Michiko Mandai

Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells.

We previously reported the differentiation of mouse embryonic stem (ES) cells into retinal progenitors. However, these progenitors rarely differentiate into photoreceptors unless they are cultured with embryonic retinal tissues. Here we show the in vitro generation of putative rod and cone photoreceptors from mouse, monkey and human ES cells by stepwise treatments under defined culture conditions, in the absence of retinal tissues. With mouse ES cells, Crx+ photoreceptor precursors were induced from Rx+ retinal progenitors by treatment with a Notch signal inhibitor. Further application of fibroblast growth factors, Shh, taurine and retinoic acid yielded a greater number of rhodopsin+ rod photoreceptors, in addition to default cone production. With monkey and human ES cells, feeder- and serum-free suspension culture combined with Wnt and Nodal inhibitors induced differentiation of Rx+ or Mitf+ retinal progenitors, which produced retinal pigment epithelial cells. Subsequent treatment with retinoic acid and taurine induced photoreceptor differentiation. These findings may facilitate the development of human ES cell–based transplantation therapies for retinal diseases.

Characterization of Human Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium Cell Sheets Aiming for Clinical Application

Summary 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.

Wnt Signaling Promotes Regeneration in the Retina of Adult Mammals

Regeneration in the mammalian CNS is severely limited. Unlike in the chick, current models hold that retinal neurons are never regenerated. Previously we demonstrated that, in the adult mammalian retina, Müller glia dedifferentiate and produce retinal cells, including photoreceptors, after acute neurotoxic injury in vivo. However, the number of newly generated retinal neurons is very limited. Here we demonstrate that Wnt (wingless-type MMTV integration site family)/β-catenin signaling promotes proliferation of Müller glia-derived retinal progenitors and neural regeneration after damage or during degeneration. Wnt3a treatment increases proliferation of dedifferentiated Müller glia >20-fold in the photoreceptor-damaged retina. Supplementation with retinoic acid or valproic acid induces differentiation of these cells primarily into Crx (cone rod homeobox)-positive and rhodopsin-positive photoreceptors. Notably, injury induces nuclear accumulation of β-catenin, cyclin D1 upregulation, and Wnt/β-catenin reporter activity. Activation of Wnt signaling by glycogen synthase kinase-3β inhibitors promotes retinal regeneration, and, conversely, inhibition of the signaling attenuates regeneration. This Wnt3a-mediated regeneration of retinal cells also occurs in rd mice, a model of retinal degeneration. These results provide evidence that Wnt/β-catenin signaling contributes to CNS regeneration in the adult mammal.

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.)

Transplantation of Embryonic and Induced Pluripotent Stem Cell-Derived 3D Retinal Sheets into Retinal Degenerative Mice

Summary In this article, we show that mouse embryonic stem cell- or induced pluripotent stem cell-derived 3D retinal tissue developed a structured outer nuclear layer (ONL) with complete inner and outer segments even in an advanced retinal degeneration model (rd1) that lacked ONL. We also observed host-graft synaptic connections by immunohistochemistry. This study provides a “proof of concept” for retinal sheet transplantation therapy for advanced retinal degenerative diseases.

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.

Dual actions of nitric oxide inN-methyl-d-aspartate receptor-mediated neurotoxicity in cultured retinal neurons

This study was performed to elucidate the role of nitric oxide (NO) in N-methyl-D-aspartate (NMDA) receptor-mediated glutamate neurotoxicity in the retina. The experiments were done with primary retinal cultures obtained from 17- to 19-day-old rat fetuses. The NOS activity measured by monitoring the conversion of [3H]arginine to [3H]citrulline was approximately 5 pmol/min/mg protein. A 10-min exposure of the cultured cells to glutamate (1 mM) or NMDA (1 mM) followed by a 1-h incubation in a normal medium consistently resulted in 60% cell death. The concomitant addition of an inhibitor of NOS, Nomega-nitro-L-arginine (300 microM), with glutamate or NMDA reduced cell death by 70%. A brief exposure of the cells to sodium nitroprusside (SNP, 500 microM) or S-nitrosocysteine (SNOC, 500 microM), NO-generating agents, caused 60% cell death. Depletion of NO by reduced hemoglobin prevented the cell death induced by either glutamate, NMDA, or NO generating agents. Fifty microM SNOC alone had no effect on the cell viability. However, pretreatment with 50 microM SNOC as well as simultaneous application of 50 microM SNOC with NMDA inhibited cell death induced by NMDA. These findings indicate that a low concentration of NO plays a protective role in glutamate neurotoxicity via closing the NMDA receptor gated ion channel. However, elevated concentrations of NO, interacting with oxygen radicals, become toxic and mediate glutamate-induced neurotoxicity in the cultured retinal neurons.

