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Shelton DA, Papania JT, Getz TE, Sellers JT, Giradot PE, Chrenek MA, Grossniklaus HE, Boatright JH, Nickerson JM. Loss of Pigment Epithelium Derived Factor Sensitizes C57BL/6J Mice to Light-Induced Retinal Damage. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.12.04.626802. [PMID: 39679905 PMCID: PMC11643041 DOI: 10.1101/2024.12.04.626802] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 12/17/2024]
Abstract
Purpose Pigment epithelium-derived factor (PEDF) is a neurotrophic glycoprotein secreted by the retinal pigment epithelium (RPE) that supports retinal photoreceptor health. Deficits in PEDF are associated with increased inflammation and retinal degeneration in aging and diabetic retinopathy. We hypothesized that light-induced stress in C57BL/6J mice deficient in PEDF would lead to increased retinal neuronal and RPE defects, impaired expression of neurotrophic factor Insulin-like growth factor 1 (IGF-1), and overactivation of Galectin-3-mediated inflammatory signaling. Methods C57BL/6J mice expressing the RPE65 M450/M450 allele were crossed to PEDF KO/KO and wildtype (PEDF +/+) littermates. Mice were exposed to 50,000 lux light for 5 hours to initiate acute damage. Changes in visual function outcomes were tracked via electroretinogram (ERG), confocal scanning laser ophthalmoscopy(cSLO), and spectral domain optical coherence tomography (SD-OCT) on days 3, 5, and 7 post-light exposure. Gene and protein expression of Galectin-3 were measured by digital drop PCR (ddPCR) and western blot. To further investigate the role of galectin-3 on visual outcomes and PEDF expression after damage, we also used a small-molecule inhibitor to reduce its activity. Results Following light damage, PEDF KO/KO mice showed more severe retinal thinning, impaired visual function (reduced a-, b-, and c-wave amplitudes), and increased Galectin-3 expressing immune cell infiltration compared to PEDF +/+. PEDF KO/KO mice had suppressed damage-associated increases in IGF-1 expression. Additionally, baseline Galectin-3 mRNA and protein expression were reduced in PEDF KO/KO mice compared to PEDF +/+. However, after light damage, Galectin-3 expression decreases in PEDF +/+ mice but increases in PEDF KO/KO mice without reaching PEDF +/+ levels. Galectin-3 inhibition worsens retinal degeneration, reduces PEDF expression in PEDF +/+ mice, and mimics the effects seen in PEDF knockouts. Conclusions Loss of PEDF alone does not elicit functional defects in C57BL/6J mice. However, under light stress, PEDF deficiency significantly increases severe retinal degeneration, visual deficits, Galectin-3 expression, and suppression of IGF-1 than PEDF +/+. PEDF deficiency reduced baseline expression of Galectin-3, and pharmacological inhibition of Galectin-3 worsens outcomes and suppresses PEDF expression in PEDF +/+, suggesting a novel co-regulatory relationship between the two proteins in mitigating light-induced retinal damage.
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Affiliation(s)
- Debresha A. Shelton
- Department of Ophthalmology, Emory University, Atlanta, Georgia, United States
| | - Jack T. Papania
- Department of Ophthalmology, Emory University, Atlanta, Georgia, United States
| | - Tatiana E. Getz
- Department of Ophthalmology, Emory University, Atlanta, Georgia, United States
| | - Jana T. Sellers
- Department of Ophthalmology, Emory University, Atlanta, Georgia, United States
| | - Preston E. Giradot
- Department of Ophthalmology, Emory University, Atlanta, Georgia, United States
| | - Micah A. Chrenek
- Department of Ophthalmology, Emory University, Atlanta, Georgia, United States
| | | | - Jeffrey H. Boatright
- Department of Ophthalmology, Emory University, Atlanta, Georgia, United States
- Atlanta Veterans Administration Center for Visual and Neurocognitive Rehabilitation, Decatur, Georgia, United States
| | - John M. Nickerson
- Department of Ophthalmology, Emory University, Atlanta, Georgia, United States
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Shelton DA, Gefke I, Summers V, Kim YK, Yu H, Getz Y, Ferdous S, Donaldson K, Liao K, Papania JT, Chrenek MA, Boatright JH, Nickerson JM. Age-Related RPE changes in Wildtype C57BL/6J Mice between 2 and 32 Months. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.01.30.574142. [PMID: 38352604 PMCID: PMC10862734 DOI: 10.1101/2024.01.30.574142] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 02/22/2024]
Abstract
Purpose This study provides a systematic evaluation of age-related changes in RPE cell structure and function using a morphometric approach. We aim to better capture nuanced predictive changes in cell heterogeneity that reflect loss of RPE integrity during normal aging. Using C57BL6/J mice ranging from P60-P730, we sought to evaluate how regional changes in RPE shape reflect incremental losses in RPE cell function with advancing age. We hypothesize that tracking global morphological changes in RPE is predictive of functional defects over time. Methods We tested three groups of C57BL/6J mice (young: P60-180; Middle-aged: P365-729; aged: 730+) for function and structural defects using electroretinograms, immunofluorescence, and phagocytosis assays. Results The largest changes in RPE morphology were evident between the young and aged groups, while the middle-aged group exhibited smaller but notable region-specific differences. We observed a 1.9-fold increase in cytoplasmic alpha-catenin expression specifically in the central-medial region of the eye between the young and aged group. There was an 8-fold increase in subretinal, IBA-1-positive immune cell recruitment and a significant decrease in visual function in aged mice compared to young mice. Functional defects in the RPE corroborated by changes in RPE phagocytotic capacity. Conclusions The marked increase of cytoplasmic alpha-catenin expression and subretinal immune cell deposition, and decreased visual output coincide with regional changes in RPE cell morphometrics when stratified by age. These cumulative changes in the RPE morphology showed predictive regional patterns of stress associated with loss of RPE integrity.
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Affiliation(s)
- Debresha A. Shelton
- Department of Ophthalmology, Emory University, Atlanta, Georgia, United States
| | - Isabelle Gefke
- Department of Ophthalmology, Emory University, Atlanta, Georgia, United States
| | - Vivian Summers
- Department of Ophthalmology, Emory University, Atlanta, Georgia, United States
| | - Yong-Kyu Kim
- Department of Ophthalmology, Emory University, Atlanta, Georgia, United States
- Department of Ophthalmology, Hallym University College of Medicine, Kangdong Sacred Heart Hospital, Seoul, South Korea
| | - Hanyi Yu
- Department of Ophthalmology, Emory University, Atlanta, Georgia, United States
- Department of Computer Science, Emory University, Atlanta, Georgia, United States
| | - Yana Getz
- Department of Ophthalmology, Emory University, Atlanta, Georgia, United States
| | - Salma Ferdous
- Department of Ophthalmology, Emory University, Atlanta, Georgia, United States
- Human Genome Sequencing Center, Baylor College of Medicine, Houston, Texas, United States
| | - Kevin Donaldson
- Department of Ophthalmology, Emory University, Atlanta, Georgia, United States
| | - Kristie Liao
- Department of Ophthalmology, Emory University, Atlanta, Georgia, United States
| | - Jack T. Papania
- Department of Ophthalmology, Emory University, Atlanta, Georgia, United States
| | - Micah A. Chrenek
- Department of Ophthalmology, Emory University, Atlanta, Georgia, United States
| | - Jeffrey H. Boatright
- Department of Ophthalmology, Emory University, Atlanta, Georgia, United States
- Atlanta VA Center for Visual and Neurocognitive Rehabilitation, Decatur, Georgia, United States
| | - John M. Nickerson
- Department of Ophthalmology, Emory University, Atlanta, Georgia, United States
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Cao M, Xu T, Song Y, Wang H, Wei S, Yin D. 2,2',4,4'-tetrabromodiphenyl ether causes depigmentation in zebrafish larvae via a light-mediated pathway. THE SCIENCE OF THE TOTAL ENVIRONMENT 2023; 897:165382. [PMID: 37422226 DOI: 10.1016/j.scitotenv.2023.165382] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/26/2023] [Revised: 07/04/2023] [Accepted: 07/05/2023] [Indexed: 07/10/2023]
Abstract
Polybrominated diphenyl ethers (PBDEs) are organic pollutants widely detected in various environmental media due to their high persistence and bioaccumulation. PBDE-induced visual impairment and neurotoxicity were previously demonstrated using zebrafish (Danio rerio) models, and recent research reported the phenotypic depigmentation effect of PBDEs at high concentrations on zebrafish, but whether those effects are still present at environment-relevant levels is still unclear. Herein, we performed both phenotypic examination and mechanism investigation in zebrafish embryos (48 hpf) and larvae (5 dpf) about their pigmentation status when exposing to PBDE congener BDE-47 (2,2',4,4'-tetrabrominated diphenyl ether) at levels from 0.25 to 25 μg/L. Results showed that low-level BDE-47 can restrain the relative melanin abundance of zebrafish larvae to 70.47% (p < 0.05) and 61.54% (p < 0.01) respectively under 2.5 and 25 μg/L BDE-47 compared with control, and the thickness of retinal pigment epithelium (RPE) remarkably reduced from 571.4 nm to 350.3 nm (p < 0.001) under 25 μg/L BDE-47 exposure. We also observed disrupted expressions of melanin synthesis genes and disorganized mitfa differentiation patterns based on Tg(mifta:EGFP), as well as visual impairment resulting from thinner RPE. Considering both processes of visual development and melanin synthesis are highly sensitive to ambient light conditions, we prolonged the light regime of maintaining zebrafish larvae from 14 hours light versus 10 hours dark (14L:10D) to 18 hours light versus 6 hours dark (18L:6D). Lengthening photoperiod successfully rescued the fluorescent level of mitfa in zebrafish epidermis and most gene expressions associated with melanin synthesis under 25 μg/L BDE-47 exposure to the normal level. In conclusion, our work reported the effects of low-level PBDEs on melanin production using zebrafish embryos and larvae, and identified the potential role of a light-mediated pathway in the neurotoxic mechanism of PBDEs.
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Affiliation(s)
- Miao Cao
- Key Laboratory of Yangtze River Water Environment, Ministry of Education, College of Environmental Science and Engineering, Tongji University, Shanghai 200092, China
| | - Ting Xu
- Key Laboratory of Yangtze River Water Environment, Ministry of Education, College of Environmental Science and Engineering, Tongji University, Shanghai 200092, China; Shanghai Institute of Pollution Control and Ecological Security, Shanghai 200092, China
| | - Yiqun Song
- Key Laboratory of Yangtze River Water Environment, Ministry of Education, College of Environmental Science and Engineering, Tongji University, Shanghai 200092, China
| | - Huan Wang
- Key Laboratory of Yangtze River Water Environment, Ministry of Education, College of Environmental Science and Engineering, Tongji University, Shanghai 200092, China
| | - Sheng Wei
- Key Laboratory of Yangtze River Water Environment, Ministry of Education, College of Environmental Science and Engineering, Tongji University, Shanghai 200092, China
| | - Daqiang Yin
- Key Laboratory of Yangtze River Water Environment, Ministry of Education, College of Environmental Science and Engineering, Tongji University, Shanghai 200092, China; Shanghai Institute of Pollution Control and Ecological Security, Shanghai 200092, China.
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Beaver D, Limnios IJ. A treatment within sight: challenges in the development of stem cell-derived photoreceptor therapies for retinal degenerative diseases. FRONTIERS IN TRANSPLANTATION 2023; 2:1130086. [PMID: 38993872 PMCID: PMC11235385 DOI: 10.3389/frtra.2023.1130086] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 12/22/2022] [Accepted: 09/07/2023] [Indexed: 07/13/2024]
Abstract
Stem cell therapies can potentially treat various retinal degenerative diseases, including age-related macular degeneration (AMD) and inherited retinal diseases like retinitis pigmentosa. For these diseases, transplanted cells may include stem cell-derived retinal pigmented epithelial (RPE) cells, photoreceptors, or a combination of both. Although stem cell-derived RPE cells have progressed to human clinical trials, therapies using photoreceptors and other retinal cell types are lagging. In this review, we discuss the potential use of human pluripotent stem cell (hPSC)-derived photoreceptors for the treatment of retinal degeneration and highlight the progress and challenges for their efficient production and clinical application in regenerative medicine.
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Affiliation(s)
- Davinia Beaver
- Clem Jones Centre for Regenerative Medicine, Bond University, Gold Coast, QL, Australia
| | - Ioannis Jason Limnios
- Clem Jones Centre for Regenerative Medicine, Bond University, Gold Coast, QL, Australia
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de Campos VS, Magalhães CF, da Rosa BG, dos Santos CM, Fragel-Madeira L, Figueiredo DP, Calaza KC, Adesse D. Maternal Toxoplasma gondii infection affects proliferation, differentiation and cell cycle regulation of retinal neural progenitor cells in mouse embryo. Front Cell Neurosci 2023; 17:1211446. [PMID: 37545879 PMCID: PMC10400775 DOI: 10.3389/fncel.2023.1211446] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2023] [Accepted: 06/29/2023] [Indexed: 08/08/2023] Open
Abstract
Background Toxoplasmosis affects one third of the world population and has the protozoan Toxoplasma gondii as etiological agent. Congenital toxoplasmosis (CT) can cause severe damage to the fetus, including miscarriages, intracranial calcification, hydrocephalus and retinochoroiditis. Severity of CT depends on the gestational period in which infection occurs, and alterations at the cellular level during retinal development have been reported. In this study, we proposed a mouse CT model to investigate the impact of infection on retinal development. Methods Pregnant females of pigmented C57BL/6 strain mice were infected intragastrically with two T. gondii cysts (ME49 strain) at embryonic day 10 (E10), and the offspring were analyzed at E18. Results Infected embryos had significantly smaller body sizes and weights than the PBS-treated controls, indicating that embryonic development was affected. In the retina, a significant increase in the number of Ki-67-positive cells (marker of proliferating cells) was found in the apical region of the NBL of infected mice compared to the control. Supporting this, cell cycle proteins Cyclin D3, Cdk6 and pChK2 were significantly altered in infected retinas. Interestingly, the immunohistochemical analysis showed a significant increase in the population of β-III-tubulin-positive cells, one of the earliest markers of neuronal differentiation. Conclusions Our data suggests that CT affects cell cycle progression in retinal progenitor cells, possibly inducing the arrest of these cells at G2/M phase. Such alterations could influence the differentiation, anticipating/increasing neuronal maturation, and therefore leading to abnormal retinal formation. Our model mimics important events observed in ocular CT.
