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Gurdal M, Baysal K, Durak I, Selver OB. Limbal explant cultures on amniotic membrane: The effects of passaging the explants on cell phenotype. Exp Eye Res 2025; 255:110392. [PMID: 40250725 DOI: 10.1016/j.exer.2025.110392] [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: 02/12/2025] [Revised: 04/04/2025] [Accepted: 04/15/2025] [Indexed: 04/20/2025]
Abstract
In vitro expansion of limbal epithelial stem cells (LESCs) while maintaining their characteristics has the potential to address the urgent need in ophthalmology clinics for the treatment of limbal stem cell deficiency (LSCD). Herein, we investigated the impact of explant passaging on the phenotype of LESCs cultured on human amniotic membrane (hAM). Following initial coverage of the hAM surface by cells (passage 0), the rabbit limbal explants underwent two additional passages. Expanded cells were then counted using a hemocytometer and examined by immunocytochemistry and RT-qPCR to assess markers associated with LESCs (ABCG2, P63, CK14, CXCR4, BMI-1, and vimentin) and differentiated LESCs (CK3 and connexin 43). The cell yield of passage 1 was the highest among all passages. Immunocytochemistry analysis revealed that the number of CK14-positive cells was similar across all passages; vimentin-positive cells were the lowest in passage 0, while vimentin-positive cells were the highest in passage 1; and CK3-positive cells were the highest in passage 0. RT-qPCR analysis revealed that CK3 and connexin 43 expression was significantly higher in passage 0 cells than in passage 2 cells; and CXCR4 and BMI-1 expressions were significantly higher in passage 1 cells than in passage 0 cells. Our data highlight that the passaging of limbal explant on hAM results in varying cell characteristics. The decrease in CK3 and increase in ABCG2 expression in cells obtained by passaging the limbal explant suggest that passaging could potentially enhance the stem cell population within the in vitro limbal explant culture on hAM.
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Affiliation(s)
- Mehmet Gurdal
- Dokuz Eylül University, Faculty of Medicine, Department of Medical Biochemistry, Izmir, Türkiye; Ege University, Faculty of Medicine, Ocular Surface Research Laboratory, Izmir, Türkiye; Limbustem R&D Medical Products Ltd., Ege University Technopark, Izmir, Türkiye.
| | - Kemal Baysal
- Dokuz Eylül University, Faculty of Medicine, Department of Medical Biochemistry, Izmir, Türkiye; Koc University, School of Medicine, Department of Biochemistry, Istanbul, Türkiye.
| | - Ismet Durak
- Dokuz Eylül University, Faculty of Medicine, Department of Ophthalmology, Izmir, Türkiye.
| | - Ozlem Barut Selver
- Ege University, Faculty of Medicine, Ocular Surface Research Laboratory, Izmir, Türkiye; Limbustem R&D Medical Products Ltd., Ege University Technopark, Izmir, Türkiye; Ege University, Faculty of Medicine, Department of Ophthalmology, Izmir, Türkiye; Department of Stem Cell, Institute of Health Sciences, Ege University, Izmir, Türkiye; Institute of Health Sciences, Department of Stem Cell, Ege University, Izmir, Türkiye; Cord Blood Cell-Tissue Application and Research Center, Ege University, Izmir, Türkiye.
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Higa K, Ishiwata M, Kimoto R, Hirayama M, Yamaguchi T, Shimmura S. Human corneal organoid has a limbal function that supplies epithelium to the cornea with limbal deficiency. Regen Ther 2025; 29:247-253. [PMID: 40230358 PMCID: PMC11995013 DOI: 10.1016/j.reth.2025.03.004] [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: 01/22/2025] [Revised: 03/03/2025] [Accepted: 03/09/2025] [Indexed: 04/16/2025] Open
Abstract
Introduction Patients with limbal dysfunction, which occurs when corneal epithelial stem cells are depleted, require the transplantation of donor corneal epithelial stem cells or donor-independent cell sources. This study aimed to establish organoids with limbal epithelial progenitor cell function from the central cornea, where stem cells do not reside in vivo. We confirmed the regenerative capacity of organoids in a rabbit limbal deficiency model. Methods After treatment with collagenase, central corneal epithelial cells were scraped from corneal tissue and seeded onto Matrigel. For comparison, cells were collected from the limbus. The cells were cultured in Limbal Phenotype Maintenance Medium (LPMM). After 1 month, the organoids were observed in terms of number and size, immunohistochemistry, cell cycle, and colony-forming efficiency. Organoids were also transplanted into a rabbit model of limbal deficiency. Results Although we were able to form organoids from the central cornea, the number of organoids from the cornea was small (approximately one tenth compared to the limbus) after 1-month culture. Cornea-derived organoids were similar in shape and size to limbal-derived organoids, and expressed keratin 15 and p63, which are characteristics of the limbal epithelium, as well as collagen type IV, laminin, and tenascin-C, which are limbal basement membrane components. Cornea-derived organoids also showed colony forming efficiency, slow-cycling cells, and label-retaining cells. Transplanted corneal organoids were observed in the limbus of a rabbit limbal deficiency model, and the presence of organoid-derived cells extending into the host cornea was confirmed by immunohistochemistry using anti-human nuclei, -K12, -collagen type IV, and -laminin antibodies. Conclusions Our data suggest that corneal organoids de-differentiated to gain a limbal phenotype and functionally supplied corneal epithelium in a rabbit limbal deficiency model for up to 1 month.
