Yasuhiko Hirami

GENERATION OF RETINAL CELLS FROM MOUSE AND HUMAN INDUCED PLURIPOTENT STEM CELLS

We previously reported a technique for generating retinal pigment epithelia (RPE) and putative photoreceptors from embryonic stem (ES) cells. Here we tested whether our procedure can promote retinal differentiation of mouse and human induced pluripotent stem cells (iPSCs). Treating iPSCs with Wnt and Nodal antagonists in suspension culture induced expression of markers of retinal progenitor cells and generated RPE cells. Subsequently, treatment with retinoic acid and taurine generated cells positive for photoreceptor markers in all but one human cell lines. We propose that iPSCs can be induced to differentiate into retinal cells which have a possibility to be used as patient-specific donor cells for transplantation therapies.

In vitro differentiation of retinal cells from human pluripotent stem cells by small-molecule induction

The use of stem-cell therapy to treat retinal degeneration holds great promise. However, definitive methods of retinal differentiation that do not depend on recombinant proteins produced in animal or Escherichia coli cells have not been devised. Here, we report a defined culture method using low-molecular-mass compounds that induce differentiation of human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells into retinal progenitors, retinal pigment epithelium cells and photoreceptors. The casein kinase I inhibitor CKI-7, the ALK4 inhibitor SB-431542 and the Rho-associated kinase inhibitor Y-27632 in serum-free and feeder-free floating aggregate culture induce retinal progenitors positive for RX, MITF, PAX6 and CHX10. The treatment induces hexagonal pigmented cells that express RPE65 and CRALBP, form ZO1-positive tight junctions and exhibit phagocytic functions. Subsequent treatment with retinoic acid and taurine induces photoreceptors that express recoverin, rhodopsin and genes involved in phototransduction. Both three-factor (OCT3/4, SOX2 and KLF4) and four-factor (OCT3/4, SOX2, KLF4 and MYC) human iPS cells could be successfully differentiated into retinal cells by small-molecule induction. This method provides a solution to the problem of cross-species antigenic contamination in cell-replacement therapy, and is also useful for in vitro modeling of development, disease and drug screening.

Autologous Induced Stem-Cell–Derived Retinal Cells for Macular Degeneration

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.)

Modeling Retinal Degeneration Using Patient-Specific Induced Pluripotent Stem Cells

Retinitis pigmentosa (RP) is the most common inherited human eye disease resulting in night blindness and visual defects. It is well known that the disease is caused by rod photoreceptor degeneration; however, it remains incurable, due to the unavailability of disease-specific human photoreceptor cells for use in mechanistic studies and drug screening. We obtained fibroblast cells from five RP patients with distinct mutations in the RP1, RP9, PRPH2 or RHO gene, and generated patient-specific induced pluripotent stem (iPS) cells by ectopic expression of four key reprogramming factors. We differentiated the iPS cells into rod photoreceptor cells, which had been lost in the patients, and found that they exhibited suitable immunocytochemical features and electrophysiological properties. Interestingly, the number of the patient-derived rod cells with distinct mutations decreased in vitro; cells derived from patients with a specific mutation expressed markers for oxidation or endoplasmic reticulum stress, and exhibited different responses to vitamin E than had been observed in clinical trials. Overall, patient-derived rod cells recapitulated the disease phenotype and expressed markers of cellular stresses. Our results demonstrate that the use of patient-derived iPS cells will help to elucidate the pathogenic mechanisms caused by genetic mutations in RP.

Hemorrhagic complications after photodynamic therapy for polypoidal choroidal vasculopathy.

Purpose: To evaluate the clinical features and risk factors of hemorrhagic complications in eyes with polypoidal choroidal vasculopathy (PCV) after photodynamic therapy (PDT). Methods: We retrospectively reviewed data for 91 consecutive eyes of 85 patients who underwent PDT for the treatment of PCV. The diagnosis of PCV was based on indocyanine green angiographic findings, showing a branching vascular network terminating in polypoidal swelling. The greatest linear dimension included all polypoidal lesions, leaking vascular network, and type 2 choroidal neovascularization. Results: During the follow-up period after PDT, postoperative subretinal hemorrhage was seen in 28 (30.8%) of 91 eyes. In 22 (78.6%) of these 28 eyes, subretinal hemorrhage was absorbed without treatment. In 6 eyes (21.4%), however, bleeding resulted in vitreous hemorrhage, and 2 eyes underwent pars plana vitrectomy. Although visual acuity was maintained or increased in 18 (81.8%) of 22 eyes with subretinal hemorrhage alone, it decreased significantly in 3 (50.0%) of 6 eyes with postoperative vitreous hemorrhage. Various systemic diseases and medication with an anticoagulant had no correlation with these hemorrhagic complications. Laser irradiation spot size for PDT was significantly larger in eyes with postoperative vitreous hemorrhage (P = 0.017) than in those without. Conclusion: Subretinal hemorrhage after PDT can be a common complication in patients with PCV and may have a minor effect on visual outcome. However, postoperative hemorrhage is occasionally so massive that it leads to vitreous hemorrhage and poor visual prognosis. When considering PDT for eyes with a large PCV lesion, ophthalmologists should be aware of the risk of serious hemorrhagic complications.