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Affiliation(s)
- Viviane Souza de Campos
- Laboratório de Neurobiologia da Retina, Instituto de Biologia, Universidade Federal Fluminense, Niterói, Brazil
- Laboratório de Biologia Estrutural, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, Brazil
| | - Camila Feitosa Magalhães
- Laboratório de Neurobiologia da Retina, Instituto de Biologia, Universidade Federal Fluminense, Niterói, Brazil
| | - Barbara Gomes da Rosa
- Laboratório de Biologia Estrutural, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, Brazil
| | | | - Lucianne Fragel-Madeira
- Laboratório de Desenvolvimento e Regeneração Neural, Instituto de Biologia, Universidade Federal Fluminense, Niterói, Brazil
| | - Danniel Pereira Figueiredo
- Laboratório de Neurobiologia da Retina, Instituto de Biologia, Universidade Federal Fluminense, Niterói, Brazil
| | - Karin C. Calaza
- Laboratório de Neurobiologia da Retina, Instituto de Biologia, Universidade Federal Fluminense, Niterói, Brazil
| | - Daniel Adesse
- Laboratório de Biologia Estrutural, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, Brazil
- Department of Biochemistry and Molecular Biology, Miller School of Medicine, University of Miami, Miami, FL, United States
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Tingaud-Sequeira A, Mercier E, Michaud V, Pinson B, Gazova I, Gontier E, Decoeur F, McKie L, Jackson IJ, Arveiler B, Javerzat S. The Dct−/− Mouse Model to Unravel Retinogenesis Misregulation in Patients with Albinism. Genes (Basel) 2022; 13:genes13071164. [PMID: 35885947 PMCID: PMC9324463 DOI: 10.3390/genes13071164] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2022] [Revised: 06/21/2022] [Accepted: 06/24/2022] [Indexed: 11/16/2022] Open
Abstract
We have recently identified DCT encoding dopachrome tautomerase (DCT) as the eighth gene for oculocutaneous albinism (OCA). Patients with loss of function of DCT suffer from eye hypopigmentation and retinal dystrophy. Here we investigate the eye phenotype in Dct−/− mice. We show that their retinal pigmented epithelium (RPE) is severely hypopigmented from early stages, contrasting with the darker melanocytic tissues. Multimodal imaging reveals specific RPE cellular defects. Melanosomes are fewer with correct subcellular localization but disrupted melanization. RPE cell size is globally increased and heterogeneous. P-cadherin labeling of Dct−/− newborn RPE reveals a defect in adherens junctions similar to what has been described in tyrosinase-deficient Tyrc/c embryos. The first intermediate of melanin biosynthesis, dihydroxyphenylalanine (L-Dopa), which is thought to control retinogenesis, is detected in substantial yet significantly reduced amounts in Dct−/− postnatal mouse eyecups. L-Dopa synthesis in the RPE alone remains to be evaluated during the critical period of retinogenesis. The Dct−/− mouse should prove useful in understanding the molecular regulation of retinal development and aging of the hypopigmented eye. This may guide therapeutic strategies to prevent vision deficits in patients with albinism.
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Affiliation(s)
- Angèle Tingaud-Sequeira
- Rare Diseases Genetics and Metabolism, INSERM U1211, SBM Department, University of Bordeaux, F-33076 Bordeaux, France; (A.T.-S.); (E.M.); (V.M.); (B.A.)
| | - Elina Mercier
- Rare Diseases Genetics and Metabolism, INSERM U1211, SBM Department, University of Bordeaux, F-33076 Bordeaux, France; (A.T.-S.); (E.M.); (V.M.); (B.A.)
| | - Vincent Michaud
- Rare Diseases Genetics and Metabolism, INSERM U1211, SBM Department, University of Bordeaux, F-33076 Bordeaux, France; (A.T.-S.); (E.M.); (V.M.); (B.A.)
- Molecular Genetics Laboratory, Bordeaux University Hospital, F-33076 Bordeaux, France
| | - Benoît Pinson
- SAM, TBMcore, CNRS UAR 3427, INSERM US005, Université Bordeaux, F-33076 Bordeaux, France;
| | - Ivet Gazova
- MRC Human Genetics Unit, University of Edinburgh, Edinburgh EH4 2XU, UK; (I.G.); (L.M.); (I.J.J.)
| | - Etienne Gontier
- Bordeaux Imaging Center, CNRS, INSERM, BIC, UMS 3420, US 4, University Bordeaux, F-33076 Bordeaux, France; (E.G.); (F.D.)
| | - Fanny Decoeur
- Bordeaux Imaging Center, CNRS, INSERM, BIC, UMS 3420, US 4, University Bordeaux, F-33076 Bordeaux, France; (E.G.); (F.D.)
| | - Lisa McKie
- MRC Human Genetics Unit, University of Edinburgh, Edinburgh EH4 2XU, UK; (I.G.); (L.M.); (I.J.J.)
| | - Ian J. Jackson
- MRC Human Genetics Unit, University of Edinburgh, Edinburgh EH4 2XU, UK; (I.G.); (L.M.); (I.J.J.)
| | - Benoît Arveiler
- Rare Diseases Genetics and Metabolism, INSERM U1211, SBM Department, University of Bordeaux, F-33076 Bordeaux, France; (A.T.-S.); (E.M.); (V.M.); (B.A.)
- Molecular Genetics Laboratory, Bordeaux University Hospital, F-33076 Bordeaux, France
| | - Sophie Javerzat
- Rare Diseases Genetics and Metabolism, INSERM U1211, SBM Department, University of Bordeaux, F-33076 Bordeaux, France; (A.T.-S.); (E.M.); (V.M.); (B.A.)
- Correspondence:
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Grigoryan EN. Self-Organization of the Retina during Eye Development, Retinal Regeneration In Vivo, and in Retinal 3D Organoids In Vitro. Biomedicines 2022; 10:1458. [PMID: 35740479 PMCID: PMC9221005 DOI: 10.3390/biomedicines10061458] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2022] [Revised: 06/16/2022] [Accepted: 06/18/2022] [Indexed: 11/23/2022] Open
Abstract
Self-organization is a process that ensures histogenesis of the eye retina. This highly intricate phenomenon is not sufficiently studied due to its biological complexity and genetic heterogeneity. The review aims to summarize the existing central theories and ideas for a better understanding of retinal self-organization, as well as to address various practical problems of retinal biomedicine. The phenomenon of self-organization is discussed in the spatiotemporal context and illustrated by key findings during vertebrate retina development in vivo and retinal regeneration in amphibians in situ. Described also are histotypic 3D structures obtained from the disaggregated retinal progenitor cells of birds and retinal 3D organoids derived from the mouse and human pluripotent stem cells. The review highlights integral parts of retinal development in these conditions. On the cellular level, these include competence, differentiation, proliferation, apoptosis, cooperative movements, and migration. On the physical level, the focus is on the mechanical properties of cell- and cell layer-derived forces and on the molecular level on factors responsible for gene regulation, such as transcription factors, signaling molecules, and epigenetic changes. Finally, the self-organization phenomenon is discussed as a basis for the production of retinal organoids, a promising model for a wide range of basic scientific and medical applications.
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Affiliation(s)
- Eleonora N Grigoryan
- Koltzov Institute of Developmental Biology, Russian Academy of Sciences, 119334 Moscow, Russia
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In vitro disease modeling of oculocutaneous albinism type 1 and 2 using human induced pluripotent stem cell-derived retinal pigment epithelium. Stem Cell Reports 2022; 17:173-186. [PMID: 35021041 PMCID: PMC8758966 DOI: 10.1016/j.stemcr.2021.11.016] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2021] [Revised: 11/29/2021] [Accepted: 11/30/2021] [Indexed: 12/18/2022] Open
Abstract
Oculocutaneous albinism (OCA) encompasses a set of autosomal recessive genetic conditions that affect pigmentation in the eye, skin, and hair. OCA patients display reduced best-corrected visual acuity, reduced to absent ocular pigmentation, abnormalities in fovea development, and/or abnormal decussation of optic nerve fibers. It has been hypothesized that improving eye pigmentation could prevent or rescue some of the vision defects. The goal of the present study was to develop an in vitro model for studying pigmentation defects in human retinal pigment epithelium (RPE). We developed a “disease in a dish” model for OCA1A and OCA2 types using induced pluripotent stem cells to generate RPE. The RPE is a monolayer of cells that are pigmented, polarized, and polygonal in shape, located between the neural retina and choroid, with an important role in vision. Here we show that RPE tissue derived in vitro from OCA patients recapitulates the pigmentation defects seen in albinism, while retaining the apical-basal polarity and normal polygonal morphology of the constituent RPE cells.
We established a human iPSC-based in vitro model for oculocutaneous albinism (OCA) iRPE derived from OCA-iPSC retains apical-basal polarity and polygonal morphology OCA-iRPE recapitulates the pigmentation defect seen in albinism Excess pre-melanosomes and scarce mature melanosomes are found in OCA-iRPE
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Mannino G, Cristaldi M, Giurdanella G, Perrotta RE, Lo Furno D, Giuffrida R, Rusciano D. ARPE-19 conditioned medium promotes neural differentiation of adipose-derived mesenchymal stem cells. World J Stem Cells 2021; 13:1783-1796. [PMID: 34909123 PMCID: PMC8641022 DOI: 10.4252/wjsc.v13.i11.1783] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/05/2021] [Revised: 06/25/2021] [Accepted: 10/15/2021] [Indexed: 02/07/2023] Open
Abstract
BACKGROUND Adipose-derived stem cells (ASCs) have been increasingly explored for cell-based medicine because of their numerous advantages in terms of easy availability, high proliferation rate, multipotent differentiation ability and low immunogenicity. In this respect, they have been widely investigated in the last two decades to develop therapeutic strategies for a variety of human pathologies including eye disease. In ocular diseases involving the retina, various cell types may be affected, such as Müller cells, astrocytes, photoreceptors and retinal pigment epithelium (RPE), which plays a fundamental role in the homeostasis of retinal tissue, by secreting a variety of growth factors that support retinal cells. AIM To test ASC neural differentiation using conditioned medium (CM) from an RPE cell line (ARPE-19). METHODS ASCs were isolated from adipose tissue, harvested from the subcutaneous region of healthy donors undergoing liposuction procedures. Four ASC culture conditions were investigated: ASCs cultured in basal Dulbecco's Modified Eagle Medium (DMEM); ASCs cultured in serum-free DMEM; ASCs cultured in serum-free DMEM/F12; and ASCs cultured in a CM from ARPE-19, a spontaneously arising cell line with a normal karyotype derived from a human RPE. Cell proliferation rate and viability were assessed by crystal violet and MTT assays at 1, 4 and 8 d of culture. At the same time points, ASC neural differentiation was evaluated by immunocytochemistry and western blot analysis for typical neuronal and glial markers: Nestin, neuronal specific enolase (NSE), protein gene product (PGP) 9.5, and glial fibrillary acidic protein (GFAP). RESULTS Depending on the culture medium, ASC proliferation rate and viability showed some significant differences. Overall, less dense populations were observed in serum-free cultures, except for ASCs cultured in ARPE-19 serum-free CM. Moreover, a different cell morphology was seen in these cultures after 8 d of treatment, with more elongated cells, often showing cytoplasmic ramifications. Immunofluorescence results and western blot analysis were indicative of ASC neural differentiation. In fact, basal levels of neural markers detected under control conditions significantly increased when cells were cultured in ARPE-19 CM. Specifically, neural marker overexpression was more marked at 8 d. The most evident increase was observed for NSE and GFAP, a modest increase was observed for nestin, and less relevant changes were observed for PGP9.5. CONCLUSION The presence of growth factors produced by ARPE-19 cells in tissue culture induces ASCs to express neural differentiation markers typical of the neuronal and glial cells of the retina.
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Affiliation(s)
- Giuliana Mannino
- Department of Biomedical and Biotechnological Sciences, University of Catania, Catania 95123, CT, Italy
| | | | - Giovanni Giurdanella
- Department of Biomedical and Biotechnological Sciences, University of Catania, Catania 95123, CT, Italy
| | - Rosario Emanuele Perrotta
- Department of General Surgery and Medical-Surgery Specialties, University of Catania, Catania 95100, CT, Italy
| | - Debora Lo Furno
- Department of Biomedical and Biotechnological Sciences, University of Catania, Catania 95123, CT, Italy
| | - Rosario Giuffrida
- Department of Biomedical and Biotechnological Sciences, University of Catania, Catania 95123, CT, Italy
| | - Dario Rusciano
- Research Center, SOOFT-Italia S.p.A., Catania 95123, CT, Italy
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Afanasyeva TAV, Corral-Serrano JC, Garanto A, Roepman R, Cheetham ME, Collin RWJ. A look into retinal organoids: methods, analytical techniques, and applications. Cell Mol Life Sci 2021; 78:6505-6532. [PMID: 34420069 PMCID: PMC8558279 DOI: 10.1007/s00018-021-03917-4] [Citation(s) in RCA: 47] [Impact Index Per Article: 11.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2021] [Revised: 07/14/2021] [Accepted: 08/09/2021] [Indexed: 12/15/2022]
Abstract
Inherited retinal diseases (IRDs) cause progressive loss of light-sensitive photoreceptors in the eye and can lead to blindness. Gene-based therapies for IRDs have shown remarkable progress in the past decade, but the vast majority of forms remain untreatable. In the era of personalised medicine, induced pluripotent stem cells (iPSCs) emerge as a valuable system for cell replacement and to model IRD because they retain the specific patient genome and can differentiate into any adult cell type. Three-dimensional (3D) iPSCs-derived retina-like tissue called retinal organoid contains all major retina-specific cell types: amacrine, bipolar, horizontal, retinal ganglion cells, Müller glia, as well as rod and cone photoreceptors. Here, we describe the main applications of retinal organoids and provide a comprehensive overview of the state-of-art analysis methods that apply to this model system. Finally, we will discuss the outlook for improvements that would bring the cellular model a step closer to become an established system in research and treatment development of IRDs.
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Affiliation(s)
- Tess A V Afanasyeva
- Department of Human Genetics and Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Geert Grooteplein 10, 6525 GA, Nijmegen, The Netherlands
| | | | - Alejandro Garanto
- Department of Pediatrics, Amalia Children's Hospital and Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands
- Department of Human Genetics and Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands
| | - Ronald Roepman
- Department of Human Genetics and Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands
| | - Michael E Cheetham
- UCL Institute of Ophthalmology, 11-43 Bath Street, London, EC1V 9EL, UK.
| | - Rob W J Collin
- Department of Human Genetics and Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Geert Grooteplein 10, 6525 GA, Nijmegen, The Netherlands.
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11
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Hanke-Gogokhia C, Lehmann GL, Benedicto I, de la Fuente-Ortega E, Arshavsky VY, Schreiner R, Rodriguez-Boulan E. Apical CLC-2 in retinal pigment epithelium is crucial for survival of the outer retina. FASEB J 2021; 35:e21689. [PMID: 34085737 PMCID: PMC8252757 DOI: 10.1096/fj.202100349r] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2021] [Revised: 04/16/2021] [Accepted: 05/06/2021] [Indexed: 12/15/2022]
Abstract
Knockout of the chloride channel protein 2 (CLC‐2; CLCN2) results in fast progressing blindness in mice. Retinal Pigment Epithelium (RPE) and photoreceptors undergo, in parallel, rapid, and profound morphological changes and degeneration. Immunohistochemistry and electron microscopy of the outer retina and electroretinography of the CLC‐2 KO mouse demonstrated normal morphology at postnatal day 2, followed by drastic changes in RPE and photoreceptor morphology and loss of vision during the first postnatal month. To investigate whether the RPE or the photoreceptors are the primary cause of the degeneration, we injected lentiviruses carrying HA‐tagged CLC‐2 with an RPE‐specific promotor in the subretinal space of CLC‐2‐KO mice at the time of eye opening. As expected, CLC‐2‐HA was expressed exclusively in RPE; strikingly, this procedure rescued the degeneration of both RPE and photoreceptors. Light response in transduced eyes was also recovered. Only a fraction of RPE was transduced with the lentivirus; however, the entire RPE monolayer appears healthy, even the RPE cells not expressing the CLC‐2‐HA. Surprisingly, in contrast with previous physiological observations that postulate that CLC‐2 has a basolateral localization in RPE, our immunofluorescence experiments demonstrated CLC‐2 has an apical distribution, facing the subretinal space and the photoreceptor outer segments. Our findings suggest that CLC‐2 does not play the postulated role in fluid transport at the basolateral membrane. Rather, they suggest that CLC‐2 performs a critical homeostatic role in the subretinal compartment involving a chloride regulatory mechanism that is critical for the survival of both RPE and photoreceptors.
