Cell Types of the Human Retina and Its Organoids at Single-Cell Resolution.

Human organoids recapitulating the cell-type diversity and function of their target organ are valuable for basic and translational research. We developed light-sensitive human retinal organoids with multiple nuclear and synaptic layers and functional synapses. We sequenced the RNA of 285,441 single cells from these organoids at seven developmental time points and from the periphery, fovea, pigment epithelium and choroid of light-responsive adult human retinas, and performed histochemistry. Cell types in organoids matured in vitro to a stable "developed" state at a rate similar to human retina development in vivo. Transcriptomes of organoid cell types converged toward the transcriptomes of adult peripheral retinal cell types. Expression of disease-associated genes was cell-type-specific in adult retina, and cell-type specificity was retained in organoids. We implicate unexpected cell types in diseases such as macular degeneration. This resource identifies cellular targets for studying disease mechanisms in organoids and for targeted repair in human retinas.

Modeling of in vivo acousto-optic two-photon imaging of the retina in the human eye.

Our aim is to establish a novel combined acousto-optical method for in vivo imaging of the human retina with the two-photon microscope. In this paper we present modeling results based on eye model samples constructed with parameters measured on patients. We used effectively the potential of the electronic compensation offered by the acousto-optic lenses to avoid the use of adaptive optical correction. Simulation predicted lateral resolution between 1.6 µm and 3 µm on the retina. This technology allows the visualization of single cells and promises real time measuring of neural activity in individual neurons, neural segments and cell assemblies with 30-100 µs temporal and subcellular spatial resolution.

Artificial vision with wirelessly powered subretinal electronic implant alpha-IMS.

This study aims at substituting the essential functions of photoreceptors in patients who are blind owing to untreatable forms of hereditary retinal degenerations. A microelectronic neuroprosthetic device, powered via transdermal inductive transmission, carrying 1500 independent microphotodiode-amplifier-electrode elements on a 9 mm(2) chip, was subretinally implanted in nine blind patients. Light perception (8/9), light localization (7/9), motion detection (5/9, angular speed up to 35 deg s(-1)), grating acuity measurement (6/9, up to 3.3 cycles per degree) and visual acuity measurement with Landolt C-rings (2/9) up to Snellen visual acuity of 20/546 (corresponding to decimal 0.037° or corresponding to 1.43 logMAR (minimum angle of resolution)) were restored via the subretinal implant. Additionally, the identification, localization and discrimination of objects improved significantly (n = 8; p < 0.05 for each subtest) in repeated tests over a nine-month period. Three subjects were able to read letters spontaneously and one subject was able to read letters after training in an alternative-force choice test. Five subjects reported implant-mediated visual perceptions in daily life within a field of 15° of visual angle. Control tests were performed each time with the implant's power source switched off. These data show that subretinal implants can restore visual functions that are useful for daily life.

Subretinal electronic chips allow blind patients to read letters and combine them to words.

A light-sensitive, externally powered microchip was surgically implanted subretinally near the macular region of volunteers blind from hereditary retinal dystrophy. The implant contains an array of 1500 active microphotodiodes ('chip'), each with its own amplifier and local stimulation electrode. At the implant's tip, another array of 16 wire-connected electrodes allows light-independent direct stimulation and testing of the neuron-electrode interface. Visual scenes are projected naturally through the eye's lens onto the chip under the transparent retina. The chip generates a corresponding pattern of 38 × 40 pixels, each releasing light-intensity-dependent electric stimulation pulses. Subsequently, three previously blind persons could locate bright objects on a dark table, two of whom could discern grating patterns. One of these patients was able to correctly describe and name objects like a fork or knife on a table, geometric patterns, different kinds of fruit and discern shades of grey with only 15 per cent contrast. Without a training period, the regained visual functions enabled him to localize and approach persons in a room freely and to read large letters as complete words after several years of blindness. These results demonstrate for the first time that subretinal micro-electrode arrays with 1500 photodiodes can create detailed meaningful visual perception in previously blind individuals.

