Stem cell sources and characterization in the development of cell-based products for treating retinal disease: An NEI Town Hall report.

National Eye Institute recently issued a new Strategic Plan outlining priority research areas for the next 5 years. Starting cell source for deriving stem cell lines is as an area with gaps and opportunities for making progress in regenerative medicine, a key area of emphasis within the NEI Strategic Plan. There is a critical need to understand how starting cell source affects the cell therapy product and what specific manufacturing capabilities and quality control standards are required for autologous vs allogeneic stem cell sources.

Transcriptional comparison of adult human primary Retinal Pigment Epithelium, human pluripotent stem cell-derived Retinal Pigment Epithelium, and ARPE19 cells.

The therapeutic potential of pluripotent stem cells is great as they promise to usher in a new era of medicine where cells or organs may be prescribed to replace dysfunctional tissue. At the forefront are efforts in the eye to develop this technology as it lends itself to monitoring and sophisticated non-invasive imaging modalities. In the retina, retinal pigment epithelium (RPE) is the most promising replacement cell as it has a single layer, is relatively simple to transplant, and is associated with several eye diseases.

Protocols for SARS-CoV-2 infection in primary ocular cells and eye organoids.

Here, we describe a series of protocols detailing the steps for evaluating SARS-CoV-2 infection in models of the human eye. Included are protocols for whole eye organoid differentiation, SARS-CoV-2 infection, and processing organoids for single-cell RNA sequencing. Additional protocols describe how to dissect and culture adult human ocular cells from cadaver donor eyes and how to compare infection of SARS-CoV-2 and the presence of SARS-CoV-2 entry factors using qPCR, immunofluorescence, and plaque assays.

A bio-functional polymer that prevents retinal scarring through modulation of NRF2 signalling pathway.

One common cause of vision loss after retinal detachment surgery is the formation of proliferative and contractile fibrocellular membranes. This aberrant wound healing process is mediated by epithelial-mesenchymal transition (EMT) and hyper-proliferation of retinal pigment epithelial (RPE) cells. Current treatment relies primarily on surgical removal of these membranes.

SARS-CoV-2 infects human adult donor eyes and hESC-derived ocular epithelium.

The SARS-CoV-2 pandemic has caused unparalleled disruption of global behavior and significant loss of life. To minimize SARS-CoV-2 spread, understanding the mechanisms of infection from all possible routes of entry is essential. While aerosol transmission is thought to be the primary route of spread, viral particles have been detected in ocular fluid, suggesting that the eye may be a vulnerable point of viral entry.

Surgical Transplantation of Human RPE Stem Cell-Derived RPE Monolayers into Non-Human Primates with Immunosuppression.

Recent trials of retinal pigment epithelium (RPE) transplantation for the treatment of disorders such as age-related macular degeneration have been promising. However, limitations of existing strategies include the uncertain survival of RPE cells delivered by cell suspension and the inherent risk of uncontrolled cell proliferation in the vitreous cavity. Human RPE stem cell-derived RPE (hRPESC-RPE) transplantation can rescue vision in a rat model of retinal dystrophy and survive in the rabbit retina for at least 1 month.

SARS-CoV-2 Infection of Ocular Cells from Human Adult Donor Eyes and hESC-Derived Eye Organoids.

The outbreak of COVID-19 caused by the SARS-CoV-2 virus has created an unparalleled disruption of global behavior and a significant loss of human lives. To minimize SARS-CoV-2 spread, understanding the mechanisms of infection from all possible viral entry routes is essential. As aerosol transmission is thought to be the primary route of spread, we sought to investigate whether the eyes are potential entry portals for SARS-CoV-2.

Epigenomic and Transcriptomic Changes During Human RPE EMT in a Stem Cell Model of Epiretinal Membrane Pathogenesis and Prevention by Nicotinamide.

Epithelial to mesenchymal transition (EMT) is a biological process involved in tissue morphogenesis and disease that causes dramatic changes in cell morphology, migration, proliferation, and gene expression. The retinal pigment epithelium (RPE), which supports the neural retina, can undergo EMT, producing fibrous epiretinal membranes (ERMs) associated with vision-impairing clinical conditions, such as macular pucker and proliferative vitreoretinopathy (PVR). We found that co-treatment with TGF-β and TNF-α (TNT) accelerates EMT in adult human RPE stem cell-derived RPE cell cultures.