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Affiliation(s)
| | | | - Ignacio Benedicto
- Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain
| | - Erwin de la Fuente-Ortega
- Departamento de Ciencias Biomédicas, Facultad de Medicina, Universidad Católica del Norte, Coquimbo, Chile
| | - Vadim Y Arshavsky
- Department of Ophthalmology, Duke University School of Medicine, Durham, NC, USA
| | - Ryan Schreiner
- Division of Regenerative Medicine, Ansary Stem Cell Institute, Department of Medicine, Weill Cornell Medicine, New York, NY, USA
| | - Enrique Rodriguez-Boulan
- Department of Ophthalmology, Margaret Dyson Vision Research Institute, Weill Cornell Medicine, New York, NY, USA
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12
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Fathi M, Ross CT, Hosseinzadeh Z. Functional 3-Dimensional Retinal Organoids: Technological Progress and Existing Challenges. Front Neurosci 2021; 15:668857. [PMID: 33958988 PMCID: PMC8095320 DOI: 10.3389/fnins.2021.668857] [Citation(s) in RCA: 24] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2021] [Accepted: 03/24/2021] [Indexed: 12/13/2022] Open
Abstract
Stem cell scientists have developed methods for the self-formation of artificial organs, often referred to as organoids. Organoids can be used as model systems for research in multiple biological disciplines. Yoshiki Sasai’s innovation for deriving mammalian retinal tissue from in vitro stem cells has had a large impact on the study of the biology of vision. New developments in retinal organoid technology provide avenues for in vitro models of human retinal diseases, studies of pathological mechanisms, and development of therapies for retinal degeneration, including electronic retinal implants and gene therapy. Moreover, these innovations have played key roles in establishing models for large-scale drug screening, studying the stages of retinal development, and providing a human model for personalized therapeutic approaches, like cell transplants to replace degenerated retinal cells. Here, we first discuss the importance of human retinal organoids to the biomedical sciences. Then, we review various functional features of retinal organoids that have been developed. Finally, we highlight the current limitations of retinal organoid technologies.
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Affiliation(s)
- Meimanat Fathi
- Department of Cell Techniques and Applied Stem Cell Biology, Faculty of Medicine, Center for Biotechnology and Biomedicine (BBZ), University of Leipzig, Leipzig, Germany.,Physiology and Pathophysiology of the Retina Group, Department of Molecular and Cellular Mechanisms of Neurodegeneration, Paul Flechsig Institute of Brain Research, University of Leipzig, Leipzig, Germany
| | - Cody T Ross
- Department of Human Behavior, Ecology and Culture, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany
| | - Zohreh Hosseinzadeh
- Physiology and Pathophysiology of the Retina Group, Department of Molecular and Cellular Mechanisms of Neurodegeneration, Paul Flechsig Institute of Brain Research, University of Leipzig, Leipzig, Germany
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13
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Mamaeva D, Jazouli Z, DiFrancesco ML, Erkilic N, Dubois G, Hilaire C, Meunier I, Boukhaddaoui H, Kalatzis V. Novel roles for voltage-gated T-type Ca 2+ and ClC-2 channels in phagocytosis and angiogenic factor balance identified in human iPSC-derived RPE. FASEB J 2021; 35:e21406. [PMID: 33724552 DOI: 10.1096/fj.202002754r] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2020] [Revised: 01/13/2021] [Accepted: 01/19/2021] [Indexed: 01/26/2023]
Abstract
Human-induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) is a powerful tool for pathophysiological studies and preclinical therapeutic screening, as well as a source for clinical cell transplantation. Thus, it must be validated for maturity and functionality to ensure correct data readouts and clinical safety. Previous studies have validated hiPSC-derived RPE as morphologically characteristic of the tissue in the human eye. However, information concerning the expression and functionality of ion channels is still limited. We screened hiPSC-derived RPE for the polarized expression of a panel of L-type (CaV 1.1, CaV 1.3) and T-type (CaV 3.1, CaV 3.3) Ca2+ channels, K+ channels (Maxi-K, Kir4.1, Kir7.1), and the Cl- channel ClC-2 known to be expressed in native RPE. We also tested the roles of these channels in key RPE functions using specific inhibitors. In addition to confirming the native expression profiles and function of certain channels, such as L-type Ca2+ channels, we show for the first time that T-type Ca2+ channels play a role in both phagocytosis and vascular endothelial growth factor (VEGF) secretion. Moreover, we demonstrate that Maxi-K and Kir7.1 channels are involved in the polarized secretion of VEGF and pigment epithelium-derived factor (PEDF). Furthermore, we show a novel localization for ClC-2 channel on the apical side of hiPSC-derived RPE, with an overexpression at the level of fluid-filled domes, and demonstrate that it plays an important role in phagocytosis, as well as VEGF and PEDF secretion. Taken together, hiPSC-derived RPE is a powerful model for advancing fundamental knowledge of RPE functions.
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Affiliation(s)
- Daria Mamaeva
- Institute for Neurosciences of Montpellier, Inserm, Montpellier University, Montpellier, France
| | - Zhour Jazouli
- Institute for Neurosciences of Montpellier, Inserm, Montpellier University, Montpellier, France
| | - Mattia L DiFrancesco
- Institute for Neurosciences of Montpellier, Inserm, Montpellier University, Montpellier, France
| | - Nejla Erkilic
- Institute for Neurosciences of Montpellier, Inserm, Montpellier University, Montpellier, France.,National Reference Centre for Inherited Sensory Diseases, Montpellier University, CHU, Montpellier, France
| | - Gregor Dubois
- Institute for Neurosciences of Montpellier, Inserm, Montpellier University, Montpellier, France
| | - Cecile Hilaire
- Institute for Neurosciences of Montpellier, Inserm, Montpellier University, Montpellier, France
| | - Isabelle Meunier
- Institute for Neurosciences of Montpellier, Inserm, Montpellier University, Montpellier, France.,National Reference Centre for Inherited Sensory Diseases, Montpellier University, CHU, Montpellier, France
| | - Hassan Boukhaddaoui
- Institute for Neurosciences of Montpellier, Inserm, Montpellier University, Montpellier, France
| | - Vasiliki Kalatzis
- Institute for Neurosciences of Montpellier, Inserm, Montpellier University, Montpellier, France
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14
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Sun WR, Ramirez S, Spiller KE, Zhao Y, Fuhrmann S. Nf2 fine-tunes proliferation and tissue alignment during closure of the optic fissure in the embryonic mouse eye. Hum Mol Genet 2020; 29:3373-3387. [PMID: 33075808 DOI: 10.1093/hmg/ddaa228] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2020] [Revised: 09/29/2020] [Accepted: 10/12/2020] [Indexed: 11/14/2022] Open
Abstract
Uveal coloboma represents one of the most common congenital ocular malformations accounting for up to 10% of childhood blindness (~1 in 5000 live birth). Coloboma originates from defective fusion of the optic fissure (OF), a transient gap that forms during eye morphogenesis by asymmetric, ventral invagination. Genetic heterogeneity combined with the activity of developmentally regulated genes suggests multiple mechanisms regulating OF closure. The tumor suppressor and FERM domain protein Neurofibromin 2 (NF2) controls diverse processes in cancer, development and regeneration, via Hippo pathway and cytoskeleton regulation. In humans, NF2 mutations can cause ocular abnormalities, including coloboma, however, its actual role in OF closure is unknown. Using conditional inactivation in the embryonic mouse eye, our data indicate that loss of Nf2 function results in a novel underlying cause for coloboma. In particular, mutant eyes show substantially increased retinal pigmented epithelium (RPE) proliferation in the fissure region with concomitant acquisition of RPE cell fate. Cells lining the OF margin can maintain RPE fate ectopically and fail to transition from neuroepithelial to cuboidal shape. In the dorsal RPE of the optic cup, Nf2 inactivation leads to a robust increase in cell number, with local disorganization of the cytoskeleton components F-actin and pMLC2. We propose that RPE hyperproliferation is the primary cause for the observed defects causing insufficient alignment of the OF margins in Nf2 mutants and failure to fuse properly, resulting in persistent coloboma. Our findings indicate that limiting proliferation particularly in the RPE layer is a critical mechanism during OF closure.
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Affiliation(s)
- Wesley R Sun
- Department of Ophthalmology and Visual Sciences, VEI, Vanderbilt University Medical Center, Nashville, TN 37232, USA
| | - Sara Ramirez
- Department of Ophthalmology and Visual Sciences, VEI, Vanderbilt University Medical Center, Nashville, TN 37232, USA.,Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37240, USA
| | - Kelly E Spiller
- Department of Ophthalmology and Visual Sciences, VEI, Vanderbilt University Medical Center, Nashville, TN 37232, USA
| | - Yan Zhao
- Department of Ophthalmology and Visual Sciences, VEI, Vanderbilt University Medical Center, Nashville, TN 37232, USA
| | - Sabine Fuhrmann
- Department of Ophthalmology and Visual Sciences, VEI, Vanderbilt University Medical Center, Nashville, TN 37232, USA.,Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37240, USA
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15
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Ghareeb AE, Lako M, Steel DH. Coculture techniques for modeling retinal development and disease, and enabling regenerative medicine. Stem Cells Transl Med 2020; 9:1531-1548. [PMID: 32767661 PMCID: PMC7695644 DOI: 10.1002/sctm.20-0201] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2020] [Revised: 06/22/2020] [Accepted: 07/05/2020] [Indexed: 12/14/2022] Open
Abstract
Stem cell-derived retinal organoids offer the opportunity to cure retinal degeneration of wide-ranging etiology either through the study of in vitro models or the generation of tissue for transplantation. However, despite much work in animals and several human pilot studies, satisfactory therapies have not been developed. Two major challenges for retinal regenerative medicine are (a) physical cell-cell interactions, which are critical to graft function, are not formed and (b) the host environment does not provide suitable queues for development. Several strategies offer to improve the delivery, integration, maturation, and functionality of cell transplantation. These include minimally invasive delivery, biocompatible material vehicles, retinal cell sheets, and optogenetics. Optimizing several variables in animal models is practically difficult, limited by anatomical and disease pathology which is often different to humans, and faces regulatory and ethical challenges. High-throughput methods are needed to experimentally optimize these variables. Retinal organoids will be important to the success of these models. In their current state, they do not incorporate a representative retinal pigment epithelium (RPE)-photoreceptor interface nor vascular elements, which influence the neural retina phenotype directly and are known to be dysfunctional in common retinal diseases such as age-related macular degeneration. Advanced coculture techniques, which emulate the RPE-photoreceptor and RPE-Bruch's-choriocapillaris interactions, can incorporate disease-specific, human retinal organoids and overcome these drawbacks. Herein, we review retinal coculture models of the neural retina, RPE, and choriocapillaris. We delineate the scientific need for such systems in the study of retinal organogenesis, disease modeling, and the optimization of regenerative cell therapies for retinal degeneration.
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Affiliation(s)
- Ali E. Ghareeb
- Sunderland Eye Infirmary, South Tyneside and Sunderland NHS Foundation TrustSunderlandUK
- Biosciences Institute, Newcastle UniversityNewcastle‐upon‐TyneUK
| | - Majlinda Lako
- Biosciences Institute, Newcastle UniversityNewcastle‐upon‐TyneUK
| | - David H. Steel
- Sunderland Eye Infirmary, South Tyneside and Sunderland NHS Foundation TrustSunderlandUK
- Biosciences Institute, Newcastle UniversityNewcastle‐upon‐TyneUK
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16
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Singh RK, Nasonkin IO. Limitations and Promise of Retinal Tissue From Human Pluripotent Stem Cells for Developing Therapies of Blindness. Front Cell Neurosci 2020; 14:179. [PMID: 33132839 PMCID: PMC7513806 DOI: 10.3389/fncel.2020.00179] [Citation(s) in RCA: 27] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2020] [Accepted: 05/25/2020] [Indexed: 12/13/2022] Open
Abstract
The self-formation of retinal tissue from pluripotent stem cells generated a tremendous promise for developing new therapies of retinal degenerative diseases, which previously seemed unattainable. Together with use of induced pluripotent stem cells or/and CRISPR-based recombineering the retinal organoid technology provided an avenue for developing models of human retinal degenerative diseases "in a dish" for studying the pathology, delineating the mechanisms and also establishing a platform for large-scale drug screening. At the same time, retinal organoids, highly resembling developing human fetal retinal tissue, are viewed as source of multipotential retinal progenitors, young photoreceptors and just the whole retinal tissue, which may be transplanted into the subretinal space with a goal of replacing patient's degenerated retina with a new retinal "patch." Both approaches (transplantation and modeling/drug screening) were projected when Yoshiki Sasai demonstrated the feasibility of deriving mammalian retinal tissue from pluripotent stem cells, and generated a lot of excitement. With further work and testing of both approaches in vitro and in vivo, a major implicit limitation has become apparent pretty quickly: the absence of the uniform layer of Retinal Pigment Epithelium (RPE) cells, which is normally present in mammalian retina, surrounds photoreceptor layer and develops and matures first. The RPE layer polarize into apical and basal sides during development and establish microvilli on the apical side, interacting with photoreceptors, nurturing photoreceptor outer segments and participating in the visual cycle by recycling 11-trans retinal (bleached pigment) back to 11-cis retinal. Retinal organoids, however, either do not have RPE layer or carry patches of RPE mostly on one side, thus directly exposing most photoreceptors in the developing organoids to neural medium. Recreation of the critical retinal niche between the apical RPE and photoreceptors, where many retinal disease mechanisms originate, is so far unattainable, imposes clear limitations on both modeling/drug screening and transplantation approaches and is a focus of investigation in many labs. Here we dissect different retinal degenerative diseases and analyze how and where retinal organoid technology can contribute the most to developing therapies even with a current limitation and absence of long and functional outer segments, supported by RPE.
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17
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Lenis TL, Gunzenhauser RC, Fung SSM, Dhindsa YK, Sarraf D, Pineles SL, Tsui I. Myopia and anterior segment optical coherence tomography findings in laser-treated retinopathy of prematurity eyes. J AAPOS 2020; 24:86.e1-86.e7. [PMID: 32224286 DOI: 10.1016/j.jaapos.2020.01.007] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/02/2019] [Revised: 01/13/2020] [Accepted: 01/16/2020] [Indexed: 11/30/2022]
Abstract
PURPOSE To evaluate structural features and visual outcomes in eyes with a prior history of laser treatment for retinopathy of prematurity (ROP). METHODS Laser-treated eyes for type 1 ROP, preterm monitored eyes, and full-term control eyes were included. LogMAR conversion of Snellen best-corrected visual acuity and spherical equivalent based on cycloplegic refraction were measured in children 5-15 years of age. Anterior segment optical coherence tomography (OCT) was used to study structural features, including anterior chamber angle (ACA) in a subset of eyes. RESULTS A total of 50 eyes of 50 patients were included (19 full-term eyes, 19 laser-treated type 1 ROP eyes, 12 preterm monitored eyes). Of these, 44 eyes had visual outcomes data, and 15 eyes had anterior segment data. There was no significant difference in sex or age at final examination between the three groups. There was no significant difference in gestational age between the laser-treated and preterm monitored groups. Compared with the full-term control group and the preterm monitored group, the laser-treated ROP group had narrower ACA and more myopic refractive error. There was a significant correlation between ACA and spherical equivalent. CONCLUSIONS Laser treatment may affect angle configuration in ROP eyes. Anterior segment OCT is an easy and useful modality that could aid in screening for visually impairing conditions such as myopia and glaucoma in children with ROP.