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Affiliation(s)
- Kazunari Higa
- Cornea Center Eye Bank, Tokyo Dental College Ichikawa General Hospital, 5-11-13 Sugano, Ichikawa, Chiba, 272-8513, Japan
| | - Mifuyu Ishiwata
- Cornea Center Eye Bank, Tokyo Dental College Ichikawa General Hospital, 5-11-13 Sugano, Ichikawa, Chiba, 272-8513, Japan
| | - Reona Kimoto
- Cornea Center Eye Bank, Tokyo Dental College Ichikawa General Hospital, 5-11-13 Sugano, Ichikawa, Chiba, 272-8513, Japan
| | - Masatoshi Hirayama
- Department of Ophthalmology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan
| | - Takefumi Yamaguchi
- Cornea Center Eye Bank, Tokyo Dental College Ichikawa General Hospital, 5-11-13 Sugano, Ichikawa, Chiba, 272-8513, Japan
- Department of Ophthalmology, Tokyo Dental College Ichikawa General Hospital, 5-11-13 Sugano, Ichikawa, Chiba, 272-8513, Japan
| | - Shigeto Shimmura
- Department of Ophthalmology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan
- Department of Clinical Regenerative Medicine, Fujita Medical Innovation Center, Fujita Health University, 1-1-4 Haneda-kukou, Ota-ku, Tokyo 144-0041, Japan
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Braunsperger MV, Martin G, Herzig T, Kußberger I, Gießl A, Steimle S, Schlötzer-Schrehardt U, Schlunck G, Reinhard T, Polisetti N. Proteomic Insights into Human Limbal Epithelial Progenitor-Derived Small Extracellular Vesicles. Stem Cell Rev Rep 2025:10.1007/s12015-025-10877-w. [PMID: 40238075 DOI: 10.1007/s12015-025-10877-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 04/04/2025] [Indexed: 04/18/2025]
Abstract
Limbal epithelial stem/progenitor cells (LEPC), supported by limbal mesenchymal stromal cells (LMSC) and limbal melanocytes (LM) within a specialized niche, are responsible for maintaining the corneal epithelium. Small extracellular vesicles (sEV) emerged as critical mediators of intercellular communication in various stem cell niches, yet their role in maintaining human limbal niche homeostasis remains poorly understood. In this study, tangential flow filtration and size exclusion chromatography were used to isolate sEV from LEPC-, LMSC- and LM-conditioned media. The isolated sEV from LEPC exhibited properties characteristic for sEV as confirmed by nanoparticle tracking analysis for size and concentration, by electron microscopy for morphology, and by western blot analysis of canonical EV markers including the cell-specific protein (cytokeratin 17/19). Quantitative and comparative proteomic profiling revealed distinct molecular signatures of LEPC-derived sEV, enriched in factors associated with keratinocyte development, extracellular matrix organization, and niche regulation. These findings suggest that LEPC-derived sEV may serve as important signaling mediators within the limbal niche microenvironment, though additional studies are needed to determine their specific functional roles in maintaining niche homeostasis.
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Affiliation(s)
- Moritz Vincent Braunsperger
- Eye Center, Medical Center - Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106, Freiburg, Germany
| | - Gottfried Martin
- Eye Center, Medical Center - Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106, Freiburg, Germany
| | - Tabea Herzig
- Eye Center, Medical Center - Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106, Freiburg, Germany
| | - Isabell Kußberger
- Eye Center, Medical Center - Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106, Freiburg, Germany
| | - Andreas Gießl
- Department of Ophthalmology, University Hospital Erlangen, Friedrich-Alexander-University of Erlan-gen-Nürnberg, Schwabachanlage 6, D-91054, Erlangen, Germany
| | - Stefan Steimle
- Cryo-EM Facility (CEF), University of Freiburg, Albertstrasse 21, 79104, Freiburg, Germany
- Institute of Physical Chemistry, University of Freiburg, Albertstrasse 21, 79104, Freiburg, Germany
| | - Ursula Schlötzer-Schrehardt
- Department of Ophthalmology, University Hospital Erlangen, Friedrich-Alexander-University of Erlan-gen-Nürnberg, Schwabachanlage 6, D-91054, Erlangen, Germany
| | - Günther Schlunck
- Eye Center, Medical Center - Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106, Freiburg, Germany
| | - Thomas Reinhard
- Eye Center, Medical Center - Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106, Freiburg, Germany
| | - Naresh Polisetti
- Eye Center, Medical Center - Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106, Freiburg, Germany.