[Vision restoration with implants in retinal degenerations]. / Látásjavító implantátumok látóhártya-degenerációkban.

Up until now there has been no available treatment for diseases causing the permanent impairment of retinal photoreceptors. Currently the development of the retinal prostheses is the earliest to promise a result that can be implemented in the clinical treatment of these patients. Implants with different operating principles and in various stages of progress are presented in details, highlighting the characteristics, as well as the Hungarian aspects of the development. This survey intends to provide an overview on retinal prostheses, implantable in case of degenerative diseases of the retina, by reviewing and assessing the papers published in relevant journals and based on personal experience. Developments in microelectronics in recent years made it possible and proved to be feasible to replace the degenerated elements in the retina with electrical stimulation. Multiple comparable approaches are running simultaneously. Two types of these implants are directly stimulating the remaining living cells in the retina. Hitherto the finest resolution has been achieved with the subretinal implants. Although the epiretinal implant offer lower resolution, but requires shorter surgery for implantation. Retinal implants in certain retinal diseases are proved to be capable of generating vision-like experiences. A number of types of retinal implants can be expected to appear in clinical practice a few years after the successful conclusion of clinical trials.

The Predictive Role of Thyroid Hormone Levels for Early Diabetic Retinal Changes in Experimental Rat and Human Diabetes.

Purpose: In diabetic subjects, early visual functional alterations such as color vision deficiencies (CVDs) are known to precede clinically apparent diabetic retinopathy. Prominent photoreceptor outer segment degeneration and an increase in the number of retinal dual cones (co-expressing S- and M-opsins simultaneously) have been described in diabetic rat models, suggesting a connection with the development of CVDs. As cone opsin expression is controlled by thyroid hormones, we investigated the diabetic retina in association with thyroid hormone alterations. Methods: In rat models of type 1 and 2 diabetes, dual cones were labeled by immunohistochemistry, and their numbers were analyzed in relation to free triiodothyronine (fT3) and free thyroxine (fT4) levels. Quantification of dual cones was also performed in human postmortem retinas. Additionally, a cross-sectional case-control study was performed where thyroid hormone levels were measured and color vision was assessed with Lanthony desaturated D15 discs. Results: A higher number of dual cones was detectable in diabetic rats, correlating with fT4 levels. Dual cones were also present in postmortem human retinas, with higher numbers in the three diabetic retinas. As expected, age was strongly associated with CVDs in human patients, and the presence of diabetes also increased the risk. However, the current study failed to detect any effect of thyroid hormones on the development of CVDs. Conclusions: Our results point toward the involvement of thyroid homeostasis in the opsin expression changes in diabetic rats and human samples. The evaluation of the possible clinical consequences warrants further research.

Changes in microchip position after implantation of a subretinal vision prosthesis in humans.

PURPOSE: Retinal prosthetic devices have been developed to partially restore very low vision in legally blind patients with end-stage hereditary retinal dystrophies. Subretinal implants, unlike epiretinal implants, are not fixated by a tack. The aim of this study was to assess and analyse possible changes over time in the subretinal position of the RETINA IMPLANT Alpha IMS and Alpha AMS (ClinicalTrials.gov NCT01024803). METHODS: Imaging studies were performed on fundus photographs using GIMP (Version 2.8.14). Postoperative photographs of the implanted eye were scaled and aligned. Landmarks were chosen and distances between landmarks were measured to then calculate the displacement of the microchip using a transformation matrix for rotational and translational movements. Analyses were performed using MATLAB 8.6 (The MathWorks Inc., Natick, MA). RESULTS: Of the 27 datasets with the Alpha IMS device, 12 (44%) remained stable without displacement of the microchip relative to the optic disc and the major blood vessels, whereas in 15 (56%), displacement occurred. The mean ± SD displacement in those 15 eyes was 0.66 ± 0.35 mm (range, 0.24-1.67 mm). Of the eight datasets with the Alpha AMS device, 1 (13%) remained stable without displacement of the microchip relative to the optic disc and the major blood vessels, whereas in 7 (87%), displacement occurred. The mean ± SD displacement in those seven eyes was 0.66 ± 0.26 mm (range, 0.32-0.97 mm). Calculated from all eyes (including those in which no displacement occurred), the mean displacement was 0.36 mm in the IMS cohort, and 0.58 mm in the AMS cohort, however, the difference was not statistically significant (p = 0.17). CONCLUSIONS: We have shown that the position of the subretinal implant changes in the majority of the cases after implantation. While the overall mean displacement of the chip was not significantly different in either of the cohorts, the maximum displacement was smaller in the Alpha AMS cohort.

