Kohei Homma

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.

Developing Rods Transplanted into the Degenerating Retina of Crx-Knockout Mice Exhibit Neural Activity Similar to Native Photoreceptors†‡§

Replacement of dysfunctional or dying photoreceptors offers a promising approach for retinal neurodegenerative diseases, including age‐related macular degeneration and retinitis pigmentosa. Several studies have demonstrated the integration and differentiation of developing rod photoreceptors when transplanted in wild‐type or degenerating retina; however, the physiology and function of the donor cells are not adequately defined. Here, we describe the physiological properties of developing rod photoreceptors that are tagged with green fluorescent protein (GFP) driven by the promoter of rod differentiation factor, Nrl. GFP‐tagged developing rods show Ca2 + responses and rectifier outward currents that are smaller than those observed in fully developed photoreceptors, suggesting their immature developmental state. These immature rods also exhibit hyperpolarization‐activated current (Ih) induced by the activation of hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels. When transplanted into the subretinal space of wild‐type or retinal degeneration mice, GFP‐tagged developing rods can integrate into the photoreceptor outer nuclear layer in wild‐type mouse retina and exhibit Ca2 + responses and membrane current comparable to native rod photoreceptors. A proportion of grafted rods develop rhodopsin‐positive outer segment‐like structures within 2 weeks after transplantation into the retina of Crx‐knockout mice and produce rectifier outward current and Ih upon membrane depolarization and hyperpolarization. GFP‐positive rods derived from induced pluripotent stem (iPS) cells also display similar membrane current Ih as native developing rod photoreceptors, express rod‐specific phototransduction genes, and HCN‐1 channels. We conclude that Nrl‐promoter‐driven GFP‐tagged donor photoreceptors exhibit physiological characteristics of rods and that iPS cell‐derived rods in vitro may provide a renewable source for cell‐replacement therapy. STEM Cells 2013;31:1149–1159

Transcriptome Dynamics of Developing Photoreceptors in Three-Dimensional Retina Cultures Recapitulates Temporal Sequence of Human Cone and Rod Differentiation Revealing Cell Surface Markers and Gene Networks.

The derivation of three‐dimensional (3D) stratified neural retina from pluripotent stem cells has permitted investigations of human photoreceptors. We have generated a H9 human embryonic stem cell subclone that carries a green fluorescent protein (GFP) reporter under the control of the promoter of cone‐rod homeobox (CRX), an established marker of postmitotic photoreceptor precursors. The CRXp‐GFP reporter replicates endogenous CRX expression in vitro when the H9 subclone is induced to form self‐organizing 3D retina‐like tissue. At day 37, CRX+ photoreceptors appear in the basal or middle part of neural retina and migrate to apical side by day 67. Temporal and spatial patterns of retinal cell type markers recapitulate the predicted sequence of development. Cone gene expression is concomitant with CRX, whereas rod differentiation factor neural retina leucine zipper protein (NRL) is first observed at day 67. At day 90, robust expression of NRL and its target nuclear receptor NR2E3 is evident in many CRX+ cells, while minimal S‐opsin and no rhodopsin or L/M‐opsin is present. The transcriptome profile, by RNA‐seq, of developing human photoreceptors is remarkably concordant with mRNA and immunohistochemistry data available for human fetal retina although many targets of CRX, including phototransduction genes, exhibit a significant delay in expression. We report on temporal changes in gene signatures, including expression of cell surface markers and transcription factors; these expression changes should assist in isolation of photoreceptors at distinct stages of differentiation and in delineating coexpression networks. Our studies establish the first global expression database of developing human photoreceptors, providing a reference map for functional studies in retinal cultures. Stem Cells 2015;33:3504–3518

Serotonin induces the increase in intracellular Ca2+ that enhances neurite outgrowth in PC12 cells via activation of 5-HT3 receptors and voltage-gated calcium channels

