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Izrael M, Chebath J, Molakandov K, Revel M. Clinical perspective on pluripotent stem cells derived cell therapies for the treatment of neurodegenerative diseases. Adv Drug Deliv Rev 2025; 218:115525. [PMID: 39880333 DOI: 10.1016/j.addr.2025.115525] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2024] [Revised: 01/09/2025] [Accepted: 01/26/2025] [Indexed: 01/31/2025]
Abstract
Self-renewal capacity and potential to differentiate into almost any cell type of the human body makes pluripotent stem cells a valuable starting material for manufacturing of clinical grade cell therapies. Neurodegenerative diseases are characterized by gradual loss of structure or function of neurons, often leading to neuronal death. This results in gradual decline of cognitive, motor, and physiological functions due to the degeneration of the central nervous systems. Over the past two decades, comprehensive preclinical efficacy (proof-of-concept) and safety studies have led to the initiation of First-in-Human phase I-II clinical trials for a range of neurodegenerative diseases. In this review, we explore the fundamentals and challenges of neural-cell therapies derived from pluripotent stem cells for treating neurodegenerative diseases. Additionally, we highlight key preclinical investigations that paved the way for regulatory approvals of these trials. Furthermore, we provide an overview on progress and status of clinical trials done so far in treating neurodegenerative diseases such as spinal cord injury (SCI), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS), as well as advances in retina diseases such as Stargardt disease (a.k.a fundus flavimaculatus), retinitis pigmentosa (RP) and age-related macular degeneration (AMD). These trials will pave the way for the development of new cell-based therapies targeting additional neurological conditions, including Alzheimer's disease and epilepsy.
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Affiliation(s)
- Michal Izrael
- Neurodegenerative Diseases Department, Kadimastem Ltd, Pinchas Sapir 7, Weizmann Science Park, Ness-Ziona, Israel.
| | - Judith Chebath
- Neurodegenerative Diseases Department, Kadimastem Ltd, Pinchas Sapir 7, Weizmann Science Park, Ness-Ziona, Israel
| | - Kfir Molakandov
- Neurodegenerative Diseases Department, Kadimastem Ltd, Pinchas Sapir 7, Weizmann Science Park, Ness-Ziona, Israel
| | - Michel Revel
- Neurodegenerative Diseases Department, Kadimastem Ltd, Pinchas Sapir 7, Weizmann Science Park, Ness-Ziona, Israel; Department of Molecular Genetics, Weizmann Institute of Science, 76100, Rehovot, Israel
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Flores-Bellver M, Canto-Soler MV. Generation of Induced-Primary Retinal Pigment Epithelium from Human Retinal Organoids. Methods Mol Biol 2025; 2848:197-214. [PMID: 39240525 DOI: 10.1007/978-1-0716-4087-6_13] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/07/2024]
Abstract
Retinal pigment epithelium (RPE) cells derived from induced pluripotent stem cells (iPSCs) serve multiple roles, including among others, modeling RPE development in normal and pathological conditions, investigating mechanisms of RPE physiology, modeling retinal diseases involving the RPE, and developing strategies for regenerative therapies. We have developed a simple and efficient protocol to generate RPE tissue from human iPSCs-derived retinal organoids. The RPE tissue present in the retinal organoids is analogous to the native human RPE in differentiation timeline, histological organization, and key features of functional maturation. Building upon this system, we established a method to generate functionally mature, polarized RPE monolayers comparable to human primary RPE. This comprehensive protocol outlines the steps for isolating and culturing RPE tissue using retinal organoids. The outcome is a pure population of cells expressing mature RPE signatures and organized in a characteristic cobblestone monolayer featuring robust ultrastructural polarization. These RPE monolayers also exhibit the functional hallmarks of bona fide mature RPE cells, providing a suitable system to mimic the biology and function of the native human RPE.
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Affiliation(s)
- Miguel Flores-Bellver
- CellSight Ocular Stem Cell and Regeneration Research Program, Department of Ophthalmology, Sue Anschutz-Rodgers Eye Center, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.
| | - M Valeria Canto-Soler
- CellSight Ocular Stem Cell and Regeneration Research Program, Department of Ophthalmology, Sue Anschutz-Rodgers Eye Center, University of Colorado Anschutz Medical Campus, Aurora, CO, USA
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Sen S, de Guimaraes TAC, Filho AG, Fabozzi L, Pearson RA, Michaelides M. Stem cell-based therapies for retinal diseases: focus on clinical trials and future prospects. Ophthalmic Genet 2024:1-14. [PMID: 39544140 DOI: 10.1080/13816810.2024.2423784] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2024] [Revised: 10/09/2024] [Accepted: 10/26/2024] [Indexed: 11/17/2024]
Abstract
Stem cell-based therapy has gained importance over the past decades due to huge advances in science and technology behind the generation and directed differentiation of pluripotent cells from embryos and adult cells. Preclinical proof-of-concept studies have been followed by clinical trials showing efficacy and safety of transplantation of stem cell-based therapy, which are beginning to establish this as a modality of treatment. Disease candidates of interest are primarily conditions that may benefit from replacing dead or dying cells, including advanced inherited retinal dystrophies and age-related macular degeneration, and predominantly seek to transplant either RPE or photoreceptors, although neurotrophic approaches have also been trialed. Whilst a consensus has yet to be reached about the best stage/type of cells for transplantation (stem cells, progenitor cells, differentiated RPE and photoreceptors) and the methods of implantation (sheet, suspension), several CTs have shown safety. There remain potential concerns regarding tumorigenicity and immune rejection; however, with ongoing improvements in cell generation, selection, and delivery, these can be minimized. Earlier studies showed efficacy with immunosuppressive drugs to prevent rejection, and recent donor-matched transplants have avoided the need for immunosuppression. Retinal regenerative medicine is a challenging field and is in a nascent stage but holds tremendous promise. This narrative review delves into the current understanding of stem cells and the latest clinical trials of retinal cell transplantation.
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Affiliation(s)
- Sagnik Sen
- Deaprtment of Genetics, Moorfields Eye Hospital, London, UK
- UCL Institute of Ophthalmology, University College London, London, UK
| | | | | | | | - Rachael A Pearson
- Ocular Cell and Gene Therapy Group, Centre for Gene Therapy and Regenerative Medicine, King's College London, London, UK
| | - Michel Michaelides
- Deaprtment of Genetics, Moorfields Eye Hospital, London, UK
- UCL Institute of Ophthalmology, University College London, London, UK
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Abbasi N, O'Neill H. Cytocompatibility of electrospun poly-L-lactic acid membranes for Bruch's membrane regeneration using human embryonic stem cell-derived retinal pigment epithelial cells. J Biomed Mater Res A 2024; 112:1902-1920. [PMID: 38726752 DOI: 10.1002/jbm.a.37736] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2023] [Revised: 04/11/2024] [Accepted: 04/30/2024] [Indexed: 09/03/2024]
Abstract
Cell replacement therapy is under development for dry age-related macular degeneration (AMD). A thin membrane resembling the Bruch's membrane is required to form a cell-on-membrane construct with retinal pigment epithelial (RPE) cells. These cells have been differentiated from human embryonic stem cells (hESCs) in vitro. A carrier membrane is required for cell implantation, which is biocompatible for cell growth and has dimensions and physical properties resembling the Bruch's membrane. Here a nanofiber electrospun poly-L-lactic acid (PLLA) membrane is tested for capacity to support cell growth and maturation. The requirements for laminin coating of the membrane are identified here. A porous electrospun nanofibrous PLLA membrane of ∼50 nm fiber diameter was developed as a prototype support for functional RPE cells grown as a monolayer. The need for laminin coating applied to the membrane following treatment with poly-L-ornithine (PLO), was identified in terms of cell growth and survival. Test membranes were compared in terms of hydrophilicity after laminin coating, mechanical properties of surface roughness and Young's modulus, porosity and ability to promote the attachment and proliferation of hESC-RPE cells in culture for up to 8 weeks. Over this time, RPE cell proliferation, morphology, and marker and gene expression, were monitored. The functional capacity of cell monolayers was identified in terms of transepithelial electrical resistance (TEER), phagocytosis of cells, as well as expression of the cytokines, vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF). PLLA polymer fibers are naturally hydrophobic, so their hydrophilicity was improved by pretreatment with PLO for subsequent coating with the bioactive protein laminin. They were then assessed for amount of laminin adsorbed, contact angle and uniformity of coating using scanning electron microscopy (SEM). Pretreatment with 100% PLO gave the best result over 10% PLO treatment or no treatment prior to laminin adsorption with significantly greater surface stiffness and modulus. By 6 weeks after cell plating, the coated membranes could support a mature RPE monolayer showing a dense apical microvillus structure and pigmented 3D polygonal cell morphology. After 8 weeks, PLO (100%)-Lam coated membranes exhibited the highest cell number, cell proliferation, and RPE barrier function measured as TEER. RPE cells showed the higher levels of specific surface marker and gene expression. Microphthalmia-associated transcription factor expression was highly upregulated indicating maturation of cells. Functionality of cells was indicated by expression of VEGF and PEDF genes as well as phagocytic capacity. In conclusion, electrospun PLLA membranes coated with PLO-Lam have the physical and biological properties to support the distribution and migration of hESC-RPE cells throughout the whole structure. They represent a good membrane candidate for preparation of hESC-RPE cells as a monolayer for implantation into the subretinal space of AMD patients.
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Affiliation(s)
- Naghmeh Abbasi
- Clem Jones Centre for Regenerative Medicine, Faculty of Health Sciences & Medicine, Bond University, Gold Coast, Queensland, Australia
| | - Helen O'Neill
- Clem Jones Centre for Regenerative Medicine, Faculty of Health Sciences & Medicine, Bond University, Gold Coast, Queensland, Australia
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DiCesare SM, Ortega AJ, Collier GE, Daniel S, Thompson KN, McCoy MK, Posner BA, Hulleman JD. GSK3 inhibition reduces ECM production and prevents age-related macular degeneration-like pathology. JCI Insight 2024; 9:e178050. [PMID: 39114980 PMCID: PMC11383595 DOI: 10.1172/jci.insight.178050] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2023] [Accepted: 06/20/2024] [Indexed: 08/22/2024] Open
Abstract
Malattia Leventinese/Doyne honeycomb retinal dystrophy (ML/DHRD) is an age-related macular degeneration-like (AMD-like) retinal dystrophy caused by an autosomal dominant R345W mutation in the secreted glycoprotein, fibulin-3 (F3). To identify new small molecules that reduce F3 production in retinal pigmented epithelium (RPE) cells, we knocked-in a luminescent peptide tag (HiBiT) into the endogenous F3 locus that enabled simple, sensitive, and high-throughput detection of the protein. The GSK3 inhibitor, CHIR99021 (CHIR), significantly reduced F3 burden (expression, secretion, and intracellular levels) in immortalized RPE and non-RPE cells. Low-level, long-term CHIR treatment promoted remodeling of the RPE extracellular matrix, reducing sub-RPE deposit-associated proteins (e.g., amelotin, complement component 3, collagen IV, and fibronectin), while increasing RPE differentiation factors (e.g., tyrosinase, and pigment epithelium-derived factor). In vivo, treatment of 8-month-old R345W+/+ knockin mice with CHIR (25 mg/kg i.p., 1 mo) was well tolerated and significantly reduced R345W F3-associated AMD-like basal laminar deposit number and size, thereby preventing the main pathological feature in these mice. This is an important demonstration of small molecule-based prevention of AMD-like pathology in ML/DHRD mice and may herald a rejuvenation of interest in GSK3 inhibition for the treatment of retinal degenerative diseases, including potentially AMD itself.
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Affiliation(s)
- Sophia M. DiCesare
- Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, Texas, USA
| | - Antonio J. Ortega
- Department of Ophthalmology and Visual Neurosciences, University of Minnesota, Minneapolis, Minnesota, USA
| | - Gracen E. Collier
- Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, Texas, USA
| | - Steffi Daniel
- Department of Ophthalmology and Visual Neurosciences, University of Minnesota, Minneapolis, Minnesota, USA
| | - Krista N. Thompson
- Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, Texas, USA
| | - Melissa K. McCoy
- Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, USA
| | - Bruce A. Posner
- Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, USA
| | - John D. Hulleman
- Department of Ophthalmology and Visual Neurosciences, University of Minnesota, Minneapolis, Minnesota, USA
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Niu Y, Ji J, Yao K, Fu Q. Regenerative treatment of ophthalmic diseases with stem cells: Principles, progress, and challenges. ADVANCES IN OPHTHALMOLOGY PRACTICE AND RESEARCH 2024; 4:52-64. [PMID: 38586868 PMCID: PMC10997875 DOI: 10.1016/j.aopr.2024.02.001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/26/2023] [Revised: 02/08/2024] [Accepted: 02/20/2024] [Indexed: 04/09/2024]
Abstract
Background Degenerate eye disorders, such as glaucoma, cataracts and age-related macular degeneration (AMD), are prevalent causes of blindness and visual impairment worldwide. Other eye disorders, including limbal stem cell deficiency (LSCD), dry eye diseases (DED), and retinitis pigmentosa (RP), result in symptoms such as ocular discomfort and impaired visual function, significantly impacting quality of life. Traditional therapies are limited, primarily focus on delaying disease progression, while emerging stem cell therapy directly targets ocular tissues, aiming to restore ocular function by reconstructing ocular tissue. Main text The utilization of stem cells for the treatment of diverse degenerative ocular diseases is becoming increasingly significant, owing to the regenerative and malleable properties of stem cells and their functional cells. Currently, stem cell therapy for ophthalmopathy involves various cell types, such as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), and retinal progenitor cells (RPCs). In the current article, we will review the current progress regarding the utilization of stem cells for the regeneration of ocular tissue covering key eye tissues from the cornea to the retina. These therapies aim to address the loss of functional cells, restore damaged ocular tissue and or in a paracrine-mediated manner. We also provide an overview of the ocular disorders that stem cell therapy is targeting, as well as the difficulties and opportunities in this field. Conclusions Stem cells can not only promote tissue regeneration but also release exosomes to mitigate inflammation and provide neuroprotection, making stem cell therapy emerge as a promising approach for treating a wide range of eye disorders through multiple mechanisms.
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Affiliation(s)
- Yifei Niu
- Eye Center of the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, China
- Zhejiang Provincial Key Lab of Ophthalmology, Hangzhou, Zhejiang Province, China
| | - Junfeng Ji
- Center of Stem Cell and Regenerative Medicine, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, China
- Zhejiang Provincial Key Laboratory of Tissue Engineering and Regenerative Medicine, Hangzhou, China
| | - Ke Yao
- Eye Center of the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, China
- Zhejiang Provincial Key Lab of Ophthalmology, Hangzhou, Zhejiang Province, China
| | - Qiuli Fu
- Eye Center of the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, China
- Zhejiang Provincial Key Lab of Ophthalmology, Hangzhou, Zhejiang Province, China
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Baqué-Vidal L, Main H, Petrus-Reurer S, Lederer AR, Beri NE, Bär F, Metzger H, Zhao C, Efstathopoulos P, Saietz S, Wrona A, Jaberi E, Willenbrock H, Reilly H, Hedenskog M, Moussaud-Lamodière E, Kvanta A, Villaescusa JC, La Manno G, Lanner F. Clinically compliant cryopreservation of differentiated retinal pigment epithelial cells. Cytotherapy 2024; 26:340-350. [PMID: 38349309 DOI: 10.1016/j.jcyt.2024.01.014] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2023] [Revised: 01/29/2024] [Accepted: 01/31/2024] [Indexed: 04/07/2024]
Abstract
BACKGROUND AIMS Age-related macular degeneration (AMD) is the most common cause of blindness in elderly patients within developed countries, affecting more than 190 million worldwide. In AMD, the retinal pigment epithelial (RPE) cell layer progressively degenerates, resulting in subsequent loss of photoreceptors and ultimately vision. There is currently no cure for AMD, but therapeutic strategies targeting the complement system are being developed to slow the progression of the disease. METHODS Replacement therapy with pluripotent stem cell-derived (hPSC) RPEs is an alternative treatment strategy. A cell therapy product must be produced in accordance with Good Manufacturing Practices at a sufficient scale to facilitate extensive pre-clinical and clinical testing. Cryopreservation of the final cell product is therefore highly beneficial, as the manufacturing, pre-clinical and clinical testing can be separated in time and location. RESULTS We found that mature hPSC-RPE cells do not survive conventional cryopreservation techniques. However, replating the cells 2-5 days before cryopreservation facilitates freezing. The replated and cryopreserved hPSC-RPE cells maintained their identity, purity and functionality as characteristic RPEs, shown by cobblestone morphology, pigmentation, transcriptional profile, RPE markers, transepithelial resistance and pigment epithelium-derived factor secretion. Finally, we showed that the optimal replating time window can be tracked noninvasively by following the change in cobblestone morphology. CONCLUSIONS The possibility of cryopreserving the hPSC-RPE product has been instrumental in our efforts in manufacturing and performing pre-clinical testing with the aim for clinical translation.