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Affiliation(s)
- Tamara L Lenis
- Stein Eye Institute, University of California Los Angeles; David Geffen School of Medicine, University of California Los Angeles
| | - Robert C Gunzenhauser
- Stein Eye Institute, University of California Los Angeles; David Geffen School of Medicine, University of California Los Angeles
| | - Simon S M Fung
- Stein Eye Institute, University of California Los Angeles; David Geffen School of Medicine, University of California Los Angeles
| | - Yasmeen K Dhindsa
- Stein Eye Institute, University of California Los Angeles; David Geffen School of Medicine, University of California Los Angeles
| | - David Sarraf
- Stein Eye Institute, University of California Los Angeles; David Geffen School of Medicine, University of California Los Angeles
| | - Stacy L Pineles
- Stein Eye Institute, University of California Los Angeles; David Geffen School of Medicine, University of California Los Angeles
| | - Irena Tsui
- Stein Eye Institute, University of California Los Angeles; David Geffen School of Medicine, University of California Los Angeles
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18
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Carrillo-Rosas S, Weber C, Fievet L, Messaddeq N, Karam A, Trottier Y. Loss of zebrafish Ataxin-7, a SAGA subunit responsible for SCA7 retinopathy, causes ocular coloboma and malformation of photoreceptors. Hum Mol Genet 2020; 28:912-927. [PMID: 30445451 DOI: 10.1093/hmg/ddy401] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2018] [Revised: 10/31/2018] [Accepted: 11/10/2018] [Indexed: 12/26/2022] Open
Abstract
Polyglutamine (polyQ) expansion in Ataxin-7 (ATXN7) results in spinocerebellar ataxia type 7 (SCA7) and causes visual impairment. SCA7 photoreceptors progressively lose their outer segments (OSs), a structure essential for their visual function. ATXN7 is a subunit of the transcriptional coactivator Spt-Ada-Gcn5 Acetyltransferase complex, implicated in the development of the visual system in flies. To determine the function of ATXN7 in the vertebrate eye, we have inactivated ATXN7 in zebrafish. While ATXN7 depletion in flies led to gross retinal degeneration, in zebrafish, it primarily results in ocular coloboma, a structural malformation responsible for pediatric visual impairment in humans. ATXN7 inactivation leads to elevated Hedgehog signaling in the forebrain, causing an alteration of proximo-distal patterning of the optic vesicle during early eye development and coloboma. At later developmental stages, malformations of photoreceptors due to incomplete formation of their OSs are observed and correlate with altered expression of crx, a key transcription factor involved in the formation of photoreceptor OS. Therefore, we propose that a primary toxic effect of polyQ expansion is the alteration of ATXN7 function in the daily renewal of OS in SCA7. Together, our data indicate that ATXN7 plays an essential role in vertebrate eye morphogenesis and photoreceptor differentiation, and its loss of function may contribute to the development of human coloboma.
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Affiliation(s)
- Samantha Carrillo-Rosas
- Institute of Genetics and Molecular and Cellular Biology (IGBMC).,Centre National de la Recherche Scientifique, UMR7104.,Institut National de la Santé et de la Recherche Médicale, U1254.,University of Strasbourg, Illkirch, 67000, France
| | - Chantal Weber
- Institute of Genetics and Molecular and Cellular Biology (IGBMC).,Centre National de la Recherche Scientifique, UMR7104.,Institut National de la Santé et de la Recherche Médicale, U1254.,University of Strasbourg, Illkirch, 67000, France
| | - Lorraine Fievet
- Institute of Genetics and Molecular and Cellular Biology (IGBMC).,Centre National de la Recherche Scientifique, UMR7104.,Institut National de la Santé et de la Recherche Médicale, U1254.,University of Strasbourg, Illkirch, 67000, France
| | - Nadia Messaddeq
- Institute of Genetics and Molecular and Cellular Biology (IGBMC).,Centre National de la Recherche Scientifique, UMR7104.,Institut National de la Santé et de la Recherche Médicale, U1254.,University of Strasbourg, Illkirch, 67000, France
| | - Alice Karam
- Institute of Genetics and Molecular and Cellular Biology (IGBMC).,Centre National de la Recherche Scientifique, UMR7104.,Institut National de la Santé et de la Recherche Médicale, U1254.,University of Strasbourg, Illkirch, 67000, France
| | - Yvon Trottier
- Institute of Genetics and Molecular and Cellular Biology (IGBMC).,Centre National de la Recherche Scientifique, UMR7104.,Institut National de la Santé et de la Recherche Médicale, U1254.,University of Strasbourg, Illkirch, 67000, France
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19
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Vigouroux RJ, Cesar Q, Chédotal A, Nguyen-Ba-Charvet KT. Revisiting the role of Dcc in visual system development with a novel eye clearing method. eLife 2020; 9:51275. [PMID: 32096760 PMCID: PMC7062470 DOI: 10.7554/elife.51275] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2019] [Accepted: 02/24/2020] [Indexed: 12/22/2022] Open
Abstract
The Deleted in Colorectal Carcinoma (Dcc) receptor plays a critical role in optic nerve development. Whilst Dcc is expressed postnatally in the eye, its function remains unknown as Dcc knockouts die at birth. To circumvent this drawback, we generated an eye-specific Dcc mutant. To study the organization of the retina and visual projections in these mice, we also established EyeDISCO, a novel tissue clearing protocol that removes melanin allowing 3D imaging of whole eyes and visual pathways. We show that in the absence of Dcc, some ganglion cell axons stalled at the optic disc, whereas others perforated the retina, separating photoreceptors from the retinal pigment epithelium. A subset of visual axons entered the CNS, but these projections are perturbed. Moreover, Dcc-deficient retinas displayed a massive postnatal loss of retinal ganglion cells and a large fraction of photoreceptors. Thus, Dcc is essential for the development and maintenance of the retina.
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Affiliation(s)
- Robin J Vigouroux
- Institut de la Vision, Sorbonne Université, INSERM, CNRS, Paris, France
| | - Quénol Cesar
- Institut de la Vision, Sorbonne Université, INSERM, CNRS, Paris, France
| | - Alain Chédotal
- Institut de la Vision, Sorbonne Université, INSERM, CNRS, Paris, France
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20
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Ma L, Ng M, van der Weele CM, Yoshizawa M, Jeffery WR. Dual roles of the retinal pigment epithelium and lens in cavefish eye degeneration. JOURNAL OF EXPERIMENTAL ZOOLOGY PART B-MOLECULAR AND DEVELOPMENTAL EVOLUTION 2020; 334:438-449. [PMID: 31930686 DOI: 10.1002/jez.b.22923] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/06/2019] [Revised: 11/04/2019] [Accepted: 12/21/2019] [Indexed: 01/03/2023]
Abstract
Astyanax mexicanus consists of two forms, a sighted surface dwelling form (surface fish) and a blind cave-dwelling form (cavefish). Embryonic eyes are initially formed in cavefish but they are subsequently arrested in growth and degenerate during larval development. Previous lens transplantation studies have shown that the lens plays a central role in cavefish eye loss. However, several lines of evidence suggest that additional factors, such as the retinal pigment epithelium (RPE), which is morphologically altered in cavefish, could also be involved in the eye regression process. To explore the role of the RPE in cavefish eye degeneration, we generated an albino eyed (AE) strain by artificial selection for hybrid individuals with large eyes and a depigmented RPE. The AE strain exhibited an RPE lacking pigment granules and showed reduced expression of the RPE specific enzyme retinol isomerase, allowing eye development to be studied by lens ablation in an RPE background resembling cavefish. We found that lens ablation in the AE strain had stronger negative effects on eye growth than in surface fish, suggesting that an intact RPE is required for normal eye development. We also found that the AE strain develops a cartilaginous sclera lacking boney ossicles, a trait similar to cavefish. Extrapolation of the results to cavefish suggests that the RPE and lens have dual roles in eye degeneration, and that deficiencies in the RPE may be associated with evolutionary changes in scleral ossification.
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Affiliation(s)
- Li Ma
- Department of Biology, University of Maryland, College Park, Maryland
| | - Mandy Ng
- Department of Biology, University of Maryland, College Park, Maryland
| | | | - Masato Yoshizawa
- Department of Biology, University of Maryland, College Park, Maryland
| | - William R Jeffery
- Department of Biology, University of Maryland, College Park, Maryland
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21
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Reprogramming of Adult Retinal Müller Glial Cells into Human-Induced Pluripotent Stem Cells as an Efficient Source of Retinal Cells. Stem Cells Int 2019; 2019:7858796. [PMID: 31396286 PMCID: PMC6664555 DOI: 10.1155/2019/7858796] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2019] [Accepted: 06/09/2019] [Indexed: 12/30/2022] Open
Abstract
The reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs) has broad applications in regenerative medicine. The generation of self-organized retinal structures from these iPSCs offers the opportunity to study retinal development and model-specific retinal disease with patient-specific iPSCs and provides the basis for cell replacement strategies. In this study, we demonstrated that the major type of glial cells of the human retina, Müller cells, can be reprogrammed into iPSCs that acquire classical signature of pluripotent stem cells. These Müller glial cell-derived iPSCs were able to differentiate toward retinal fate and generate concomitantly retinal pigmented epithelial cells and self-forming retinal organoid structures containing retinal progenitor cells. Retinal organoids recapitulated retinal neurogenesis with differentiation of retinal progenitor cells into all retinal cell types in a sequential overlapping order. With a modified retinal maturation protocol characterized by the presence of serum and high glucose levels, our study revealed that the retinal organoids contained pseudolaminated neural retina with important features reminiscent of mature photoreceptors, both rod and cone subtypes. This advanced maturation of photoreceptors not only supports the possibility to use 3D retinal organoids for studying photoreceptor development but also offers a novel opportunity for disease modeling, particularly for inherited retinal diseases.
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22
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Akhtar T, Xie H, Khan MI, Zhao H, Bao J, Zhang M, Xue T. Accelerated photoreceptor differentiation of hiPSC-derived retinal organoids by contact co-culture with retinal pigment epithelium. Stem Cell Res 2019; 39:101491. [PMID: 31326746 DOI: 10.1016/j.scr.2019.101491] [Citation(s) in RCA: 50] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/12/2018] [Revised: 06/19/2019] [Accepted: 06/25/2019] [Indexed: 12/29/2022] Open
Abstract
Retinal organoids (ROs) derived from human-induced pluripotent stem cells recapitulate the three-dimensional structure of retina, mimic human retinal development, and provide cell sources for pre-clinical retinal transplantation. Retinal pigment epithelium (RPE) is crucial for normal outer retinal physiology, including phagocytosis of shed photoreceptor outer segments and secretion of neurotrophic and vasculotrophic growth factors. However, whether ROs-RPE co-culture can improve the differentiation of photoreceptors in ROs in vitro remains unknown. Herein, primary mouse RPE cells were contact co-cultured with ROs at different time points. Our results revealed that the RPE cells accelerated photoreceptor differentiation in ROs, as the cross talk between the RPE and ROs promoted the stage specific expression of photoreceptor markers at different differentiation stages. Thus, we established an improved co-culture system based on modeling of human retina-RPE dynamics during retinogenesis for the evaluation of ocular therapies.
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Affiliation(s)
- Tasneem Akhtar
- Eye Center, The First Affiliated Hospital of USTC, Hefei National Laboratory for Physical Sciences at the Microscale, Neurodegenerative Disorder Research Center, Chinese Academy of Sciences Key Laboratory of Brain Function and Disease, School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei 230026, China
| | - Haohuan Xie
- Eye Center, The First Affiliated Hospital of USTC, Hefei National Laboratory for Physical Sciences at the Microscale, Neurodegenerative Disorder Research Center, Chinese Academy of Sciences Key Laboratory of Brain Function and Disease, School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei 230026, China
| | - Muhammad Imran Khan
- Eye Center, The First Affiliated Hospital of USTC, Hefei National Laboratory for Physical Sciences at the Microscale, Neurodegenerative Disorder Research Center, Chinese Academy of Sciences Key Laboratory of Brain Function and Disease, School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei 230026, China
| | - Huan Zhao
- Department of Biological and Environmental Engineering, Hefei University, Hefei 230601, China
| | - Jin Bao
- Eye Center, The First Affiliated Hospital of USTC, Hefei National Laboratory for Physical Sciences at the Microscale, Neurodegenerative Disorder Research Center, Chinese Academy of Sciences Key Laboratory of Brain Function and Disease, School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei 230026, China
| | - Mei Zhang
- Eye Center, The First Affiliated Hospital of USTC, Hefei National Laboratory for Physical Sciences at the Microscale, Neurodegenerative Disorder Research Center, Chinese Academy of Sciences Key Laboratory of Brain Function and Disease, School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei 230026, China.
| | - Tian Xue
- Eye Center, The First Affiliated Hospital of USTC, Hefei National Laboratory for Physical Sciences at the Microscale, Neurodegenerative Disorder Research Center, Chinese Academy of Sciences Key Laboratory of Brain Function and Disease, School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei 230026, China; Chinese Academy of Sciences Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai 200031, China; Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China.
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23
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Liu B, Calton MA, Abell NS, Benchorin G, Gloudemans MJ, Chen M, Hu J, Li X, Balliu B, Bok D, Montgomery SB, Vollrath D. Genetic analyses of human fetal retinal pigment epithelium gene expression suggest ocular disease mechanisms. Commun Biol 2019; 2:186. [PMID: 31123710 PMCID: PMC6527609 DOI: 10.1038/s42003-019-0430-6] [Citation(s) in RCA: 20] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2018] [Accepted: 04/17/2019] [Indexed: 02/07/2023] Open
Abstract
The retinal pigment epithelium (RPE) serves vital roles in ocular development and retinal homeostasis but has limited representation in large-scale functional genomics datasets. Understanding how common human genetic variants affect RPE gene expression could elucidate the sources of phenotypic variability in selected monogenic ocular diseases and pinpoint causal genes at genome-wide association study (GWAS) loci. We interrogated the genetics of gene expression of cultured human fetal RPE (fRPE) cells under two metabolic conditions and discovered hundreds of shared or condition-specific expression or splice quantitative trait loci (e/sQTLs). Co-localizations of fRPE e/sQTLs with age-related macular degeneration (AMD) and myopia GWAS data suggest new candidate genes, and mechanisms by which a common RDH5 allele contributes to both increased AMD risk and decreased myopia risk. Our study highlights the unique transcriptomic characteristics of fRPE and provides a resource to connect e/sQTLs in a critical ocular cell type to monogenic and complex eye disorders.