Immunological Molecular Responses of Human Retinal Pigment Epithelial Cells to Infection With .

Ocular toxoplasmosis is the commonest clinical manifestation of infection with obligate intracellular parasite, . Active ocular toxoplasmosis is characterized by replication of tachyzoites in the retina, with reactive inflammation. The multifunctional retinal pigment epithelium is a key target cell population for .

P38 inhibition reverses TGFβ1 and TNFα-induced contraction in a model of proliferative vitreoretinopathy.

Proliferative vitreoretinopathy (PVR) is a metaplasia in the vitreous of the eye manifested by the transformation of retinal pigment epithelial (RPE) cells and the development of contracting epiretinal membranes (ERM), which lead to retinal detachment and vision loss. While TGFβ1 and TNFα have been associated with PVR, here we show that these cytokines act synergistically to induce an aggressive contraction phenotype on adult human (ah)RPE. Connected RPE detach upon contraction and form motile membranes that recruit more cells.

Complex spike synchrony dependent modulation of rat deep cerebellar nuclear activity.

Unlabelled: The rules governing cerebellar output are not fully understood, but must involve Purkinje cell (PC) activity, as PCs are the major input to deep cerebellar nuclear (DCN) cells (which form the majority of cerebellar output). Here, the influence of PC complex spikes (CSs) was investigated by simultaneously recording DCN activity with CSs from PC arrays in anesthetized rats. Crosscorrelograms were used to identify PCs that were presynaptic to recorded DCN cells (presynaptic PCs).

Aberrant early endosome biogenesis mediates complement activation in the retinal pigment epithelium in models of macular degeneration.

Abnormally enlarged early endosomes (EEs) are pathological features of neurodegenerative diseases, yet insight into the mechanisms and consequences of EE expansion remains elusive. Here, we report swollen apical EEs in the retinal pigment epithelium (RPE) of aged human donors and in the pigmented mouse model of Stargardt early-onset macular degeneration. Using high-resolution live-cell imaging, we show that age-related and pathological accumulation of lipofuscin bisretinoids increases ceramide at the apical surface of the RPE, which promotes inward budding and homotypic fusion of EEs.

Stem Cell-Derived Retinal Pigment Epithelial Layer Model from Adult Human Globes Donated for Corneal Transplants.

An adult human retinal pigment epithelial layer (ahRPE) model derived from stem cells isolated from native RPE monolayers (ahRPE-SCs) exhibits key physiological characteristics of native tissue and therefore provides the means to create a human "disease in a dish" model to study RPE diseases. Traditionally, RPE lines are established from whole globes dedicated to research. Here we describe a new technique for establishing primary RPE lines from the posterior poles of globes used for corneal transplants.

The Developmental Stage of Adult Human Stem Cell-Derived Retinal Pigment Epithelium Cells Influences Transplant Efficacy for Vision Rescue.

Age-related macular degeneration (AMD) is a common cause of central visual loss in the elderly. Retinal pigment epithelial (RPE) cell loss occurs early in the course of AMD and RPE cell transplantation holds promise to slow disease progression. We report that subretinal transplantation of RPE stem cell (RPESC)-derived RPE cells (RPESC-RPE) preserved vision in a rat model of RPE cell dysfunction.

Nicotinamide Ameliorates Disease Phenotypes in a Human iPSC Model of Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) affects the retinal pigment epithelium (RPE), a cell monolayer essential for photoreceptor survival, and is the leading cause of vision loss in the elderly. There are no disease-altering therapies for dry AMD, which is characterized by accumulation of subretinal drusen deposits and complement-driven inflammation. We report the derivation of human-induced pluripotent stem cells (hiPSCs) from patients with diagnosed AMD, including two donors with the rare ARMS2/HTRA1 homozygous genotype.

Automated Measurement of Cobblestone Morphology for Characterizing Stem Cell Derived Retinal Pigment Epithelial Cell Cultures.

Purpose: Assessing the morphologic properties of cells in microscopy images is an important task to evaluate cell health, identity, and purity. Typically, subjective visual assessments are accomplished by an experienced researcher. This subjective human step makes transfer of the evaluation process from the laboratory to the cell manufacturing facility difficult and time consuming.