Targeting neuronal and glial cell types with synthetic promoter AAVs in mice, non-human primates and humans.

Targeting genes to specific neuronal or glial cell types is valuable for both understanding and repairing brain circuits. Adeno-associated viruses (AAVs) are frequently used for gene delivery, but targeting expression to specific cell types is an unsolved problem. We created a library of 230 AAVs, each with a different synthetic promoter designed using four independent strategies. We show that a number of these AAVs specifically target expression to neuronal and glial cell types in the mouse and non-human primate retina in vivo and in the human retina in vitro. We demonstrate applications for recording and stimulation, as well as the intersectional and combinatorial labeling of cell types. These resources and approaches allow economic, fast and efficient cell-type targeting in a variety of species, both for fundamental science and for gene therapy.

Optical Coherence Tomography in Patients With the Subretinal Implant Retina Implant Alpha IMS.

BACKGROUND AND OBJECTIVE: The aim of this study was to assess changes in retinal structure and thickness after subretinal implantation of the Retina Implant Alpha IMS (Retina Implant AG, Reutlingen, Germany). PATIENTS AND METHODS: Spectral-domain optical coherence tomography (SD-OCT) imaging was performed to assess the structure and thickness of the retina anterior to the microphotodiode array preoperatively, within 6 weeks and 6 months ± 1 month after implantation. Thickness measurements were performed using the distance tool of the built-in software. Three thickness measurements were performed in each of the four quadrants of the retina on the microchip within 6 weeks and 6 months ± 1 month after implantation. RESULTS: The mean ± standard deviation change in retinal thickness from within 6 weeks to 6 months ± 1 month after implantation in all four quadrants combined was 24 µm ± 68 µm. None of the tested variables (location, time, or their interaction) had a statistically significant effect on the mean retinal thickness (P = .961, P = .131, and P = .182, respectively; n = 19). CONCLUSION: The authors report on qualitative and quantitative findings in retinal structure in 27 patients after subretinal implantation of the Retina Implant Alpha IMS using OCT technology. No significant changes of retinal thickness could be observed in a period of 6 months after surgery. With more patients receiving subretinal implants and with advanced OCT technology, the data set will be extended to study possible changes in retinal structure in finer detail. [Ophthalmic Surg Lasers Imaging Retina. 2017;48:993-999.].

Congenital Nystagmus Gene FRMD7 Is Necessary for Establishing a Neuronal Circuit Asymmetry for Direction Selectivity.

Neuronal circuit asymmetries are important components of brain circuits, but the molecular pathways leading to their establishment remain unknown. Here we found that the mutation of FRMD7, a gene that is defective in human congenital nystagmus, leads to the selective loss of the horizontal optokinetic reflex in mice, as it does in humans. This is accompanied by the selective loss of horizontal direction selectivity in retinal ganglion cells and the transition from asymmetric to symmetric inhibitory input to horizontal direction-selective ganglion cells. In wild-type retinas, we found FRMD7 specifically expressed in starburst amacrine cells, the interneuron type that provides asymmetric inhibition to direction-selective retinal ganglion cells. This work identifies FRMD7 as a key regulator in establishing a neuronal circuit asymmetry, and it suggests the involvement of a specific inhibitory neuron type in the pathophysiology of a neurological disease.

Subretinal Visual Implant Alpha IMS--Clinical trial interim report.