As a neurotransmitter and neuromodulator, serotonin (5‐HT) influences neuronal outgrowth in the nervous systems of several species. In PC12 cells, 5‐HT is known to have neuritogenic effects, although the signal transduction pathway responsible for these effects is not understood. In this study, we hypothesized that a 5‐HT‐induced increase in intracellular Ca2+ concentration ([Ca2+]i) could be involved in mediating the effects of 5‐HT. Application of 5‐HT to PC12 cells enhanced nerve growth factor (NGF)‐induced neurite outgrowth in a dose‐dependent manner, and the sensitivity of this neuritogenic effect was increased in differentiated PC12 cells. In accordance, an increase in [Ca2+]i was observed following application of 5‐HT in differentiated PC12 cells. This increase was amplified by further NGF treatment. 5‐HT‐induced increases in [Ca2+]i were inhibited by MDL 72222, a selective 5‐HT3 receptor antagonist, and nifedipine, an L‐type calcium channel blocker, but not by ketanserin, a 5‐HT2 receptor antagonist, or thapsigargin, a specific inhibitor of endoplasmic reticulum Ca2+‐ATPase. These pharmacological tests indicated that 5‐HT‐induced increases in [Ca2+]i are mediated by activation of voltage‐gated calcium channels via 5‐HT3 receptors and that 5‐HT‐induced increases in [Ca2+]i are likely to be independent of activation of 5‐HT2 receptors in PC12 cells. Furthermore, the neuritogenic effect of 5‐HT was suppressed by MDL 72222, nifedipine, calmodulin (CaM) inhibitor, and calcineurin inhibitors. Taken together, our results indicate that 5‐HT‐induced increases in [Ca2+]i, which are mediated via 5‐HT3 receptors and L‐type calcium channels in PC12 cells, and subsequent activation of CaM and calcineurin enhance NGF‐induced neurite outgrowth.

Detection of localized retinal malfunction in retinal degeneration model using a multielectrode array system.

Light stimulation inhibits the retinal dark current through phototransduction signals in the photoreceptors. Electroretinography (ERG) detects the blockage of the dark current as the a‐wave of the ERG. However, standard ERGs represent the summed neural activity of the retina, and information on localized functions cannot be obtained. In this study, we used a multielectrode array (MEA) system and directly recorded the focal activities of the photoreceptors of the retina. Retinas were isolated from dark‐adapted rodents and were draped over the electrode array with vitreal surface of the retina on the electrode array. After light stimulation, negative waves were recorded from each electrode. Adding aminobutyric acid, a selective agonist of mGluR6 expressed on ON‐bipolar cells, to the media did not block the generation of the responses. The amplitude of the response increased with increasing retinal development. When the retina was locally injured, light‐elicited responses were diminished only in the injured areas of the retina. Retinas isolated from rats given N‐methyl‐N‐nitrosourea (MNU) were also tested. In central retinas of MNU‐treated rats, the responses were progressively decreased following injection of MNU. In contrast, in the peripheral retinas, amplitude of the responses was relatively retained, consistent with the retinal thickness observed by immunohistochemistry. In conclusion, light‐evoked responses were recorded with the MEA system. The MEA system was useful for detecting subtle and focal activation of photoreceptors. This spatial information should be valuable in investigating local functional recovery in therapeutically treated areas, such as in gene transfer or cell transplantation.

Treatment Paradigms for Retinal and Macular Diseases Using 3-D Retina Cultures Derived From Human Reporter Pluripotent Stem Cell Lines

We discuss the use of pluripotent stem cell lines carrying fluorescent reporters driven by retinal promoters to derive three-dimensional (3-D) retina in culture and how this system can be exploited for elucidating human retinal biology, creating disease models in a dish, and designing targeted drug screens for retinal and macular degeneration. Furthermore, we realize that stem cell investigations are labor-intensive and require extensive resources. To expedite scientific discovery by sharing of resources and to avoid duplication of efforts, we propose the formation of a Retinal Stem Cell Consortium. In the field of vision, such collaborative approaches have been enormously successful in elucidating genetic susceptibility associated with age-related macular degeneration.

