Efficacy of valproic acid for retinitis pigmentosa patients: a pilot study

Purpose: The purpose of this study was to examine the efficacy and safety of valproic acid (VPA) use in patients with retinitis pigmentosa (RP). Patients and methods: This was a prospective, interventional, noncomparative case study. In total, 29 eyes from 29 patients with RP whose best-corrected visual acuities (BCVAs) in logarithm of the minimum angle of resolution (logMAR) ranged from 1.0 to 0.16 with visual fields (VFs) of ≤10° (measured using Goldmann perimeter with I4) were recruited. The patients received oral supplementation with 400 mg of VPA daily for 6 months and were followed for an additional 6 months. BCVAs, VFs (measured with the Humphrey field analyzer central 10-2 program), and subjective questionnaires were examined before, during, and after the cessation of VPA supplementation. Results: The changes in BCVA and VF showed statistically significant differences during the internal use of VPA, compared with after cessation (P=0.001). With VPA intake, BCVA in logMAR significantly improved from baseline to 6 months (P=0.006). The mean deviation value of the VF significantly improved from baseline to 1 month (P=0.001), 3 months (P=0.004), and 6 months (P=0.004). These efficacies, however, were reversed to the baseline levels after the cessation of VPA intake. There were no significant relations between the mean blood VPA concentrations of each patient and the changes in BCVA and VF. During the internal use of VPA, 15 of 29 patients answered “easier to see”, whereas blurred vision was registered in 21 of 29 patients on cessation. No systemic drug-related adverse events were observed. Conclusion: While in use, oral intake of VPA indicated a short-term benefit to patients with RP. It is necessary to examine the effect of a longer VPA supplementation in a controlled study design

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

Protocol for Obtaining Mouse iPS-RPE

Purpose To establish a novel protocol for differentiation of retinal pigment epithelium (RPE) with high purity from mouse induced pluripotent stem cells (iPSC). Methods Retinal progenitor cells were differentiated from mouse iPSC, and RPE differentiation was then enhanced by activation of the Wnt signaling pathway, inhibition of the fibroblast growth factor signaling pathway, and inhibition of the Rho-associated, coiled-coil containing protein kinase signaling pathway. Expanded pigmented cells were purified by plate adhesion after Accutase® treatment. Enriched cells were cultured until they developed a cobblestone appearance with cuboidal shape. The characteristics of iPS-RPE were confirmed by gene expression, immunocytochemistry, and electron microscopy. Functions and immunologic features of the iPS-RPE were also evaluated. Results We obtained iPS-RPE at high purity (approximately 98%). The iPS-RPE showed apical-basal polarity and cellular structure characteristic of RPE. Expression levels of several RPE markers were lower than those of freshly isolated mouse RPE but comparable to those of primary cultured RPE. The iPS-RPE could form tight junctions, phagocytose photoreceptor outer segments, express immune antigens, and suppress lymphocyte proliferation. Conclusion We successfully developed a differentiation/purification protocol to obtain mouse iPS-RPE. The mouse iPS-RPE can serve as an attractive tool for functional and morphological studies of RPE

Establishment of Immunodeficient Retinal Degeneration Model Mice and Functional Maturation of Human ESC-Derived Retinal Sheets after Transplantation

Increasing demand for clinical retinal degeneration therapies featuring human ESC/iPSC-derived retinal tissue and cells warrants proof-of-concept studies. Here, we established two mouse models of end-stage retinal degeneration with immunodeficiency, NOG-rd1-2J and NOG-rd10, and characterized disease progress and immunodeficient status. We also transplanted human ESC-derived retinal sheets into NOG-rd1-2J and confirmed their long-term survival and maturation of the structured graft photoreceptor layer, without rejection or tumorigenesis. We recorded light responses from the host ganglion cells using a multi-electrode array system; this result was consistent with whole-mount immunostaining suggestive of host-graft synapse formation at the responding sites. This study demonstrates an application of our mouse models and provides a proof of concept for the clinical use of human ESC-derived retinal sheets

Combined analysis of single cell RNA-Seq and ATAC-Seq data reveals putative regulatory toggles operating in native and iPS-derived retina.

We report the generation and analysis of single-cell RNA-Seq data (> 38,000 cells) from native and iPSC-derived murine retina at four matched developmental stages spanning the emergence of the major retinal cell types. We combine information from temporal sampling, visualization of 3D UMAP manifolds, pseudo-time and RNA velocity analyses, to show that iPSC-derived 3D retinal aggregates broadly recapitulate the native developmental trajectories. However, we show relaxation of spatial and temporal transcriptome control, premature emergence and dominance of photoreceptor precursor cells, and susceptibility of dynamically regulated pathways and transcription factors to culture conditions in iPSC-derived retina. We generate bulk ATAC-Seq data for native and iPSC-derived murine retina identifying ~125,000 peaks. We combine single-cell RNA-Seq with ATAC-Seq information and obtain evidence that approximately half the transcription factors that are dynamically regulated during retinal development may act as repressors rather than activators. We propose that sets of activators and repressors with cell-type specific expression constitute regulatory toggles that lock cells in distinct transcriptome states underlying differentiation. We provide evidence supporting our hypothesis from the analysis of publicly available single-cell ATAC-Seq data for adult mouse retina. We identify subtle but noteworthy differences in the operation of such toggles between native and iPSC-derived retina particularly for the Etv1, Etv5, Hes1 and Zbtb7a group of transcription factors

Pluripotent stem cell-derived retinal organoid/cells for retinal regeneration therapies: A review

