Three-dimensional automated reporter quantification (3D-ARQ) technology enables quantitative screening in retinal organoids.

The advent of stem cell-derived retinal organoids has brought forth unprecedented opportunities for developmental and physiological studies, while presenting new therapeutic promise for retinal degenerative diseases. From a translational perspective, organoid systems provide exciting new prospects for drug discovery, offering the possibility to perform compound screening in a three-dimensional (3D) human tissue context that resembles the native histoarchitecture and to some extent recapitulates cellular interactions. However, inherent variability issues and a general lack of robust quantitative technologies for analyzing organoids on a large scale pose severe limitations for their use in translational applications. To address this need, we have developed a screening platform that enables accurate quantification of fluorescent reporters in complex human iPSC-derived retinal organoids. This platform incorporates a fluorescence microplate reader that allows xyz-dimensional detection and fine-tuned wavelength selection. We have established optimal parameters for fluorescent reporter signal detection, devised methods to compensate for organoid size variability, evaluated performance and sensitivity parameters, and validated this technology for functional applications.

Identifying Treatments for Taste and Smell Disorders: Gaps and Opportunities.

The chemical senses of taste and smell play a vital role in conveying information about ourselves and our environment. Tastes and smells can warn against danger and also contribute to the daily enjoyment of food, friends and family, and our surroundings. Over 12% of the US population is estimated to experience taste and smell (chemosensory) dysfunction. Yet, despite this high prevalence, long-term, effective treatments for these disorders have been largely elusive. Clinical successes in other sensory systems, including hearing and vision, have led to new hope for developments in the treatment of chemosensory disorders. To accelerate cures, we convened the "Identifying Treatments for Taste and Smell Disorders" conference, bringing together basic and translational sensory scientists, health care professionals, and patients to identify gaps in our current understanding of chemosensory dysfunction and next steps in a broad-based research strategy. Their suggestions for high-yield next steps were focused in 3 areas: increasing awareness and research capacity (e.g., patient advocacy), developing and enhancing clinical measures of taste and smell, and supporting new avenues of research into cellular and therapeutic approaches (e.g., developing human chemosensory cell lines, stem cells, and gene therapy approaches). These long-term strategies led to specific suggestions for immediate research priorities that focus on expanding our understanding of specific responses of chemosensory cells and developing valuable assays to identify and document cell development, regeneration, and function. Addressing these high-priority areas should accelerate the development of novel and effective treatments for taste and smell disorders.

Human iPSC-derived retinal organoids develop robust Alzheimer's disease neuropathology.

Alzheimer's disease (AD), characterized by memory loss and cognitive decline, affects nearly 50 million people worldwide. Amyloid beta (Aß) plaques and intracellular neurofibrillary tangles (NFTs) of phosphorylated Tau protein (pTau) are key histopathological features of the disease in the brain, and recent advances have also identified AD histopathology in the retina. Thus, the retina represents a central nervous system (CNS) tissue highly amenable to non-invasive diagnostic imaging that shows promise as a biomarker for early AD. Given the devastating effects of AD on patients, their families, and society, new treatment modalities that can significantly alter the disease course are urgently needed. In this study, we have developed and characterized a novel human retinal organoid (RO) model derived from induced pluripotent stem cells (iPSCs) from patients with familial AD due to mutations in the amyloid precursor protein gene (APP). Using immunofluorescence and histological staining, we evaluated the cellular composition and AD histopathological features of AD-ROs compared to control ROs from healthy individuals. We found that AD-ROs largely resemble their healthy control counterparts in cellular composition but display increased levels of Aß and pTau. We also present proof of principle of an assay to quantify amyloid levels in whole ROs. This in vitro model of the human AD retina constitutes a new tool for drug screening, biomarker discovery, and pathophysiological studies.

Ex vivo electroporation of retinal cells: a novel, high efficiency method for functional studies in primary retinal cultures.