Human RPE Stem Cell-Derived RPE Preserves Photoreceptors in the Royal College of Surgeons Rat: Method for Quantifying the Area of Photoreceptor Sparing.

Purpose: Numerous preclinical studies have shown that transplantation of stem cell-derived retinal pigment epithelial cell (RPE) preserves photoreceptor cell anatomy in the dystrophic Royal College of Surgeons (RCS) rat. How rescue is spatially distributed over the eye, relative to the transplantation site, is less clear. To understand spatial variations in transplant efficacy, we have developed a method to measure the spatial distribution of rescued photoreceptor cells.

Synchrony is Key: Complex Spike Inhibition of the Deep Cerebellar Nuclei.

The control of deep cerebellar nuclear (DCN) neuronal firing is central to cerebellar function but is not well understood. The large majority of synapses onto DCN neurons derive from Purkinje cells (PCs), suggesting that PC activity is an important determinant of DCN firing; however, PCs fire both simple and complex spikes (CSs), and little is known about how the latter's action affects DCN activity. Thus, here, we explored the effects of CSs on DCN activity.

Human Adult Retinal Pigment Epithelial Stem Cell-Derived RPE Monolayers Exhibit Key Physiological Characteristics of Native Tissue.

Purpose: We tested what native features have been preserved with a new culture protocol for adult human RPE.

Methods: We cultured RPE from adult human eyes. Standard protocols for immunohistochemistry, electron microscopy, electrophysiology, fluid transport, and ELISA were used.

Efficiency of Membrane Protein Expression Following Infection with Recombinant Adenovirus of Polarized Non-Transformed Human Retinal Pigment Epithelial Cells.

Transient expression of exogenous proteins facilitates studies of molecular mechanisms and utility for transplantation of retinal pigment epithelial (RPE) cells in culture. Here, we compared expression of the membrane protein β5 integrin-GFP (β5-GFP) in two recently established models of differentiated human RPE, adult RPE stem cell-derived RPE and primary fetal RPE, upon infection with recombinant adenovirus or transfection with DNA in liposomes. We varied viral titer and duration of virus incubation and examined β5-GFP and the tight junction marker ZO-1 in manipulated cells by confocal microscopy.

Modulation of Purkinje cell complex spike waveform by synchrony levels in the olivocerebellar system.

Purkinje cells (PCs) generate complex spikes (CSs) when activated by the olivocerebellar system. Unlike most spikes, the CS waveform is highly variable, with the number, amplitude, and timing of the spikelets that comprise it varying with each occurrence. This variability suggests that CS waveform could be an important control parameter of olivocerebellar activity.

Human RPE stem cells grown into polarized RPE monolayers on a polyester matrix are maintained after grafting into rabbit subretinal space.

Transplantation of the retinal pigment epithelium (RPE) is being developed as a cell-replacement therapy for age-related macular degeneration. Human embryonic stem cell (hESC) and induced pluripotent stem cell (iPSC)-derived RPE are currently translating toward clinic. We introduce the adult human RPE stem cell (hRPESC) as an alternative RPE source.

Chronic oxidative stress upregulates Drusen-related protein expression in adult human RPE stem cell-derived RPE cells: a novel culture model for dry AMD.

Purpose: The goal of this study was to examine changes in the expression of transcripts and proteins associated with drusen in Age-related Macular Degeneration (AMD) after exposing human retinal pigment epithelium (hRPE) cells to chronic oxidative stress.

Methods: Primary adult human RPE cells were isolated from cadaveric donor eyes. The subpopulation of RPE stem cells (RPESCs) was activated, expanded, and then differentiated into RPE progeny.

Ophthalmologic stem cell transplantation therapies.

Vision loss is a major social issue, with more than 20 million people over the age of 18 years affected in the USA alone. Loss of vision is feared more than premature death or cardiovascular disease, according to a recent Society for Consumer Research group survey. The annual direct cost of medical care for the most prevalent eye disease, age-related macular degeneration, was estimated at US$255 billion in 2010 with an additional economic impact of US$88 billion due to lost productivity and the burden of family and community care for visual disability.