Protective Effects of Human iPS-Derived Retinal Pigmented Epithelial Cells in Comparison with Human Mesenchymal Stromal Cells and Human Neural Stem Cells on the Degenerating Retina in rd1 mice.

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.

Host-Graft Synapses Form Functional Microstructures and Shape the Host Light Responses After Stem Cell-Derived Retinal Sheet Transplantation.

Purpose: Retinitis pigmentosa represents a leading cause of blindness in developed countries, yet effective treatments for the disease remain unestablished. Previous studies have demonstrated the potential of stem cell-derived retinal organoid (SC-RO) sheet transplantation to form host-graft synapses and to improve light responsiveness in animal models of retinal degeneration. However, the detailed microstructures of these de novo synapses and their functional contribution have not been well elucidated. This study aims to (1) elucidate the microstructures of the host-graft synapse, and (2) investigate the overall distribution and contribution of these synapses to host retinal light responses. Methods: We identified host-graft synapses using a reporter system in mouse SC-RO and rd1 mice, a well-established model of end-stage retinal degeneration. Correlative array tomography was used to reveal the microstructure of host-graft synapses. Furthermore, we developed a semi-automated algorithm that robustly detects the host-graft photoreceptor synapses in the overall grafted area using the same reporter system in flat-mount retinas. We then integrated the spatial distribution of the host-graft synapses with light responses detected by multi-electrode array recording. Results: Correlative array tomography revealed that host-graft synapses recapitulate the developmental process of photoreceptor synapse formation involving horizontal cells first and then rod bipolar cells. By integrating the spatial distribution of host-graft synapse and multi-electrode array recording, we showed that the number of light-responsive host retinal ganglion cells is positively correlated with the local density of host-graft synapses. Conclusions: De novo host-graft synapses recapitulate the developmental microstructure of the photoreceptor synapse, and their formation contributes to the light responsiveness after SC-RO transplantation.

A Genetic modification that reduces ON-bipolar cells in hESC-derived retinas enhances functional integration after transplantation.

Pluripotent stem cell (PSC)-derived retinal sheet transplanted in vivo can form structured photoreceptor layers, contact with host bipolar cells, and transmit light signals to host retinas. However, a major concern is the presence of graft bipolar cells that may impede host-graft interaction. In this study, we used human ESC-retinas with the deletion of Islet-1 (ISL1) gene to achieve the reduced graft ON-bipolar cells after xenotransplantation into end-stage retinal degeneration model rats. Compared with wild-type graft, ISL1 -/- hESC-retinas showed better host-graft contact, with indication of host-graft synapse formation and significant restoration of light responsiveness in host ganglion cells. We further analyzed to find out that improved functional integration of ISL1 -/- hESC-retinas seemed attributed by a better host-graft contact and a better preservation of host inner retina. ISL1 -/- hESC-retinas are promising for the efficient reconstruction of a degenerated retinal network in future clinical application.

Transplanted Mouse Embryonic Stem Cell-Derived Retinal Ganglion Cells Integrate and Form Synapses in a Retinal Ganglion Cell-Depleted Mouse Model.

Purpose: Retinal ganglion cell (RGC) transplantation is a therapeutic approach to replace irreversibly degenerated RGCs in diseases such as glaucoma. However, the application of primary RGCs is limited by the availability of tissues. The goal of this study was to evaluate whether transplanted mouse embryonic stem cell (mESC)-derived RGCs can integrate into the host retina and form cell connectivity with host cells. Methods: In this study, we prepared small retinal fragments containing RGC as THY1-enhanced green fluorescent protein (EGFP)+ cells from mESCs and placed them near the retinal surface in the air-injected mouse eyes with or without N-methyl-d-aspartate (NMDA)-induced RGC depletion. After transplantation, THY1-EGFP+ cell integration was observed in whole-mounts and with immunostaining for synaptic markers. Results: Transplanted THY1-EGFP+ cells survived for 12 weeks and extended neurites into the inner plexiform layer (IPL) of the host retina. Presumptive synapse formation was identified between grafted RGCs and host bipolar cells. The ratio of transplanted eyes with integration of THY1-EGFP+ neurites in the host IPL was higher in RGC-injured mice compared with healthy controls. Conclusions: This report shows the potential for therapeutic use of pluripotent cell-derived RGCs by grafting the cells in healthy conditions and with an appropriate technical approach.

Genetically engineered stem cell-derived retinal grafts for improved retinal reconstruction after transplantation.

ESC/iPSC-retinal sheet transplantation, which supplies photoreceptors as well as other retinal cells, has been shown to be able to restore visual function in mice with end-stage retinal degeneration. Here, by introducing a novel type of genetically engineered mouse ESC/iPSC-retinal sheet with reduced numbers of secondary retinal neurons but intact photoreceptor cell layer structure, we reinforced the evidence that ESC/iPSC-retinal sheet transplantation can establish synaptic connections with the host, restore light responsiveness, and reduce aberrant retinal ganglion cell spiking in mice. Furthermore, we show that genetically engineered grafts can substantially improve the outcome of the treatment by improving neural integration. We speculate that this leads to reduced spontaneous activity in the host which in turn contributes to a better visual recovery.

Prolyl 4-hydroxylation of alpha-fibrinogen: a novel protein modification revealed by plasma proteomics.