In recent decades, many researchers have attempted to restore vision via transplantation of retina/retinal cells in eyes with retinal degeneration. The advent of induced pluripotent stem cells (iPSC) and retinal organoid induction technologies has boosted research on retinal regeneration therapy. Although the recognition of functional integration of graft photoreceptor cells in the host retina from 2006 has been disputed a decade later by the newly evidenced phenomenon denoted as “material transfer,” several reports support possible reconstruction of the host-graft network in the retinas of both end-stage degeneration and in progressing degeneration cases. Based on proof of concept (POC) studies in animal models, a clinical study was conducted in Kobe, Japan in 2020 and showed the feasibility of cell-based therapy using iPSC retinal organoid technology. Although the graft potency of human embryonic stem (ES)/iPS cell-derived retinal organoid/retinal cells has been suggested by previous studies, much is still unknown regarding host capability, that is, how long-standing human degenerating retinas are capable of rewiring with transplanted cells. This review summarizes past POC studies on photoreceptor replacement therapy and introduces some new challenges to maximize the possible efficacy in future human clinical studies of regenerative therapy

Possible vitreous involvement in a case with rapidly progressing choroidal neovascularization

A 65-year-old man with subfoveal choroidal neovascularization (CNV) underwent photodynamic therapy (PDT). Despite the sequential treatments, the CNV grew larger and finally penetrated the retina. Vitreous adhesion was observed at the edge of the supraretinal fibrotic tissue. The case highlighted the possible unexpected side-effect of PDT. The upregulation of the vascular endothelial growth factor or the enhanced vitreous traction was considered to be responsible for the event

Proliferation potential of Müller glia after retinal damage varies between mouse strains.

Retinal Müller glia can serve as a source for regeneration of damaged retinal neurons in fish, birds and mammals. However, the proliferation rate of Müller glia has been reported to be low in the mammalian retina. To overcome this problem, growth factors and morphogens have been studied as potent promoters of Müller glial proliferation, but the molecular mechanisms that limit the proliferation of Müller glia in the mammalian retina remain unknown. In the present study, we found that the degree of damage-induced Müller glia proliferation varies across mouse strains. In mouse line 129×1/SvJ (129), there was a significantly larger proliferative response compared with that observed in C57BL/6 (B6) after photoreceptor cell death. Treatment with a Glycogen synthase kinase 3 (GSK3) inhibitor enhanced the proliferation of Müller glia in 129 but not in B6 mouse retinas. We therefore focused on the different gene expression patterns during retinal degeneration between B6 and 129. Expression levels of Cyclin D1 and Nestin correlated with the degree of Müller glial proliferation. A comparison of genome-wide gene expression between B6 and 129 showed that distinct sets of genes were upregulated in the retinas after damage, including immune response genes and chromatin remodeling factors

Recovery of photoreceptor outer segments after anti-VEGF therapy for age-related macular degeneration.

[Purpose]To evaluate whether the status of the external limiting membrane (ELM) or inner segment/outer segment junction (IS/OS) improves after intravitreal injection of ranibizumab for age-related macular degeneration (AMD). We also evaluated whether the pre-operative values of these parameters are associated with the visual prognosis. [Methods]This was a hospital-based, cross-sectional study. Seventy-six eyes of 76 treatment-naive AMD patients who received three monthly intravitreal injections of ranibizumab followed for more than 6 months with additional as-needed injections were investigated. Spectral domain OCT was used to evaluate the length of ELM, IS/OS, and foveal thickness pre- and post-operatively. Changes of ELM and IS/OS length were evaluated postoperatively. Correlation coefficients between pre-operative parameters and post-operative visual acuity were also analyzed. [Results]Significant changes were noted in mean logMAR (0.66 to 0.53), foveal thickness (231.1 to 151.1 μm), and IS/OS length (514.9 to 832.3 μm) after the treatment. ELM length did not improve significantly (1,312.4 to 1,376.7 μm). Restoration of IS/OS occured where ELM is retained. Although pre-operative ELM length, IS/OS length, and foveal thickness showed correlation with post-operative logMAR (R=-0.51, -0.39, and 0.46, respectively), the most powerful predictive factor for visual prognosis was pre-operative logMAR (R=0.77, p<0.001). [Conclusions] IS/OS status improves in response to anti-VEGF therapy but ELM seems to have less plasticity. The status of IS/OS and ELM can be used as prognostic factors but the predictive power is inferior to that of baseline visual acuity

Induction of Functional 3D Ciliary Epithelium?Like Structure From Mouse Induced Pluripotent Stem Cells

PURPOSE. To generate ciliary epithelium (CE) from mouse induced pluripotent stem (iPS) cells. METHODS. Recently, a protocol for self-organizing optic cup morphogenesis in threedimensional culture was reported, and it was suggested that ocular tissue derived from neural ectoderm could be differentiated. We demonstrated that a CE-like double-layered structure could be induced in simple culture by using a modified Eiraku differentiation protocol. RESULTS. Differentiation of a CE-like double-layered structure could be promoted by glycogen synthase kinase 3β (GSK-3β) inhibitor. Connexin43 and aquaporin1 were expressed in both thin layers, and induced CE-like cells expressed ciliary marker genes, such as cyclinD2, zic1, tgfb2, aldh1a3, wfdc1, otx1, BMP4, and BMP7. Increases in cytoplasmic and nuclear β-catenin in aggregates of the CE-like double-layered structure were confirmed by Western blot analysis. In addition, tankyrase inhibitor prevented the induction of the CE-like double-layered structure by GSK-3β inhibitor. Dye movement from pigmented cells to nonpigmented cells in the mouse iPS cell?derived CE-like structure was observed in a fluid movement experiment, consistent with the physiological function of CE in vivo. CONCLUSIONS. We could differentiate CE from mouse iPS cells in the present study. In the future, we hope that this CE-like complex will become useful as a graft for transplantation therapy in pathologic ocular hypotension due to CE dysfunction, and as a screening tool for the development of drugs for diseases associated with CE function