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Affiliation(s)
- Boxiang Liu
- Department of Biology, Stanford University, Stanford, CA 94305 USA
| | - Melissa A. Calton
- Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305 USA
| | - Nathan S. Abell
- Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305 USA
| | - Gillie Benchorin
- Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305 USA
| | - Michael J. Gloudemans
- Program in Biomedical Informatics, Stanford University School of Medicine, Stanford, 94305 CA USA
| | - Ming Chen
- Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305 USA
| | - Jane Hu
- Department of Ophthalmology, Jules Stein Eye Institute, UCLA, Los Angeles, 90095 CA USA
| | - Xin Li
- Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305 USA
| | - Brunilda Balliu
- Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305 USA
| | - Dean Bok
- Department of Ophthalmology, Jules Stein Eye Institute, UCLA, Los Angeles, 90095 CA USA
| | - Stephen B. Montgomery
- Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305 USA
- Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305 USA
| | - Douglas Vollrath
- Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305 USA
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24
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Mason C, Guillery R. Conversations with Ray Guillery on albinism: linking Siamese cat visual pathway connectivity to mouse retinal development. Eur J Neurosci 2019; 49:913-927. [PMID: 30801828 DOI: 10.1111/ejn.14396] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2018] [Revised: 01/23/2019] [Accepted: 02/12/2019] [Indexed: 02/06/2023]
Abstract
In albinism of all species, perturbed melanin biosynthesis in the eye leads to foveal hypoplasia, retinal ganglion cell misrouting, and, consequently, altered binocular vision. Here, written before he died, Ray Guillery chronicles his discovery of the aberrant circuitry from eye to brain in the Siamese cat. Ray's characterization of visual pathway anomalies in this temperature sensitive mutation of tyrosinase and thus melanin synthesis in domestic cats opened the exploration of albinism and simultaneously, a genetic approach to the organization of neural circuitry. I follow this account with a remembrance of Ray's influence on my work. Beginning with my postdoc research with Ray on the cat visual pathway, through my own work on the mechanisms of retinal axon guidance in the developing mouse, Ray and I had a continuous and rich dialogue about the albino visual pathway. I will present the questions Ray posed and clues we have to date on the still-elusive link between eye pigment and the proper balance of ipsilateral and contralateral retinal ganglion cell projections to the brain.
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Affiliation(s)
- Carol Mason
- Departments of Pathology and Cell Biology, Neuroscience, and Ophthalmology, Mortimer B. Zuckerman Mind Brain Behavior Institute, Columbia University, Jerome L. Greene Science Center, 3227 Broadway, Room L3-043, Quad 3C, New York, NY, 10027, USA
| | - Ray Guillery
- Departments of Pathology and Cell Biology, Neuroscience, and Ophthalmology, Mortimer B. Zuckerman Mind Brain Behavior Institute, Columbia University, Jerome L. Greene Science Center, 3227 Broadway, Room L3-043, Quad 3C, New York, NY, 10027, USA
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25
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Complement factor H regulates retinal development and its absence may establish a footprint for age related macular degeneration. Sci Rep 2019; 9:1082. [PMID: 30705315 PMCID: PMC6355813 DOI: 10.1038/s41598-018-37673-6] [Citation(s) in RCA: 26] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2018] [Accepted: 11/29/2018] [Indexed: 01/22/2023] Open
Abstract
Age related macular degeneration (AMD) is the most common blinding disease in those over 60 years. In 50% of cases it is associated with polymorphisms of complement factor H (FH), implicating immune vulnerability. But such individuals may exhibit abnormal outer retinal blood flow decades before disease initiation, suggesting an early disease footprint. FH is expressed in the retinal pigmented epithelium (RPE). During development the RPE is adjacent to the site of retinal mitosis and complex regulatory interactions occur between the relatively mature RPE and retinal neuronal precursors that control the cell cycle. Here we ask if the absence of FH from the RPE influences retinal development using a mouse CFH knockout (Cfh−/−) with an aged retinal degenerative phenotype. We reveal that from birth, these mice have significantly disrupted and delayed retinal development. However, once development is complete, their retinae appear relatively normal, although many photoreceptor and RPE mitochondria are abnormally large, suggesting dysfunction consistent with premature ATP decline in Cfh−/−. Total retinal mtDNA is also reduced and these deficits are associated shortly after with reduced retinal function. Cfh−/+ mice also show significant abnormal patterns of cell production but not as great as in Cfh−/−. These results reveal that not only is FH an important player in sculpting retinal development but also that the developmental abnormality in Cfh−/− likely establishes critical vulnerability for later aged retinal degeneration.
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26
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Dorgau B, Felemban M, Hilgen G, Kiening M, Zerti D, Hunt NC, Doherty M, Whitfield P, Hallam D, White K, Ding Y, Krasnogor N, Al-Aama J, Asfour HZ, Sernagor E, Lako M. Decellularised extracellular matrix-derived peptides from neural retina and retinal pigment epithelium enhance the expression of synaptic markers and light responsiveness of human pluripotent stem cell derived retinal organoids. Biomaterials 2019; 199:63-75. [PMID: 30738336 DOI: 10.1016/j.biomaterials.2019.01.028] [Citation(s) in RCA: 58] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2018] [Revised: 01/11/2019] [Accepted: 01/20/2019] [Indexed: 12/13/2022]
Abstract
Tissue specific extracellular matrices (ECM) provide structural support and enable access to molecular signals and metabolites, which are essential for directing stem cell renewal and differentiation. To mimic this phenomenon in vitro, tissue decellularisation approaches have been developed, resulting in the generation of natural ECM scaffolds that have comparable physical and biochemical properties of the natural tissues and are currently gaining traction in tissue engineering and regenerative therapies due to the ease of standardised production, and constant availability. In this manuscript we report the successful generation of decellularised ECM-derived peptides from neural retina (decel NR) and retinal pigment epithelium (decel RPE), and their impact on differentiation of human pluripotent stem cells (hPSCs) to retinal organoids. We show that culture media supplementation with decel RPE and RPE-conditioned media (CM RPE) significantly increases the generation of rod photoreceptors, whilst addition of decel NR and decel RPE significantly enhances ribbon synapse marker expression and the light responsiveness of retinal organoids. Photoreceptor maturation, formation of correct synapses between retinal cells and recording of robust light responses from hPSC-derived retinal organoids remain unresolved challenges for the field of regenerative medicine. Enhanced rod photoreceptor differentiation, synaptogenesis and light response in response to addition of decellularised matrices from RPE and neural retina as shown herein provide a novel and substantial advance in generation of retinal organoids for drug screening, tissue engineering and regenerative medicine.
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Affiliation(s)
- Birthe Dorgau
- Institute of Genetic Medicine, Newcastle University, UK
| | | | | | | | - Darin Zerti
- Institute of Genetic Medicine, Newcastle University, UK
| | | | | | | | - Dean Hallam
- Institute of Genetic Medicine, Newcastle University, UK
| | | | - Yuchun Ding
- Interdisciplinary Computing and Complex Biosystems (ICOS) Research Group, Newcastle University, UK
| | - Natalio Krasnogor
- Interdisciplinary Computing and Complex Biosystems (ICOS) Research Group, Newcastle University, UK
| | - Jumana Al-Aama
- Department of Genetic Medicine and Princess Al-Jawhara Center of Excellence in Research of Hereditary Disorders, Faculty of Medicine, King Abdulaziz University, Saudi Arabia
| | - Hani Z Asfour
- Department of Medical Microbiology and Parasitology, Faculty of Medicine, Princess Al-Jawhara Center of Excellence in Research o Hereditary Disorders, King Abdulaziz University, Jeddah, Saudi Arabia
| | | | - Majlinda Lako
- Institute of Genetic Medicine, Newcastle University, UK.
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27
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Chen Y, Yang J, Geng H, Li L, Li J, Cheng B, Ma X, Li H, Hou L. Photoreceptor degeneration in microphthalmia ( Mitf) mice: partial rescue by pigment epithelium-derived factor. Dis Model Mech 2019; 12:12/1/dmm035642. [PMID: 30651300 PMCID: PMC6361154 DOI: 10.1242/dmm.035642] [Citation(s) in RCA: 25] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2018] [Accepted: 12/04/2018] [Indexed: 12/13/2022] Open
Abstract
Dysfunction and loss of the retinal pigment epithelium (RPE) are hallmarks of retinal degeneration, but the underlying pathogenetic processes are only partially understood. Using mice with a null mutation in the transcription factor gene Mitf, in which RPE deficiencies are associated with retinal degeneration, we evaluated the role of trophic factors secreted by the RPE in retinal homeostasis. In such mice, the thickness of the outer nuclear layer (ONL) is as in wild type up to postnatal day 10, but then is progressively reduced, associated with a marked increase in the number of apoptotic cells and a decline in staining for rhodopsin. We show that retinal degeneration and decrease in rhodopsin staining can be prevented partially in three different ways: first, by recombining mutant-derived postnatal retina with postnatal wild-type RPE in tissue explant cultures; second, by adding to cultured mutant retina the trophic factor pigment epithelium-derived factor (PEDF; also known as SERPINF1), which is normally produced in RPE under the control of Mitf; and third, by treating the eyes of Mitf mutant mice in vivo with drops containing a bioactive PEDF 17-mer peptide. This latter treatment also led to marked increases in a number of rod and cone genes. The results indicate that RPE-derived trophic factors, in particular PEDF, are instrumental in retinal homeostasis, and suggest that PEDF or its bioactive fragments may have therapeutic potential in RPE deficiency-associated retinal degeneration.
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Affiliation(s)
- Yu Chen
- Laboratory of Developmental Cell Biology and Disease, School of Ophthalmology and Optometry and Eye Hospital, State Key Laboratory of Optometry, Ophthalmology, and Vision Science and Zhejiang Provincial Key Laboratory of Ophthalmology, Wenzhou Medical University, Wenzhou 325003, China
| | - Juan Yang
- Laboratory of Developmental Cell Biology and Disease, School of Ophthalmology and Optometry and Eye Hospital, State Key Laboratory of Optometry, Ophthalmology, and Vision Science and Zhejiang Provincial Key Laboratory of Ophthalmology, Wenzhou Medical University, Wenzhou 325003, China
| | - Huiqin Geng
- Laboratory of Developmental Cell Biology and Disease, School of Ophthalmology and Optometry and Eye Hospital, State Key Laboratory of Optometry, Ophthalmology, and Vision Science and Zhejiang Provincial Key Laboratory of Ophthalmology, Wenzhou Medical University, Wenzhou 325003, China
| | - Liping Li
- Laboratory of Developmental Cell Biology and Disease, School of Ophthalmology and Optometry and Eye Hospital, State Key Laboratory of Optometry, Ophthalmology, and Vision Science and Zhejiang Provincial Key Laboratory of Ophthalmology, Wenzhou Medical University, Wenzhou 325003, China
| | - Jinyang Li
- Laboratory of Developmental Cell Biology and Disease, School of Ophthalmology and Optometry and Eye Hospital, State Key Laboratory of Optometry, Ophthalmology, and Vision Science and Zhejiang Provincial Key Laboratory of Ophthalmology, Wenzhou Medical University, Wenzhou 325003, China
| | - Bing Cheng
- Laboratory of Developmental Cell Biology and Disease, School of Ophthalmology and Optometry and Eye Hospital, State Key Laboratory of Optometry, Ophthalmology, and Vision Science and Zhejiang Provincial Key Laboratory of Ophthalmology, Wenzhou Medical University, Wenzhou 325003, China
| | - Xiaoyin Ma
- Laboratory of Developmental Cell Biology and Disease, School of Ophthalmology and Optometry and Eye Hospital, State Key Laboratory of Optometry, Ophthalmology, and Vision Science and Zhejiang Provincial Key Laboratory of Ophthalmology, Wenzhou Medical University, Wenzhou 325003, China
| | - Huirong Li
- Laboratory of Developmental Cell Biology and Disease, School of Ophthalmology and Optometry and Eye Hospital, State Key Laboratory of Optometry, Ophthalmology, and Vision Science and Zhejiang Provincial Key Laboratory of Ophthalmology, Wenzhou Medical University, Wenzhou 325003, China
| | - Ling Hou
- Laboratory of Developmental Cell Biology and Disease, School of Ophthalmology and Optometry and Eye Hospital, State Key Laboratory of Optometry, Ophthalmology, and Vision Science and Zhejiang Provincial Key Laboratory of Ophthalmology, Wenzhou Medical University, Wenzhou 325003, China
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28
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Hua J, Chen H, Chen Y, Zheng G, Li F, Qu J, Ma X, Hou L. MITF acts as an anti-oxidant transcription factor to regulate mitochondrial biogenesis and redox signaling in retinal pigment epithelial cells. Exp Eye Res 2018; 170:138-147. [PMID: 29486165 DOI: 10.1016/j.exer.2018.02.023] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2017] [Revised: 01/08/2018] [Accepted: 02/23/2018] [Indexed: 10/18/2022]
Abstract
There is increasing evidence that the mechanisms protecting the retinal pigment epithelium (RPE) against oxidative stress are important for preventing retinal degenerative diseases. Little, however, is known about these mechanisms. Here we show that MITF, a transcription factor responsible for RPE development and function, regulates redox signaling by acting through PGC1α, a master regulator of mitochondrial biogenesis. Mitf deficiency in mice leads to significantly higher levels of reactive oxygen species (ROS) in both RPE and retina, suggesting that Mitf dysfunction might lead to oxidative damage in the RPE and, by extension, in the retina. Furthermore, overexpression of MITF in the human RPE cell line ARPE-19 indicates that MITF up-regulates antioxidant gene expression and mitochondrial biogenesis by regulating PGC1α and protects cells against oxidative stress. Our findings provide new insights into understanding the redox function of MITF in RPE cells and its potential contribution to prevention of RPE-associated retinal degenerations.
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Affiliation(s)
- Jiajia Hua
- Labratory of Developmental Cell Biology and Disease, School of Ophthalmology and Optometry and Eye Hospital, Wenzhou Medical University, China
| | - Huaicheng Chen
- Labratory of Developmental Cell Biology and Disease, School of Ophthalmology and Optometry and Eye Hospital, Wenzhou Medical University, China
| | - Yu Chen
- Labratory of Developmental Cell Biology and Disease, School of Ophthalmology and Optometry and Eye Hospital, Wenzhou Medical University, China; State Key Laboratory of Ophthalmology, Optometry and Vision Science and Key Laboratory of Vision Science of Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology, Wenzhou, 325003, China
| | - Guoxiao Zheng
- Labratory of Developmental Cell Biology and Disease, School of Ophthalmology and Optometry and Eye Hospital, Wenzhou Medical University, China
| | - Fang Li
- Labratory of Developmental Cell Biology and Disease, School of Ophthalmology and Optometry and Eye Hospital, Wenzhou Medical University, China
| | - Jia Qu
- State Key Laboratory of Ophthalmology, Optometry and Vision Science and Key Laboratory of Vision Science of Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology, Wenzhou, 325003, China.
| | - Xiaoyin Ma
- Labratory of Developmental Cell Biology and Disease, School of Ophthalmology and Optometry and Eye Hospital, Wenzhou Medical University, China; State Key Laboratory of Ophthalmology, Optometry and Vision Science and Key Laboratory of Vision Science of Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology, Wenzhou, 325003, China.
| | - Ling Hou
- Labratory of Developmental Cell Biology and Disease, School of Ophthalmology and Optometry and Eye Hospital, Wenzhou Medical University, China; State Key Laboratory of Ophthalmology, Optometry and Vision Science and Key Laboratory of Vision Science of Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology, Wenzhou, 325003, China.