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Affiliation(s)
- Laura Baqué-Vidal
- Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, Stockholm, Sweden; Division of Obstetrics and Gynecology, Karolinska Universitetssjukhuset, Stockholm, Sweden
| | - Heather Main
- Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, Stockholm, Sweden; Division of Obstetrics and Gynecology, Karolinska Universitetssjukhuset, Stockholm, Sweden
| | - Sandra Petrus-Reurer
- Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, Stockholm, Sweden; Division of Obstetrics and Gynecology, Karolinska Universitetssjukhuset, Stockholm, Sweden; Department of Clinical Neuroscience, Division of Eye and Vision, St. Erik Eye Hospital, Karolinska Institutet, Stockholm, Sweden; Department of Surgery, University of Cambridge, NIHR Cambridge Biomedical Research Centre, Cambridge, UK
| | - Alex R Lederer
- Laboratory of Neurodevelopmental Systems Biology, Brain Mind Institute, School of Life Sciences, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland
| | - Nefeli-Eirini Beri
- Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, Stockholm, Sweden; Division of Obstetrics and Gynecology, Karolinska Universitetssjukhuset, Stockholm, Sweden
| | - Frederik Bär
- Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, Stockholm, Sweden; Division of Obstetrics and Gynecology, Karolinska Universitetssjukhuset, Stockholm, Sweden
| | - Hugo Metzger
- Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, Stockholm, Sweden; Division of Obstetrics and Gynecology, Karolinska Universitetssjukhuset, Stockholm, Sweden
| | - Cheng Zhao
- Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, Stockholm, Sweden; Division of Obstetrics and Gynecology, Karolinska Universitetssjukhuset, Stockholm, Sweden
| | | | - Sarah Saietz
- Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, Stockholm, Sweden; Division of Obstetrics and Gynecology, Karolinska Universitetssjukhuset, Stockholm, Sweden
| | | | - Elham Jaberi
- Cell Therapy R&D, Novo Nordisk A/S, Måløv, Denmark
| | | | - Hazel Reilly
- Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, Stockholm, Sweden; Division of Obstetrics and Gynecology, Karolinska Universitetssjukhuset, Stockholm, Sweden
| | - Mona Hedenskog
- Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, Stockholm, Sweden; Division of Obstetrics and Gynecology, Karolinska Universitetssjukhuset, Stockholm, Sweden
| | - Elisabeth Moussaud-Lamodière
- Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, Stockholm, Sweden; Division of Obstetrics and Gynecology, Karolinska Universitetssjukhuset, Stockholm, Sweden
| | - Anders Kvanta
- Department of Clinical Neuroscience, Division of Eye and Vision, St. Erik Eye Hospital, Karolinska Institutet, Stockholm, Sweden
| | | | - Gioele La Manno
- Laboratory of Neurodevelopmental Systems Biology, Brain Mind Institute, School of Life Sciences, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland
| | - Fredrik Lanner
- Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, Stockholm, Sweden; Division of Obstetrics and Gynecology, Karolinska Universitetssjukhuset, Stockholm, Sweden; Ming Wai Lau Center for Reparative Medicine, Karolinska Institutet, Stockholm, Sweden.
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Pollalis D, Calle AG, Martinez-Camarillo JC, Ahluwalia K, Hinman C, Mitra D, Lebkowski J, Lee SY, Thomas BB, Ahmed F, Chan V, Junge JA, Fraser S, Louie S, Humayun M. Scaling up polarized RPE cell supernatant production on parylene membrane. Exp Eye Res 2024; 240:109789. [PMID: 38242423 DOI: 10.1016/j.exer.2024.109789] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2023] [Revised: 01/09/2024] [Accepted: 01/10/2024] [Indexed: 01/21/2024]
Abstract
Age-related macular degeneration (AMD), a leading cause of vision loss, primarily arises from the degeneration of retinal pigment epithelium (RPE) and photoreceptors. Current therapeutic options for dry AMD are limited. Encouragingly, cultured RPE cells on parylene-based biomimetic Bruch's membrane demonstrate characteristics akin to the native RPE layer. In this study, we cultivated human embryonic stem cell-derived polarized RPE (hESC-PRPE) cells on parylene membranes at both small- and large-scale settings, collecting conditioned supernatant, denoted as PRPE-SF. We conducted a comprehensive analysis of the morphology of the cultured hESC-RPE cells and the secreted growth factors in PRPE-SF. To evaluate the in vivo efficacy of these products, the product was administered via intravitreal injections of PRPE-SF in immunodeficient Royal College of Surgeons (iRCS) rats, a model for retinal degeneration. Our study not only demonstrated the scalability of PRPE-SF production while maintaining RPE cell phenotype but also showed consistent protein concentrations between small- and large-scale batches. We consistently identified 10 key factors in PRPE-SF, including BMP-7, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6, MANF, PEDF, PDGF-AA, TGFβ1, and VEGF. Following intravitreal administration of PRPE-SF, we observed a significant increase in the thickness of the outer nuclear layer (ONL) and photoreceptor preservation in iRCS rats. Furthermore, correlation analysis revealed that IGFBP-3, IGFBP-4, MANF, PEDF, and TGFβ1 displayed positive associations with in vivo bioactivity, while GDF-15 exhibited a negative correlation. Overall, this study highlights the feasibility of scaling up PRPE-SF production on parylene membranes without compromising its essential constituents. The outcomes of PRPE-SF administration in an animal model of retinal degeneration present substantial potential for photoreceptor preservation. Moreover, the identification of candidate surrogate potency markers, showing strong positive associations with in vivo bioactivity, lays a solid foundation for the development of a promising therapeutic intervention for retinal degenerative diseases.
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Affiliation(s)
- Dimitrios Pollalis
- USC Roski Eye Institute, Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA; USC Ginsburg Institute for Biomedical Therapeutics, University of Southern California, Los Angeles, CA 90033, USA
| | - Alejandra Gonzalez Calle
- USC Roski Eye Institute, Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA; USC Ginsburg Institute for Biomedical Therapeutics, University of Southern California, Los Angeles, CA 90033, USA
| | - Juan Carlos Martinez-Camarillo
- USC Roski Eye Institute, Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA; USC Ginsburg Institute for Biomedical Therapeutics, University of Southern California, Los Angeles, CA 90033, USA
| | - Kabir Ahluwalia
- USC Ginsburg Institute for Biomedical Therapeutics, University of Southern California, Los Angeles, CA 90033, USA; USC Mann School of Pharmacy and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90089, USA
| | - Cassidy Hinman
- USC Ginsburg Institute for Biomedical Therapeutics, University of Southern California, Los Angeles, CA 90033, USA
| | - Debbie Mitra
- USC Ginsburg Institute for Biomedical Therapeutics, University of Southern California, Los Angeles, CA 90033, USA
| | - Jane Lebkowski
- Regenerative Patch Technologies LLC, Menlo Park, CA 94028, USA
| | - Sun Young Lee
- USC Roski Eye Institute, Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA; USC Ginsburg Institute for Biomedical Therapeutics, University of Southern California, Los Angeles, CA 90033, USA
| | - Biju B Thomas
- USC Ginsburg Institute for Biomedical Therapeutics, University of Southern California, Los Angeles, CA 90033, USA
| | - Faizah Ahmed
- USC Ginsburg Institute for Biomedical Therapeutics, University of Southern California, Los Angeles, CA 90033, USA
| | - Victoria Chan
- USC Ginsburg Institute for Biomedical Therapeutics, University of Southern California, Los Angeles, CA 90033, USA
| | - Jason A Junge
- Translational Imaging Center, University of Southern California, Los Angeles, CA 90089, USA
| | - Scott Fraser
- Translational Imaging Center, University of Southern California, Los Angeles, CA 90089, USA
| | - Stan Louie
- USC Ginsburg Institute for Biomedical Therapeutics, University of Southern California, Los Angeles, CA 90033, USA; USC Mann School of Pharmacy and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90089, USA
| | - Mark Humayun
- USC Roski Eye Institute, Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA; USC Ginsburg Institute for Biomedical Therapeutics, University of Southern California, Los Angeles, CA 90033, USA.
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DiCesare SM, Ortega AJ, Collier GE, Daniel S, Thompson KN, McCoy MK, Posner BA, Hulleman JD. GSK3 inhibition reduces ECM production and prevents age-related macular degeneration-like pathology. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.12.14.571757. [PMID: 38168310 PMCID: PMC10760106 DOI: 10.1101/2023.12.14.571757] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/05/2024]
Abstract
Malattia Leventinese/Doyne Honeycomb Retinal Dystrophy (ML/DHRD) is an age-related macular degeneration (AMD)-like retinal dystrophy caused by an autosomal dominant R345W mutation in the secreted glycoprotein, fibulin-3 (F3). To identify new small molecules that reduce F3 production from retinal pigmented epithelium (RPE) cells, we knocked-in a luminescent peptide tag (HiBiT) into the endogenous F3 locus which enabled simple, sensitive, and high throughput detection of the protein. The GSK3 inhibitor, CHIR99021 (CHIR), significantly reduced F3 burden (expression, secretion, and intracellular levels) in immortalized RPE and non-RPE cells. Low-level, long-term CHIR treatment promoted remodeling of the RPE extracellular matrix (ECM), reducing sub-RPE deposit-associated proteins (e.g., amelotin, complement component 3, collagen IV, and fibronectin), while increasing RPE differentiation factors (e.g., tyrosinase, and pigment epithelium derived factor). In vivo, treatment of 8 mo R345W+/+ knockin mice with CHIR (25 mg/kg i.p., 1 mo) was well tolerated and significantly reduced R345W F3-associated AMD-like basal laminar deposit number and size, thereby preventing the main pathological feature in these mice. This is the first demonstration of small molecule-based prevention of AMD-like pathology in ML/DHRD mice and may herald a rejuvenation of interest in GSK3 inhibition for the treatment of neurodegenerative diseases, including, potentially AMD itself.
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Affiliation(s)
- Sophia M. DiCesare
- Department of Ophthalmology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, Texas, 75390, United States
| | - Antonio J. Ortega
- Department of Ophthalmology and Visual Neurosciences, University of Minnesota, 2001 6 St. SE, Minneapolis, Minnesota, 55455, United States
| | - Gracen E. Collier
- Department of Ophthalmology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, Texas, 75390, United States
| | - Steffi Daniel
- Department of Ophthalmology and Visual Neurosciences, University of Minnesota, 2001 6 St. SE, Minneapolis, Minnesota, 55455, United States
| | - Krista N. Thompson
- Department of Ophthalmology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, Texas, 75390, United States
| | - Melissa K. McCoy
- Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, Texas, United States
| | - Bruce A. Posner
- Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, Texas, United States
| | - John D. Hulleman
- Department of Ophthalmology and Visual Neurosciences, University of Minnesota, 2001 6 St. SE, Minneapolis, Minnesota, 55455, United States
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10
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Ahluwalia K, Martinez-Camarillo JC, Thomas BB, Naik A, Gonzalez-Calle A, Pollalis D, Lebkowski J, Lee SY, Mitra D, Louie SG, Humayun MS. Polarized RPE Secretome Preserves Photoreceptors in Retinal Dystrophic RCS Rats. Cells 2023; 12:1689. [PMID: 37443724 PMCID: PMC10340490 DOI: 10.3390/cells12131689] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2023] [Revised: 06/19/2023] [Accepted: 06/20/2023] [Indexed: 07/15/2023] Open
Abstract
Retinal degenerative diseases, including age-related macular degeneration (AMD) and retinitis pigmentosa, lack effective therapies. Conventional monotherapeutic approaches fail to target the multiple affected pathways in retinal degeneration. However, the retinal pigment epithelium (RPE) secretes several neurotrophic factors addressing diverse cellular pathways, potentially preserving photoreceptors. This study explored human embryonic stem cell-derived, polarized RPE soluble factors (PRPE-SF) as a combination treatment for retinal degeneration. PRPE-SF promoted retinal progenitor cell survival, reduced oxidative stress in ARPE-19 cells, and demonstrated critical antioxidant and anti-inflammatory effects for preventing retinal degeneration in the Royal College of Surgeons (RCS) rat model. Importantly, PRPE-SF treatment preserved retinal structure and scotopic b-wave amplitudes, suggesting therapeutic potential for delaying retinal degeneration. PRPE-SF is uniquely produced using biomimetic membranes for RPE polarization and maturation, promoting a protective RPE secretome phenotype. Additionally, PRPE-SF is produced without animal serum to avoid immunogenicity in future clinical development. Lastly, PRPE-SF is a combination of neurotrophic factors, potentially ameliorating multiple dysfunctions in retinal degenerations. In conclusion, PRPE-SF offers a promising therapeutic candidate for retinal degenerative diseases, advancing the development of effective therapeutic strategies for these debilitating conditions.
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Affiliation(s)
- Kabir Ahluwalia
- Mann School of Pharmacy and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90089, USA; (K.A.); (A.N.)
| | - Juan-Carlos Martinez-Camarillo
- USC Ginsburg Institute of for Biomedical Therapeutics, University of Southern California, Los Angeles, CA 90033, USA; (J.-C.M.-C.); (B.B.T.); (A.G.-C.); (D.P.); (S.Y.L.); (D.M.)
- USC Roski Eye Institute, Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA
| | - Biju B. Thomas
- USC Ginsburg Institute of for Biomedical Therapeutics, University of Southern California, Los Angeles, CA 90033, USA; (J.-C.M.-C.); (B.B.T.); (A.G.-C.); (D.P.); (S.Y.L.); (D.M.)
- USC Roski Eye Institute, Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA
| | - Aditya Naik
- Mann School of Pharmacy and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90089, USA; (K.A.); (A.N.)
| | - Alejandra Gonzalez-Calle
- USC Ginsburg Institute of for Biomedical Therapeutics, University of Southern California, Los Angeles, CA 90033, USA; (J.-C.M.-C.); (B.B.T.); (A.G.-C.); (D.P.); (S.Y.L.); (D.M.)
- USC Roski Eye Institute, Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA
| | - Dimitrios Pollalis
- USC Ginsburg Institute of for Biomedical Therapeutics, University of Southern California, Los Angeles, CA 90033, USA; (J.-C.M.-C.); (B.B.T.); (A.G.-C.); (D.P.); (S.Y.L.); (D.M.)
- USC Roski Eye Institute, Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA
| | - Jane Lebkowski
- Regenerative Patch Technologies LLC, Menlo Park, CA 94028, USA;
| | - Sun Young Lee
- USC Ginsburg Institute of for Biomedical Therapeutics, University of Southern California, Los Angeles, CA 90033, USA; (J.-C.M.-C.); (B.B.T.); (A.G.-C.); (D.P.); (S.Y.L.); (D.M.)
- USC Roski Eye Institute, Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA
- Department of Physiology & Neuroscience, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA
| | - Debbie Mitra
- USC Ginsburg Institute of for Biomedical Therapeutics, University of Southern California, Los Angeles, CA 90033, USA; (J.-C.M.-C.); (B.B.T.); (A.G.-C.); (D.P.); (S.Y.L.); (D.M.)
| | - Stan G. Louie
- Mann School of Pharmacy and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90089, USA; (K.A.); (A.N.)
- USC Ginsburg Institute of for Biomedical Therapeutics, University of Southern California, Los Angeles, CA 90033, USA; (J.-C.M.-C.); (B.B.T.); (A.G.-C.); (D.P.); (S.Y.L.); (D.M.)
| | - Mark S. Humayun
- USC Ginsburg Institute of for Biomedical Therapeutics, University of Southern California, Los Angeles, CA 90033, USA; (J.-C.M.-C.); (B.B.T.); (A.G.-C.); (D.P.); (S.Y.L.); (D.M.)
- USC Roski Eye Institute, Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA
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11
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Daniele E, Bosio L, Hussain NA, Ferrari B, Ferrari S, Barbaro V, McArdle B, Rassu N, Mura M, Parmeggiani F, Ponzin D. Denuded Descemet's membrane supports human embryonic stem cell-derived retinal pigment epithelial cell culture. PLoS One 2023; 18:e0281404. [PMID: 36745611 PMCID: PMC9901769 DOI: 10.1371/journal.pone.0281404] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2022] [Accepted: 01/22/2023] [Indexed: 02/07/2023] Open
Abstract
Recent clinical studies suggest that retinal pigment epithelial (RPE) cell replacement therapy may preserve vision in retinal degenerative diseases. Scaffold-based methods are being tested in ongoing clinical trials for delivering pluripotent-derived RPE cells to the back of the eye. The aim of this study was to investigate human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells survival and behaviour on a decellularized Descemet's Membrane (DM), which may be of clinical relevance in retinal transplantation. DMs were isolated from human donor corneas and treated with thermolysin. The DM surface topology and the efficiency of the denudation method were evaluated by atomic force microscope, scanning electron microscopy and histology. hESC-RPE cells were seeded onto the endothelial-side surface of decellularized DM in order to determine the potential of the membrane to support hESC-RPE cell culture, alongside maintaining their viability. Integrity of the hESC-RPE monolayer was assessed by measuring transepithelial resistance. RPE-specific gene expression and growth factors secretion were assessed to confirm maturation and functionality of the cells over the new substrate. Thermolysin treatment did not affect the integrity of the tissue, thus ensuring a reliable method to standardize the preparation of decellularized DM. 24 hours post-seeding, hESC-RPE cell attachment and initial proliferation rate over the denuded DM were higher than hESC-RPE cells cultured on tissue culture inserts. On the new matrix, hESC-RPE cells succeeded in forming an intact monolayer with mature tight junctions. The resulting cell culture showed characteristic RPE cell morphology and proper protein localization. Gene expression analysis and VEGF secretion demonstrate DM provides supportive scaffolding and inductive properties to enhance hESC-RPE cells maturation. Decellularized DM was shown to be capable of sustaining hESC-RPE cells culture, thus confirming to be potentially a suitable candidate for retinal cell therapy.