Primary retinal cultures constitute valuable tools not only for basic research on retinal cell development and physiology, but also for the identification of factors or drugs that promote cell survival and differentiation. In order to take full advantage of the benefits of this system it is imperative to develop efficient and reliable techniques for the manipulation of gene expression. However, achieving appropriate transfection efficiencies in these cultures has remained challenging. The purpose of this work was to develop and optimize a technique that would allow the transfection of chick retinal cells with high efficiency and reproducibility for multiple applications. We developed an ex vivo electroporation method applied to dissociated retinal cell cultures that offers a significant improvement over other currently available transfection techniques, increasing efficiency by five-fold. In this method, eyes were enucleated, devoid of RPE, and electroporated with GFP-encoding plasmids using custom-made electrodes. Electroporated retinas were then dissociated into single cells and plated in low density conditions, to be analyzed after 4 days of incubation. Parameters such as voltage and number of electric pulses, as well as plasmid concentration and developmental stage of the animal were optimized for efficiency. The characteristics of the cultures were assessed by morphology and immunocytochemistry, and cell viability was determined by ethidium homodimer staining. Cell imaging and counting was performed using an automated high-throughput system. This procedure resulted in transfection efficiencies in the order of 22-25% of cultured cells, encompassing both photoreceptors and non-photoreceptor neurons, and without affecting normal cell survival and differentiation. Finally, the feasibility of the technique for cell-autonomous studies of gene function in a biologically relevant context was tested by carrying out gain and loss-of-function experiments for the transcription factor PAX6. Electroporation of a plasmid construct expressing PAX6 resulted in a marked upregulation in the expression levels of this protein that could be measured in the whole culture as well as cell-intrinsically. This was accompanied by a significant decrease in the percentage of cells differentiating as photoreceptors among the transfected population. Conversely, electroporation of an RNAi construct targeting PAX6 resulted in a significant decrease in the levels of this protein, with a concomitant increase in the proportion of photoreceptors. Taken together these results provide strong proof-of-principle of the suitability of this technique for genetic studies in retinal cultures. The combination of the high transfection efficiency obtained by this method with automated high-throughput cell analysis supplies the scientific community with a powerful system for performing functional studies in a cell-autonomous manner.

Retinal organoid light responsivity: current status and future opportunities.

The ability to generate human retinas in vitro from pluripotent stem cells opened unprecedented opportunities for basic science and for the development of therapeutic approaches for retinal degenerative diseases. Retinal organoid models not only mimic the histoarchitecture and cellular composition of the native retina, but they can achieve a remarkable level of maturation that allows them to respond to light stimulation. However, studies evaluating the nature, magnitude, and properties of light-evoked responsivity from each cell type, in each retinal organoid layer, have been sparse. In this review we discuss the current understanding of retinal organoid function, the technologies used for functional assessment in human retinal organoids, and the challenges and opportunities that lie ahead.

BMP inhibition-driven regulation of six-3 underlies induction of newt lens regeneration.

Lens regeneration in adult newts is a classic example of how cells can faithfully regenerate a complete organ through the process of transdifferentiation. After lens removal, the pigment epithelial cells of the dorsal, but not the ventral, iris dedifferentiate and then differentiate to form a new lens. Understanding how this process is regulated might provide clues about why lens regeneration does not occur in higher vertebrates. The genes six-3 and pax-6 are known to induce ectopic lenses during embryogenesis. Here we tested these genes, as well as members of the bone morphogenetic protein (BMP) pathway that regulate establishment of the dorsal-ventral axis in embryos, for their ability to induce lens regeneration. We show that the lens can be regenerated from the ventral iris when the BMP pathway is inhibited and when the iris is transfected with six-3 and treated with retinoic acid. In intact irises, six-3 is expressed at higher levels in the ventral than in the dorsal iris. During regeneration, however, only expression in the dorsal iris is significantly increased. Such an increase is seen in ventral irises only when they are induced to transdifferentiate by six-3 and retinoic acid or by BMP inhibitors. These data suggest that lens regeneration can be achieved in noncompetent adult tissues and that this regeneration occurs through a gene regulatory mechanism that is more complex than the dorsal expression of lens regeneration-specific genes.

Organoids for the Study of Retinal Development and Developmental Abnormalities.

The cumulative knowledge of retina development has been instrumental in the generation of retinal organoid systems from pluripotent stem cells; and these three-dimensional organoid models, in turn, have provided unprecedented opportunities for retinal research and translational applications, including the ability to model disease in a human setting and to apply these models to the development and validation of therapeutic drugs. In this review article, we examine how retinal organoids can also contribute to our understanding of retinal developmental mechanisms, how this knowledge can be applied to modeling developmental abnormalities, and highlight some of the avenues that remain to be explored.

New Drug Discovery Paradigms for Retinal Diseases: A Focus on Retinal Organoids.

