generation-retinal-organoids-from-healthy-retinal-disease-specific

Generation of Retinal Organoids from Healthy and Retinal Disease-Specific Human-Induced Pluripotent Stem Cells

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative ...

LV Prasad Eye Institute

To establish primary cultures of human nasal mucosal fibroblasts (HNMFs) and to test the effect of varying concentrations of mitomycin C (MMC) and treatment durations on cellular proliferation and viability of the fibroblasts.

Mitomycin C in dacryocystorhinostomy: the search for the right concentration and duration--a fundamental study on human nasal mucosa fibroblasts.

Stem cells at the limbus mediate corneal epithelial regeneration and regulate normal tissue homeostasis. Ex vivo cultured limbal epithelial transplantations are being widely practiced in the treatment of limbal stem cell deficiency. In this report, we examined whether the limbal niche cells that nurture and regulate epithelial stem cells coexist in ex vivo limbal cultures. We also compared the inherent differences between explant and suspension culture systems in terms of spatial distribution of niche cells and their effect on epithelial stem cell proliferation, migration, and differentiation in vitro. We report that the stem cell content of both culture systems was similar, explaining the comparable clinical outcomes reported using these two methods. We also showed that the niche cells get expanded in culture and the nestin-positive cells migrate at the leading edges to direct epithelial cell migration in suspension cultures, whereas they are limited to the intact niche in explant cultures. We provide evidence that C/EBPδ-positive, p15-positive, and quiescent, label-retaining, early activated stem cells migrate at the leading edges to regulate epithelial cell proliferation in explant cultures, and this position effect is lost in early suspension cultures. However, in confluent suspension cultures, the stem cells and niche cells interact with each another, migrate in spiraling patterns, and self-organize to form three-dimensional niche-like compartments resembling the limbal crypts and thereby reestablish the position effect. These 3D-sphere clusters are enriched with nestin-, vimentin-, S100-, and p27-positive niche cells and p15-, p21-, p63α-, C/EBPδ-, ABCG2-, and Pax6-positive quiescent epithelial stem cells.

Spatial distribution of niche and stem cells in ex vivo human limbal cultures.

Corneal epithelial stem cells residing within the annular limbal crypts regulate adult tissue homeostasis. Autologous limbal grafts and tissue-engineered corneal epithelial cell sheets have been widely used in the treatment of various ocular surface defects. In the case of bilateral limbal defects, pluripotent stem cell (PSC)-derived corneal epithelial cells are now being explored as an alternative to allogeneic limbal grafts. Here, we report an efficient method to generate complex three-dimensional corneal organoids from human PSCs. The eye field primordial clusters that emerged from differentiating PSCs developed into whole eyeball-like, self-organized, three-dimensional, miniature structures consisting of retinal primordia, corneal primordia, a primitive eyelid-like outer covering and ciliary margin zone-like adnexal tissues in a stepwise maturation process within 15 weeks. These minicorneal organoids recapitulate the early developmental events  and display similar anatomical features and marker expression profiles to adult corneal tissues. They offer an alternative tissue source for regenerating different layers of the cornea and eliminate the need for complicated cell enrichment procedures.

Generating minicorneal organoids from human induced pluripotent stem cells.

The sub-retinal injections are not very commonly performed procedures in vitreoretina, but form a crucial step in any cell replacement therapy for retinal diseases. The purpose of this study is to describe the learning curve of a trained vitreo-retinal surgeon in sub-retinal injections in a rat model and its implications in future clinical trials.

Learning curve of a trained vitreo-retinal surgeon in sub-retinal injections in a rat model: Implications for future clinical trials.

Over 2 million people worldwide are suffering from gene-related retinal diseases, inherited or acquired, and over 270 genes have been identified which are found to be responsible for these conditions. This review article touches upon the mechanisms of gene therapy, various enzymes of the visual cycle responsible for different genetic diseases, Luxturna-the first US Food and Drug Administration (FDA)-approved therapeutic gene product, and several ongoing trials of gene therapy for age-related macular degeneration. Gene therapy has tremendous potential for retinal conditions due to its ease of accessibility, immune-privileged status, and tight blood-retinal barriers, limiting systemic side effects of the drug. In recent years, advances in gene therapy in retinal conditions have increasing significantly, with progress in cell-specific targeting and transduction efficiency of gene products through the use of adeno-associated viral vectors (AAVs), suggesting that even greater success in future clinical trials is possible.

Gene therapy in retinal diseases: A review.

Stargardt retinopathy is an inherited form of macular degeneration caused by mutations in gene ABCA4 and characterized by the accumulation of lipid-rich deposits in the retinal pigment epithelium (RPE), RPE atrophy, and photoreceptor cell death. Inadequate mechanistic insights into pathophysiological changes occurring in Stargardt RPE have hindered disease treatments. Here, we show that ABCA4 knockout and induced pluripotent stem cell-derived RPE (STGD1-iRPE) from patients with Stargardt differentiate normally but display intracellular lipid and ceramide deposits reminiscent of the disease phenotype. STGD1-iRPE also shows defective photoreceptor outer segment (POS) processing and reduced cathepsin B activity-indicating higher lysosomal pH. Lipid deposits in STGD1-iRPE are lowered by increasing the activity of ABCA1, a lipid transporter, and ABCA4 ortholog. Our work suggests that ABCA4 is involved in POS and lipid handling in RPE cells and provides guidance for ongoing gene therapy approaches to target both RPE and photoreceptor cells for an effective treatment.

Article	Year
Mitomycin C in dacryocystorhinostomy: the search for the right concentration and duration--a fundamental study on human nasal mucosa fibroblasts. Ophthalmic plastic and reconstructive surgery\| PubMed ID: 24217477	2013
Spatial distribution of niche and stem cells in ex vivo human limbal cultures. Stem cells translational medicine\| PubMed ID: 25232182	2014
Generating minicorneal organoids from human induced pluripotent stem cells. Development (Cambridge, England)\| PubMed ID: 28559289	2017
Learning curve of a trained vitreo-retinal surgeon in sub-retinal injections in a rat model: Implications for future clinical trials. Indian journal of ophthalmology\| PubMed ID: 31436191	2019
Gene therapy in retinal diseases: A review. Indian journal of ophthalmology\| PubMed ID: 34427196	2021
Cell-autonomous lipid-handling defects in Stargardt iPSC-derived retinal pigment epithelium cells. Stem cell reports\| PubMed ID: 36306781	2022

Indumathi Mariappan

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