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Affiliation(s)
- Elena Daniele
- Department of Translational Medicine, University of Ferrara, Ferrara, Italy
- Veneto Eye Bank Foundation, Venice, Italy
- * E-mail:
| | | | - Noor Ahmed Hussain
- Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom
| | | | | | | | - Brian McArdle
- The Eye-Bank for Sight Restoration, Inc., New York City, New York, United States of America
| | - Nicolò Rassu
- Ophthalmic Unit, Ospedale dell’Angelo, Venice, Italy
| | - Marco Mura
- Department of Translational Medicine, University of Ferrara, Ferrara, Italy
| | - Francesco Parmeggiani
- Department of Translational Medicine, University of Ferrara, Ferrara, Italy
- ERN-EYE Network - Center for Retinitis Pigmentosa of Veneto Region, Camposampiero Hospital, Padua, Italy
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12
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Álvarez-Barrios A, Álvarez L, Artime E, García M, Lengyel I, Pereiro R, González-Iglesias H. Altered zinc homeostasis in a primary cell culture model of the retinal pigment epithelium. Front Nutr 2023; 10:1124987. [PMID: 37139441 PMCID: PMC10149808 DOI: 10.3389/fnut.2023.1124987] [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: 12/15/2022] [Accepted: 03/22/2023] [Indexed: 05/05/2023] Open
Abstract
The retinal pigment epithelium (RPE) is progressively degenerated during age-related macular degeneration (AMD), one of the leading causes of irreversible blindness, which clinical hallmark is the buildup of sub-RPE extracellular material. Clinical observations indicate that Zn dyshomeostasis can initiate detrimental intracellular events in the RPE. In this study, we used a primary human fetal RPE cell culture model producing sub-RPE deposits accumulation that recapitulates features of early AMD to study Zn homeostasis and metalloproteins changes. RPE cell derived samples were collected at 10, 21 and 59 days in culture and processed for RNA sequencing, elemental mass spectrometry and the abundance and cellular localization of specific proteins. RPE cells developed processes normal to RPE, including intercellular unions formation and expression of RPE proteins. Punctate deposition of apolipoprotein E, marker of sub-RPE material accumulation, was observed from 3 weeks with profusion after 2 months in culture. Zn cytoplasmic concentrations significantly decreased 0.2 times at 59 days, from 0.264 ± 0.119 ng·μg-1 at 10 days to 0.062 ± 0.043 ng·μg-1 at 59 days (p < 0.05). Conversely, increased levels of Cu (1.5-fold in cytoplasm, 5.0-fold in cell nuclei and membranes), Na (3.5-fold in cytoplasm, 14.0-fold in cell nuclei and membranes) and K (6.8-fold in cytoplasm) were detected after 59-days long culture. The Zn-regulating proteins metallothioneins showed significant changes in gene expression over time, with a potent down-regulation at RNA and protein level of the most abundant isoform in primary RPE cells, from 0.141 ± 0.016 ng·mL-1 at 10 days to 0.056 ± 0.023 ng·mL-1 at 59 days (0.4-fold change, p < 0.05). Zn influx and efflux transporters were also deregulated, along with an increase in oxidative stress and alterations in the expression of antioxidant enzymes, including superoxide dismutase, catalase and glutathione peroxidase. The RPE cell model producing early accumulation of extracellular deposits provided evidences on an altered Zn homeostasis, exacerbated by changes in cytosolic Zn-binding proteins and Zn transporters, along with variations in other metals and metalloproteins, suggesting a potential role of altered Zn homeostasis during AMD development.
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Affiliation(s)
- Ana Álvarez-Barrios
- Fundación de Investigación Oftalmológica, Oviedo, Spain
- Department of Physical and Analytical Chemistry, University of Oviedo, Julián Clavería, 8, Oviedo, Spain
| | - Lydia Álvarez
- Fundación de Investigación Oftalmológica, Oviedo, Spain
- Lydia Álvarez,
| | - Enol Artime
- Fundación de Investigación Oftalmológica, Oviedo, Spain
| | | | - Imre Lengyel
- Wellcome-Wolfson Institute for Experimental Medicine, School of Medicine, Dentistry and Biomedical Science, Queen’s University Belfast, Belfast, Northern Ireland, United Kingdom
| | - Rosario Pereiro
- Department of Physical and Analytical Chemistry, University of Oviedo, Julián Clavería, 8, Oviedo, Spain
| | - Héctor González-Iglesias
- Instituto de Productos Lácteos de Asturias, Consejo Superior de Investigaciones Científicas (IPLA-CSIC), Villaviciosa, Spain
- *Correspondence: Héctor González-Iglesias,
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13
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The retinal pigmentation pathway in human albinism: Not so black and white. Prog Retin Eye Res 2022; 91:101091. [PMID: 35729001 DOI: 10.1016/j.preteyeres.2022.101091] [Citation(s) in RCA: 36] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2022] [Revised: 05/24/2022] [Accepted: 05/26/2022] [Indexed: 12/16/2022]
Abstract
Albinism is a pigment disorder affecting eye, skin and/or hair. Patients usually have decreased melanin in affected tissues and suffer from severe visual abnormalities, including foveal hypoplasia and chiasmal misrouting. Combining our data with those of the literature, we propose a single functional genetic retinal signalling pathway that includes all 22 currently known human albinism disease genes. We hypothesise that defects affecting the genesis or function of different intra-cellular organelles, including melanosomes, cause syndromic forms of albinism (Hermansky-Pudlak (HPS) and Chediak-Higashi syndrome (CHS)). We put forward that specific melanosome impairments cause different forms of oculocutaneous albinism (OCA1-8). Further, we incorporate GPR143 that has been implicated in ocular albinism (OA1), characterised by a phenotype limited to the eye. Finally, we include the SLC38A8-associated disorder FHONDA that causes an even more restricted "albinism-related" ocular phenotype with foveal hypoplasia and chiasmal misrouting but without pigmentation defects. We propose the following retinal pigmentation pathway, with increasingly specific genetic and cellular defects causing an increasingly specific ocular phenotype: (HPS1-11/CHS: syndromic forms of albinism)-(OCA1-8: OCA)-(GPR143: OA1)-(SLC38A8: FHONDA). Beyond disease genes involvement, we also evaluate a range of (candidate) regulatory and signalling mechanisms affecting the activity of the pathway in retinal development, retinal pigmentation and albinism. We further suggest that the proposed pigmentation pathway is also involved in other retinal disorders, such as age-related macular degeneration. The hypotheses put forward in this report provide a framework for further systematic studies in albinism and melanin pigmentation disorders.
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14
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Rebustini IT, Crawford SE, Becerra SP. PEDF Deletion Induces Senescence and Defects in Phagocytosis in the RPE. Int J Mol Sci 2022; 23:7745. [PMID: 35887093 PMCID: PMC9316002 DOI: 10.3390/ijms23147745] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2022] [Revised: 07/05/2022] [Accepted: 07/09/2022] [Indexed: 02/01/2023] Open
Abstract
The retinal pigment epithelium (RPE) expresses the Serpinf1 gene to produce pigment epithelium-derived factor (PEDF), a retinoprotective protein that is downregulated with cell senescence, aging and retinal degenerations. We determined the expression of senescence-associated genes in the RPE of 3-month-old mice that lack the Serpinf1 gene and found that Serpinf1 deletion induced H2ax for histone H2AX protein, Cdkn1a for p21 protein, and Glb1 gene for β-galactosidase. Senescence-associated β-galactosidase activity increased in the Serpinf1 null RPE when compared with wild-type RPE. We evaluated the subcellular morphology of the RPE and found that ablation of Serpinf1 increased the volume of the nuclei and the nucleoli number of RPE cells, implying chromatin reorganization. Given that the RPE phagocytic function declines with aging, we assessed the expression of the Pnpla2 gene, which is required for the degradation of photoreceptor outer segments by the RPE. We found that both the Pnpla2 gene and its protein PEDF-R declined with the Serpinf1 gene ablation. Moreover, we determined the levels of phagocytosed rhodopsin and lipids in the RPE of the Serpinf1 null mice. The RPE of the Serpinf1 null mice accumulated rhodopsin and lipids compared to littermate controls, implying an association of PEDF deficiency with RPE phagocytosis dysfunction. Our findings establish PEDF loss as a cause of senescence-like changes in the RPE, highlighting PEDF as both a retinoprotective and a regulatory protein of aging-like changes associated with defective degradation of the photoreceptor outer segment in the RPE.
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Affiliation(s)
- Ivan T. Rebustini
- Section of Protein Structure and Function, Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA;
| | - Susan E. Crawford
- Department of Surgery, North Shore University Research Institute, University of Chicago Pritzker School of Medicine, Chicago, IL 60201, USA;
| | - S. Patricia Becerra
- Section of Protein Structure and Function, Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA;
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15
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Faynus MA, Bailey JK, Pennington BO, Katsura M, Proctor DA, Yeh AK, Menon S, Choi DG, Lebkowski JS, Johnson LV, Clegg DO. Microcarrier-Based Culture of Human Pluripotent Stem-Cell-Derived Retinal Pigmented Epithelium. Bioengineering (Basel) 2022; 9:bioengineering9070297. [PMID: 35877348 PMCID: PMC9311890 DOI: 10.3390/bioengineering9070297] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2022] [Revised: 06/28/2022] [Accepted: 07/01/2022] [Indexed: 11/16/2022] Open
Abstract
Dry age-related macular degeneration (AMD) is estimated to impact nearly 300 million individuals globally by 2040. While no treatment options are currently available, multiple clinical trials investigating retinal pigmented epithelial cells derived from human pluripotent stem cells (hPSC-RPE) as a cellular replacement therapeutic are currently underway. It has been estimated that a production capacity of >109 RPE cells annually would be required to treat the afflicted population, but current manufacturing protocols are limited, being labor-intensive and time-consuming. Microcarrier technology has enabled high-density propagation of many adherent mammalian cell types via monolayer culture on surfaces of uM-diameter matrix spheres; however, few studies have explored microcarrier-based culture of RPE cells. Here, we provide an approach to the growth, maturation, and differentiation of hPSC-RPE cells on Cytodex 1 (C1) and Cytodex 3 (C3) microcarriers. We demonstrate that hPSC-RPE cells adhere to microcarriers coated with Matrigel, vitronectin or collagen, and mature in vitro to exhibit characteristic epithelial cell morphology and pigmentation. Microcarrier-grown hPSC-RPE cells (mcRPE) are viable; metabolically active; express RPE signature genes including BEST1, RPE65, TYRP1, and PMEL17; secrete the trophic factors PEDF and VEGF; and demonstrate phagocytosis of photoreceptor outer segments. Furthermore, we show that undifferentiated hESCs also adhere to Matrigel-coated microcarriers and are amenable to directed RPE differentiation. The capacity to support hPSC-RPE cell cultures using microcarriers enables efficient large-scale production of therapeutic RPE cells sufficient to meet the treatment demands of a large AMD patient population.
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Affiliation(s)
- Mohamed A. Faynus
- Program for Biomolecular Science and Engineering, University of California, Santa Barbara, CA 93106, USA
- Center for Stem Cell Biology and Engineering, Neuroscience Research Institute, University of California, Santa Barbara, CA 93106, USA; (J.K.B.); (B.O.P.); (M.K.); (D.A.P.); (A.K.Y.); (S.M.); (D.G.C.); (D.O.C.)
- Correspondence:
| | - Jeffrey K. Bailey
- Center for Stem Cell Biology and Engineering, Neuroscience Research Institute, University of California, Santa Barbara, CA 93106, USA; (J.K.B.); (B.O.P.); (M.K.); (D.A.P.); (A.K.Y.); (S.M.); (D.G.C.); (D.O.C.)
| | - Britney O. Pennington
- Center for Stem Cell Biology and Engineering, Neuroscience Research Institute, University of California, Santa Barbara, CA 93106, USA; (J.K.B.); (B.O.P.); (M.K.); (D.A.P.); (A.K.Y.); (S.M.); (D.G.C.); (D.O.C.)
| | - Mika Katsura
- Center for Stem Cell Biology and Engineering, Neuroscience Research Institute, University of California, Santa Barbara, CA 93106, USA; (J.K.B.); (B.O.P.); (M.K.); (D.A.P.); (A.K.Y.); (S.M.); (D.G.C.); (D.O.C.)
| | - Duncan A. Proctor
- Center for Stem Cell Biology and Engineering, Neuroscience Research Institute, University of California, Santa Barbara, CA 93106, USA; (J.K.B.); (B.O.P.); (M.K.); (D.A.P.); (A.K.Y.); (S.M.); (D.G.C.); (D.O.C.)
- Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, CA 93106, USA
| | - Ashley K. Yeh
- Center for Stem Cell Biology and Engineering, Neuroscience Research Institute, University of California, Santa Barbara, CA 93106, USA; (J.K.B.); (B.O.P.); (M.K.); (D.A.P.); (A.K.Y.); (S.M.); (D.G.C.); (D.O.C.)
- Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, CA 93106, USA
| | - Sneha Menon
- Center for Stem Cell Biology and Engineering, Neuroscience Research Institute, University of California, Santa Barbara, CA 93106, USA; (J.K.B.); (B.O.P.); (M.K.); (D.A.P.); (A.K.Y.); (S.M.); (D.G.C.); (D.O.C.)
- Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, CA 93106, USA
| | - Dylan G. Choi
- Center for Stem Cell Biology and Engineering, Neuroscience Research Institute, University of California, Santa Barbara, CA 93106, USA; (J.K.B.); (B.O.P.); (M.K.); (D.A.P.); (A.K.Y.); (S.M.); (D.G.C.); (D.O.C.)
- College of Creative Studies, Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106, USA
| | - Jane S. Lebkowski
- Program in Biological Engineering, University of California, Santa Barbara, CA 93106, USA
| | - Lincoln V. Johnson
- Program in Biological Engineering, University of California, Santa Barbara, CA 93106, USA
| | - Dennis O. Clegg
- Center for Stem Cell Biology and Engineering, Neuroscience Research Institute, University of California, Santa Barbara, CA 93106, USA; (J.K.B.); (B.O.P.); (M.K.); (D.A.P.); (A.K.Y.); (S.M.); (D.G.C.); (D.O.C.)
- Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, CA 93106, USA
- Regenerative Patch Technologies LLC, Portola Valley, CA 94028, USA; (J.S.L.); (L.V.J.)
- Program in Biological Engineering, University of California, Santa Barbara, CA 93106, USA
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16
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Kashani AH. Stem cell-derived retinal pigment epithelium transplantation in age-related macular degeneration: recent advances and challenges. Curr Opin Ophthalmol 2022; 33:211-218. [PMID: 35200164 DOI: 10.1097/icu.0000000000000838] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
Abstract
PURPOSE OF REVIEW Age-related macular degeneration (AMD) is one of the leading causes of irreversible vision loss in the world with more than 80% of the prevalence accounted for by the nonneovascular (NNAMD) or 'dry' form of the disease. NNAMD does not have any definitive treatment once vision loss has ensued and presents a major unmet medical need. This review will highlight stem cell-based therapies that are a promising form of treatment for advanced NNAMD. RECENT FINDINGS In the past decade, clinical trials utilizing both induced pluripotent stem cell-derived RPE and human embryonic stem cell-derived RPE have been aggressively pursued as potential treatments of RPE loss and prevention of overlying neurosensory atrophy. While promising preliminary results demonstrating safety and potential efficacy have been published, new challenges have also been identified. These include selecting the most appropriate cell-based therapy, identifying and managing potential immune response as well as characterizing anatomic and functional efficacy. In this review, we will discuss some of these challenges in light of the available data from several early phase clinical trials and discuss the strategies that are being considered to further advance the field. SUMMARY Cell-based therapies demonstrate promising potential to treat advanced stages of NNAMD. Several early phase clinical trials using both induced pluripotent stem cells (iPSC) and human embryonic stem cell derived (hESC) have demonstrated safety and preliminary signs of efficacy and highlighted remaining challenges which appear surmountable. These challenges include development of selection criteria for use of cell suspensions versus RPE sheets, especially in light of immunological properties of RPE that are intrinsic to the status of RPE differentiation in each of these cell formulations.
