Mutations in pre-mRNA processing factors (PRPFs) cause autosomal-dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed genes cause non-syndromic retinal disease. Here, we generate transcriptome profiles from RP11 (PRPF31-mutated) patient-derived retinal organoids and retinal pigment epithelium (RPE), as well as Prpf31+/− mouse tissues, which revealed that disrupted alternative splicing occurred for specific splicing programmes. Mis-splicing of genes encoding pre-mRNA splicing proteins was limited to patient-specific retinal cells and Prpf31+/− mouse retinae and RPE. Mis-splicing of genes implicated in ciliogenesis and cellular adhesion was associated with severe RPE defects that include disrupted apical – basal polarity, reduced trans-epithelial resistance and phagocytic capacity, and decreased cilia length and incidence. Disrupted cilia morphology also occurred in patient-derived photoreceptors, associated with progressive degeneration and cellular stress. In situ gene editing of a pathogenic mutation rescued protein expression and key cellular phenotypes in RPE and photoreceptors, providing proof of concept for future therapeutic strategies.
The availability of in vitro models of the human retina in which to perform pharmacological and toxicological studies is an urgent and unmet need. An essential step for developing in vitro models of human retina is the ability to generate laminated, physiologically functional, and light-responsive retinal organoids from renewable and patient specific sources. We investigated five different human-induced pluripotent stem cell (iPSC) lines and showed a significant variability in their efficiency to generate retinal organoids. Despite this variability, by month 5 of differentiation, all iPSC-derived retinal organoids were able to generate light responses, albeit immature, comparable to the earliest light responses recorded from the neonatal mouse retina, close to the period of eye opening. All iPSC-derived retinal organoids exhibited at this time a well-formed outer nuclear like layer containing photoreceptors with inner segments, connecting cilium, and outer like segments. The differentiation process was highly dependent on seeding cell density and nutrient availability determined by factorial experimental design. We adopted the differentiation protocol to a multiwell plate format, which enhanced generation of retinal organoids with retinal-pigmented epithelium (RPE) and improved ganglion cell development and the response to physiological stimuli. We tested the response of iPSC-derived retinal organoids to Moxifloxacin and showed that similarly to in vivo adult mouse retina, the primary affected cell types were photoreceptors. Together our data indicate that light responsive retinal organoids derived from carefully selected and differentiation efficient iPSC lines can be generated at the scale needed for pharmacology and drug screening purposes. Stem Cells 2018;36:1535-1551.
Graphical Abstract Highlights d An automated spike sorting method for dense, large-scale recordings is presented d Efficient data representation enables sorting of thousands of channels d Automated unit selection through model-based quality control d Conventional spike sorting frequently fails under non-optimal signal conditions Correspondence m.hennig@ed.ac.uk In Brief Data volume and complexity make spike sorting for large-scale extracellular recordings computationally extremely challenging. Hilgen et al. introduce a method enabling analysis of recordings with thousands of channels and provide tools for automated quality control and unit selection. SUMMARY We present a method for automated spike sorting for recordings with high-density, large-scale multielec-trode arrays. Exploiting the dense sampling of single neurons by multiple electrodes, an efficient, low-dimensional representation of detected spikes consisting of estimated spatial spike locations and dominant spike shape features is exploited for fast and reliable clustering into single units. Millions of events can be sorted in minutes, and the method is parallel-ized and scales better than quadratically with the number of detected spikes. Performance is demonstrated using recordings with a 4,096-channel array and validated using anatomical imaging, optoge-netic stimulation, and model-based quality control. A comparison with semi-automated, shape-based spike sorting exposes significant limitations of conventional methods. Our approach demonstrates that it is feasible to reliably isolate the activity of up to thousands of neurons and that dense, multi-channel probes substantially aid reliable spike sorting.
Mouse horizontal cells are coupled by gap junctions composed of connexin57. These gap junctions are regulated by ambient light via multiple neuromodulators including dopamine. In order to analyze the distribution and structure of horizontal cell gap junctions in the mouse retina, and examine the effects of light adaptation on gap junction density, we developed antibodies that detect mouse retinal connexin57. Using immunohistochemistry in retinal slices, flat-mounted retinas, and dissociated retinal cells, we showed that connexin57 is expressed in the dendrites and axon terminal processes of mouse horizontal cells. No staining was found in retinas of connexin57-deficient mice. Significantly more connexin57-positive puncta were found in the distal than in the proximal outer plexiform layer, indicating a higher level of expression in axon terminal processes than in the dendrites. We also examined the gap junctions using immunoelectron microscopy and showed that connexin57 does not form hemichannels in the horizontal cell dendritic tips. Light adaptation resulted in a significant increase in the number of connexin57-immunoreactive plaques in the outer plexiform layer, consistent with previously reported effects of light adaptation on connexin57 expression in the mouse retina. This study shows for the first time the detailed location of connexin57 expression within mouse horizontal cells, and provides the first ultrastructural data on mouse horizontal cell gap junctions.
A major goal in the stem cell field is to generate tissues that can be utilized as a universal tool for in vitro models of development and disease, drug development, or as a resource for patients suffering from disease or injury. Great efforts are being made to differentiate human pluripotent stem cells in vitro toward retinal tissue, which is akin to native human retina in its cytoarchitecture and function, yet the numerous existing retinal induction protocols remain variable in their efficiency and do not routinely produce morphologically or functionally mature photoreceptors. Herein, we determine the impact that the method of embryoid body (EB) formation and maintenance as well as cell line background has on retinal organoid differentiation from human embryonic stem cells and human induced pluripotent stem cells. Our data indicate that cell line‐specific differences dominate the variables that underline the differentiation efficiency in the early stages of differentiation. In contrast, the EB generation method and maintenance conditions determine the later differentiation and maturation of retinal organoids. Of the latter, the mechanical method of EB generation under static conditions, accompanied by media supplementation with Y27632 for the first 48 hours of differentiation, results in the most consistent formation of laminated retinal neuroepithelium containing mature and electrophysiologically responsive photoreceptors. Collectively, our data provide substantive evidence for stage‐specific differences in the ability to give rise to laminated retinae, which is determined by cell line‐specific differences in the early stages of differentiation and EB generation/organoid maintenance methods at later stages.
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