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29
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Llonch S, Carido M, Ader M. Organoid technology for retinal repair. Dev Biol 2017; 433:132-143. [PMID: 29291970 DOI: 10.1016/j.ydbio.2017.09.028] [Citation(s) in RCA: 112] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2017] [Revised: 09/05/2017] [Accepted: 09/21/2017] [Indexed: 02/07/2023]
Abstract
A major cause for vision impairment and blindness in industrialized countries is the loss of the light-sensing retinal tissue in the eye. Photoreceptor damage is one of the main characteristics found in retinal degeneration diseases, such as Retinitis Pigmentosa or age-related macular degeneration. The lack of effective therapies to stop photoreceptor loss together with the absence of significant intrinsic regeneration in the human retina converts such degenerative diseases into permanent conditions that are currently irreversible. Cell replacement by means of photoreceptor transplantation has been proposed as a potential approach to tackle cell loss in the retina. Since the first attempt of photoreceptor transplantation in humans, about twenty years ago, several research groups have focused in the development and improvement of technologies necessary to bring cell transplantation for retinal degeneration diseases to reality. Progress in recent years in the generation of human tissue derived from pluripotent stem cells (PSCs) has significantly improved our tools to study human development and disease in the dish. Particularly the availability of 3D culture systems for the generation of PSC-derived organoids, including the human retina, has dramatically increased access to human material for basic and medical research. In this review, we focus on important milestones towards the generation of transplantable photoreceptor precursors from PSC-derived retinal organoids and discuss recent pre-clinical transplantation studies using organoid-derived photoreceptors in context to related in vivo work using primary photoreceptors as donor material. Additionally, we summarize remaining challenges for developing photoreceptor transplantation towards clinical application.
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Affiliation(s)
- Sílvia Llonch
- CRTD/Center for Regenerative Therapies Dresden, Center for Molecular and Cellular Bioengineering (CMCB), Technische Universität Dresden, Fetscherstraße 105, 01307 Dresden, Germany
| | - Madalena Carido
- CRTD/Center for Regenerative Therapies Dresden, Center for Molecular and Cellular Bioengineering (CMCB), Technische Universität Dresden, Fetscherstraße 105, 01307 Dresden, Germany; German Center for Neurodegenerative Diseases Dresden (DZNE), Arnoldstraße 18, 01307 Dresden, Germany
| | - Marius Ader
- CRTD/Center for Regenerative Therapies Dresden, Center for Molecular and Cellular Bioengineering (CMCB), Technische Universität Dresden, Fetscherstraße 105, 01307 Dresden, Germany.
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30
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Photoreceptor Outer Segment-like Structures in Long-Term 3D Retinas from Human Pluripotent Stem Cells. Sci Rep 2017; 7:766. [PMID: 28396597 PMCID: PMC5429674 DOI: 10.1038/s41598-017-00774-9] [Citation(s) in RCA: 182] [Impact Index Per Article: 22.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2016] [Accepted: 03/14/2017] [Indexed: 12/22/2022] Open
Abstract
The retinal degenerative diseases, which together constitute a leading cause of hereditary blindness worldwide, are largely untreatable. Development of reliable methods to culture complex retinal tissues from human pluripotent stem cells (hPSCs) could offer a means to study human retinal development, provide a platform to investigate the mechanisms of retinal degeneration and screen for neuroprotective compounds, and provide the basis for cell-based therapeutic strategies. In this study, we describe an in vitro method by which hPSCs can be differentiated into 3D retinas with at least some important features reminiscent of a mature retina, including exuberant outgrowth of outer segment-like structures and synaptic ribbons, photoreceptor neurotransmitter expression, and membrane conductances and synaptic vesicle release properties consistent with possible photoreceptor synaptic function. The advanced outer segment-like structures reported here support the notion that 3D retina cups could serve as a model for studying mature photoreceptor development and allow for more robust modeling of retinal degenerative disease in vitro.
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31
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Richard M, Bauer R, Tavosanis G, Hoch M. The gap junction protein Innexin3 is required for eye disc growth in Drosophila. Dev Biol 2017; 425:191-207. [PMID: 28390801 DOI: 10.1016/j.ydbio.2017.04.001] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2016] [Revised: 03/30/2017] [Accepted: 04/03/2017] [Indexed: 12/23/2022]
Abstract
The Drosophila compound eye develops from a bilayered epithelial sac composed of an upper peripodial epithelium layer and a lower disc proper, the latter giving rise to the eye itself. During larval stages, complex signalling events between the layers contribute to the control of cell proliferation and differentiation in the disc. Previous work in our lab established the gap junction protein Innexin2 (Inx2) as crucial for early larval eye disc growth. By analysing the contribution of other Innexins to eye size control, we have identified Innexin3 (Inx3) as an important growth regulator. Depleting inx3 during larval eye development reduces eye size, while elevating inx3 levels increases eye size, thus phenocopying the inx2 loss- and gain-of-function situation. As demonstrated previously for inx2, inx3 regulates disc cell proliferation and interacts genetically with the Dpp pathway, being required for the proper activation of the Dpp pathway transducer Mad at the furrow and the expression of Dpp receptor Punt in the eye disc. At the developmental timepoint corresponding to eye disc growth, Inx3 colocalises with Inx2 in disc proper and peripodial epithelium cell membranes. In addition, we show that Inx3 protein levels critically depend on inx2 throughout eye development and that inx3 modulates Inx2 protein levels in the larval eye disc. Rescue experiments demonstrate that Inx3 and Inx2 cooperate functionally to enable eye disc growth in Drosophila. Finally, we demonstrate that expression of Inx3 and Inx2 is not only needed in the disc proper but also in the peripodial epithelium to regulate growth of the eye disc. Our data provide a functional demonstration that putative Inx2/Inx3 heteromeric channels regulate organ size.
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Affiliation(s)
- Mélisande Richard
- Life & Medical Sciences Institute (LIMES) Development, Genetics & Molecular Physiology Unit University of Bonn, Carl-Troll-Straße, 31 53115 Bonn, Germany
| | - Reinhard Bauer
- Life & Medical Sciences Institute (LIMES) Development, Genetics & Molecular Physiology Unit University of Bonn, Carl-Troll-Straße, 31 53115 Bonn, Germany
| | - Gaia Tavosanis
- German Center for Neurodegenerative Diseases (DZNE), Dendrite Differentiation Unit, Sigmund-Freud-Str. 27, 53127 Bonn, Germany
| | - Michael Hoch
- Life & Medical Sciences Institute (LIMES) Development, Genetics & Molecular Physiology Unit University of Bonn, Carl-Troll-Straße, 31 53115 Bonn, Germany.
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32
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Toler SM. Oxidative Stress Plays an Important Role in the Pathogenesis of Drug-Induced Retinopathy. Exp Biol Med (Maywood) 2016; 229:607-15. [PMID: 15229354 DOI: 10.1177/153537020422900704] [Citation(s) in RCA: 46] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022] Open
Abstract
Several pharmaceutical agents have been associated with rare but serious retinopathies, some resulting in blindness. Little is known of the mechanism(s) that produce these injuries. Mechanisms proposed thus far have not been embraced by the medical and scientific communities. However, preclinical and clinical data indicate that oxidative stress may contribute substantially to iatrogenic retinal disease. Retinal oxidative stress may be precipitated by the interaction of putative retinal toxins with the ocular redox system. The retina, replete with cytochromes P450 and myeloperoxidase, may serve to activate xenobiotics to oxidants, resulting in ocular injury. These activated agents may directly form retinal adducts or may diminish ocular reduced glutathione concentrations. Data are reviewed that suggest that indomethacin, tamoxifen, thioridazine, and chloroquine all produce retinopathies via a common mechanism—they produce ocular oxidative stress.
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Affiliation(s)
- Steven M Toler
- Clinical Safety and Risk Management, Pfizer Inc., Pfizer Global Research and Development, 50 Pequot Avenue, New London, CT 06320, USA.
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33
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Gross JB, Powers AK, Davis EM, Kaplan SA. A pleiotropic interaction between vision loss and hypermelanism in Astyanax mexicanus cave x surface hybrids. BMC Evol Biol 2016; 16:145. [PMID: 27363593 PMCID: PMC4929771 DOI: 10.1186/s12862-016-0716-y] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2016] [Accepted: 06/28/2016] [Indexed: 12/18/2022] Open
Abstract
BACKGROUND Cave-dwelling animals evolve various traits as a consequence of life in darkness. Constructive traits (e.g., enhanced non-visual sensory systems) presumably arise under strong selective pressures. The mechanism(s) driving regression of features, however, are not well understood. Quantitative trait locus (QTL) analyses in Astyanax mexicanus Pachón cave x surface hybrids revealed phenotypic effects associated with vision and pigmentation loss. Vision QTL were uniformly associated with reductions in the homozygous cave condition, however pigmentation QTL demonstrated mixed phenotypic effects. This implied pigmentation might be lost through both selective and neutral forces. Alternatively, in this report, we examined if a pleiotropic interaction may exist between vision and pigmentation since vision loss has been shown to result in darker skin in other fish and amphibian model systems. RESULTS We discovered that certain members of Pachón x surface pedigrees are significantly darker than surface-dwelling fish. All of these "hypermelanic" individuals demonstrated severe visual system malformations suggesting they may be blind. A vision-mediated behavioral assay revealed that these fish, in stark contrast to surface fish, behaved the same as blind cavefish. Further, hypermelanic melanophores were larger and more dendritic in morphology compared to surface fish melanophores. However, hypermelanic melanophores responded normally to melanin-concentrating hormone suggesting darkening stemmed from vision loss, rather than a defect in pigment cell function. Finally, a number of genomic regions were coordinately associated with both reduced vision and increased pigmentation. CONCLUSIONS This work suggests hypermelanism in hybrid Astyanax results from blindness. This finding provides an alternative explanation for phenotypic effect studies of pigmentation QTL as stemming (at least in part) from environmental, rather than exclusively genetic, interactions between two regressive phenotypes. Further, this analysis reveals persistence of background adaptation in Astyanax. As the eye was lost in cave-dwelling forms, enhanced pigmentation resulted. Given the extreme cave environment, which is often devoid of nutrition, enhanced pigmentation may impose an energetic cost. Such an energetic cost would be selected against, as a means of energy conservation. Thus, the pleiotropic interaction between vision loss and pigmentation may reveal an additional selective pressure favoring the loss of pigmentation in cave-dwelling animals.
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Affiliation(s)
- Joshua B. Gross
- />Department of Biological Sciences, University of Cincinnati, Cincinnati, Ohio 45223 USA
- />Department of Biological Sciences, University of Cincinnati, Rieveschl Hall Room 711B, 312 Clifton Court, Cincinnati, Ohio 45221 USA
| | - Amanda K. Powers
- />Department of Biological Sciences, University of Cincinnati, Cincinnati, Ohio 45223 USA
| | - Erin M. Davis
- />Department of Biological Sciences, University of Cincinnati, Cincinnati, Ohio 45223 USA
| | - Shane A. Kaplan
- />Department of Biological Sciences, University of Cincinnati, Cincinnati, Ohio 45223 USA
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Iwai-Takekoshi L, Ramos A, Schaler A, Weinreb S, Blazeski R, Mason C. Retinal pigment epithelial integrity is compromised in the developing albino mouse retina. J Comp Neurol 2016; 524:3696-3716. [PMID: 27097562 DOI: 10.1002/cne.24025] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2015] [Revised: 04/15/2016] [Accepted: 04/18/2016] [Indexed: 12/22/2022]
Abstract
In the developing murine eye, melanin synthesis in the retinal pigment epithelium (RPE) coincides with neurogenesis of retinal ganglion cells (RGCs). Disruption of pigmentation in the albino RPE is associated with delayed neurogenesis in the ventrotemporal retina, the source of ipsilateral RGCs, and a reduced ipsilateral RGC projection. To begin to unravel how melanogenesis and the RPE regulate RGC neurogenesis and cell subpopulation specification, we compared the features of albino and pigmented mouse RPE cells during the period of RGC neurogenesis (embryonic day, E, 12.5 to 18.5) when the RPE is closely apposed to developing RGC precursors. At E12.5 and E15.5, although albino and pigmented RPE cells express RPE markers Otx2 and Mitf similarly, albino RPE cells are irregularly shaped and have fewer melanosomes compared with pigmented RPE cells. The adherens junction protein P-cadherin appears loosely distributed within the albino RPE cells rather than tightly localized on the cell membrane, as in pigmented RPE. Connexin 43 (gap junction protein) is expressed in pigmented and albino RPE cells at E13.5 but at E15.5 albino RPE cells have fewer small connexin 43 puncta, and a larger fraction of phosphorylated connexin 43 at serine 368. These results suggest that the lack of pigment in the RPE results in impaired RPE cell integrity and communication via gap junctions between RPE and neural retina during RGC neurogenesis. Our findings should pave the way for further investigation of the role of RPE in regulating RGC development toward achieving proper RGC axon decussation. J. Comp. Neurol. 524:3696-3716, 2016. © 2016 Wiley Periodicals, Inc.
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Affiliation(s)
- Lena Iwai-Takekoshi
- Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, New York, USA
| | - Anna Ramos
- Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, New York, USA
| | - Ari Schaler
- Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, New York, USA
| | - Samuel Weinreb
- Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, New York, USA
| | - Richard Blazeski
- Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, New York, USA
| | - Carol Mason
- Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, New York, USA. .,Department of Neuroscience, College of Physicians and Surgeons, Columbia University, New York, New York, USA. .,Department of Ophthalmology, College of Physicians and Surgeons, Columbia University, New York, New York, USA.
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Wen B, Li S, Li H, Chen Y, Ma X, Wang J, Lu F, Qu J, Hou L. Microphthalmia-associated transcription factor regulates the visual cycle genes Rlbp1 and Rdh5 in the retinal pigment epithelium. Sci Rep 2016; 6:21208. [PMID: 26876013 PMCID: PMC4753414 DOI: 10.1038/srep21208] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2015] [Accepted: 01/19/2016] [Indexed: 12/14/2022] Open
Abstract
Regeneration of the visual pigment by cells of the retinal pigment epithelium (RPE) is fundamental to vision. Here we show that the microphthalmia-associated transcription factor, MITF, which plays a central role in the development and function of RPE cells, regulates the expression of two visual cycle genes, Rlbp1 which encodes retinaldehyde binding protein-1 (RLBP1), and Rdh5, which encodes retinol dehydrogenase-5 (RDH5). First, we found that Rlbp1 and Rdh5 are downregulated in optic cups and presumptive RPEs of Mitf-deficient mouse embryos. Second, experimental manipulation of MITF levels in human RPE cells in culture leads to corresponding modulations of the endogenous levels of RLBP1 and RDH5. Third, the retinal degeneration associated with the disruption of the visual cycle in Mitf-deficient mice can be partially corrected both structurally and functionally by an exogenous supply of 9-cis-retinal. We conclude that the expression of Rlbp1 and Rdh5 critically depends on functional Mitf in the RPE and suggest that MITF has an important role in controlling retinoid processing in the RPE.