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Affiliation(s)
- Amir H Kashani
- Department of Ophthalmology and Biomedical Engineering, T Boone Pickens Professorship in Ophthalmology, Wilmer Eye Institute, Johns Hopkins University, Baltimore, Maryland, USA
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17
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German OL, Vallese-Maurizi H, Soto TB, Rotstein NP, Politi LE. Retina stem cells, hopes and obstacles. World J Stem Cells 2021; 13:1446-1479. [PMID: 34786153 PMCID: PMC8567457 DOI: 10.4252/wjsc.v13.i10.1446] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/28/2021] [Revised: 07/14/2021] [Accepted: 09/17/2021] [Indexed: 02/07/2023] Open
Abstract
Retinal degeneration is a major contributor to visual dysfunction worldwide. Although it comprises several eye diseases, loss of retinal pigment epithelial (RPE) and photoreceptor cells are the major contributors to their pathogenesis. Early therapies included diverse treatments, such as provision of anti-vascular endothelial growth factor and many survival and trophic factors that, in some cases, slow down the progression of the degeneration, but do not effectively prevent it. The finding of stem cells (SC) in the eye has led to the proposal of cell replacement strategies for retina degeneration. Therapies using different types of SC, such as retinal progenitor cells (RPCs), embryonic SC, pluripotent SCs (PSCs), induced PSCs (iPSCs), and mesenchymal stromal cells, capable of self-renewal and of differentiating into multiple cell types, have gained ample support. Numerous preclinical studies have assessed transplantation of SC in animal models, with encouraging results. The aim of this work is to revise the different preclinical and clinical approaches, analyzing the SC type used, their efficacy, safety, cell attachment and integration, absence of tumor formation and immunorejection, in order to establish which were the most relevant and successful. In addition, we examine the questions and concerns still open in the field. The data demonstrate the existence of two main approaches, aimed at replacing either RPE cells or photoreceptors. Emerging evidence suggests that RPCs and iPSC are the best candidates, presenting no ethical concerns and a low risk of immunorejection. Clinical trials have already supported the safety and efficacy of SC treatments. Serious concerns are pending, such as the risk of tumor formation, lack of attachment or integration of transplanted cells into host retinas, immunorejection, cell death, and also ethical. However, the amazing progress in the field in the last few years makes it possible to envisage safe and effective treatments to restore vision loss in a near future.
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Affiliation(s)
- Olga L German
- Department of Biology, Biochemistry and Pharmacy, Universidad Nacional del Sur, Bahia blanca 8000, Buenos Aires, Argentina
- Department of Biology, Biochemistry and Pharmacy, Universidad Nacional del Sur, and Neurobiology Department, Instituto de Investigaciones Bioquímicas de Bahía Blanca (INIBIBB) Conicet, Bahía Blanca 8000, Buenos Aires, Argentina
| | - Harmonie Vallese-Maurizi
- Department of Biology, Biochemistry and Pharmacy, Universidad Nacional del Sur, Bahia blanca 8000, Buenos Aires, Argentina
- Department of Biology, Biochemistry and Pharmacy, Universidad Nacional del Sur, and Neurobiology Department, Instituto de Investigaciones Bioquímicas de Bahía Blanca (INIBIBB) Conicet, Bahía Blanca 8000, Buenos Aires, Argentina
| | - Tamara B Soto
- Department of Biology, Biochemistry and Pharmacy, Universidad Nacional del Sur, and Neurobiology Department, Instituto de Investigaciones Bioquímicas de Bahía Blanca (INIBIBB) Conicet, Bahía Blanca 8000, Buenos Aires, Argentina
| | - Nora P Rotstein
- Department of Biology, Biochemistry and Pharmacy, Universidad Nacional del Sur, Bahia blanca 8000, Buenos Aires, Argentina
- Department of Biology, Biochemistry and Pharmacy, Universidad Nacional del Sur, and Neurobiology Department, Instituto de Investigaciones Bioquímicas de Bahía Blanca (INIBIBB) Conicet, Bahía Blanca 8000, Buenos Aires, Argentina
| | - Luis Enrique Politi
- Department of Biology, Biochemistry and Pharmacy, Universidad Nacional del Sur, and Neurobiology Department, Instituto de Investigaciones Bioquímicas de Bahía Blanca (INIBIBB) Conicet, Bahía Blanca 8000, Buenos Aires, Argentina
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18
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Al-Ani A, Toms D, Sunba S, Giles K, Touahri Y, Schuurmans C, Ungrin M. Scaffold-Free Retinal Pigment Epithelium Microtissues Exhibit Increased Release of PEDF. Int J Mol Sci 2021; 22:11317. [PMID: 34768747 PMCID: PMC8583603 DOI: 10.3390/ijms222111317] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2021] [Revised: 10/14/2021] [Accepted: 10/16/2021] [Indexed: 12/26/2022] Open
Abstract
The retinal pigmented epithelium (RPE) plays a critical role in photoreceptor survival and function. RPE deficits are implicated in a wide range of diseases that result in vision loss, including age-related macular degeneration (AMD) and Stargardt disease, affecting millions worldwide. Subretinal delivery of RPE cells is considered a promising avenue for treatment, and encouraging results from animal trials have supported recent progression into the clinic. However, the limited survival and engraftment of transplanted RPE cells delivered as a suspension continues to be a major challenge. While RPE delivery as epithelial sheets exhibits improved outcomes, this comes at the price of increased complexity at both the production and transplant stages. In order to combine the benefits of both approaches, we have developed size-controlled, scaffold-free RPE microtissues (RPE-µTs) that are suitable for scalable production and delivery via injection. RPE-µTs retain key RPE molecular markers, and interestingly, in comparison to conventional monolayer cultures, they show significant increases in the transcription and secretion of pigment-epithelium-derived factor (PEDF), which is a key trophic factor known to enhance the survival and function of photoreceptors. Furthermore, these microtissues readily spread in vitro on a substrate analogous to Bruch's membrane, suggesting that RPE-µTs may collapse into a sheet upon transplantation. We anticipate that this approach may provide an alternative cell delivery system to improve the survival and integration of RPE transplants, while also retaining the benefits of low complexity in production and delivery.
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Affiliation(s)
- Abdullah Al-Ani
- Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada; (A.A.-A.); (S.S.); (K.G.)
- Alberta Children’s Hospital Research Institute, University of Calgary, Calgary, AB T2N 4N1, Canada
- Alberta Diabetes Institute, University of Alberta, Edmonton, AB T6G 2E1, Canada
- Biomedical Engineering Graduate Program, University of Calgary, Calgary, AB T2N 1N4, Canada
- Cumming School of Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada
| | - Derek Toms
- Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada; (A.A.-A.); (S.S.); (K.G.)
- Alberta Children’s Hospital Research Institute, University of Calgary, Calgary, AB T2N 4N1, Canada
| | - Saud Sunba
- Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada; (A.A.-A.); (S.S.); (K.G.)
- Alberta Children’s Hospital Research Institute, University of Calgary, Calgary, AB T2N 4N1, Canada
| | - Kayla Giles
- Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada; (A.A.-A.); (S.S.); (K.G.)
| | - Yacine Touahri
- Sunnybrook Research Institute, Toronto, ON M4N 3M5, Canada; (Y.T.); (C.S.)
- Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada
| | - Carol Schuurmans
- Sunnybrook Research Institute, Toronto, ON M4N 3M5, Canada; (Y.T.); (C.S.)
- Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada
| | - Mark Ungrin
- Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada; (A.A.-A.); (S.S.); (K.G.)
- Alberta Children’s Hospital Research Institute, University of Calgary, Calgary, AB T2N 4N1, Canada
- Alberta Diabetes Institute, University of Alberta, Edmonton, AB T6G 2E1, Canada
- Biomedical Engineering Graduate Program, University of Calgary, Calgary, AB T2N 1N4, Canada
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19
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Ahmed I, Johnston RJ, Singh MS. Pluripotent stem cell therapy for retinal diseases. ANNALS OF TRANSLATIONAL MEDICINE 2021; 9:1279. [PMID: 34532416 PMCID: PMC8421932 DOI: 10.21037/atm-20-4747] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/16/2020] [Accepted: 12/04/2020] [Indexed: 12/20/2022]
Abstract
Pluripotent stem cells (PSCs), which include human embryonic stem cells (hESCs) and induced pluripotent stem cell (iPSC), have been used to study development of disease processes, and as potential therapies in multiple organ systems. In recent years, there has been increasing interest in the use of PSC-based transplantation to treat disorders of the retina in which retinal cells have been functionally damaged or lost through degeneration. The retina, which consists of neuronal tissue, provides an excellent system to test the therapeutic utility of PSC-based transplantation due to its accessibility and the availability of high-resolution imaging technology to evaluate effects. Preclinical trials in animal models of retinal diseases have shown improvement in visual outcomes following subretinal transplantation of PSC-derived photoreceptors or retinal pigment epithelium (RPE) cells. This review focuses on preclinical studies and clinical trials exploring the use of PSCs for retinal diseases. To date, several phase I/II clinical trials in patients with age-related macular degeneration (AMD) and Stargardt disease (STGD1) have demonstrated the safety and feasibility of PSC-derived RPE transplantation. Additional phase I/II clinical trials using PSC-derived RPE or photoreceptor cells for the treatment of AMD, STGD1, and also retinitis pigmentosa (RP) are currently in the pipeline. As this field continues to evolve, additional technologies may enhance PSC-derived cell transplantation through gene-editing of autologous cells, transplantation of more complex cellular structures such as organoids, and monitoring of transplanted cells through novel imaging technologies.
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Affiliation(s)
- Ishrat Ahmed
- Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | | | - Mandeep S Singh
- Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA
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20
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Huang D, Heath Jeffery RC, Aung-Htut MT, McLenachan S, Fletcher S, Wilton SD, Chen FK. Stargardt disease and progress in therapeutic strategies. Ophthalmic Genet 2021; 43:1-26. [PMID: 34455905 DOI: 10.1080/13816810.2021.1966053] [Citation(s) in RCA: 23] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Background: Stargardt disease (STGD1) is an autosomal recessive retinal dystrophy due to mutations in ABCA4, characterized by subretinal deposition of lipofuscin-like substances and bilateral centrifugal vision loss. Despite the tremendous progress made in the understanding of STGD1, there are no approved treatments to date. This review examines the challenges in the development of an effective STGD1 therapy.Materials and Methods: A literature review was performed through to June 2021 summarizing the spectrum of retinal phenotypes in STGD1, the molecular biology of ABCA4 protein, the in vivo and in vitro models used to investigate the mechanisms of ABCA4 mutations and current clinical trials.Results: STGD1 phenotypic variability remains an challenge for clinical trial design and patient selection. Pre-clinical development of therapeutic options has been limited by the lack of animal models reflecting the diverse phenotypic spectrum of STDG1. Patient-derived cell lines have facilitated the characterization of splice mutations but the clinical presentation is not always predicted by the effect of specific mutations on retinoid metabolism in cellular models. Current therapies primarily aim to delay vision loss whilst strategies to restore vision are less well developed.Conclusions: STGD1 therapy development can be accelerated by a deeper understanding of genotype-phenotype correlations.
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Affiliation(s)
- Di Huang
- Centre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Western Australia, Australia.,Centre for Ophthalmology and Visual Science (Incorporating Lions Eye Institute), the University of Western Australia, Nedlands, Western Australia, Australia.,Perron Institute for Neurological and Translational Science & the University of Western Australia, Nedlands, Western Australia, Australia
| | - Rachael C Heath Jeffery
- Centre for Ophthalmology and Visual Science (Incorporating Lions Eye Institute), the University of Western Australia, Nedlands, Western Australia, Australia
| | - May Thandar Aung-Htut
- Centre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Western Australia, Australia.,Perron Institute for Neurological and Translational Science & the University of Western Australia, Nedlands, Western Australia, Australia
| | - Samuel McLenachan
- Centre for Ophthalmology and Visual Science (Incorporating Lions Eye Institute), the University of Western Australia, Nedlands, Western Australia, Australia
| | - Sue Fletcher
- Centre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Western Australia, Australia.,Perron Institute for Neurological and Translational Science & the University of Western Australia, Nedlands, Western Australia, Australia
| | - Steve D Wilton
- Centre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Western Australia, Australia.,Perron Institute for Neurological and Translational Science & the University of Western Australia, Nedlands, Western Australia, Australia
| | - Fred K Chen
- Centre for Ophthalmology and Visual Science (Incorporating Lions Eye Institute), the University of Western Australia, Nedlands, Western Australia, Australia.,Australian Inherited Retinal Disease Registry and DNA Bank, Department of Medical Technology and Physics, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia.,Department of Ophthalmology, Royal Perth Hospital, Perth, Western Australia, Australia.,Department of Ophthalmology, Perth Children's Hospital, Nedlands, Western Australia, Australia
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21
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Coco-Martin RM, Pastor-Idoate S, Pastor JC. Cell Replacement Therapy for Retinal and Optic Nerve Diseases: Cell Sources, Clinical Trials and Challenges. Pharmaceutics 2021; 13:pharmaceutics13060865. [PMID: 34208272 PMCID: PMC8230855 DOI: 10.3390/pharmaceutics13060865] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2021] [Revised: 06/05/2021] [Accepted: 06/07/2021] [Indexed: 12/15/2022] Open
Abstract
The aim of this review was to provide an update on the potential of cell therapies to restore or replace damaged and/or lost cells in retinal degenerative and optic nerve diseases, describing the available cell sources and the challenges involved in such treatments when these techniques are applied in real clinical practice. Sources include human fetal retinal stem cells, allogenic cadaveric human cells, adult hippocampal neural stem cells, human CNS stem cells, ciliary pigmented epithelial cells, limbal stem cells, retinal progenitor cells (RPCs), human pluripotent stem cells (PSCs) (including both human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs)) and mesenchymal stem cells (MSCs). Of these, RPCs, PSCs and MSCs have already entered early-stage clinical trials since they can all differentiate into RPE, photoreceptors or ganglion cells, and have demonstrated safety, while showing some indicators of efficacy. Stem/progenitor cell therapies for retinal diseases still have some drawbacks, such as the inhibition of proliferation and/or differentiation in vitro (with the exception of RPE) and the limited long-term survival and functioning of grafts in vivo. Some other issues remain to be solved concerning the clinical translation of cell-based therapy, including (1) the ability to enrich for specific retinal subtypes; (2) cell survival; (3) cell delivery, which may need to incorporate a scaffold to induce correct cell polarization, which increases the size of the retinotomy in surgery and, therefore, the chance of severe complications; (4) the need to induce a localized retinal detachment to perform the subretinal placement of the transplanted cell; (5) the evaluation of the risk of tumor formation caused by the undifferentiated stem cells and prolific progenitor cells. Despite these challenges, stem/progenitor cells represent the most promising strategy for retinal and optic nerve disease treatment in the near future, and therapeutics assisted by gene techniques, neuroprotective compounds and artificial devices can be applied to fulfil clinical needs.
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Affiliation(s)
- Rosa M. Coco-Martin
- Instituto de Oftalmobiologia Aplicada (IOBA), Medical School, Universidad de Valladolid, 47011 Valladolid, Spain; (S.P.-I.); (J.C.P.)
- National Institute of Health Carlos III (ISCIII), (RETICS) Cooperative Health Network for Research in Ophthalmology (Oftared), 28040 Madrid, Spain
- Correspondence: ; Tel.: +34-983423559
| | - Salvador Pastor-Idoate
- Instituto de Oftalmobiologia Aplicada (IOBA), Medical School, Universidad de Valladolid, 47011 Valladolid, Spain; (S.P.-I.); (J.C.P.)
- National Institute of Health Carlos III (ISCIII), (RETICS) Cooperative Health Network for Research in Ophthalmology (Oftared), 28040 Madrid, Spain
- Department of Ophthalmology, Hospital Clinico Universitario of Valladolid, 47003 Valladolid, Spain
| | - Jose Carlos Pastor
- Instituto de Oftalmobiologia Aplicada (IOBA), Medical School, Universidad de Valladolid, 47011 Valladolid, Spain; (S.P.-I.); (J.C.P.)
- National Institute of Health Carlos III (ISCIII), (RETICS) Cooperative Health Network for Research in Ophthalmology (Oftared), 28040 Madrid, Spain
- Department of Ophthalmology, Hospital Clinico Universitario of Valladolid, 47003 Valladolid, Spain
- Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, Fundacion del Instituto de Estudios de Ciencias de la Salud de Castilla y León (ICSCYL), 42002 Soria, Spain
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22
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Singh D, Chen X, Xia T, Ghiassi-Nejad M, Tainsh L, Adelman RA, Rizzolo LJ. Partially Differentiated Neuroretinal Cells Promote Maturation of the Retinal Pigment Epithelium. Invest Ophthalmol Vis Sci 2021; 61:9. [PMID: 33151282 PMCID: PMC7671856 DOI: 10.1167/iovs.61.13.9] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
Purpose Many studies have demonstrated the ability of the retinal pigment epithelium (RPE) to foster the maturation of the developing retina. Few studies have examined the reciprocal effects of developing retina on the RPE. Methods RPE isolated from human fetal RPE or differentiated from human stem cells was cultured on Transwell filter inserts. Retinal progenitor cells (RPCs) were differentiated from human stem cells and cultured on a planar scaffold composed of gelatin, chondroitin sulfate, hyaluronic acid, and laminin-521. Cultures were analyzed by quantitative RT-PCR, immunofluorescence, immunoblotting, and transepithelial electrical resistance (TER). Results RPCs initially differentiated into several retina-like cell types that segregated from one another and formed loosely organized layers or zones. With time, the presumptive photoreceptor and ganglion cell layers persisted, but the intervening zone became dominated by cells that expressed glial markers with no evidence of bipolar cells or interneurons. Co-culture of this underdeveloped retinoid with the RPE resulted in a thickened layer of recoverin-positive cells but did not prevent the loss of interneuron markers in the intervening zone. Although photoreceptor inner and outer segments were not observed, immunoblots revealed that co-culture increased expression of rhodopsin and red/green opsin. Co-culture of the RPE with this underdeveloped retinal culture increased the TER of the RPE and the expression of RPE signature genes. Conclusions These studies indicated that an immature neurosensory retina can foster maturation of the RPE; however, the ability of RPE alone to foster maturation of the neurosensory retina is limited.
