xenotransplantation to mouse remodeled the iRPE methylome to demethylate a subset of suppressed genes and accumulate non-CpG methylation, but failed to resolve PMDs and hypermethylated CpG islands. Although the impacts of these alterations remain elusive, our findings should provide a useful guide for methylome analyses of other iPSC-derived cells.
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http://dx.doi.org/10.1016/j.stemcr.2019.08.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6829753PMC
October 2019

Retinal stem cell transplantation: Balancing safety and potential.

Prog Retin Eye Res 2020 03 5;75:100779. Epub 2019 Sep 5.

National Eye Institute, National Institutes of Health, Bethesda, MD, 90892, USA.

Stem cell transplantation holds great promise as a potential treatment for currently incurable retinal degenerative diseases that cause poor vision and blindness. Recently, safety data have emerged from several Phase I/II clinical trials of retinal stem cell transplantation. These clinical trials, usually run in partnership with academic institutions, are based on sound preclinical studies and are focused on patient safety. However, reports of serious adverse events arising from cell therapy in other poorly regulated centers have now emerged in the lay and scientific press. While progress in stem cell research for blindness has been greeted with great enthusiasm by patients, scientists, doctors and industry alike, these adverse events have raised concerns about the safety of retinal stem cell transplantation and whether patients are truly protected from undue harm. The aim of this review is to summarize and appraise the safety of human retinal stem cell transplantation in the context of its potential to be developed into an effective treatment for retinal degenerative diseases.
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http://dx.doi.org/10.1016/j.preteyeres.2019.100779DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7056514PMC
March 2020

Optical Coherence Tomography-Based Deep-Learning Models for Classifying Normal and Age-Related Macular Degeneration and Exudative and Non-Exudative Age-Related Macular Degeneration Changes.

Ophthalmol Ther 2019 Dec 12;8(4):527-539. Epub 2019 Aug 12.

Department of Ophthalmology, Kobe City Eye Hospital, Kobe, Japan.

Introduction: The use of optical coherence tomography (OCT) images is increasing in the medical treatment of age-related macular degeneration (AMD), and thus, the amount of data requiring analysis is increasing. Advances in machine-learning techniques may facilitate processing of large amounts of medical image data. Among deep-learning methods, convolution neural networks (CNNs) show superior image recognition ability. This study aimed to build deep-learning models that could distinguish AMD from healthy OCT scans and to distinguish AMD with and without exudative changes without using a segmentation algorithm.

Methods: This was a cross-sectional observational clinical study. A total of 1621 spectral domain (SD)-OCT images of patients with AMD and a healthy control group were studied. The first CNN model was trained and validated using 1382 AMD images and 239 normal images. The second transfer-learning model was trained and validated with 721 AMD images with exudative changes and 661 AMD images without any exudate. The attention area of the CNN was described as a heat map by class activation mapping (CAM). In the second model, which classified images into AMD with or without exudative changes, we compared the learning stabilization of models using or not using transfer learning.

Results: Using the first CNN model, we could classify AMD and normal OCT images with 100% sensitivity, 91.8% specificity, and 99.0% accuracy. In the second, transfer-learning model, we could classify AMD as having or not having exudative changes, with 98.4% sensitivity, 88.3% specificity, and 93.9% accuracy. CAM successfully described the heat-map area on the OCT images. Including the transfer-learning model in the second model resulted in faster stabilization than when the transfer-learning model was not included.

Conclusion: Two computational deep-learning models were developed and evaluated here; both models showed good performance. Automation of the interpretation process by using deep-learning models can save time and improve efficiency.

Trial Registration: No15073.
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http://dx.doi.org/10.1007/s40123-019-00207-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6858411PMC
December 2019

Induced 2C Expression and Implantation-Competent Blastocyst-like Cysts from Primed Pluripotent Stem Cells.

Stem Cell Reports 2019 09 8;13(3):485-498. Epub 2019 Aug 8.

Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA; Department of Pharmacology, Faculty of Medicine, Osaka Medical College, Osaka 569-8686, Japan. Electronic address:

Soon after fertilization, the few totipotent cells of mammalian embryos diverge to form a structure called the blastocyst (BC). Although numerous cell types, including germ cells and extended-pluripotency stem cells, have been developed from pluripotent stem cells (PSCs) in vitro, generating functional BCs only from PSCs remains elusive. Here, we describe induced self-organizing 3D BC-like cysts (iBLCs) generated from mouse PSC culture. Resembling natural BCs, iBLCs have a blastocoel-like cavity and were formed with outer cells expressing trophectoderm lineage markers and with inner cells expressing pluripotency markers. iBLCs transplanted to pseudopregnant mice uteruses implanted, induced decidualization, and exhibited growth and development before resorption, demonstrating that iBLCs are implantation competent. iBLC precursor intermediates required the transcription factor Prdm14 and concomitantly activated the totipotency-related cleavage-stage MERVL reporter and 2C genes. Thus, our system may contribute to the understanding of molecular mechanisms underpinning totipotency, embryogenesis, and implantation.
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http://dx.doi.org/10.1016/j.stemcr.2019.07.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6739768PMC
September 2019

Evaluation of Transplanted Autologous Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium in Exudative Age-Related Macular Degeneration.