Retinal disease represents a growing global problem, both in terms of quality of life and economic impact, yet new therapies are not being developed at a sufficient rate to meet this mounting need. In this context, retinal organoids derived from human induced pluripotent stem cells hold significant promise for improving upon the current drug development process, increasing the speed and efficiency of moving potential therapeutic agents from bench to bedside. These organoid systems display the cell-cell and cell-matrix interactions, cellular heterogeneity, and physiological responses reflective of human biology and, thus, have the ability to replicate retinal disease pathology in a way that 2-dimensional cell cultures and animal models have been heretofore unable to achieve. However, organoid technology is not yet mature enough to meet the high-throughput demands of the first stages of drug screening. Hence, the augmentation of the existing drug development pipeline with retinal organoids, rather than the replacement of existing pathway components, may provide a way to harness the benefits of this improved pathological modeling. In this study, we outline the possible benefits of such a symbiosis, discuss other potential uses, and highlight barriers that remain to be overcome.

Retinal regeneration in the Xenopus laevis tadpole: a new model system.

PURPOSE: Retinal regeneration research holds potential for providing new avenues for the treatment of degenerative diseases of the retina. Various animal models have been used to study retinal regeneration over the years, providing insights into different aspects of this process. However the mechanisms that drive this important phenomenon remain to be fully elucidated. In the present study, we introduce and characterize a new model system for retinal regeneration research that uses the tadpole of the African clawed frog, Xenopus laevis. METHODS: The neural retina was surgically removed from Xenopus laevis tadpoles at stages 51-54, and a heparin-coated bead soaked in fibroblast growth factor 2 (FGF-2) was introduced in the eyes to induce regeneration. Histological and immunohistochemical analyses as well as DiI tracing were performed to characterize the regenerate. A similar surgical approach but with concomitant removal of the anterior portion of the eye was used to assess the capacity of the retinal pigmented epithelium (RPE) to regenerate a retina. Immunohistochemistry for FGF receptors 1 and 2 and phosphorylated extracellular signal-regulated protein kinase (pERK) was performed to start elucidating the intracellular mechanisms involved in this process. The role of the mitogen activated protein kinase (MAPK) pathway was confirmed through a pharmacological approach using the MAPK kinase (MEK) inhibitor U0126. RESULTS: We observed that Xenopus laevis tadpoles were able to regenerate a neural retina upon induction with FGF-2 in vivo. The regenerated tissue has the characteristics of a differentiated retina, as assessed by the presence and distribution of different retinal cell markers, and DiI tracing indicated that it is able to form an optic nerve. We also showed that retinal regeneration in this system could take place independently of the presence of the anterior eye tissues. Finally, we demonstrated that FGF-2 treatment induces ERK phosphorylation in the pigmented epithelia 10 days after retinectomy, and that inhibition of the MAPK pathway significantly decreases the amount of retina regenerated at 30 days post-operation. CONCLUSIONS: Regeneration of a complete neural retina can be achieved in larval Xenopus laevis through activation of the MAPK signaling pathway by administering exogenous FGF-2. This mechanism is conserved in other animal models, which can regenerate their retina via pigmented epithelium transdifferentiation. Our results provide an alternative approach to retinal regeneration studies, capitalizing on the advantages of the Xenopus laevis tadpole as a model system.

The alpha1 isoform of the Na+/K+ ATPase is up-regulated in dedifferentiated progenitor cells that mediate lens and retina regeneration in adult newts.

Adult newts are able to regenerate their retina and lens after injury or complete removal through transdifferentiation of the pigmented epithelial tissues of the eye. This process needs to be tightly controlled, and several different mechanisms are likely to be recruited for this function. The Na(+)/K(+) ATPase is a transmembrane protein that establishes electrochemical gradients through the transport of Na(+) and K(+) and has been implicated in the modulation of key cellular processes such as cell division, migration and adhesion. Even though it is expressed in all cells, its isoform composition varies with cell type and is tightly controlled during development and regeneration. In the present study we characterize the expression pattern of Na(+)/K(+) ATPase alpha1 in the adult newt eye and during the process of lens and retina regeneration. We show that this isoform is up-regulated in undifferentiated cells during transdifferentiation. Such change in composition could be one of the mechanisms that newt cells utilize to modulate this process.

Lens regeneration: a historical perspective.

The idea of regenerating injured body parts has captivated human imagination for centuries, and the topic still remains an area of extensive scientific research. This review focuses on the process of lens regeneration: its history, our current knowledge, and the questions that remain unanswered. By highlighting some of the milestones that have shaped our understanding of this phenomenon and the contributions of scientists who have dedicated their lives to investigating these questions, we explore how regeneration enquiry evolved into the science it is today, and how technological advances accelerated our understanding of these remarkable processes.