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Affiliation(s)
- Deepti Singh
- Department of Surgery, Yale School of Medicine, Yale University, New Haven, Connecticut, United States.,Department of Ophthalmology and Visual Sciences, Yale School of Medicine, Yale University, New Haven, Connecticut, United States
| | - Xiaoyu Chen
- Department of Surgery, Yale School of Medicine, Yale University, New Haven, Connecticut, United States.,Department of Ophthalmology and Visual Sciences, Yale School of Medicine, Yale University, New Haven, Connecticut, United States.,Department of Ophthalmology, Second Xiangya Hospital, Central South University, Changsha, Hunan, China
| | - Tina Xia
- Department of Surgery, Yale School of Medicine, Yale University, New Haven, Connecticut, United States.,Department of Ophthalmology and Visual Sciences, Yale School of Medicine, Yale University, New Haven, Connecticut, United States
| | - Maryam Ghiassi-Nejad
- Department of Surgery, Yale School of Medicine, Yale University, New Haven, Connecticut, United States.,Department of Ophthalmology and Visual Sciences, Yale School of Medicine, Yale University, New Haven, Connecticut, United States
| | - Laurel Tainsh
- Department of Surgery, Yale School of Medicine, Yale University, New Haven, Connecticut, United States.,Department of Ophthalmology and Visual Sciences, Yale School of Medicine, Yale University, New Haven, Connecticut, United States
| | - Ron A Adelman
- Department of Ophthalmology and Visual Sciences, Yale School of Medicine, Yale University, New Haven, Connecticut, United States
| | - Lawrence J Rizzolo
- Department of Surgery, Yale School of Medicine, Yale University, New Haven, Connecticut, United States.,Department of Ophthalmology and Visual Sciences, Yale School of Medicine, Yale University, New Haven, Connecticut, United States
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23
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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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24
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Kim J, Park JY, Kong JS, Lee H, Won JY, Cho DW. Development of 3D Printed Bruch's Membrane-Mimetic Substance for the Maturation of Retinal Pigment Epithelial Cells. Int J Mol Sci 2021; 22:ijms22031095. [PMID: 33499245 PMCID: PMC7865340 DOI: 10.3390/ijms22031095] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2020] [Revised: 01/20/2021] [Accepted: 01/21/2021] [Indexed: 12/15/2022] Open
Abstract
Retinal pigment epithelium (RPE) is a monolayer of the pigmented cells that lies on the thin extracellular matrix called Bruch's membrane. This monolayer is the main component of the outer blood-retinal barrier (BRB), which plays a multifunctional role. Due to their crucial roles, the damage of this epithelium causes a wide range of diseases related to retinal degeneration including age-related macular degeneration, retinitis pigmentosa, and Stargardt disease. Unfortunately, there is presently no cure for these diseases. Clinically implantable RPE for humans is under development, and there is no practical examination platform for drug development. Here, we developed porcine Bruch's membrane-derived bioink (BM-ECM). Compared to conventional laminin, the RPE cells on BM-ECM showed enhanced functionality of RPE. Furthermore, we developed the Bruch's membrane-mimetic substrate (BMS) via the integration of BM-ECM and 3D printing technology, which revealed structure and extracellular matrix components similar to those of natural Bruch's membrane. The developed BMS facilitated the appropriate functions of RPE, including barrier and clearance functions, the secretion of anti-angiogenic growth factors, and enzyme formation for phototransduction. Moreover, it could be used as a basement frame for RPE transplantation. We established BMS using 3D printing technology to grow RPE cells with functions that could be used for an in vitro model and RPE transplantation.
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Affiliation(s)
- Jongmin Kim
- Department of Mechanical Engineering, Pohang University of Science and Technology (POSTECH), Pohang 37673, Korea; (J.K.); (J.Y.P.); (H.L.)
| | - Ju Young Park
- Department of Mechanical Engineering, Pohang University of Science and Technology (POSTECH), Pohang 37673, Korea; (J.K.); (J.Y.P.); (H.L.)
| | - Jeong Sik Kong
- School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology (POSTECH), Pohang 37673, Korea;
| | - Hyungseok Lee
- Department of Mechanical Engineering, Pohang University of Science and Technology (POSTECH), Pohang 37673, Korea; (J.K.); (J.Y.P.); (H.L.)
- Department of Mechanical and Biomedical Engineering, Kangwon National University, Chuncheon 24341, Korea
| | - Jae Yon Won
- Department of Ophthalmology and Visual Science, Eunpyeong St. Mary’s Hospital, The Catholic University of Korea, Seoul 03312, Korea
- Catholic Institute for Visual Science, College of Medicine, The Catholic University of Korea, Seoul 14662, Korea
- Correspondence: (J.Y.W.); (D.W.C.)
| | - Dong Woo Cho
- Department of Mechanical Engineering, Pohang University of Science and Technology (POSTECH), Pohang 37673, Korea; (J.K.); (J.Y.P.); (H.L.)
- School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology (POSTECH), Pohang 37673, Korea;
- Institute of Convergence Science, Yonsei University, Seoul 03722, Korea
- Correspondence: (J.Y.W.); (D.W.C.)
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25
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Yeh SI, Yu SH, Chu HS, Huang CT, Tsao YP, Cheng CM, Chen WL. Pigment Epithelium-Derived Factor Peptide Promotes Corneal Nerve Regeneration: An In Vivo and In Vitro Study. Invest Ophthalmol Vis Sci 2021; 62:23. [PMID: 33481984 PMCID: PMC7838554 DOI: 10.1167/iovs.62.1.23] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2020] [Accepted: 12/28/2020] [Indexed: 12/17/2022] Open
Abstract
Purpose To investigate the potential of a pigment epithelium-derived factor (PEDF) peptide 44-mer to promote nerve regeneration in a rabbit corneal nerve injury model to demonstrate its neurotrophic ability in cultivated mouse trigeminal neuron cells. Methods Subconjunctival or intrastromal injection of 44-mer on the cornea was performed in a rabbit model of corneal nerve injury created by corneal epithelial debridement. Immunocytochemical analysis (44-mer, anti-tubulin III, SMI312, CD11b, and α-SMA) and in vivo confocal microscopy were performed. Corneal sensation was estimated using a Cochet-Bonnet corneal esthesiometer. Primary cultivated mouse trigeminal neurons were used to examine the in vitro neurotrophic ability of 44-mer. The cellular morphology and the immunocytochemical staining with anti-tubulin III and SMI312 in different concentrations of 44-mer were compared, and a quantitative assessment of neurite outgrowth was performed. Results Immunohistochemical staining showed the retention of 44-mer in the corneal stroma for at least 7 days after a single dose of corneal intrastromal injection and promoted corneal nerve regeneration revealed by in vivo confocal microscopy. Corneal esthesiometer demonstrated gradual recovery of the corneal sensation in 44-mer-treated eyes with a lower corneal touch threshold than wounded vehicles and closer to baseline at 3 weeks after corneal injury (P < 0.001). In vitro studies showed a dose-dependent neurotrophic effect of 44-mer in cultivated trigeminal neuron cells. Conclusions The 44-mer showed in vivo and in vitro corneal neurotrophic abilities. Our results suggest that intrastromal injection of 44-mer into the corneal stroma may have a potential role in treating diseases related to corneal nerve damage.
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Affiliation(s)
- Shu-I Yeh
- Department of Ophthalmology, Mackay Memorial Hospital, Taipei, Taiwan
- Department of Medicine, Mackay Medical College, New Taipei City, Taiwan
| | - Sung-Hsun Yu
- Department of Ophthalmology, National Taiwan University Hospital, Taipei, Taiwan
- Advanced Ocular Surface and Corneal Nerve Research Center, National Taiwan University, Taipei, Taiwan
| | - Hsiao-Sang Chu
- Department of Ophthalmology, National Taiwan University Hospital, Taipei, Taiwan
- Advanced Ocular Surface and Corneal Nerve Research Center, National Taiwan University, Taipei, Taiwan
| | - Chin-Te Huang
- Department of Ophthalmology, National Taiwan University Hospital, Taipei, Taiwan
- Advanced Ocular Surface and Corneal Nerve Research Center, National Taiwan University, Taipei, Taiwan
- Department of Ophthalmology, Chung Shan Medical University Hospital, School of Medicine, Chung Shan Medical University, Taichung, Taiwan
| | - Yeou-Ping Tsao
- Department of Ophthalmology, Mackay Memorial Hospital, Taipei, Taiwan
- Department of Medicine, Mackay Medical College, New Taipei City, Taiwan
- Department of Medical Research, Mackay Memorial Hospital, Taipei, Taiwan
| | - Chao-Min Cheng
- Institute of Biomedical Engineering, National Tsing Hua University, Hsinchu, Taiwan
| | - Wei-Li Chen
- Department of Ophthalmology, National Taiwan University Hospital, Taipei, Taiwan
- Advanced Ocular Surface and Corneal Nerve Research Center, National Taiwan University, Taipei, Taiwan
- Department of Ophthalmology, College of Medicine, National Taiwan University; Taipei, Taiwan
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26
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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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Udry F, Decembrini S, Gamm DM, Déglon N, Kostic C, Arsenijevic Y. Lentiviral mediated RPE65 gene transfer in healthy hiPSCs-derived retinal pigment epithelial cells markedly increased RPE65 mRNA, but modestly protein level. Sci Rep 2020; 10:8890. [PMID: 32483256 PMCID: PMC7264209 DOI: 10.1038/s41598-020-65657-y] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2019] [Accepted: 05/08/2020] [Indexed: 12/15/2022] Open
Abstract
The retinal pigment epithelium (RPE) is a monolayer of cobblestone-like epithelial cells that accomplishes critical functions for the retina. Several protocols have been published to differentiate pluripotent stem cells into RPE cells suitable for disease modelling and therapy development. In our study, the RPE identity of human induced pluripotent stem cell (hiPSC)-derived RPE (iRPE) was extensively characterized, and then used to test a lentiviral-mediated RPE65 gene augmentation therapy. A dose study of the lentiviral vector revealed a dose-dependent effect of the vector on RPE65 mRNA levels. A marked increase of the RPE65 mRNA was also observed in the iRPE (100-fold) as well as in an experimental set with RPE derived from another hiPSC source and from foetal human RPE. Although iRPE displayed features close to bona fide RPE, no or a modest increase of the RPE65 protein level was observed depending on the protein detection method. Similar results were observed with the two other cell lines. The mechanism of RPE65 protein regulation remains to be elucidated, but the current work suggests that high vector expression will not produce an excess of the normal RPE65 protein level.
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Affiliation(s)
- Florian Udry
- Department of ophthalmology, Unit of Retinal Degeneration and Regeneration, University of Lausanne, Hôpital ophtalmique Jules-Gonin, 1004, Lausanne, Switzerland
| | - Sarah Decembrini
- Department of ophthalmology, Unit of Retinal Degeneration and Regeneration, University of Lausanne, Hôpital ophtalmique Jules-Gonin, 1004, Lausanne, Switzerland
- Department of Biomedicine, University Hospital Basel & University Basel, Hebelstr. 20, 4031, Basel, Switzerland
| | - David M Gamm
- McPherson Eye Research Institute, Waisman Center and Department of Ophthalmology and Visual Sciences, and University of Wisconsin-Madison, Madison, USA
| | - Nicole Déglon
- Neuroscience Research Center, Laboratory of Neurotherapies and Neuromodulation, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland
| | - Corinne Kostic
- Department of ophthalmology, Unit of Retinal Degeneration and Regeneration, University of Lausanne, Hôpital ophtalmique Jules-Gonin, 1004, Lausanne, Switzerland
| | - Yvan Arsenijevic
- Department of ophthalmology, Unit of Retinal Degeneration and Regeneration, University of Lausanne, Hôpital ophtalmique Jules-Gonin, 1004, Lausanne, Switzerland.
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28
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de Diego-Otero Y, Giráldez-Pérez RM, Lima-Cabello E, Heredia-Farfan R, Calvo Medina R, Sanchez-Salido L, Pérez Costillas L. Pigment epithelium-derived factor (PEDF) and PEDF-receptor in the adult mouse brain: Differential spatial/temporal localization pattern. J Comp Neurol 2020; 529:141-158. [PMID: 32427349 DOI: 10.1002/cne.24940] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2019] [Revised: 04/20/2020] [Accepted: 04/22/2020] [Indexed: 12/11/2022]
Abstract
Pigment epithelium-derived factor (PEDF) is a multifunctional protein which was initially described in the retina, although it is also present in other tissues. It functions as an antioxidant agent promoting neuronal survival. Recently, a PEDF receptor has shown an elevated binding affinity for PEDF. There are no relevant data regarding the distribution of both proteins in the brain, therefore the main goal of this work was to investigate the spatiotemporal presence of PEDF and PEDFR in the adult mouse brain, and to determine the PEDF blood level in mouse and human. The localization of both proteins was analyzed by different experimental methods such as immunohistochemistry, western-blotting, and also by enzyme-linked immunosorbent assay. Differential expression was found in some telencephalic structures and positive signals for both proteins were detected in the cerebellum. The magnitude of the PEDFR labeling pattern was higher than PEDF and included some cortical and subventricular areas. Age-dependent changes in intensity of both protein immunoreactions were found in the cortical and hippocampal areas with greater reactivity between 4 and 8 months of age, whilst others, like the subventricular zones, these differences were more evident for PEDFR. Although ubiquitous presence was not found in the brain for these two proteins, their relevant functions must not be underestimated. It has been described that PEDF plays an important role in neuroprotection and data provided in the present work represents the first extensive study to understand the relevance of these two proteins in specific brain areas.
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Affiliation(s)
- Yolanda de Diego-Otero
- Research Laboratory, Hospital Civil, Institute of Biomedical Research in Malaga (IBIMA), Málaga, Spain.,Mental Health Clinic Unit, .Regional University Hospital, Hospital Civil, Málaga, Spain.,Research Unit, International Institute of Innovation and Attention to Neurodevelopment and Language, Málaga, Spain
| | - Rosa María Giráldez-Pérez
- Cellular Biology, Physiology and Immunology Department, University of Cordoba, Edificio Charles Darwin, Córdoba, Spain
| | - Elena Lima-Cabello
- Research Laboratory, Hospital Civil, Institute of Biomedical Research in Malaga (IBIMA), Málaga, Spain
| | - Raúl Heredia-Farfan
- Research Laboratory, Hospital Civil, Institute of Biomedical Research in Malaga (IBIMA), Málaga, Spain
| | - Rocío Calvo Medina
- Pediatric Clinic Unit. Regional University Hospital, Hospital Materno-Infantil Avd, Arroyo de los Angeles, Málaga, Spain
| | - Lourdes Sanchez-Salido
- Research Laboratory, Hospital Civil, Institute of Biomedical Research in Malaga (IBIMA), Málaga, Spain
| | - Lucía Pérez Costillas
- Mental Health Clinic Unit, .Regional University Hospital, Hospital Civil, Málaga, Spain.,Psychiatry and Physiotherapy Department, University of Malaga. Medical School, Málaga, Spain
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29
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Kashani AH, Uang J, Mert M, Rahhal F, Chan C, Avery RL, Dugel P, Chen S, Lebkowski J, Clegg DO, Hinton DR, Humayun MS. Surgical Method for Implantation of a Biosynthetic Retinal Pigment Epithelium Monolayer for Geographic Atrophy: Experience from a Phase 1/2a Study. Ophthalmol Retina 2019; 4:264-273. [PMID: 31786135 DOI: 10.1016/j.oret.2019.09.017] [Citation(s) in RCA: 49] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2019] [Revised: 09/24/2019] [Accepted: 09/27/2019] [Indexed: 11/27/2022]
Abstract
PURPOSE To report the intraoperative methods and anatomic results for subretinal implantation of an investigational human embryonic stem cell-derived retinal pigment epithelium (RPE) monolayer seeded on a synthetic substrate (California Project to Cure Blindness Retinal Pigment Epithelium 1 [CPCB-RPE1]) in geographic atrophy (GA). DESIGN Single-arm, open label, prospective, nonrandomized, Phase 1/2a study. PARTICIPANTS Advanced non-neovascular age-related macular degeneration (NNAMD). METHODS The worse-seeing eye (≤20/200) of each subject underwent subretinal implantation of a single 3.5×6.25 mm CPCB-RPE1 implant with a preplanned primary end point of safety and efficacy at 365 days. Commercially available 23-gauge vitrectomy equipment, custom surgical forceps, and operating microscope with or without intraoperative OCT (iOCT) were used. Exact Wilcoxon rank-sum tests and Spearman rank correlation coefficients were used to assess the association of the percentage of the GA area covered by the implant with patient and surgery characteristics. The partial Spearman correlation coefficient was calculated for the correlation between duration of surgery and baseline GA size after adjustment for surgeon experience. MAIN OUTCOME MEASURES Intraoperative exploratory measures are reported, including area of GA covered by implant, subretinal position of implant, duration of surgery, and incidence of adverse events. Operative recordings and reports were used to determine exploratory outcome measures. RESULTS Sixteen subjects were enrolled with a median age of 78 years (range, 69-85 years). Median duration of the surgery for all subjects was 160 minutes (range, 121-466 minutes). Intraoperative OCT was used to guide subretinal placement in 9 cases. Intraoperative OCT was potentially useful in identifying pathology not evident with standard intraoperative visualization. Median GA area at baseline was 13.8 mm2 (range, 6.0-46.4 mm2), and median GA area left uncovered by the implant was 1.7 mm2 (range, 0-20.4 mm2). On average, 86.9% of the baseline GA area was covered by the implant. In 5 subjects, >90% of the GA area was covered. Baseline GA size was inversely correlated with percentage of GA area covered by the implant (rs=-0.72; P = 0.002). No unanticipated serious adverse events related to the implant or surgery were reported. CONCLUSIONS Surgical implantation of CPCB-RPE1 targeted to the area of GA in subjects with advanced NNAMD is feasible in an outpatient setting. Intraoperative OCT is not necessary but potentially useful in identifying subretinal pathology and confirming implant location.