Ophthalmol Retina 2019 10 26;3(10):850-859. Epub 2019 Apr 26.

Department of Ophthalmology, Kobe City Eye Hospital, Kobe, Japan; Laboratory for Retinal Regeneration, Center for Biosystems Dynamics Research, Kobe, Japan.

Purpose: 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.

Design: Follow-up of a single case.

Participant: A patient with exudative age-related macular degeneration and polypoidal choroidal vasculopathy.

Methods: Transplantation of an autologous iPSC-derived RPE cell sheet after removal of choroidal neovascularization (CNV) in September 2014.

Main Outcome Measures: 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.

Results: 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.

Conclusions: 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.
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http://dx.doi.org/10.1016/j.oret.2019.04.021DOI Listing
October 2019

Assessment of the deformation of the outer nuclear layer in the Epiretinal membrane using spectral-domain optical coherence tomography.

BMC Ophthalmol 2019 May 17;19(1):113. Epub 2019 May 17.

Department of Ophthalmology, Teikyo University, University Hospital Mizonokuchi, 5-1-1 Futago, Takatsu-ku, Kawasaki, Kanagawa, 213-8507, Japan.

Background: We aimed to investigate the deformation of the outer nuclear layer using optical coherence tomography in patients with epiretinal membrane (ERM) and its relationship with metamorphopsia.

Methods: Thirty-nine eyes from 39 patients with ERM were included in the study. Patients with the subtypes of pseudo macula hole and lamellar hole were excluded. Twenty-one fellow eyes without macular disease were included as normal controls. Forty-nine B-scan images were obtained in the range of 20 degrees around the macula using SD-OCT. The outer nuclear layer (ONL) was evaluated as a three-dimensional image (3D-ONL) reconstructed using the distance between the ONL and retinal pigment epithelium (RPE) line. The deformation of the ONL was figured at the reference plane and evaluation plane (ONL-B). The characteristic parameters of the ONL-B were defined as circularity, area ratio, and axis ratio. The correlations between these parameters and visual acuity and MCHART score ratio (MH/MV) were then evaluated.

Results: ONL height was significantly higher in ERM patients than in normal controls (54.1 ± 5.3 μm and 84.1 ± 12.9 μm, respectively; P < 0.001). In ERM patients, the MV score was 0.53 ± 0.50, the MH score was 0.71 ± 0.61, and the distance from the RPE line to the ONL-B was 153.5 ± 13.5 μm. The axis of the ONL-B in normal controls and ERM patients was - 6.25 ± 21.8 and - 1.28 ± 29.1, respectively, which indicates that the ONL is horizontally long in both normal individuals and ERM patients. The circularity and area ratio were significantly smaller in ERM patients than in normal controls. In all ERM patients, MH/MV had a significant correlation with axis (r = - 0.29, p = 0.034), circularity (r = - 0.28, p = 0.044), and area ratio (r = - 0.47, p = 0.001). Moreover, we found that the correlation was more significant if the subjects had an axis of the ONL within ±10 degrees (n = 16); the correlations of MH/MV with axis (r = - 0.29, p = 0.034), circularity (r = - 0.53, p = 0.021), and area ratio were more significant (r = - 0.78, P < 0.0001).

Conclusion: The ONL is horizontally long in normal individuals and ERM patients. The direction of metamorphopsia is correlated with the direction of ONL deformation.
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http://dx.doi.org/10.1186/s12886-019-1124-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6525344PMC
May 2019

Gene and Induced Pluripotent Stem Cell Therapy for Retinal Diseases.

Annu Rev Genomics Hum Genet 2019 08 24;20:201-216. Epub 2019 Apr 24.

Laboratory for Retinal Regeneration, Center for Biosystems Dynamics Research, RIKEN, Kobe, Hyogo 650-0047, Japan; email:

Given the importance of visual information to many daily activities, retinal degenerative diseases-which include both inherited conditions (such as retinitis pigmentosa) and acquired conditions (such as age-related macular degeneration)-can have a dramatic impact on human lives. The therapeutic options for these diseases remain limited. Since the discovery of the first causal gene for retinitis pigmentosa almost three decades ago, more than 250 genes have been identified, and gene therapies have been rapidly developed. Simultaneously, stem cell technologies such as induced pluripotent stem cell-based transplantation have advanced and have been applied to the treatment of retinal degenerative diseases. Here, we review recent progress in these expanding fields and discuss the potential for precision medicine in ophthalmic care.
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http://dx.doi.org/10.1146/annurev-genom-083118-015043DOI Listing
August 2019