Plasma proteome analysis requires sufficient power to compare numerous samples and detect changes in protein modification, because the protein content of human samples varies significantly among individuals, and many plasma proteins undergo changes in the bloodstream. A label-free proteomics platform developed in our laboratory, termed "Two-Dimensional Image Converted Analysis of Liquid chromatography and mass spectrometry (2DICAL)," is capable of these tasks. Here, we describe successful detection of novel prolyl hydroxylation of alpha-fibrinogen using 2DICAL, based on comparison of plasma samples of 38 pancreatic cancer patients and 39 healthy subjects. Using a newly generated monoclonal antibody 11A5, we confirmed the increase in prolyl-hydroxylated alpha-fibrinogen plasma levels and identified prolyl 4-hydroxylase A1 as a key enzyme for the modification. Competitive enzyme-linked immunosorbent assay of 685 blood samples revealed dynamic changes in prolyl-hydroxylated alpha-fibrinogen plasma level depending on clinical status. Prolyl-hydroxylated alpha-fibrinogen is presumably controlled by multiple biological mechanisms, which remain to be clarified in future studies.

Quantitative and Qualitative Evaluation of Photoreceptor Synapses in Developing, Degenerating and Regenerating Retinas.

Quantitative and qualitative evaluation of synapses is crucial to understand neural connectivity. This is particularly relevant now, in view of the recent advances in regenerative biology and medicine. There is an urgent need to evaluate synapses to access the extent and functionality of reconstructed neural network. Most of the currently used synapse evaluation methods provide only all-or-none assessments. However, very often synapses appear in a wide spectrum of transient states such as during synaptogenesis or neural degeneration. Robust evaluation of synapse quantity and quality is therefore highly sought after. In this paper we introduce QUANTOS, a new method that can evaluate the number, likelihood, and maturity of photoreceptor ribbon synapses based on graphical properties of immunohistochemistry images. QUANTOS is composed of ImageJ Fiji macros, and R scripts which are both open-source and free software. We used QUANTOS to evaluate synaptogenesis in developing and degenerating retinas, as well as de novo synaptogenesis of mouse iPSC-retinas after transplantation to a retinal degeneration mouse model. Our analysis shows that while mouse iPSC-retinas are largely incapable of forming synapses in vitro, they can form extensive synapses following transplantation. The de novo synapses detected after transplantation seem to be in an intermediate state between mature and immature compared to wildtype retina. Furthermore, using QUANTOS we tested whether environmental light can affect photoreceptor synaptogenesis. We found that the onset of synaptogenesis was earlier under cyclic light (LD) condition when compared to constant dark (DD), resulting in more synapses at earlier developmental stages. The effect of light was also supported by micro electroretinography showing larger responses under LD condition. The number of synapses was also increased after transplantation of mouse iPSC-retinas to rd1 mice under LD condition. Our new probabilistic assessment of synapses may prove to be a valuable tool to gain critical insights into neural-network reconstruction and help develop treatments for neurodegenerative disorders.

Medium- to long-term survival and functional examination of human iPSC-derived retinas in rat and primate models of retinal degeneration.

BACKGROUND: We have previously reported that xeno-transplanted human ESC-derived retinas are able to mature in the immunodeficient retinal degeneration rodent models, similar to allo-transplantations using mouse iPSC-derived retina. The photoreceptors in the latter developed outer segments and formed synapses with host bipolar cells, driving light responses of host retinal ganglion cells. In view of clinical application, here we further confirmed the competency of human iPSC-derived retina (hiPSC-retina) to mature in the degenerated retinas of rat and monkey models. METHODS: Human iPSC-retinas were transplanted in rhodopsin mutant SD-Foxn1 Tg(S334ter)3LavRrrc nude rats and two monkeys with laser-induced photoreceptor degeneration. Graft maturation was studied by immunohistochemistry and its function was examined by multi-electrode array (MEA) recording in rat retinas and visually-guided saccade (VGS) in a monkey. FINDINGS: A substantial amount of mature photoreceptors in hiPSC-retina graft survived well in the host retinas for at least 5 months (rat) to over 2 years (monkey). In 4 of 7 transplanted rat retinas, RGC light responses were detected at the grafted area. A mild recovery of light perception was also suggested by the VGS performance 1.5 years after transplantation in that monkey. INTERPRETATION: Our results support the competency of hiPSC-derived retinas to be clinically applied for transplantation therapy in retinal degeneration, although the light responses observed in the present models were not conclusively distinguishable from residual functions of degenerating host retinas. The functional analysis may be further elaborated using other models with more advanced retinal degeneration.

iPSC-Derived Retina Transplants Improve Vision in rd1 End-Stage Retinal-Degeneration Mice.

Recent success in functional recovery by photoreceptor precursor transplantation in dysfunctional retina has led to an increased interest in using embryonic stem cell (ESC) or induced pluripotent stem cell (iPSC)-derived retinal progenitors to treat retinal degeneration. However, cell-based therapies for end-stage degenerative retinas that have lost the outer nuclear layer (ONL) are still a big challenge. In the present study, by transplanting mouse iPSC-derived retinal tissue (miPSC retina) in the end-stage retinal-degeneration model (rd1), we visualized the direct contact between host bipolar cell terminals and the presynaptic terminal of graft photoreceptors by gene labeling, showed light-responsive behaviors in transplanted rd1 mice, and recorded responses from the host retina with transplants by ex vivo micro-electroretinography and ganglion cell recordings using a multiple-electrode array system. Our data provides a proof of concept for transplanting ESC/iPSC retinas to restore vision in end-stage retinal degeneration.

Inhibition of T-cell activation by retinal pigment epithelial cells derived from induced pluripotent stem cells.

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.