Allelic Copy Number Variation in FSCN2 Detected Using Allele-Specific Genotyping and Multiplex Real-Time PCRs

PURPOSE Allelic copy number variation (CNV) may alter the functional effects of a heterozygous mutation. The underlying mechanisms and their roles in hereditary diseases, however, are largely unknown. In the present study an FSCN2 mutation was examined that has been reported, not only in patients with retinitis pigmentosa (RP), but also in the normal population. METHODS Experiments were performed to investigate the gene and allele copy numbers of FSCN2 in patients with RP who have the c.72delG mutation as well as healthy subjects with or without the mutation. A real-time PCR-based genotyping approach was established that used a real-time PCR assay to qualify the copy numbers of both the wild-type and mutant alleles of the FSCN2 gene. RESULTS Three patients with RP and three normal subjects had an equal ratio of the alleles. Of interest, another patient had an asymmetric allele ratio (4:1) of the copy number of the wild-type allele, compared with that of the mutant allele. These findings were further verified using quantitative assays. An allele-specific methylation assay demonstrated a random methylation pattern in the FSCN2 gene. CONCLUSIONS The copy numbers of the FSNC2 gene and of each allele in the mutant samples were quantified. The findings excluded the possibility that allelic CNV was associated with RP, suggesting that the c.72delG variant is not the primary cause of RP. It is not likely that the FSCN2 gene is imprinted differentially. The real-time PCR-based genotyping method developed in this study is useful for investigations of allelic asymmetries within genomic regions with CNVs.

Adequate Time Window and Environmental Factors Supporting Retinal Graft Cell Survival in rd Mice.

Postnatal photoreceptor cells can be integrated into the wild-type adult retina in mice, and retinal transplantation is now one therapeutic option for retinal degenerative diseases when photoreceptor degeneration is the primary cause of the disease. The aim of this study was to specify the optimal time window during the course of retinal degeneration and to modulate the host and/or graft environment for a successful transplantation. We first studied the background features of the mice with phosphodiesterase 6b (PDE6b) gene mutation (rd; C3H/Hej) and found that the infiltration of microglia and glial fibrillary acidic protein (GFAP) expression once increased at the peak of rod death (∼2-3 weeks of age) but then reduced for a following period until gliosis began to take place with enhanced GFAP expression (∼8 weeks of age). The postnatal retinal cells (p4-p7) were successfully transplanted during this period with neurite extension into the host retina. In later transplantations (6 or 8 weeks of age), graft cells survived better in the presence of chondroitinase ABC (ChABC), which digests chondroitin sulfate proteoglycan (CSPG), an essential component of gliosis. In contrast, in earlier transplantations (4 weeks of age), graft cells survived better in the presence of valproic acid (VPA), a neural differentiating reagent, or glatiramer acetate, an immune modulator. These suggest that, immediately after the outer nuclear layer (ONL) degeneration, an inflammatory reaction may be easily induced but the host neurons may be more able to accept donor cells in the presence of neural differentiating factor. These results will help optimize transplantation conditions when we consider clinical application.

Detection of neuronal activities of transplanted retinal cells in retinal degeneration models

We propose a discrete cable model to describe the conduction of retinal graded-voltage photoresponses, which propagate along a two-dimensional syncytium of transient amacrine cells of carp retina, coupled via gap junctions between adjacent cells. The model was simplified by transforming the syncytium into a one-dimensional cable, where a slit of light was used as stimulus. The cable equation was solved analytically and the conduction velocity v for the response peak travelling along the syncytium was described by (2D/Cm)(gjgm), where D is the cell spacing, Cm the membrane capacitance, gj the intercellular gap junctional conductance and gm the non-gap junctional membrane conductance of the cell at the light response peak. By using a pair of experimentally obtained parameters, the conduction velocity and a space constant (L) expressed by D(gj/gm) for the exponential spread of the receptive field, we could experimentally determine the absolute values of the electric conductances gj and gm, written as (Cm/2D)vL and (Cm/2)v/L, respectively. The receptive field of OFF response was 28% larger than that of ON response, due to a 43% larger OFF-gj value, as estimated by the model. The model thus enabled us to determine the rapid increase in gj induced by the used light stimulus. We also found that an application of 30 M 8-bromo-cAMP slowed down the conduction velocities of responses with 50% and in a spatially asymmetrical way, which could be ascribed to a spatially asymmetric suppression of both gj and gm, which decreased with ca 50%. This finding might be related to spatially asymmetric suppression of amacrine cells, similar to starburst cells suggested for direction selective ganglion cells in mammalian retinae. The model also enabled us to estimate the number of opened connexons at gap junctions. Furthermore, the model provided an explanation of the displacement of amplitude centre as shown previously (Djupsund et al., 2003).