A subretinal visual implant (Alpha IMS, Retina Implant AG, Reutlingen, Germany) was implanted in 29 blind participants with outer retinal degeneration in an international multicenter clinical trial. Primary efficacy endpoints of the study protocol were a significant improvement of activities of daily living and mobility to be assessed by activities of daily living tasks, recognition tasks, mobility, or a combination thereof. Secondary efficacy endpoints were a significant improvement of visual acuity/light perception and/or object recognition (clinicaltrials.gov, NCT01024803). During up to 12 months observation time twenty-one participants (72%) reached the primary endpoints, of which thirteen participants (45%) reported restoration of visual function which they use in daily life. Additionally, detection, localization, and identification of objects were significantly better with the implant power switched on in the first 3 months. Twenty-five participants (86%) reached the secondary endpoints. Measurable grating acuity was up to 3.3 cycles per degree, visual acuities using standardized Landolt C-rings were 20/2000, 20/2000, 20/606 and 20/546. Maximal correct motion perception ranged from 3 to 35 degrees per second. These results show that subretinal implants can restore very-low-vision or low vision in blind (light perception or less) patients with end-stage hereditary retinal degenerations.

Functional outcome in subretinal electronic implants depends on foveal eccentricity.

PURPOSE: An active microelectronic subretinal implant, developed to replace the photoreceptive function in hereditary degenerations of the outer retina, has been applied in a pilot and clinical study in patients with end-stage retinal degeneration. METHODS: The study population comprised 20 blind patients, all of whom lost vision as result of a hereditary retinal disease. An active visual implant was placed surgically within the subretinal space of each patient: subfoveal placement in eight patients (group 1) and parafoveal placement in 12 (group 2). Standardized low-vision tests, including light perception, light localization, movement detection, grating acuity, and visual acuity by Landolt C-rings, were used under masked, randomized implant-OFF and implant-ON conditions. For the chip-mediated vision functional results of both subject groups were compared. RESULTS: Three of 20 patients were excluded from analysis because of surgical or technical implant issues. Among patients with nonfoveal placement of the implant, 80% could perceive light, 10% recognized location, and 10% correctly distinguished stripe patterns up to a resolution of 0.33 cycles/degree. No nonfoveal placement patient passed the motion or Landolt C-ring tests. When the implant was placed subfoveally, 100% of patients could perceive light and determine light localization, 75% could resolve motion up to 35°/s, 88% correctly distinguished stripe patterns up to a resolution of 3.3 cycles/degree, and 38% passed a Landolt C-ring test with a decimal visual acuity of up to 20/546 (logMAR 1.43). CONCLUSIONS: Subfoveal placement of active subretinal visual implants allows superior measurable outcomes compared to para- or nonfoveal placement locations. (ClinicalTrials.gov numbers, NCT01024803, NCT00515814.).

Safety and efficacy of subretinal visual implants in humans: methodological aspects.

BACKGROUND: Replacing the function of visual pathway neurons by electronic implants is a novel approach presently explored by various groups in basic research and clinical trials. The novelty raises unexplored methodological aspects of clinical trial design that may require adaptation and validation. METHODS: We present procedures of efficacy and safety testing for subretinal visual implants in humans, as developed during our pilot trial 2005 to 2009 and multi-centre clinical trial since 2010. RESULTS: Planning such a trial requires appropriate inclusion and exclusion criteria. For subretinal electronic visual implants, patients with photoreceptor degeneration are the target patient group, whereas presence of additional diseases affecting clear optic media or the visual pathway must be excluded. Because sham surgery is not possible, a masked study design with implant power ON versus OFF is necessary. Prior to the efficacy testing by psychophysical tests, the implant's technical characteristics have to be controlled via electroretinography (ERG). Moreover the testing methods require adaptation to the particular technology. We recommend standardised tasks first to determine the light perception thresholds, light localisation and movement detection, followed by grating acuity and vision acuity test via Landolt C rings. A laboratory setup for assessing essential activities of daily living is presented. Subjective visual experiences with the implant in a natural environment, as well as questionnaires and psychological counselling are further important aspects. CONCLUSIONS: A clinical trial protocol for artificial vision in humans, which leads a patient from blindness to the state of very low vision is a challenge and cannot be defined completely prior to the study. Available tests of visual function may not be sufficiently suited for efficacy testing of artificial vision devices. A protocol based on experience with subretinal visual implants in 22 patients is presented that has been found adequate to monitor safety and efficacy.