Stem Cell Sources and Their Potential for the Treatment of Retinal Degenerations.

Stem cells offer unprecedented opportunities for the development of strategies geared toward the treatment of retinal degenerative diseases. A variety of cellular sources have been investigated for various potential clinical applications, including tissue regeneration, disease modeling, and screening for non-cell-based therapeutic agents. As the field transitions from more than a decade of preclinical research to the first phase I/II clinical trials, we provide a concise overview of the stem cell sources most commonly used, weighing their therapeutic potential on the basis of their technical strengths/limitations, their ethical implications, and the extent of the progress achieved to date. This article serves as a framework for further in-depth analyses presented in the following chapters of this Special Issue.

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach.

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols(1). In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

Generation of three-dimensional retinal tissue with functional photoreceptors from human iPSCs.

Many forms of blindness result from the dysfunction or loss of retinal photoreceptors. Induced pluripotent stem cells (iPSCs) hold great potential for the modelling of these diseases or as potential therapeutic agents. However, to fulfill this promise, a remaining challenge is to induce human iPSC to recreate in vitro key structural and functional features of the native retina, in particular the presence of photoreceptors with outer-segment discs and light sensitivity. Here we report that hiPSC can, in a highly autonomous manner, recapitulate spatiotemporally each of the main steps of retinal development observed in vivo and form three-dimensional retinal cups that contain all major retinal cell types arranged in their proper layers. Moreover, the photoreceptors in our hiPSC-derived retinal tissue achieve advanced maturation, showing the beginning of outer-segment disc formation and photosensitivity. This success brings us one step closer to the anticipated use of hiPSC for disease modelling and open possibilities for future therapies.

Rediscovering the chick embryo as a model to study retinal development.

The embryonic chick occupies a privileged place among animal models used in developmental studies. Its rapid development and accessibility for visualization and experimental manipulation are just some of the characteristics that have made it a vertebrate model of choice for more than two millennia. Until a few years ago, the inability to perform genetic manipulations constituted a major drawback of this system. However, the completion of the chicken genome project and the development of techniques to manipulate gene expression have allowed this classic animal model to enter the molecular age. Such techniques, combined with the embryological manipulations that this system is well known for, provide a unique toolkit to study the genetic basis of neural development. A major advantage of these approaches is that they permit targeted gene misexpression with extremely high spatiotemporal resolution and over a large range of developmental stages, allowing functional analysis at a level, speed and ease that is difficult to achieve in other systems. This article provides a general overview of the chick as a developmental model focusing more specifically on its application to the study of eye development. Special emphasis is given to the state of the art of the techniques that have made gene gain- and loss-of-function studies in this model a reality. In addition, we discuss some methodological considerations derived from our own experience that we believe will be beneficial to researchers working with this system.

CNS regeneration: a morphogen's tale.

Tissue regeneration will soon become an avenue for repair of damaged or diseased tissues as stem cell niches have been found in almost every organ of the vertebrate body including the CNS. In addition, different animals display an array of regenerative capabilities that are currently being researched to dissect the molecular mechanisms involved. This review concentrates on the different ways in which CNS tissues such as brain, spinal cord and retina can regenerate or display neurogenic potential and how these abilities are modulated by morphogens.

A novel role of the hedgehog pathway in lens regeneration.

Lens regeneration in the adult newt is a classic example of replacing a lost organ by the process of transdifferentiation. After lens removal, the pigmented epithelial cells of the dorsal iris proliferate and dedifferentiate to form a lens vesicle, which subsequently differentiates to form a new lens. In searching for factors that control this remarkable process, we investigated the expression and role of hedgehog pathway members. These molecules are known to affect retina and pigment epithelium morphogenesis and have been recently shown to be involved in repair processes. Here we show that Shh, Ihh, ptc-1, and ptc-2 are expressed during lens regeneration. The expression of Shh and Ihh is quite unique since these genes have never been detected in lens. Interestingly, both Shh and Ihh are only expressed in the regenerating and developing lens, but not in the intact lens. Interfering with the hedgehog pathway results in considerable inhibition of the process of lens regeneration, including decreased cell proliferation as well as interference with lens fiber differentiation in the regenerating lens vesicle. Down-regulation of ptc-1 was also observed when inhibiting the pathway. These results provide the first evidence of a novel role for the hedgehog pathway in specific regulation of the regenerating lens.