Identifying Pathogenic Genetic Background of Simplex or Multiplex Retinitis Pigmentosa Patients: A Large-Scale Mutation Screening Study

Background and purpose: More than half of the retinitis pigmentosa (RP) cases are genetically simplex or multiplex. To date, 37 causative genes of RP have been identified; however, the elucidation of gene defects in simplex or multiplex RP patients/families remains problematic. The aim of our study was to identify the genetic causes of RP in patients with unknown or non-Mendelian inheritance. Methods and results: Since 2003, 52 simplex RP patients, 151 patients from 141 multiplex RP families, and six sporadic patients with retinal degeneration were studied. A total of 108 exons of 30 RP-causing genes that harboured the reported mutations were screened by an efficient denaturing high performance liquid chromatography (dHPLC) based assay. Aberrant fragments were subsequently analysed by automatic sequencing. Twenty-six mutations, including two frameshift mutations, one single amino acid deletion, and 23 missense mutations, were identified in 28 probands (14.07%). Eighteen mutations have not been reported to date. Three pairs of combined mutations in different genes were identified in two sporadic cases and one multiplex family, indicating the possibility of novel digenic patterns. Of the 23 missense mutations, 21 were predicted as deleterious mutations by computational methods using PolyPhen, SIFT, PANTHER, and PMut programs. Conclusion: We elucidated the mutation spectrum in Japanese RP patients and demonstrated the validity of the mutation detection system using dHPLC sequencing for genetic diagnosis in RP patients independent of familial incidence, which may provide a model strategy for identifying genetic causes in other diseases linked to a wide range of genes.

Alterations of retinal pigment epithelium in central serous chorioretinopathy

Background:  To examine with en face optical coherence tomography (OCT) the alterations of retinal pigment epithelium (RPE) in central serous chorioretinopathy (CSC) and their role in the pathophysiology of CSC.

Two novel mutations in the EYS gene are possible major causes of autosomal recessive retinitis pigmentosa in the Japanese population.

Retinitis pigmentosa (RP) is a highly heterogeneous genetic disease including autosomal recessive (ar), autosomal dominant (ad), and X-linked inheritance. Recently, arRP has been associated with mutations in EYS (Eyes shut homolog), which is a major causative gene for this disease. This study was conducted to determine the spectrum and frequency of EYS mutations in 100 Japanese arRP patients. To determine the prevalence of EYS mutations, all EYS exons were screened for mutations by polymerase chain reaction amplification, and sequence analysis was performed. We detected 67 sequence alterations in EYS, of which 21 were novel. Of these, 7 were very likely pathogenic mutations, 6 were possible pathogenic mutations, and 54 were predicted non-pathogenic sequence alterations. The minimum observed prevalence of distinct EYS mutations in our study was 18% (18/100, comprising 9 patients with 2 very likely pathogenic mutations and the remaining 9 with only one such mutation). Among these mutations, 2 novel truncating mutations, c.4957_4958insA (p.S1653KfsX2) and c.8868C>A (p.Y2956X), were identified in 16 patients and accounted for 57.1% (20/35 alleles) of the mutated alleles. Although these 2 truncating mutations were not detected in Japanese patients with adRP or Lebers congenital amaurosis, we detected them in Korean arRP patients. Similar to Japanese arRP results, the c.4957_4958insA mutation was more frequently detected than the c.8868C>A mutation. The 18% estimated prevalence of very likely pathogenic mutations in our study suggests a major involvement of EYS in the pathogenesis of arRP in the Japanese population. Mutation spectrum of EYS in 100 Japanese patients, including 13 distinct very likely and possible pathogenic mutations, was largely different from the previously reported spectrum in patients from non-Asian populations. Screening for c.4957_4958insA and c.8868C>A mutations in the EYS gene may therefore be very effective for the genetic testing and counseling of RP patients in Japan.

Successful Transplantation of Retinal Pigment Epithelial Cells from MHC Homozygote iPSCs in MHC-Matched Models

Summary 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.

Association of clinical characteristics with disease subtypes, initial visual acuity, and visual prognosis in neovascular age-related macular degeneration

PurposeTo comprehend the clinical characteristics and factors related to visual prognosis in two major subtypes of neovascular age-related macular degeneration (AMD)—traditional, typical AMD and polypoidal choroidal vasculopathy (PCV).MethodsMedical records of 272 eyes of 216 patients diagnosed with neovascular AMD at Kyoto University Hospital between January 2000 and March 2003 were retrospectively reviewed for up to 3 years. Ophthalmoscopic, angiographic, and optical coherence tomography (OCT) findings were collected, and univariate and multivariate analyses were performed to determine the characteristic factors, the factors associated with initial visual acuity (VA), and visual prognosis in typical AMD and PCV.ResultsWe studied 154 eyes with typical AMD and 117 eyes with PCV. The presence of classic choroidal neovascularization (CNV) and posterior confluent drusen were characteristic of typical AMD, whereas PCV was characterized by a larger number of retinal pigment epithelial detachments and a small number of drusen. Poor initial VA (<0.1) was significantly associated with subfoveal classic CNV, hard exudates, late fibrosis staining of >−1 disc area in AMD, and with blood and cystoid macular edema in PCV. Maintenance of good VA was associated with better initial VA in typical AMD and with smaller lesions in PCV.ConclusionTypical AMD and PCV revealed statistical differences both in their clinical characteristics and in the factors associated with visual prognosis.