Positioning of electronic subretinal implants in blind retinitis pigmentosa patients through multimodal assessment of retinal structures.

PURPOSE: To optimize methods for positioning subretinal visual implants, customizing their cable length, guiding them to the predetermined retinal position, and evaluating their performance. METHODS: Ten eyes of 10 patients (6 male, 4 female, mean age 46.4 years) were investigated before implantation of a subretinal visual implant. The structural characteristics of the retina as well as the ocular dimensions were determined. Topographic images of the prospective implantation site were subdivided into grids of squares. Each square received a weighted score for suitability. The sum of the scores was calculated, and the region with the highest score was chosen for the implant. In each case, the implant's power supply cable length was calculated by means of magnetic resonance imaging. The planned and achieved positions before and after implantation were compared. RESULTS: The mean light sensitivity ratio between the area actually covered by the chip and that of the planned position was 90.8% with an SD of 11.4%. In two cases with almost perfect positioning, the computed ratio was 100%. Measurements showed that to achieve a 95% sensitivity rate the difference between the planned and achieved chip position must be less than 1.7 mm. Preoperative calculations of the intraocular cable length proved accurate in all cases. CONCLUSIONS: Preoperative evaluation of retinal structures and eye morphology is useful for guiding a retinal implant to the designated area. It is a meaningful tool for planning and performing retinal chip implantation, and it optimizes personalized implantation. (ClinicalTrials.gov numbers, NCT00515814, NCT01024803.).

Spatial resolution and perception of patterns mediated by a subretinal 16-electrode array in patients blinded by hereditary retinal dystrophies.

PURPOSE: The perception of 11 persons blinded by hereditary retinal degeneration elicited by a subretinally implanted 16-electrode array used for light-independent direct stimulation of the retina is described. This device is part of the Tübingen retina implant, which also employs a light-sensitive, multiphotodiode array (MPDA). The ability to reliably recognize complex spatial percepts was investigated. METHODS: Eleven blind volunteers received implants and participated in standardized psychophysical tests investigating the size and shape of perceptions elicited by single-electrode activation, multiple-electrode activation, and activation of compound patterns such as simplified letters. RESULTS: Visual percepts were elicited reliably in 8 of 11 patients. On single-electrode activation, percepts were generally described as round spots of light of distinguishable localization in the visual field. On activation of a pattern of electrodes, percepts matched that pattern when electrodes were activated sequentially. Patterns such as horizontal or vertical bars were identified reliably; the most recent participant was able to recognize simplified letters presented on the 16-electrode array. The smallest distance between sites of concurrent retinal stimulation still yielding discernible spots of light was assessed to be 280 µm, corresponding to a logMAR of 1.78. CONCLUSIONS: Subretinal electric stimulation can yield reliable, predictable percepts. Patterned perception is feasible, enabling blind persons to recognize shapes and discriminate different letters. Stimulation paradigms must be optimized, to further increase spatial resolution, demanding a better understanding of physical and biological effects of single versus repetitive stimulation (ClinicalTrials.gov number, NCT00515814).

Restoration of useful vision up to letter recognition capabilities using subretinal microphotodiodes.

Our group has developed a subretinal microphotodiode array for restoration of vision. In a clinical pilot study the array has been implanted in 11 patients suffering from photoreceptor degenerations. Here we present promising results from some of those patients where the retinal tissue above the chip was functional and the implant fulfilled its expected function. A spatial resolution of approximately 0.3 cycles/degree could be achieved with fine stripe patterns. In one subject where the implant had been placed directly under the macular region of the retina a visual acuity of 20/1000 could be measured. Artificially restored visual acuity of this quality has not been reported previously. Finally, we present images illustrating an approximation of how the visual perceptions might have appeared to the subjects, based on a mathematical model and patient reports.