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Affiliation(s)
- Bin Wen
- Laboratory of Developmental Cell Biology and Disease, School of Ophthalmology and Optometry and Eye Hospital, Wenzhou Medical University, Wenzhou, 325003, China.,State Key Laboratory Cultivation Base and Key Laboratory of Vision Science of Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology, Wenzhou Medical University, Wenzhou, 325003, China
| | - Shuang Li
- Laboratory of Developmental Cell Biology and Disease, School of Ophthalmology and Optometry and Eye Hospital, Wenzhou Medical University, Wenzhou, 325003, China
| | - Huirong Li
- Laboratory of Developmental Cell Biology and Disease, School of Ophthalmology and Optometry and Eye Hospital, Wenzhou Medical University, Wenzhou, 325003, China
| | - Yu Chen
- Laboratory of Developmental Cell Biology and Disease, School of Ophthalmology and Optometry and Eye Hospital, Wenzhou Medical University, Wenzhou, 325003, China
| | - Xiaoyin Ma
- Laboratory of Developmental Cell Biology and Disease, School of Ophthalmology and Optometry and Eye Hospital, Wenzhou Medical University, Wenzhou, 325003, China
| | - Jing Wang
- Laboratory of Developmental Cell Biology and Disease, School of Ophthalmology and Optometry and Eye Hospital, Wenzhou Medical University, Wenzhou, 325003, China
| | - Fan Lu
- State Key Laboratory Cultivation Base and Key Laboratory of Vision Science of Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology, Wenzhou Medical University, Wenzhou, 325003, China
| | - Jia Qu
- State Key Laboratory Cultivation Base and Key Laboratory of Vision Science of Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology, Wenzhou Medical University, Wenzhou, 325003, China
| | - Ling Hou
- Laboratory of Developmental Cell Biology and Disease, School of Ophthalmology and Optometry and Eye Hospital, Wenzhou Medical University, Wenzhou, 325003, China.,State Key Laboratory Cultivation Base and Key Laboratory of Vision Science of Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology, Wenzhou Medical University, Wenzhou, 325003, China
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Guo X, Wang SB, Xu H, Ribic A, Mohns EJ, Zhou Y, Zhu X, Biederer T, Crair MC, Chen B. A short N-terminal domain of HDAC4 preserves photoreceptors and restores visual function in retinitis pigmentosa. Nat Commun 2015; 6:8005. [PMID: 26272629 PMCID: PMC4538705 DOI: 10.1038/ncomms9005] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2015] [Accepted: 07/07/2015] [Indexed: 11/09/2022] Open
Abstract
Retinitis pigmentosa is a leading cause of inherited blindness, with no effective treatment currently available. Mutations primarily in genes expressed in rod photoreceptors lead to early rod death, followed by a slower phase of cone photoreceptor death. Rd1 mice provide an invaluable animal model to evaluate therapies for the disease. We previously reported that overexpression of histone deacetylase 4 (HDAC4) prolongs rod survival in rd1 mice. Here we report a key role of a short N-terminal domain of HDAC4 in photoreceptor protection. Expression of this domain suppresses multiple cell death pathways in photoreceptor degeneration, and preserves even more rd1 rods than the full-length HDAC4 protein. Expression of a short N-terminal domain of HDAC4 as a transgene in mice carrying the rd1 mutation also prolongs the survival of cone photoreceptors, and partially restores visual function. Our results may facilitate the design of a small protein therapy for some forms of retinitis pigmentosa.
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Affiliation(s)
- Xinzheng Guo
- Department of Ophthalmology and Visual Science, Yale University School of Medicine, 300 George Street, Suite 8100, New Haven, Connecticut 06511, USA
| | - Shao-Bin Wang
- Department of Ophthalmology and Visual Science, Yale University School of Medicine, 300 George Street, Suite 8100, New Haven, Connecticut 06511, USA
| | - Hongping Xu
- Department of Neurobiology, Yale University School of Medicine, 333 Cedar Street, SHM B301, New Haven, Connecticut 06510, USA
| | - Adema Ribic
- Department of Neuroscience, Tufts University School of Medicine, Boston, Massachusetts 02111, USA
| | - Ethan J Mohns
- Department of Neurobiology, Yale University School of Medicine, 333 Cedar Street, SHM B301, New Haven, Connecticut 06510, USA
| | - Yu Zhou
- 1] Sichuan Provincial Key Laboratory for Human Disease Gene Study and Institute of Laboratory Medicine, Sichuan Academy of Medical Sciences and Sichuan Provincial People's Hospital, Chengdu, Sichuan 610072, China [2] Hospital of University of Electronic Science and Technology of China (UESTC) &Sichuan Provincial People's Hospital, Chengdu, Sichuan 610072, China
| | - Xianjun Zhu
- 1] Sichuan Provincial Key Laboratory for Human Disease Gene Study and Institute of Laboratory Medicine, Sichuan Academy of Medical Sciences and Sichuan Provincial People's Hospital, Chengdu, Sichuan 610072, China [2] Hospital of University of Electronic Science and Technology of China (UESTC) &Sichuan Provincial People's Hospital, Chengdu, Sichuan 610072, China
| | - Thomas Biederer
- Department of Neuroscience, Tufts University School of Medicine, Boston, Massachusetts 02111, USA
| | - Michael C Crair
- Department of Neurobiology, Yale University School of Medicine, 333 Cedar Street, SHM B301, New Haven, Connecticut 06510, USA
| | - Bo Chen
- 1] Department of Ophthalmology and Visual Science, Yale University School of Medicine, 300 George Street, Suite 8100, New Haven, Connecticut 06511, USA [2] Department of Neurobiology, Yale University School of Medicine, 333 Cedar Street, SHM B301, New Haven, Connecticut 06510, USA
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Ohana R, Weiman-Kelman B, Raviv S, Tamm ER, Pasmanik-Chor M, Rinon A, Netanely D, Shamir R, Solomon AS, Ashery-Padan R. MicroRNAs are essential for differentiation of the retinal pigmented epithelium and maturation of adjacent photoreceptors. Development 2015; 142:2487-98. [PMID: 26062936 DOI: 10.1242/dev.121533] [Citation(s) in RCA: 43] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/24/2014] [Accepted: 06/03/2015] [Indexed: 12/21/2022]
Abstract
Dysfunction of the retinal pigmented epithelium (RPE) results in degeneration of photoreceptors and vision loss and is correlated with common blinding disorders in humans. Although many protein-coding genes are known to be expressed in RPE and are important for its development and maintenance, virtually nothing is known about the in vivo roles of non-coding transcripts. The expression patterns of microRNAs (miRNAs) have been analyzed in a variety of ocular tissues, and a few were implicated to play role in RPE based on studies in cell lines. Here, through RPE-specific conditional mutagenesis of Dicer1 or Dgcr8 in mice, the importance of miRNAs for RPE differentiation was uncovered. miRNAs were found to be dispensable for maintaining RPE fate and survival, and yet they are essential for the acquisition of important RPE properties such as the expression of genes involved in the visual cycle pathway, pigmentation and cell adhesion. Importantly, miRNAs of the RPE are required for maturation of adjacent photoreceptors, specifically for the morphogenesis of the outer segments. The alterations in the miRNA and mRNA profiles in the Dicer1-deficient RPE point to a key role of miR-204 in regulation of the RPE differentiation program in vivo and uncover the importance of additional novel RPE miRNAs. This study reveals the combined regulatory activity of miRNAs that is required for RPE differentiation and for the development of the adjacent neuroretina.
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Affiliation(s)
- Reut Ohana
- Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine and Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 69978, Israel
| | - Benjamin Weiman-Kelman
- Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine and Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 69978, Israel
| | - Shaul Raviv
- Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine and Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 69978, Israel
| | - Ernst R Tamm
- Institute of Human Anatomy and Embryology, University of Regensburg, D-93053 Regensburg, Germany
| | - Metsada Pasmanik-Chor
- Bioinformatics Unit, Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel
| | - Ariel Rinon
- Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine and Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 69978, Israel
| | - Dvir Netanely
- Blavatnik School of Computer Science, Tel Aviv University, Tel Aviv 69978, Israel
| | - Ron Shamir
- Blavatnik School of Computer Science, Tel Aviv University, Tel Aviv 69978, Israel
| | - Arie S Solomon
- The Goldschleger Eye Research Institute, Sackler Faculty of Medicine, Tel Aviv University Sheba Medical Center, Tel Hashomer 52621, Israel
| | - Ruth Ashery-Padan
- Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine and Sagol School of Neuroscience, Tel Aviv University, Tel Aviv 69978, Israel
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Liu GH, Lin B, Sun XQ, He ZF, Li JR, Zhou R, Liu XL. Focal choroidal excavation: a preliminary interpretation based on clinic and review. Int J Ophthalmol 2015; 8:513-21. [PMID: 26086000 DOI: 10.3980/j.issn.2222-3959.2015.03.14] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2014] [Accepted: 11/18/2014] [Indexed: 11/02/2022] Open
Abstract
AIM To describe the clinical and imaging characteristics associated with focal choroidal excavation (FCE), analyze the possible complication, and interpret its probable etiopathogenesis. METHODS Retrospective descriptive case series of 37 eyes of 32 patients with FCE. Findings of spectral-domain optical coherence tomography (SD-OCT), fluorescein angiography, indocyanine green angiography, and clinical features were analyzed. RESULTS All patients were Chinese. Five patients (15.6%) were bilaterally involved. Patients' ages ranged from 7 to 66y. Refractive error ranged between +2.0 D and -11.0 D. Mean best-corrected visual acuity was 0.6 (range, 0.1 to 1.2). Fundus examinations exhibited mild-moderate localized pigmentary disturbances in the corresponding area of 17 eyes. Fluorescein angiography performed in 18 patients showed varying degrees of hyperfluorescence and hypofluorescence related to a range of retinal pigment epithelium (RPE) alterations. Indocyanine green angiography performed in 7 patients showed hypofluorescence at the excavation. SD-OCT demonstrated choroidal excavation in all 37 eyes. Twenty-nine eyes showed a single lesion of FCE, and three eyes showed 2-3 separated lesions. Fifteen eyes showed separation between the photoreceptor tips and RPE consistent with nonconforming FCE. Central serous chorioretinopathy (CSC, n=1) and choroidal neovascularization (CNV, n=1) developed during follow-up. CONCLUSION FCE could be interpreted as congenital focal choroidal dysplasia involving the RPE, choriocapillaris, and photoreceptor associated with the faulty anatomy. The abnormal anatomy of FCE was similar to anatomy at risk of CSC and CNV.
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Affiliation(s)
- Guang-Hui Liu
- School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou 325000, Zhejiang Province, China ; Department of Ophthalmology, Affiliated People's Hospital to Fujian University of Traditional Chinese Medicine, Fuzhou 350004, Fujian Province, China
| | - Bing Lin
- School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou 325000, Zhejiang Province, China
| | - Xin-Quan Sun
- Department of Ophthalmology, China-Japan Friendship Hospital, Beijing 100029, China
| | - Zi-Fang He
- School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou 325000, Zhejiang Province, China
| | - Ji-Rong Li
- School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou 325000, Zhejiang Province, China
| | - Rong Zhou
- School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou 325000, Zhejiang Province, China
| | - Xiao-Ling Liu
- School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou 325000, Zhejiang Province, China
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Hartenstein V, Reh TA. Homologies between vertebrate and invertebrate eyes. Results Probl Cell Differ 2015; 37:219-55. [PMID: 25707078 DOI: 10.1007/978-3-540-45398-7_14] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/24/2023]
Affiliation(s)
- Volker Hartenstein
- Department of Biology, University of California, Los Angeles, California, USA
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40
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Fernández-Sánchez L, de Sevilla Müller LP, Brecha NC, Cuenca N. Loss of outer retinal neurons and circuitry alterations in the DBA/2J mouse. Invest Ophthalmol Vis Sci 2014; 55:6059-72. [PMID: 25118265 DOI: 10.1167/iovs.14-14421] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
PURPOSE The DBA/2J mouse line develops essential iris atrophy, pigment dispersion, and glaucomatous age-related changes, including an increase of IOP, optic nerve atrophy, and retinal ganglion cell (RGC) death. The aim of this study was to evaluate possible morphological changes in the outer retina of the DBA/2J mouse concomitant with disease progression and aging, based on the reduction of both the a- and b-waves and photopic flicker ERGs in this mouse line. METHODS Vertically sectioned DBA/2J mice retinas were evaluated at 3, 8, and 16 months of age using photoreceptor, horizontal, and bipolar cell markers. Sixteen-month-old C57BL/6 mice retinas were used as controls. RESULTS The DBA/2J mice had outer retinal degeneration at all ages, with the most severe degeneration in the oldest retinas. At 3 months of age, the number of photoreceptor cells and the thickness of the OPL were reduced. In addition, there was a loss of horizontal and ON-bipolar cell processes. At 8 months of age, RGC degeneration occurred in patches, and in the outer retina overlying these patches, cone morphology was impaired with a reduction in size as well as loss of outer segments and growth of horizontal and bipolar cell processes into the outer nuclear layer. At 16 months of age, connectivity between photoreceptors and horizontal and bipolar cell processes overlying these patches was lost. CONCLUSIONS Retinal degeneration in DBA/2J mice includes photoreceptor death, loss of bipolar and horizontal cell processes, and loss of synaptic contacts in an aging-dependent manner.
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Affiliation(s)
- Laura Fernández-Sánchez
- Department of Physiology, Genetics, and Microbiology, University of Alicante, San Vicente del Raspeig, Spain
| | - Luis Pérez de Sevilla Müller
- Department of Neurobiology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, California, United States
| | - Nicholas C Brecha
- Department of Neurobiology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, California, United States
| | - Nicolás Cuenca
- Department of Physiology, Genetics, and Microbiology, University of Alicante, San Vicente del Raspeig, Spain
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Generation of three-dimensional retinal tissue with functional photoreceptors from human iPSCs. Nat Commun 2014; 5:4047. [PMID: 24915161 PMCID: PMC4370190 DOI: 10.1038/ncomms5047] [Citation(s) in RCA: 672] [Impact Index Per Article: 61.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2013] [Accepted: 05/05/2014] [Indexed: 12/17/2022] Open
Abstract
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.
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Hägglund AC, Berghard A, Carlsson L. Canonical Wnt/β-catenin signalling is essential for optic cup formation. PLoS One 2013; 8:e81158. [PMID: 24324671 PMCID: PMC3852023 DOI: 10.1371/journal.pone.0081158] [Citation(s) in RCA: 49] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2013] [Accepted: 10/09/2013] [Indexed: 12/17/2022] Open
Abstract
A multitude of signalling pathways are involved in the process of forming an eye. Here we demonstrate that β-catenin is essential for eye development as inactivation of β-catenin prior to cellular specification in the optic vesicle caused anophthalmia in mice. By achieving this early and tissue-specific β-catenin inactivation we find that retinal pigment epithelium (RPE) commitment was blocked and eye development was arrested prior to optic cup formation due to a loss of canonical Wnt signalling in the dorsal optic vesicle. Thus, these results show that Wnt/β-catenin signalling is required earlier and play a more central role in eye development than previous studies have indicated. In our genetic model system a few RPE cells could escape β-catenin inactivation leading to the formation of a small optic rudiment. The optic rudiment contained several neural retinal cell classes surrounded by an RPE. Unlike the RPE cells, the neural retinal cells could be β-catenin-negative revealing that differentiation of the neural retinal cell classes is β-catenin-independent. Moreover, although dorsoventral patterning is initiated in the mutant optic vesicle, the neural retinal cells in the optic rudiment displayed almost exclusively ventral identity. Thus, β-catenin is required for optic cup formation, commitment to RPE cells and maintenance of dorsal identity of the retina.