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Affiliation(s)
- Amir H Kashani
- USC Roski Eye Institute, USC Institute for Biomedical Therapeutics and Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, California.
| | - Jeremy Uang
- USC Roski Eye Institute, USC Institute for Biomedical Therapeutics and Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, California
| | - Melissa Mert
- Southern California Clinical and Translational Science Institute, University of Southern California, Department of Preventative Medicine (Biostatistics), Los Angeles, California
| | - Firas Rahhal
- Retina-Vitreous Associates Medical Group, Beverly Hills, California
| | - Clement Chan
- Southern California Desert Retina Consultants, Palm Desert, California
| | - Robert L Avery
- California Retinal Consultants, Santa Barbara, California
| | - Pravin Dugel
- Retinal Consultants of Arizona, Phoenix, Arizona
| | | | - Jane Lebkowski
- Regenerative Patch Technologies LLC, Portola Valley, California
| | - Dennis O Clegg
- Center for Stem Cell Biology and Engineering, University of California, Santa Barbara, California
| | - David R Hinton
- Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, California
| | - Mark S Humayun
- USC Roski Eye Institute, USC Institute for Biomedical Therapeutics and Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, California; Department of Biomedical Engineering, Denney Research Center, University of Southern California, Los Angeles, California.
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30
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Guo Y, Wang P, Ma JH, Cui Z, Yu Q, Liu S, Xue Y, Zhu D, Cao J, Li Z, Tang S, Chen J. Modeling Retinitis Pigmentosa: Retinal Organoids Generated From the iPSCs of a Patient With the USH2A Mutation Show Early Developmental Abnormalities. Front Cell Neurosci 2019; 13:361. [PMID: 31481876 PMCID: PMC6709881 DOI: 10.3389/fncel.2019.00361] [Citation(s) in RCA: 57] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2019] [Accepted: 07/23/2019] [Indexed: 11/21/2022] Open
Abstract
Retinitis pigmentosa (RP) represents a group of inherited retinopathies with early-onset nyctalopia followed by progressive photoreceptor degeneration causing irreversible vision loss. Mutations in USH2A are the most common cause of non-syndromic RP. Here, we reprogrammed induced pluripotent stem cells (iPSCs) from a RP patient with a mutation in USH2A (c.8559-2A > G/c.9127_9129delTCC). Then, multilayer retinal organoids including neural retina (NR) and retinal pigment epithelium (RPE) were generated by three-step “induction-reversal culture.” The early retinal organoids derived from the RP patient with the USH2A mutation exhibited significant defects in terms of morphology, immunofluorescence staining and transcriptional profiling. To the best of our knowledge, the pathogenic mutation (c.9127_9129delTCC) in USH2A has not been reported previously among RP patients. Notably, the expression of laminin in the USH2A mutation organoids was significantly lower than in the iPSCs derived from healthy, age- and sex-matched controls during the retinal organogenesis. We also observed that abnormal retinal neuroepithelium differentiation and polarization caused defective retinal progenitor cell development and retinal layer formation, disordered organization of NRs in the presence of the USH2A mutation. Furthermore, the USH2A mutation bearing RPE cells presented abnormal morphology, lacking pigmented foci and showing an apoptotic trend and reduced expression of specific makers, such as MITF, PEDF, and RPE65. In addition, the USH2A mutation organoids had lower expression of cilium-associated (especially CFAP43, PIFO) and dopaminergic synapse-related genes (including DLGAP1, GRIK1, SLC17A7, and SLC17A8), while there was higher expression of neuron apoptotic process-related genes (especially HIF1A, ADARB1, and CASP3). This study may provide essential assistance in the molecular diagnosis and screening of RP. This work recapitulates the pathogenesis of USH2A using patient-specific organoids and demonstrated that alterations in USH2A function due to mutations may lead to cellular and molecular abnormalities.
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Affiliation(s)
- Yonglong Guo
- Ophthalmology Department, The First Affiliated Hospital of Jinan University, Guangzhou, China.,Key Laboratory for Regenerative Medicine of Ministry of Education, Jinan University, Guangzhou, China
| | - Peiyuan Wang
- Ophthalmology Department, The First Affiliated Hospital of Jinan University, Guangzhou, China
| | - Jacey Hongjie Ma
- Aier School of Ophthalmology, Central South University, Changsha, China.,Shenzhen Aier Eye Hospital, Shenzhen, China
| | - Zekai Cui
- Aier School of Ophthalmology, Central South University, Changsha, China.,Aier Eye Institute, Changsha, China
| | - Quan Yu
- Centric Laboratory, Medical College, Jinan University, Guangzhou, China
| | - Shiwei Liu
- Ophthalmology Department, The First Affiliated Hospital of Jinan University, Guangzhou, China
| | - Yunxia Xue
- Institute of Ophthalmology, Medical College, Jinan University, Guangzhou, China
| | - Deliang Zhu
- Key Laboratory for Regenerative Medicine of Ministry of Education, Jinan University, Guangzhou, China
| | - Jixing Cao
- Ophthalmology Department, The First Affiliated Hospital of Jinan University, Guangzhou, China
| | - Zhijie Li
- Institute of Ophthalmology, Medical College, Jinan University, Guangzhou, China
| | - Shibo Tang
- Aier School of Ophthalmology, Central South University, Changsha, China.,Aier Eye Institute, Changsha, China
| | - Jiansu Chen
- Ophthalmology Department, The First Affiliated Hospital of Jinan University, Guangzhou, China.,Key Laboratory for Regenerative Medicine of Ministry of Education, Jinan University, Guangzhou, China.,Aier School of Ophthalmology, Central South University, Changsha, China.,Aier Eye Institute, Changsha, China.,Institute of Ophthalmology, Medical College, Jinan University, Guangzhou, China
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31
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PEDF peptides promote photoreceptor survival in rd10 retina models. Exp Eye Res 2019; 184:24-29. [PMID: 30980815 DOI: 10.1016/j.exer.2019.04.008] [Citation(s) in RCA: 26] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2018] [Revised: 12/19/2018] [Accepted: 04/05/2019] [Indexed: 12/20/2022]
Abstract
The purpose of the study is to evaluate the protective properties of PEDF peptide fragments on rd10 mouse models of retinal degeneration ex vivo. Human recombinant PEDF and synthetic peptides were used. Rd10 retinal explants as well as wild-type retinal explants treated with zaprinast to mimic the rd10 photoreceptor cell death were employed. PEDF protein was intravitreally administered into rd10 mice. Outer nuclear layer thickness measurements in retinal sections, TUNEL labeling in retinal explants, western blots and immunofluorescence with retinal samples were performed. PEDF protein levels in the RPE of rd10 mice decreased with age (P15 - P25). Levels of PEDF receptor PEDF-R declined in the photoreceptor inner segments from rd10 relative to wild-type mice at P25. PEDF administration increased the outer nuclear layer thickness of rd10 retinas in vivo and decreased the number of TUNEL+ nuclei of photoreceptors in rd10 retinal explant cultures, both relative to untreated controls. Peptides containing the PEDF neurotrophic region decreased the number of TUNEL+ photoreceptors in both rd10 and zaprinast-induced cell death ex vivo models, while peptides without the neurotrophic region and/or lacking affinity for PEDF-R were ineffective in protecting photoreceptors. Thus, retinal explants are a valuable system to evaluate PEDF activity. Short peptides with the photoreceptor-protective property of PEDF may prove useful for the development of therapeutic agents for photoreceptor protection in retinal degenerations.
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32
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Wang M, Lau LI, Sreekumar PG, Spee C, Hinton DR, Sadda SR, Kannan R. Characterization and Regulation of Carrier Proteins of Mitochondrial Glutathione Uptake in Human Retinal Pigment Epithelium Cells. Invest Ophthalmol Vis Sci 2019; 60:500-516. [PMID: 30707752 PMCID: PMC6360990 DOI: 10.1167/iovs.18-25686] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/03/2022] Open
Abstract
Purpose To characterize two mitochondrial membrane transporters 2-oxoglutarate (OGC) and dicarboxylate (DIC) in human RPE (hRPE) and to elucidate their role in the regulation of mitochondrial glutathione (mGSH) uptake and cell death in oxidative stress. Methods The localization of OGC and DIC proteins in confluent hRPE, polarized hRPE monolayers and mouse retina was assessed by immunoblotting and confocal microscopy. Time- and dose-dependent expression of the two carriers were determined after treatment of hRPE with H2O2, phenyl succinate (PS), and butyl malonate (BM), respectively, for 24 hours. The effect of inhibition of OGC and DIC on apoptosis (TUNEL), mGSH, and mtDNA was determined. Silencing of OGC by siRNA knockdown on RPE cell death was studied. Kinetics of caspase 3/7 activation with OGC and DIC inhibitors and effect of cotreatment with glutathione monoethyl ester (GSH-MEE) was determined using the IncuCyte live cell imaging. Results OGC and DIC are expressed in hRPE mitochondria and exhibited a time- and dose-dependent decrease with stress. Pharmacologic inhibition caused a decrease in OGC and DIC in mitochondria without changes in mtDNA and resulted in increased apoptosis and mGSH depletion. GSH-MEE prevented apoptosis through restoration of mGSH. OGC siRNA exacerbated apoptotic cell death in stressed RPE which was inhibited by increased mGSH from GSH-MEE cotreatment. Conclusions Characterization and mechanism of action of two carrier proteins of mGSH uptake in RPE are reported. Regulation of OGC and DIC will be of value in devising therapeutic strategies for retinal disorders such as AMD.
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Affiliation(s)
- Mo Wang
- Department of Ophthalmology, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China
- The Stephen J. Ryan Initiative for Macular Research (RIMR), Doheny Eye Institute, Los Angeles, California, United States
| | - Lin-Ing Lau
- The Stephen J. Ryan Initiative for Macular Research (RIMR), Doheny Eye Institute, Los Angeles, California, United States
- Institute of Clinical Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan
| | - Parameswaran G Sreekumar
- The Stephen J. Ryan Initiative for Macular Research (RIMR), Doheny Eye Institute, Los Angeles, California, United States
| | - Christine Spee
- Department of Pathology and Ophthalmology, USC Roski Eye Institute, Keck School of Medicine of the University of Southern California, Los Angeles, California, United States
| | - David R Hinton
- Department of Pathology and Ophthalmology, USC Roski Eye Institute, Keck School of Medicine of the University of Southern California, Los Angeles, California, United States
| | - Srinivas R Sadda
- The Stephen J. Ryan Initiative for Macular Research (RIMR), Doheny Eye Institute, Los Angeles, California, United States
| | - Ram Kannan
- The Stephen J. Ryan Initiative for Macular Research (RIMR), Doheny Eye Institute, Los Angeles, California, United States
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Artero-Castro A, Popelka S, Jendelova P, Motlik J, Ardan T, Rodriguez Jimenez FJ, Erceg S. The identification of small molecules that stimulate retinal pigment epithelial cells: potential novel therapeutic options for treating retinopathies. Expert Opin Drug Discov 2019; 14:169-177. [PMID: 30616395 DOI: 10.1080/17460441.2019.1559148] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
Abstract
INTRODUCTION Combinatory strategies using pharmacology and stem cell therapy have emerged due to their potential in the treatment of retinal pigment epithelium (RPE) cell related diseases, and a variety of different stem cell sources have been evaluated both in animal models and in humans. RPE cells derived from human embryonic stem cells (hESCs) and human induced pluripotent cells (hiPSCs) are already in clinical trials, holding great promise for the treatment of age-related macular disease (AMD) and hereditary RPE-related retinal dystrophies. Highly efficient protocol for RPE generations have been developed, but they are still time-consuming and laborious. Areas covered: The authors review RPE related diseases, as well as the known functions of RPE cells in retinal homeostasis. The authors also discuss small molecules that target RPE in vivo as well as in vitro to aid RPE differentiation from pluripotent stem cells clinically. The authors base this review on literature searches performed through PubMed. Expert opinion: Using high-throughput systems, technology will provide the possibility of identifying and optimizing molecules/drugs that could lead to faster and simpler protocols for RPE differentiation. This could be crucial in moving forward to create safer and more efficient RPE-based personalized therapies.
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Affiliation(s)
- Ana Artero-Castro
- a Stem Cell Therapies in Neurodegenerative Diseases Lab , Research Center "Principe Felipe" , Valencia , Spain
| | - Stepan Popelka
- b Institute of Macromolecular Chemistry , Czech Academy of Sciences , Praha 6 , Czech Republic
| | - Pavla Jendelova
- c Institute of Experimental Medicine, Department of Tissue Cultures and Stem Cells , Czech Academy of Sciences , Prague , Czech Republic
| | - Jan Motlik
- d Laboratory of Cell Regeneration and Plasticity, Research Center PIGMOD , Institute of Animal Physiology and Genetics, Czech Academy of Sciences , Libechov , Czech Republic
| | - Taras Ardan
- d Laboratory of Cell Regeneration and Plasticity, Research Center PIGMOD , Institute of Animal Physiology and Genetics, Czech Academy of Sciences , Libechov , Czech Republic
| | | | - Slaven Erceg
- a Stem Cell Therapies in Neurodegenerative Diseases Lab , Research Center "Principe Felipe" , Valencia , Spain.,c Institute of Experimental Medicine, Department of Tissue Cultures and Stem Cells , Czech Academy of Sciences , Prague , Czech Republic.,e National Stem Cell Bank-Valencia Node, Biomolecular and Bioinformatics Resources Platform PRB2,ISCIII , Research Center "Principe Felipe" , Valencia , Spain
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Kashani AH, Martynova A, Koss M, Brant R, Zhu DH, Lebkowski J, Hinton D, Clegg D, Humayun MS. Subretinal Implantation of a Human Embryonic Stem Cell-Derived Retinal Pigment Epithelium Monolayer in a Porcine Model. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2019; 1185:569-574. [PMID: 31884672 DOI: 10.1007/978-3-030-27378-1_93] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
The goal of this study was to quantitatively assess retinal thickness using spectral domain optical coherence tomography (SD-OCT) after subretinal implantation of human embryonic stem cell-derived retinal pigment epithelium in a porcine model. The implant is called CPCB-RPE1 for the California Project to Cure Blindness-Retinal Pigment Epithelium 1. Data were derived from previous experiments on 14 minipigs that received either subretinal implantation of CPCB-RPE1 (n = 11) or subretinal bleb formation alone (sham; n = 3) using previously described methods and procedures (Brant Fernandes et al. Ophthalmic Surg Lasers Imaging Retina 47:342-51, 2016; Martynova et al. (2016) Koss et al. Graefes Arch Clin Exp Ophthalmol 254:1553-65, 2016; Hu et al. Ophthalmic Res 48:186-91, 2016; Martynova et al. ARVO Abstract 2016. SD-OCT retinal thickness (RT) and sublayer thickness over the implant were compared with topographically similar preimplantation regions as described previously Martynova et al. ARVO Abstract 2016. Imaging results were compared to postmortem histology using hematoxylin-eosin staining. RT overlying the CPCB-RPE1 postimplantation was not significantly different from preimplantation (308 ± 72 μm vs 292 ± 41 μm; p = 0.44). RT was not significantly different before and after implantation in any retinal sublayer at 1 month. Histology demonstrated grossly normal retinal anatomy as well as photoreceptor interdigitation with RPE.
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Affiliation(s)
- Amir H Kashani
- Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA. .,USC Roski Eye Institute and Institute for Biomedical Therapeutics, Los Angeles, CA, USA.
| | - Ana Martynova
- Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
| | - Michael Koss
- Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
| | - Rodrigo Brant
- Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
| | - Dan Hong Zhu
- Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
| | - Jane Lebkowski
- Regenerative Patch Technologies (RPT), Menlo Park, CA, USA
| | - David Hinton
- Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
| | - Dennis Clegg
- Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, CA, USA
| | - Mark S Humayun
- Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA. .,USC Roski Eye Institute and Institute for Biomedical Therapeutics, Los Angeles, CA, USA.