The Significance of External Limiting Membrane Status for Visual Acuity in Age-Related Macular Degeneration

PURPOSE To evaluate status of the external limiting membrane (ELM) as a contributor of visual acuity (VA) in age-related macular degeneration (AMD). DESIGN Hospital-based, cross-sectional study. METHODS We retrospectively reviewed spectral-domain optical coherence tomography images of 158 patients with AMD who had undergone photodynamic therapy and classified them based on the status of the ELM: absent, discontinuous, or complete. We simultaneously assessed foveal thickness, presence or absence of subretinal fluid/mass, presence or absence of subretinal pigment epithelium fluid/mass, status of the inner segment/outer segment (IS/OS) junction, and status of the intermediate line between the IS/OS junction and retinal pigment epithelium. Correlation coefficients between each parameter and VA were analyzed. RESULTS There was a strong correlation between ELM status and VA (r = -0.75, P < .001), and that was higher than that of the IS/OS (r = -0.69, P < .001). Multivariate analysis showed that ELM status is the most important factor for VA. Other parameters that correlated with VA included age, status of the intermediate line, and presence of subretinal or subretinal pigment epithelium fibrosis. Foveal thickness showed V-shaped correlation, with the dividing line around 200 mum. CONCLUSION ELM status may be more useful than is IS/OS status in evaluation of retinal morphology and function in patients with AMD.

Tumorigenicity Studies of Induced Pluripotent Stem Cell (iPSC)-Derived Retinal Pigment Epithelium (RPE) for the Treatment of Age-Related Macular Degeneration

Basic studies of human pluripotential stem cells have advanced rapidly and stem cell products are now seeing therapeutic applications. However, questions remain regarding the tumorigenic potential of such cells. Here, we report the tumorigenic potential of induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) for the treatment of wet-type, age-related macular degeneration (AMD). First, immunodeficient mouse strains (nude, SCID, NOD-SCID and NOG) were tested for HeLa cells’ tumor-forming capacity by transplanting various cell doses subcutaneously with or without Matrigel. The 50% Tumor Producing Dose (TPD50 value) is the minimal dose of transplanted cells that generated tumors in 50% of animals. For HeLa cells, the TPD50 was the lowest when cells were embedded in Matrigel and transplanted into NOG mice (TPD50 = 101.1, n = 75). The TPD50 for undifferentiated iPSCs transplanted subcutaneously to NOG mice in Matrigel was 102.12; (n = 30). Based on these experiments, 1×106 iPSC-derived RPE were transplanted subcutaneously with Matrigel, and no tumor was found during 15 months of monitoring (n = 65). Next, to model clinical application, we assessed the tumor-forming potential of HeLa cells and iPSC 201B7 cells following subretinal transplantation of nude rats. The TPD50 for iPSCs was 104.73 (n = 20) and for HeLa cells 101.32 (n = 37) respectively. Next, the tumorigenicity of iPSC-derived RPE was tested in the subretinal space of nude rats by transplanting 0.8–1.5×104 iPSC-derived RPE in a collagen-lined (1 mm×1 mm) sheet. No tumor was found with iPSC-derived RPE sheets during 6–12 months of monitoring (n = 26). Considering the number of rodents used, the monitoring period, the sensitivity of detecting tumors via subcutaneous and subretinal administration routes and the incidence of tumor formation from the iPSC-derived RPE, we conclude that the tumorigenic potential of the iPSC-derived RPE was negligible.

Transplantation of human embryonic stem cell-derived retinal tissue in two primate models of retinal degeneration

Significance We first confirmed the ability of human embryonic stem cell-derived retina (hESC-retina) to form structured mature photoreceptor layers after transplantation into nude rats. We then developed two monkey models of retinal degeneration and evaluated their utility as host models for transplantation studies. Finally, we performed a pilot study of hESC-retina transplantation in the developed models and conducted in vivo monitoring studies using clinical devices and subsequently confirmed structured graft maturation and the potential formation of synaptic contacts between graft and host cells. This study demonstrates the competency of hESC-retina as a graft source and the eligibility of two newly developed monkey models that may be useful in future, long-term, functional studies of retinal transplantation. Retinal transplantation therapy for retinitis pigmentosa is increasingly of interest due to accumulating evidence of transplantation efficacy from animal studies and development of techniques for the differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells into retinal tissues or cells. In this study, we aimed to assess the potential clinical utility of hESC-derived retinal tissues (hESC-retina) using newly developed primate models of retinal degeneration to obtain preparatory information regarding the potential clinical utility of these hESC-retinas in transplantation therapy. hESC-retinas were first transplanted subretinally into nude rats with or without retinal degeneration to confirm their competency as a graft to mature to form highly specified outer segment structure and to integrate after transplantation. Two focal selective photoreceptor degeneration models were then developed in monkeys by subretinal injection of cobalt chloride or 577-nm optically pumped semiconductor laser photocoagulation. The utility of the developed models and a practicality of visual acuity test developed for monkeys were evaluated. Finally, feasibility of hESC-retina transplantation was assessed in the developed monkey models under practical surgical procedure and postoperational examinations. Grafted hESC-retina was observed differentiating into a range of retinal cell types, including rod and cone photoreceptors that developed structured outer nuclear layers after transplantation. Further, immunohistochemical analyses suggested the formation of host–graft synaptic connections. The findings of this study demonstrate the clinical feasibility of hESC-retina transplantation and provide the practical tools for the optimization of transplantation strategies for future clinical applications.