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Affiliation(s)
| | - Anna Berghard
- Department of Molecular Biology, Umeå University, Umeå, Sweden
| | - Leif Carlsson
- Umeå Center for Molecular Medicine, Umeå University, Umeå, Sweden
- * E-mail:
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Nakamoto C, Kuan SL, Findlay AS, Durward E, Ouyang Z, Zakrzewska ED, Endo T, Nakamoto M. Nel positively regulates the genesis of retinal ganglion cells by promoting their differentiation and survival during development. Mol Biol Cell 2013; 25:234-44. [PMID: 24258025 PMCID: PMC3890344 DOI: 10.1091/mbc.e13-08-0453] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023] Open
Abstract
For correct functioning of the nervous system, the appropriate number and complement of neuronal cell types must be produced during development. However, the molecular mechanisms that regulate the production of individual classes of neurons are poorly understood. In this study, we investigate the function of the thrombospondin-1-like glycoprotein, Nel (neural epidermal growth factor [EGF]-like), in the generation of retinal ganglion cells (RGCs) in chicks. During eye development, Nel is strongly expressed in the presumptive retinal pigment epithelium and RGCs. Nel overexpression in the developing retina by in ovo electroporation increases the number of RGCs, whereas the number of displaced amacrine cells decreases. Conversely, knockdown of Nel expression by transposon-mediated introduction of RNA interference constructs results in decrease in RGC number and increase in the number of displaced amacrine cells. Modifications of Nel expression levels do not appear to affect proliferation of retinal progenitor cells, but they significantly alter the progression rate of RGC differentiation from the central retina to the periphery. Furthermore, Nel protects RGCs from apoptosis during retinal development. These results indicate that Nel positively regulates RGC production by promoting their differentiation and survival during development.
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Affiliation(s)
- Chizu Nakamoto
- Aberdeen Developmental Biology Group, School of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, United Kingdom Department of Neurosciences, Lerner Research Institute, Cleveland Clinic Foundation, and Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine, Case Western Reserve University, Cleveland, OH 44195
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Fuhrmann S, Zou C, Levine EM. Retinal pigment epithelium development, plasticity, and tissue homeostasis. Exp Eye Res 2013; 123:141-50. [PMID: 24060344 DOI: 10.1016/j.exer.2013.09.003] [Citation(s) in RCA: 180] [Impact Index Per Article: 15.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2013] [Revised: 09/05/2013] [Accepted: 09/07/2013] [Indexed: 12/13/2022]
Abstract
The retinal pigment epithelium (RPE) is a simple epithelium interposed between the neural retina and the choroid. Although only 1 cell-layer in thickness, the RPE is a virtual workhorse, acting in several capacities that are essential for visual function and preserving the structural and physiological integrities of neighboring tissues. Defects in RPE function, whether through chronic dysfunction or age-related decline, are associated with retinal degenerative diseases including age-related macular degeneration. As such, investigations are focused on developing techniques to replace RPE through stem cell-based methods, motivated primarily because of the seemingly limited regeneration or self-repair properties of mature RPE. Despite this, RPE cells have an unusual capacity to transdifferentiate into various cell types, with the particular fate choices being highly context-dependent. In this review, we describe recent findings elucidating the mechanisms and steps of RPE development and propose a developmental framework for understanding the apparent contradiction in the capacity for low self-repair versus high transdifferentiation.
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Affiliation(s)
- Sabine Fuhrmann
- Department of Ophthalmology & Visual Sciences, John A. Moran Eye Center, University of Utah, 65 Mario Capecchi Drive, Salt Lake City, UT 84132, USA.
| | - ChangJiang Zou
- Department of Ophthalmology & Visual Sciences, John A. Moran Eye Center, University of Utah, 65 Mario Capecchi Drive, Salt Lake City, UT 84132, USA.
| | - Edward M Levine
- Department of Ophthalmology & Visual Sciences, John A. Moran Eye Center, University of Utah, 65 Mario Capecchi Drive, Salt Lake City, UT 84132, USA.
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Nasonkin IO, Merbs SL, Lazo K, Oliver VF, Brooks M, Patel K, Enke RA, Nellissery J, Jamrich M, Le YZ, Bharti K, Fariss RN, Rachel RA, Zack DJ, Rodriguez-Boulan EJ, Swaroop A. Conditional knockdown of DNA methyltransferase 1 reveals a key role of retinal pigment epithelium integrity in photoreceptor outer segment morphogenesis. Development 2013; 140:1330-41. [PMID: 23406904 DOI: 10.1242/dev.086603] [Citation(s) in RCA: 65] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Dysfunction or death of photoreceptors is the primary cause of vision loss in retinal and macular degenerative diseases. As photoreceptors have an intimate relationship with the retinal pigment epithelium (RPE) for exchange of macromolecules, removal of shed membrane discs and retinoid recycling, an improved understanding of the development of the photoreceptor-RPE complex will allow better design of gene- and cell-based therapies. To explore the epigenetic contribution to retinal development we generated conditional knockout alleles of DNA methyltransferase 1 (Dnmt1) in mice. Conditional Dnmt1 knockdown in early eye development mediated by Rx-Cre did not produce lamination or cell fate defects, except in cones; however, the photoreceptors completely lacked outer segments despite near normal expression of phototransduction and cilia genes. We also identified disruption of RPE morphology and polarization as early as E15.5. Defects in outer segment biogenesis were evident with Dnmt1 exon excision only in RPE, but not when excision was directed exclusively to photoreceptors. We detected a reduction in DNA methylation of LINE1 elements (a measure of global DNA methylation) in developing mutant RPE as compared with neural retina, and of Tuba3a, which exhibited dramatically increased expression in mutant retina. These results demonstrate a unique function of DNMT1-mediated DNA methylation in controlling RPE apicobasal polarity and neural retina differentiation. We also establish a model to study the epigenetic mechanisms and signaling pathways that guide the modulation of photoreceptor outer segment morphogenesis by RPE during retinal development and disease.
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Affiliation(s)
- Igor O Nasonkin
- 1Neurobiology-Neurodegeneration and Repair Laboratory (N-NRL), National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA
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Schmidt K, Cavodeassi F, Feng Y, Stephens DJ. Early stages of retinal development depend on Sec13 function. Biol Open 2013; 2:256-66. [PMID: 23519012 PMCID: PMC3603407 DOI: 10.1242/bio.20133251] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2012] [Accepted: 11/22/2012] [Indexed: 01/17/2023] Open
Abstract
ER-to-Golgi transport of proteins destined for the extracellular space or intracellular compartments depends on the COPII vesicle coat and is constitutive in all translationally active cells. Nevertheless, there is emerging evidence that this process is regulated on a cell- and tissue-specific basis, which means that components of the COPII coat will be of differential importance to certain cell types. The COPII coat consists of an inner layer, Sec23/24 and an outer shell, Sec13/31. We have shown previously that knock-down of Sec13 results in concomitant loss of Sec31. In zebrafish and cultured human cells this leads to impaired trafficking of large cargo, namely procollagens, and is causative for defects in craniofacial and gut development. It is now widely accepted that the outer COPII coat is key to the architecture and stability of ER export vesicles containing large, unusual cargo proteins. Here, we investigate zebrafish eye development following Sec13 depletion. We find that photoreceptors degenerate or fail to develop from the onset. Impaired collagen trafficking from the retinal pigment epithelium and defects in overall retinal lamination also seen in Sec13-depleted zebrafish might have been caused by increased apoptosis and reduced topical proliferation in the retina. Our data show that the outer layer of the COPII coat is also necessary for the transport of large amounts of cargo proteins, in this case rhodopsin, rather than just large cargo as previously thought.
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Affiliation(s)
- Katy Schmidt
- Cell Biology Laboratories, School of Biochemistry, Medical Sciences Building, University of Bristol, University Walk , Bristol BS8 1TD , UK ; Present address: Max F. Perutz Laboratories, University of Vienna and Medical University of Vienna, Dr-Bohr-Gasse 9/3, 1030 Wien, Austria
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Eberle D, Kurth T, Santos-Ferreira T, Wilson J, Corbeil D, Ader M. Outer segment formation of transplanted photoreceptor precursor cells. PLoS One 2012; 7:e46305. [PMID: 23029471 PMCID: PMC3460822 DOI: 10.1371/journal.pone.0046305] [Citation(s) in RCA: 54] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2012] [Accepted: 08/30/2012] [Indexed: 12/30/2022] Open
Abstract
Transplantation of photoreceptor precursor cells (PPCs) into the retina represents a promising treatment for cell replacement in blinding diseases characterized by photoreceptor loss. In preclinical studies, we and others demonstrated that grafted PPCs integrate into the host outer nuclear layer (ONL) and develop into mature photoreceptors. However, a key feature of light detecting photoreceptors, the outer segment (OS) with natively aligned disc membrane staples, has not been studied in detail following transplantation. Therefore, we used as donor cells PPCs isolated from neonatal double transgenic reporter mice in which OSs are selectively labeled by green fluorescent protein while cell bodies are highlighted by red fluorescent protein. PPCs were enriched using CD73-based magnetic associated cell sorting and subsequently transplanted into either adult wild-type or a model of autosomal-dominant retinal degeneration mice. Three weeks post-transplantation, donor photoreceptors were identified based on fluorescent-reporter expression and OS formation was monitored at light and electron microscopy levels. Donor cells that properly integrated into the host wild-type retina developed OSs with the formation of a connecting cilium and well-aligned disc membrane staples similar to the surrounding native cells of the host. Surprisingly, the majority of not-integrated PPCs that remained in the sub-retinal space also generated native-like OSs in wild-type mice and those affected by retinal degeneration. Moreover, they showed an improved photoreceptor maturation and OS formation by comparison to donor cells located on the vitreous side suggesting that environmental cues influence the PPC differentiation and maturation. We conclude that transplanted PPCs, whether integrated or not into the host ONL, are able to generate the cellular structure for effective light detection, a phenomenon observed in wild-type as well as in degenerated retinas. Given that patients suffering from retinitis pigmentosa lose almost all photoreceptors, our findings are of utmost importance for the development of cell-based therapies.
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Affiliation(s)
- Dominic Eberle
- DFG-Center for Regenerative Therapies Dresden (CRTD), Cluster of Excellence/TU-Dresden, Dresden, Germany
| | - Thomas Kurth
- DFG-Center for Regenerative Therapies Dresden (CRTD), Cluster of Excellence/TU-Dresden, Dresden, Germany
| | - Tiago Santos-Ferreira
- DFG-Center for Regenerative Therapies Dresden (CRTD), Cluster of Excellence/TU-Dresden, Dresden, Germany
| | - John Wilson
- Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, United States of America
| | - Denis Corbeil
- DFG-Center for Regenerative Therapies Dresden (CRTD), Cluster of Excellence/TU-Dresden, Dresden, Germany
- Biotechnology Center, TU-Dresden, Dresden, Germany
| | - Marius Ader
- DFG-Center for Regenerative Therapies Dresden (CRTD), Cluster of Excellence/TU-Dresden, Dresden, Germany
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Abstract
The original hedgehog (hh) gene was found in Drosophila and named for the appearance of a mutant phenotype which causes an embryo to be covered with pointy denticles, thus resembling a hedgehog. The hedgehog family consists of sonic hedgehog (Shh), desert hedgehog (Dhh), and Indian hedgehog (Ihh). Shh is found in vertebrates and the best studied ligand of the hedgehog signaling pathway (Gilbert, 2000). It plays an important role in regulating vertebrate organogenesis, such as in the growth of digits on limbs and organization of the brain, and earlier studies also show that it is important during retinal development (for a review, see Amato et al., 2004). Hedgehog expression drives waves of neurogenesis in animal retina, although genetic programs that control its expression are poorly elucidated. Recently, a novel transcriptional cascade which involves the atonal and Iroquois gene family was proposed in the regulation of hedgehog waves during vertebrate retinal development (Choy et al., 2010). This chapter will focus on Shh by addressing its signaling mechanisms and roles in vertebrate eye development, as well as a novel finding in retinogenesis.
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Lange CAK, Luhmann UFO, Mowat FM, Georgiadis A, West EL, Abrahams S, Sayed H, Powner MB, Fruttiger M, Smith AJ, Sowden JC, Maxwell PH, Ali RR, Bainbridge JWB. Von Hippel-Lindau protein in the RPE is essential for normal ocular growth and vascular development. Development 2012; 139:2340-50. [PMID: 22627278 DOI: 10.1242/dev.070813] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023]
Abstract
Molecular oxygen is essential for the development, growth and survival of multicellular organisms. Hypoxic microenvironments and oxygen gradients are generated physiologically during embryogenesis and organogenesis. In the eye, oxygen plays a crucial role in both physiological vascular development and common blinding diseases. The retinal pigment epithelium (RPE) is a monolayer of cells essential for normal ocular development and in the mature retina provides support for overlying photoreceptors and their vascular supply. Hypoxia at the level of the RPE is closely implicated in pathogenesis of age-related macular degeneration. Adaptive tissue responses to hypoxia are orchestrated by sophisticated oxygen sensing mechanisms. In particular, the von Hippel-Lindau tumour suppressor protein (pVhl) controls hypoxia-inducible transcription factor (HIF)-mediated adaptation. However, the role of Vhl/Hif1a in the RPE in the development of the eye and its vasculature is unknown. In this study we explored the function of Vhl and Hif1a in the developing RPE using a tissue-specific conditional-knockout approach. We found that deletion of Vhl in the RPE results in RPE apoptosis, aniridia and microphthalmia. Increased levels of Hif1a, Hif2a, Epo and Vegf are associated with a highly disorganised retinal vasculature, chorioretinal anastomoses and the persistence of embryonic vascular structures into adulthood. Additional inactivation of Hif1a in the RPE rescues the RPE morphology, aniridia, microphthalmia and anterior vasoproliferation, but does not rescue retinal vasoproliferation. These data demonstrate that Vhl-dependent regulation of Hif1a in the RPE is essential for normal RPE and iris development, ocular growth and vascular development in the anterior chamber, whereas Vhl-dependent regulation of other downstream pathways is crucial for normal development and maintenance of the retinal vasculature.
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Affiliation(s)
- Clemens A K Lange
- Department of Genetics, Institute of Ophthalmology, NIHR Biomedical Research Centre for Ophthalmology, University College London, London EC1V 9EL, UK
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Huang C, Zhang J, Ao M, Li Y, Zhang C, Xu Y, Li X, Wang W. Combination of retinal pigment epithelium cell-conditioned medium and photoreceptor outer segments stimulate mesenchymal stem cell differentiation toward a functional retinal pigment epithelium cell phenotype. J Cell Biochem 2012; 113:590-8. [PMID: 21948619 DOI: 10.1002/jcb.23383] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Abstract
Recent studies have suggested that bone marrow-derived mesenchymal stem cells (BMMSCs) are capable of retinal tissue-specific differentiation but not retinal pigment epithelium (RPE) cell-specific differentiation. Photoreceptor outer segments (POS) contribute to RPE development and maturation. However, there has been no standard culture system that fosters the differentiation of BMMSCs into mature RPE cells in vitro. In this study, we investigated if the soluble factors from RPE cells and POS could differentiate BMMSCs into cells having a phenotype characteristic of RPE cells. Rat BMMSCs were separately co-cultured with RPE cells, or they were exposed to either control medium, RPE cell-conditioned medium (RPECM), POS, or a combination of RPECM and POS (RPECM-POS). After 7 days, the cells were analyzed for morphology and the expression of RPE markers (cytokeratin 8, CRALBP, and RPE65) to assess the RPE differentiation. Significantly higher pigment accumulation and increased protein expression of the three markers were seen in cells cultured in RPECM-POS than in other treated cultures. Furthermore, the RPECM-POS-treated cultures displayed ultrastructural features typical of RPE cells, expressed RPE cell functional proteins, and had the capability to phagocytose POS. Together, theses results suggest the combination of RPECM and POS stimulate BMMSCs differentiation toward a functional RPE phenotype. Our results provide the foundation for a new route to RPE regenerative therapy involving BMMSCs. Future work isolating the active agent in RPECM and POS would be useful in therapies for RPE diseases or in developing appropriately pre-differentiated BMMSCs for tissue-engineered RPE reconstruction.
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Affiliation(s)
- Chen Huang
- Department of Ophthalmology, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191, China
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