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Cao L, Liu J, Pu J, Milne G, Chen M, Xu H, Shipley A, Forrester JV, McCaig CD, Lois N. Polarized retinal pigment epithelium generates electrical signals that diminish with age and regulate retinal pathology. J Cell Mol Med 2018; 22:5552-5564. [PMID: 30160348 PMCID: PMC6201363 DOI: 10.1111/jcmm.13829] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2017] [Revised: 07/06/2018] [Accepted: 07/10/2018] [Indexed: 12/21/2022] Open
Abstract
The transepithelial potential difference (TEP) across the retinal pigment epithelial (RPE) is dependent on ionic pumps and tight junction "seals" between epithelial cells. RPE cells release neurotrophic growth factors such as pigment epithelial derived factor (PEDF), which is reduced in age-related macular degeneration (AMD). The mechanisms that control the secretion of PEDF from RPE cells are not well understood. Using the CCL2/CX3CR1 double knockout mouse model (DKO), which demonstrates RPE damage and retinal degeneration, we uncovered an interaction between PEDF and the TEP which is likely to play an important role in retinal ageing and in the pathogenesis of AMD. We found that: (a) the expression of ATP1B1 (the Na+ /K+ -ATPase β1 subunit) was reduced significantly in RPE from aged mice, in patients with CNV (Choroidal Neovascularization) and in DKO mice; (b) the expression of PEDF also was decreased in aged persons and in DKO mice; (c) the TEP across RPE was reduced markedly in RPE cells from DKO mice and (d) an applied electric field (EF) of 50-100 mV/mm, used to mimic the natural TEP, increased the expression and secretion of PEDF in primary RPE cells. In conclusion, the TEP across the RPE depends on the expression of ATP1B1 and this regulates the secretion of PEDF by RPE cells and so may regulate the onset of retinal disease. Increasing the expression of PEDF using an applied EF to replenish a disease or age-reduced TEP may offer a new way of preventing or reversing retinal dysfunction.
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Affiliation(s)
- Lin Cao
- School of MedicineMedical Sciences and NutritionInstitute of Medical SciencesUniversity of AberdeenAberdeenUK
- Yizhou International Proton Medical Centre and Cancer HospitalHe BeiChina
| | - Jie Liu
- Department of OphthalmologyFrist Hospital Affiliated to the Chinese PLA General HospitalBeijingChina
| | - Jin Pu
- School of MedicineMedical Sciences and NutritionInstitute of Medical SciencesUniversity of AberdeenAberdeenUK
| | - Gillian Milne
- School of MedicineMedical Sciences and NutritionInstitute of Medical SciencesUniversity of AberdeenAberdeenUK
| | - Mei Chen
- Wellcome‐Wolfson Institute for Experimental MedicineQueen's UniversityBelfastUK
| | - Heping Xu
- Wellcome‐Wolfson Institute for Experimental MedicineQueen's UniversityBelfastUK
| | - Alan Shipley
- Biological Research & DevelopmentUniversity of New EnglandBiddefordMaine
| | - John V Forrester
- School of MedicineMedical Sciences and NutritionInstitute of Medical SciencesUniversity of AberdeenAberdeenUK
| | - Colin D McCaig
- School of MedicineMedical Sciences and NutritionInstitute of Medical SciencesUniversity of AberdeenAberdeenUK
| | - Noemi Lois
- Wellcome‐Wolfson Institute for Experimental MedicineQueen's UniversityBelfastUK
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36
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Pavan B, Dalpiaz A. Retinal pigment epithelial cells as a therapeutic tool and target against retinopathies. Drug Discov Today 2018; 23:1672-1679. [DOI: 10.1016/j.drudis.2018.06.009] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2018] [Revised: 05/15/2018] [Accepted: 06/08/2018] [Indexed: 01/19/2023]
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Kharitonov AE, Surdina AV, Lebedeva OS, Bogomazova AN, Lagarkova MA. Possibilities for Using Pluripotent Stem Cells for Restoring Damaged Eye Retinal Pigment Epithelium. Acta Naturae 2018; 10:30-39. [PMID: 30397524 PMCID: PMC6209409] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2018] [Indexed: 12/03/2022] Open
Abstract
The retinal pigment epithelium is a monolayer of pigmented, hexagonal cells connected by tight junctions. These cells compose part of the outer blood-retina barrier, protect the eye from excessive light, have important secretory functions, and support the function of photoreceptors, ensuring the coordination of a variety of regulatory mechanisms. It is the degeneration of the pigment epithelium that is the root cause of many retinal degenerative diseases. The search for reliable cell sources for the transplantation of retinal pigment epithelium is of extreme urgency. Pluripotent stem cells (embryonic stem or induced pluripotent) can be differentiated with high efficiency into the pigment epithelium of the retina, which opens up possibilities for cellular therapy in macular degeneration and can slow down the development of pathology and, perhaps, restore a patient's vision. Pioneering clinical trials on transplantation of retinal pigment epithelial cells differentiated from pluripotent stem cells in the United States and Japan confirmed the need for developing and optimizing such approaches to cell therapy. For effective use, pigment epithelial cells differentiated from pluripotent stem cells should have a set of functional properties characteristic of such cells in vivo. This review summarizes the current state of preclinical and clinical studies in the field of retinal pigment epithelial transplantation therapy. We also discuss different differentiation protocols based on data in the literature and our own data, and the problems holding back the widespread therapeutic application of retinal pigment epithelium differentiated from pluripotent stem cells.
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Affiliation(s)
- A. E. Kharitonov
- Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Malaya Pirogovskaya Str., 1a, Moscow, 119435, Russia
| | - A. V. Surdina
- Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Malaya Pirogovskaya Str., 1a, Moscow, 119435, Russia
| | - O. S. Lebedeva
- Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Malaya Pirogovskaya Str., 1a, Moscow, 119435, Russia
| | - A. N. Bogomazova
- Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Malaya Pirogovskaya Str., 1a, Moscow, 119435, Russia
| | - M. A. Lagarkova
- Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Malaya Pirogovskaya Str., 1a, Moscow, 119435, Russia
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38
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Peng CH, Chuang JH, Wang ML, Jhan YY, Chien KH, Chung YC, Hung KH, Chang CC, Lee CK, Tseng WL, Hwang DK, Hsu CH, Lin TC, Chiou SH, Chen SJ. Laminin modification subretinal bio-scaffold remodels retinal pigment epithelium-driven microenvironment in vitro and in vivo. Oncotarget 2018; 7:64631-64648. [PMID: 27564261 PMCID: PMC5323104 DOI: 10.18632/oncotarget.11502] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2016] [Accepted: 07/19/2016] [Indexed: 11/25/2022] Open
Abstract
Advanced age-related macular degeneration (AMD) may lead to geographic atrophy or fibrovascular scar at macular, dysfunctional retinal microenvironment, and cause profound visual loss. Recent clinical trials have implied the potential application of pluripotent cell-differentiated retinal pigment epithelial cells (dRPEs) and membranous scaffolds implantation in repairing the degenerated retina in AMD. However, the efficacy of implanted membrane in immobilization and supporting the viability and functions of dRPEs, as well as maintaining the retinal microenvironment is still unclear. Herein we generated a biomimetic scaffold mimicking subretinal Bruch's basement from plasma modified polydimethylsiloxane (PDMS) sheet with laminin coating (PDMS-PmL), and investigated its potential functions to provide a subretinal environment for dRPE-monolayer grown on it. Firstly, compared to non-modified PDMS, PDMS-PmL enhanced the attachment, proliferation, polarization, and maturation of dRPEs. Second, PDMS-PmL increased the polarized tight junction, PEDF secretion, melanosome pigment deposit, and phagocytotic-ability of dRPEs. Third, PDMS-PmL was able to carry a dRPEs/photoreceptor-precursors multilayer retina tissue. Finally, the in vivo subretinal implantation of PDMS-PmL in porcine eyes showed well-biocompatibility up to 2-year follow-up. Notably, multifocal ERGs at 2-year follow-up revealed well preservation of macular function in PDMS-PmL, but not PDMS, transplanted porcine eyes. Trophic PEDF secretion of macular retina in PDMS-PmL group was also maintained to preserve retinal microenvironment in PDMS-PmL eyes at 2 year. Taken together, these data indicated that PDMS-PmL is able to sustain the physiological morphology and functions of polarized RPE monolayer, suggesting its potential of rescuing macular degeneration in vivo.
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Affiliation(s)
- Chi-Hsien Peng
- Department of Ophthalmology, Taipei Veterans General Hospital, Taipei, Taiwan.,Department of Ophthalmology, Shin Kong Wu Ho-Su Memorial Hospital & Fu-Jen Catholic University, Taipei Taiwan.,Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan
| | - Jen-Hua Chuang
- Department of Medical Research, Taipei Veterans General Hospital, Taipei, Taiwan.,Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan
| | - Mong-Lien Wang
- Department of Medical Research, Taipei Veterans General Hospital, Taipei, Taiwan.,School of Medicine, National Yang-Ming University, Taipei, Taiwan
| | - Yong-Yu Jhan
- Department of Ophthalmology, Taipei Veterans General Hospital, Taipei, Taiwan.,Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan
| | - Ke-Hung Chien
- Department of Ophthalmology, Tri-Service General Hospital & National Defense Medical Center, Taipei, Taiwan.,Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan
| | - Yu-Chien Chung
- Department of Ophthalmology, Taipei Veterans General Hospital, Taipei, Taiwan.,School of Medicine, National Yang-Ming University, Taipei, Taiwan
| | - Kuo-Hsuan Hung
- Department of Ophthalmology, Taipei Veterans General Hospital, Taipei, Taiwan.,Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan.,School of Medicine, National Yang-Ming University, Taipei, Taiwan
| | - Chia-Ching Chang
- Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taipei, Taiwan
| | - Chao-Kuei Lee
- Department of Photonics, National Sun Yat-sen University, Kaohsiung, Taiwan
| | - Wei-Lien Tseng
- Department of Medical Research, Taipei Veterans General Hospital, Taipei, Taiwan.,Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan
| | - De-Kuang Hwang
- Department of Ophthalmology, Taipei Veterans General Hospital, Taipei, Taiwan.,School of Medicine, National Yang-Ming University, Taipei, Taiwan
| | | | - Tai-Chi Lin
- Department of Ophthalmology, Taipei Veterans General Hospital, Taipei, Taiwan.,Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan
| | - Shih-Hwa Chiou
- Department of Ophthalmology, Taipei Veterans General Hospital, Taipei, Taiwan.,Department of Medical Research, Taipei Veterans General Hospital, Taipei, Taiwan.,Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan.,Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan.,School of Medicine, National Yang-Ming University, Taipei, Taiwan
| | - Shih-Jen Chen
- Department of Ophthalmology, Taipei Veterans General Hospital, Taipei, Taiwan.,School of Medicine, National Yang-Ming University, Taipei, Taiwan
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Luo M, Chen Y. Application of stem cell-derived retinal pigmented epithelium in retinal degenerative diseases: present and future. Int J Ophthalmol 2018; 11:150-159. [PMID: 29376004 DOI: 10.18240/ijo.2018.01.23] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2017] [Accepted: 08/08/2017] [Indexed: 12/12/2022] Open
Abstract
As a constituent of blood-retinal barrier and retinal outer segment (ROS) scavenger, retinal pigmented epithelium (RPE) is fundamental to normal function of retina. Malfunctioning of RPE contributes to the onset and advance of retinal degenerative diseases. Up to date, RPE replacement therapy is the only possible method to completely reverse retinal degeneration. Transplantation of human RPE stem cell-derived RPE (hRPESC-RPE) has shown some good results in animal models. With promising results in terms of safety and visual improvement, human embryonic stem cell-derived RPE (hESC-RPE) can be expected in clinical settings in the near future. Despite twists and turns, induced pluripotent stem cell-derived RPE (iPSC-RPE) is now being intensely investigated to overcome genetic and epigenetic instability. By far, only one patient has received iPSC-RPE transplant, which is a hallmark of iPSC technology development. During follow-up, no major complications such as immunogenicity or tumorigenesis have been observed. Future trials should keep focusing on the safety of stem cell-derived RPE (SC-RPE) especially in long period, and better understanding of the nature of stem cell and the molecular events in the process to generate SC-RPE is necessary to the prosperity of SC-RPE clinical application.
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Affiliation(s)
- Mingyue Luo
- Department of Ophthalmology, Peking Union Medical College Hospital, Beijing 100730, China.,Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China
| | - Youxin Chen
- Department of Ophthalmology, Peking Union Medical College Hospital, Beijing 100730, China.,Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China
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40
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Enzmann V, Lecaudé S, Kruschinski A, Vater A. CXCL12/SDF-1-Dependent Retinal Migration of Endogenous Bone Marrow-Derived Stem Cells Improves Visual Function after Pharmacologically Induced Retinal Degeneration. Stem Cell Rev Rep 2017; 13:278-286. [PMID: 27924617 DOI: 10.1007/s12015-016-9706-0] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022]
Abstract
Mobilized bone marrow-derived stem cells (BMSC) have been discussed as an alternative strategy for endogenous repair. Thereby, different approaches for BMSC mobilization have been pursued. Herein, the role of a newly discovered oligonucleotide for retinal homing and regeneration capability of BMSCs was investigated in the sodium iodate (NaIO3) model of retinal degeneration. Mobilization was achieved in GFP-chimera with NOX-A12, a CXC-motif chemokine ligand 12 (CXCL12)/stromal cell-derived factor 1 (SDF-1)-neutralizing L-aptamer. BMSC homing was directed by intravitreal SDF-1 injection. Visual acuity was measured using the optokinetic reflex. Paraffin cross sections were stained with hematoxylin and eosin for retinal thickness measurements. Immunohistochemistry was performed to investigate the expression of cell-specific markers after mobilization. A single dose of NOX-A12 induced significant mobilization of GFP+ cells which were found in all layers within the degenerating retina. An additional intravitreal injection of SDF-1 increased migration towards the site of injury. Thereby, the number of BMSCs (Sca-1+) found in the damaged retina increased whereas a decrease of activated microglia (Iba-1+) was found. The mobilization led to significantly increased visual acuity. However, no significant changes in retinal thickness or differentiation towards retinal cell types were detected. Systemic mobilization by a single dose of NOX-A12 showed increased homing of BMSCs into the degenerated retina, which was associated with improved visual function when injection of SDF-1 was additionally performed. The redistribution of the cells to the site of injury combined with their observed beneficial effects support the endogenous therapeutic strategy for retinal repair.
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Affiliation(s)
- Volker Enzmann
- Department of Ophthalmology, University Hospital, University of Bern, Freiburgstrasse 14, 3010, Bern, Switzerland. .,Department of Clinical Research, University of Bern, Bern, Switzerland.
| | - Stéphanie Lecaudé
- Department of Clinical Research, University of Bern, Bern, Switzerland
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41
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Fernandes RAB, Stefanini FR, Falabella P, Koss MJ, Wells T, Diniz B, Ribeiro R, Schor P, Maia M, Penha FM, Hinton DR, Tai YC, Humayun M. Development of a new tissue injector for subretinal transplantation of human embryonic stem cell derived retinal pigmented epithelium. Int J Retina Vitreous 2017; 3:41. [PMID: 29093829 PMCID: PMC5662097 DOI: 10.1186/s40942-017-0095-6] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2017] [Accepted: 09/26/2017] [Indexed: 11/23/2022] Open
Abstract
Background
Subretinal cell transplantation is a challenging surgical maneuver. This paper describes the preliminary findings of a new tissue injector for subretinal implantation of an ultrathin non-absorbable substrate seeded with human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE). Methods Ultrathin Parylene-C substrates measuring 3.5 mm × 6.0 mm seeded with hESC-RPE (implant referred to as CPCB-RPE1) were implanted into the subretinal space of 12 Yucatan minipigs. Animals were euthanized immediately after the procedure and underwent spectral domain optical coherence tomography (SD-OCT) and histological analysis to assess the subretinal placement of the implant. Evaluation of the hESC-RPE cells seeded on the substrate was carried out before and after implantation using standard cell counting techniques. Results The tissue injector delivered the CPCB-RPE1 implant through a 1.5 mm sclerotomy and a 1.0–1.5 mm retinectomy. SD-OCT scans and histological examination revealed that substrates were precisely placed in the subretinal space, and that the hESC-RPE cell monolayer continued to cover the surface of the substrate after the surgical procedure. Conclusion This innovative tissue injector was able to efficiently deliver the implant in the subretinal space of Yucatan minipigs, preventing significant hESC-RPE cell loss, minimizing tissue trauma, surgical complications and postoperative inflammation.
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Affiliation(s)
- Rodrigo A Brant Fernandes
- Department of Ophthalmology, USC Roski Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA USA.,Department of Ophthalmology and Visual Sciences, Federal University of São Paulo, Rua Botucatu, 822, São Paulo, SP 04023-062 Brazil
| | - Francisco R Stefanini
- Department of Ophthalmology, USC Roski Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA USA.,Department of Ophthalmology and Visual Sciences, Federal University of São Paulo, Rua Botucatu, 822, São Paulo, SP 04023-062 Brazil
| | - Paulo Falabella
- Department of Ophthalmology, USC Roski Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA USA.,Department of Ophthalmology and Visual Sciences, Federal University of São Paulo, Rua Botucatu, 822, São Paulo, SP 04023-062 Brazil
| | - Michael J Koss
- Department of Ophthalmology, USC Roski Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA USA.,Augenzentrum Nymphenburger Hoefe, Herzog Carl Theodor Augenklinik, Munich, Germany
| | - Trent Wells
- Department of Ophthalmology, USC Roski Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA USA
| | - Bruno Diniz
- Department of Ophthalmology, USC Roski Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA USA.,Department of Ophthalmology and Visual Sciences, Federal University of São Paulo, Rua Botucatu, 822, São Paulo, SP 04023-062 Brazil
| | - Ramiro Ribeiro
- Department of Ophthalmology, USC Roski Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA USA.,Department of Ophthalmology and Visual Sciences, Federal University of São Paulo, Rua Botucatu, 822, São Paulo, SP 04023-062 Brazil
| | - Paulo Schor
- Department of Ophthalmology and Visual Sciences, Federal University of São Paulo, Rua Botucatu, 822, São Paulo, SP 04023-062 Brazil
| | - Mauricio Maia
- Department of Ophthalmology and Visual Sciences, Federal University of São Paulo, Rua Botucatu, 822, São Paulo, SP 04023-062 Brazil
| | - Fernando M Penha
- Department of Ophthalmology and Visual Sciences, Federal University of São Paulo, Rua Botucatu, 822, São Paulo, SP 04023-062 Brazil.,Fundação Universidade Regional de Blumenau, Blumenau, Santa Catarina Brazil
| | - David R Hinton
- Department of Ophthalmology, USC Roski Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA USA.,Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA USA
| | - Yu-Chong Tai
- Electrical Engineering, California Institute of Technology, Pasadena, CA USA
| | - Mark Humayun
- Department of Ophthalmology, USC Roski Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA USA.,USC Institute for Biomedical Therapeutics, Keck School of Medicine, University of Southern California, Los Angeles, CA USA
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Lidgerwood GE, Lim SY, Crombie DE, Ali R, Gill KP, Hernández D, Kie J, Conquest A, Waugh HS, Wong RCB, Liang HH, Hewitt AW, Davidson KC, Pébay A. Defined Medium Conditions for the Induction and Expansion of Human Pluripotent Stem Cell-Derived Retinal Pigment Epithelium. Stem Cell Rev Rep 2017; 12:179-88. [PMID: 26589197 DOI: 10.1007/s12015-015-9636-2] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
We demonstrate that a combination of Noggin, Dickkopf-1, Insulin Growth Factor 1 and basic Fibroblast Growth Factor, promotes the differentiation of human pluripotent stem cells into retinal pigment epithelium (RPE) cells. We describe an efficient one-step approach that allows the generation of RPE cells from both human embryonic stem cells and human induced pluripotent stem cells within 40-60 days without the need for manual excision, floating aggregates or imbedded cysts. Compared to methods that rely on spontaneous differentiation, our protocol results in faster differentiation into RPE cells. This pro-retinal culture medium promotes the growth of functional RPE cells that exhibit key characteristics of the RPE including pigmentation, polygonal morphology, expression of mature RPE markers, electrophysiological membrane potential and the ability to phagocytose photoreceptor outer segments. This protocol can be adapted for feeder, feeder-free and serum-free conditions. This method thereby provides a rapid and simplified production of RPE cells for downstream applications such as disease modelling and drug screening.
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Affiliation(s)
- Grace E Lidgerwood
- Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital; Ophthalmology, University of Melbourne, Department of Surgery, 32 Gisborne Street, East Melbourne, VIC, 3002, Australia
| | - Shiang Y Lim
- O'Brien Institute Department, St Vincent's Institute of Medical Research, Fitzroy, VIC, Australia
| | - Duncan E Crombie
- Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital; Ophthalmology, University of Melbourne, Department of Surgery, 32 Gisborne Street, East Melbourne, VIC, 3002, Australia
| | - Ray Ali
- School of Medicine, Menzies Institute for Medical Research, University of Tasmania, TAS, Australia
| | - Katherine P Gill
- Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital; Ophthalmology, University of Melbourne, Department of Surgery, 32 Gisborne Street, East Melbourne, VIC, 3002, Australia
| | - Damián Hernández
- O'Brien Institute Department, St Vincent's Institute of Medical Research, Fitzroy, VIC, Australia
| | - Josh Kie
- Department of Anatomy and Neuroscience, University of Melbourne, Parkville, VIC, Australia
| | - Alison Conquest
- Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital; Ophthalmology, University of Melbourne, Department of Surgery, 32 Gisborne Street, East Melbourne, VIC, 3002, Australia
| | - Hayley S Waugh
- Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital; Ophthalmology, University of Melbourne, Department of Surgery, 32 Gisborne Street, East Melbourne, VIC, 3002, Australia
| | - Raymond C B Wong
- Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital; Ophthalmology, University of Melbourne, Department of Surgery, 32 Gisborne Street, East Melbourne, VIC, 3002, Australia
| | - Helena H Liang
- Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital; Ophthalmology, University of Melbourne, Department of Surgery, 32 Gisborne Street, East Melbourne, VIC, 3002, Australia
| | - Alex W Hewitt
- Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital; Ophthalmology, University of Melbourne, Department of Surgery, 32 Gisborne Street, East Melbourne, VIC, 3002, Australia
- School of Medicine, Menzies Institute for Medical Research, University of Tasmania, TAS, Australia
| | - Kathryn C Davidson
- Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital; Ophthalmology, University of Melbourne, Department of Surgery, 32 Gisborne Street, East Melbourne, VIC, 3002, Australia
- Australian Regenerative Medicine Institute, Monash University, Clayton, VIC, Australia
| | - Alice Pébay
- Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital; Ophthalmology, University of Melbourne, Department of Surgery, 32 Gisborne Street, East Melbourne, VIC, 3002, Australia.
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Skottman H, Muranen J, Lähdekorpi H, Pajula E, Mäkelä K, Koivusalo L, Koistinen A, Uusitalo H, Kaarniranta K, Juuti-Uusitalo K. Contacting co-culture of human retinal microvascular endothelial cells alters barrier function of human embryonic stem cell derived retinal pigment epithelial cells. Exp Cell Res 2017; 359:101-111. [DOI: 10.1016/j.yexcr.2017.08.004] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2017] [Revised: 07/31/2017] [Accepted: 08/02/2017] [Indexed: 10/19/2022]
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The stem cell regulator PEDF is dispensable for maintenance and function of hematopoietic stem cells. Sci Rep 2017; 7:10134. [PMID: 28860613 PMCID: PMC5579195 DOI: 10.1038/s41598-017-09452-2] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2017] [Accepted: 07/25/2017] [Indexed: 01/31/2023] Open
Abstract
Pigment epithelium derived factor (PEDF), a ubiquitously expressed 50 kDa secreted glycoprotein, was recently discovered to regulate self-renewal of neural stem cells and have a supportive effect on human embryonic stem cell growth. Here, we analyzed expression of PEDF in the murine hematopoietic stem cell (HSC) compartments and found that PEDF is highly expressed in primary long-term HSCs. Therefore, we characterized the hematopoietic system in a knockout mouse model for PEDF and using this model we surprisingly found that PEDF is dispensable for HSC regulation. PEDF knockout mice exhibit normal hematopoiesis in steady state conditions and the absence of PEDF lead to normal regeneration capacity in a serial competitive transplantation setting. Additionally, PEDF-deficient cells exhibit unaltered lineage distribution upon serial transplantations. When human cord blood stem and progenitor cells were cultured in media supplemented with recombinant PEDF they did not show changes in growth potential. Taken together, we report that PEDF is not a critical regulatory factor for HSC function during regeneration in vivo or growth of human stem/progenitor cells in vitro.
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Bhattacharya S, Yin J, Winborn CS, Zhang Q, Yue J, Chaum E. Prominin-1 Is a Novel Regulator of Autophagy in the Human Retinal Pigment Epithelium. Invest Ophthalmol Vis Sci 2017; 58:2366-2387. [PMID: 28437526 PMCID: PMC5403116 DOI: 10.1167/iovs.16-21162] [Citation(s) in RCA: 51] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
Purpose Prominin-1 (Prom1) is a transmembrane glycoprotein, which is expressed in stem cell lineages, and has recently been implicated in cancer stem cell survival. Mutations in the Prom1 gene have been shown to disrupt photoreceptor disk morphogenesis and cause an autosomal dominant form of Stargardt-like macular dystrophy (STGD4). Despite the apparent structural role of Prom1 in photoreceptors, its role in other cells of the retina is unknown. The purpose of this study is to investigate the role of Prom1 in the highly metabolically active cells of the retinal pigment epithelium (RPE). Methods Lentiviral siRNA and the genome editing CRISPR/Cas9 system were used to knockout Prom1 in primary RPE and ARPE-19 cells, respectively. Western blotting, confocal microscopy, and flow sight imaging cytometry assays were used to quantify autophagy flux. Immunoprecipitation was used to detect Prom1 interacting proteins. Results Our studies demonstrate that Prom1 is primarily a cytosolic protein in the RPE. Stress signals and physiological aging robustly increase autophagy with concomitant upregulation of Prom1 expression. Knockout of Prom1 increased mTORC1 and mTORC2 signaling, decreased autophagosome trafficking to the lysosome, increased p62 accumulation, and inhibited autophagic puncta induced by activators of autophagy. Conversely, ectopic overexpression of Prom1 inhibited mTORC1 and mTORC2 activities, and potentiated autophagy flux. Through interactions with p62 and HDAC6, Prom1 regulates autophagosome maturation and trafficking, suggesting a new cytoplasmic role of Prom1 in RPE function. Conclusions Our results demonstrate that Prom1 plays a key role in the regulation of autophagy via upstream suppression of mTOR signaling and also acting as a component of a macromolecular scaffold involving p62 and HDAC6.
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Affiliation(s)
- Sujoy Bhattacharya
- Department of Ophthalmology, University of Tennessee Health Science Center, Memphis, Tennessee, United States
| | - Jinggang Yin
- Department of Ophthalmology, University of Tennessee Health Science Center, Memphis, Tennessee, United States
| | - Christina S Winborn
- Department of Ophthalmology, University of Tennessee Health Science Center, Memphis, Tennessee, United States
| | - Qiuhua Zhang
- Department of Ophthalmology, University of Tennessee Health Science Center, Memphis, Tennessee, United States
| | - Junming Yue
- Department of Pathology, University of Tennessee Health Science Center, Memphis, Tennessee, United States
| | - Edward Chaum
- Department of Ophthalmology, University of Tennessee Health Science Center, Memphis, Tennessee, United States 3Department of Anatomy and Neurobiology, University of Tennessee Health Science Center, Memphis, Tennessee, United States
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Abstract
Purpose of review Progress in stem cell research for blinding diseases over the past decade is now being applied to patients with retinal degenerative diseases and soon perhaps, glaucoma. However, the field still has much to learn about the conversion of stem cells into various retinal cell types, and the potential delivery methods that will be required to optimize the clinical efficacy of stem cells delivered into the eye. Recent findings Recent groundbreaking human clinical trials have demonstrated both the opportunities and current limitations of stem cell transplantation for retinal diseases. New progress in developing in vitro retinal organoids, coupled with the maturation of bio-printing technology, and non-invasive high-resolution imaging have created new possibilities for repairing and regenerating the diseased retina and rigorously validating its clinical impact in vivo. Summary While promising progress is being made, meticulous clinical trials with cells derived using good manufacturing practice, novel surgical methods, and improved methods to derive all of the neuronal cell types present in the retina will be indispensable for developing stem cell transplantation as a paradigm shift for the treatment of blinding diseases.
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Autophagy Regulates Proteasome Inhibitor-Induced Pigmentation in Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells. Int J Mol Sci 2017; 18:ijms18051089. [PMID: 28534814 PMCID: PMC5454998 DOI: 10.3390/ijms18051089] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2017] [Revised: 05/11/2017] [Accepted: 05/12/2017] [Indexed: 02/06/2023] Open
Abstract
The impairment of autophagic and proteasomal cleansing together with changes in pigmentation has been documented in retinal pigment epithelial (RPE) cell degeneration. However, the function and co-operation of these mechanisms in melanosome-containing RPE cells is still unclear. We show that inhibition of proteasomal degradation with MG-132 or autophagy with bafilomycin A1 increased the accumulation of premelanosomes and autophagic structures in human embryonic stem cell (hESC)-derived RPE cells. Consequently, upregulation of the autophagy marker p62 (also known as sequestosome-1, SQSTM1) was confirmed in Western blot and perinuclear staining. Interestingly, cells treated with the adenosine monophosphatedependent protein kinase activator, AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide), decreased the proteasome inhibitor-induced accumulation of premelanosomes, increased the amount of autophagosomes and eradicated the protein expression of p62 and LC3 (microtubule-associated protein 1A/1B-light chain 3). These results revealed that autophagic machinery is functional in hESC-RPE cells and may regulate cellular pigmentation with proteasomes.
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Jones MK, Lu B, Girman S, Wang S. Cell-based therapeutic strategies for replacement and preservation in retinal degenerative diseases. Prog Retin Eye Res 2017; 58:1-27. [PMID: 28111323 PMCID: PMC5441967 DOI: 10.1016/j.preteyeres.2017.01.004] [Citation(s) in RCA: 68] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2016] [Revised: 01/08/2017] [Accepted: 01/17/2017] [Indexed: 12/13/2022]
Abstract
Cell-based therapeutics offer diverse options for treating retinal degenerative diseases, such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP). AMD is characterized by both genetic and environmental risks factors, whereas RP is mainly a monogenic disorder. Though treatments exist for some patients with neovascular AMD, a majority of retinal degenerative patients have no effective therapeutics, thus indicating a need for universal therapies to target diverse patient populations. Two main cell-based mechanistic approaches are being tested in clinical trials. Replacement therapies utilize cell-derived retinal pigment epithelial (RPE) cells to supplant lost or defective host RPE cells. These cells are similar in morphology and function to native RPE cells and can potentially supplant the responsibilities of RPE in vivo. Preservation therapies utilize supportive cells to aid in visual function and photoreceptor preservation partially by neurotrophic mechanisms. The goal of preservation strategies is to halt or slow the progression of disease and maintain remaining visual function. A number of clinical trials are testing the safety of replacement and preservation cell therapies in patients; however, measures of efficacy will need to be further evaluated. In addition, a number of prevailing concerns with regards to the immune-related response, longevity, and functionality of the grafted cells will need to be addressed in future trials. This review will summarize the current status of cell-based preclinical and clinical studies with a focus on replacement and preservation strategies and the obstacles that remain regarding these types of treatments.
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Affiliation(s)
- Melissa K Jones
- Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Los Angeles, CA 90048, USA; Department of Biomedical Sciences, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Los Angeles, CA 90048, USA
| | - Bin Lu
- Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Los Angeles, CA 90048, USA; Department of Biomedical Sciences, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Los Angeles, CA 90048, USA
| | - Sergey Girman
- Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Los Angeles, CA 90048, USA; Department of Biomedical Sciences, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Los Angeles, CA 90048, USA
| | - Shaomei Wang
- Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Los Angeles, CA 90048, USA; Department of Biomedical Sciences, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Los Angeles, CA 90048, USA; David Geffen School of Medicine, University of California Los Angeles, 10833 Le Conte Ave., Los Angeles, CA 90095, USA.
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49
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Combes RD, Shah AB. The use of in vivo, ex vivo, in vitro, computational models and volunteer studies in vision research and therapy, and their contribution to the Three Rs. Altern Lab Anim 2017; 44:187-238. [PMID: 27494623 DOI: 10.1177/026119291604400302] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Much is known about mammalian vision, and considerable progress has been achieved in treating many vision disorders, especially those due to changes in the eye, by using various therapeutic methods, including stem cell and gene therapy. While cells and tissues from the main parts of the eye and the visual cortex (VC) can be maintained in culture, and many computer models exist, the current non-animal approaches are severely limiting in the study of visual perception and retinotopic imaging. Some of the early studies with cats and non-human primates (NHPs) are controversial for animal welfare reasons and are of questionable clinical relevance, particularly with respect to the treatment of amblyopia. More recently, the UK Home Office records have shown that attention is now more focused on rodents, especially the mouse. This is likely to be due to the perceived need for genetically-altered animals, rather than to knowledge of the similarities and differences of vision in cats, NHPs and rodents, and the fact that the same techniques can be used for all of the species. We discuss the advantages and limitations of animal and non-animal methods for vision research, and assess their relative contributions to basic knowledge and clinical practice, as well as outlining the opportunities they offer for implementing the principles of the Three Rs (Replacement, Reduction and Refinement).
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Affiliation(s)
| | - Atul B Shah
- Ophthalmic Surgeon, National Eye Registry Ltd, Leicester, UK
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50
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Grob SR, Finn A, Papakostas TD, Eliott D. Clinical Trials in Retinal Dystrophies. Middle East Afr J Ophthalmol 2016; 23:49-59. [PMID: 26957839 PMCID: PMC4759904 DOI: 10.4103/0974-9233.173135] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
Research development is burgeoning for genetic and cellular therapy for retinal dystrophies. These dystrophies are the focus of many research efforts due to the unique biology and accessibility of the eye, the transformative advances in ocular imaging technology that allows for in vivo monitoring, and the potential benefit people would gain from success in the field – the gift of renewed sight. Progress in the field has revealed the immense complexity of retinal dystrophies and the challenges faced by researchers in the development of this technology. This study reviews the current trials and advancements in genetic and cellular therapy in the treatment of retinal dystrophies and also discusses the current and potential future challenges.
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Affiliation(s)
- Seanna R Grob
- Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, USA; Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, USA
| | - Avni Finn
- Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, USA; Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, USA
| | - Thanos D Papakostas
- Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, USA; Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, USA; Retina, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, USA
| | - Dean Eliott
- Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, USA; Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, USA; Retina, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, USA
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