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Garg A, Goel K, Gour A, Sapra M, Sangwan VS, Tripathi R, Tiwari A. Unveiling the Molecular Mechanisms Underlying the Success of Simple Limbal Epithelial Transplantation (SLET). Cells 2025; 14:200. [PMID: 39936991 PMCID: PMC11817669 DOI: 10.3390/cells14030200] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/26/2024] [Revised: 01/17/2025] [Accepted: 01/19/2025] [Indexed: 02/13/2025] Open
Abstract
Simple limbal epithelial transplantation (SLET) has emerged as an effective treatment option for limbal stem cell deficiency (LSCD). However, the precise molecular mechanisms underlying its success remain incompletely understood. This review delves into the proposed mechanisms involving the donor limbus, host microenvironment, and the amniotic membrane as a scaffold in SLET. The donor limbus contributes to SLET efficacy through various factors secreted by limbal epithelial stem cells, including hepatocyte growth factor (HGF), soluble Fms-like tyrosine kinase-1 (sFLT-1), and pigment epithelium-derived factor (PEDF), which support corneal healing and transparency. Additionally, the presence of melanocytes, immune cells, limbal fibroblasts, and adhesion molecules within the donor tissue helps preserve the integrity of the limbal niche. The host environment plays a critical role in supporting the transplanted stem cells, with mesenchymal stem cell-secreted factors promoting proliferation and differentiation. Although the amniotic membrane has traditionally been used as a scaffold, emerging evidence suggests that it may not always be necessary. Further studies are needed to validate this scaffold-free approach and to evaluate the vitality and functional contributions of individual components used in SLET. Understanding these complex interactions and molecular mechanisms sheds light on the importance of the donor tissue, host microenvironment, and scaffold in SLET, paving the way for the optimization of this technique for the effective treatment of LSCD.
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Affiliation(s)
- Aastha Garg
- Department of Cornea and Anterior Segment Services, Dr. Shroff’s Charity Eye Hospital, New Delhi 110002, India; (A.G.); (A.G.); (V.S.S.)
- Department of Stem Cells Research, Dr. Shroff’s Charity Eye Hospital, New Delhi 110002, India; (K.G.); (M.S.)
| | - Kartik Goel
- Department of Stem Cells Research, Dr. Shroff’s Charity Eye Hospital, New Delhi 110002, India; (K.G.); (M.S.)
| | - Abha Gour
- Department of Cornea and Anterior Segment Services, Dr. Shroff’s Charity Eye Hospital, New Delhi 110002, India; (A.G.); (A.G.); (V.S.S.)
- Department of Stem Cells Research, Dr. Shroff’s Charity Eye Hospital, New Delhi 110002, India; (K.G.); (M.S.)
| | - Mehak Sapra
- Department of Stem Cells Research, Dr. Shroff’s Charity Eye Hospital, New Delhi 110002, India; (K.G.); (M.S.)
| | - Virender Singh Sangwan
- Department of Cornea and Anterior Segment Services, Dr. Shroff’s Charity Eye Hospital, New Delhi 110002, India; (A.G.); (A.G.); (V.S.S.)
- Department of Stem Cells Research, Dr. Shroff’s Charity Eye Hospital, New Delhi 110002, India; (K.G.); (M.S.)
| | - Ratnakar Tripathi
- Department of Veterinary Medicine, University of Missouri, Columbia, MO 65201, USA
| | - Anil Tiwari
- Department of Stem Cells Research, Dr. Shroff’s Charity Eye Hospital, New Delhi 110002, India; (K.G.); (M.S.)
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Koc AC, Sari V, Kocak G, Recber T, Nemutlu E, Aberdam D, Güven S. Patient-derived cornea organoid model to study metabolomic characterization of rare disease: aniridia-associated keratopathy. BMC Ophthalmol 2025; 25:14. [PMID: 39794714 PMCID: PMC11724546 DOI: 10.1186/s12886-024-03831-w] [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: 09/16/2024] [Accepted: 12/23/2024] [Indexed: 01/13/2025] Open
Abstract
BACKGROUND Aniridia is a rare panocular disease caused by gene mutation in the PAX6, which is essential for eye development. Aniridia is inherited in an autosomal dominant manner, but its phenotype can vary significantly among individuals with the same mutation. Animal models, such as drosophila, zebrafish, and rodents, have been used to study aniridia through Pax6 deletions. Recently, patient-derived limbal epithelial stem cells (LESCs) and human-induced pluripotent stem cells (hiPSCs) have been used to model the disease in vitro, providing new insights into therapeutic strategies. METHODS In this study, corneal organoids were generated from hiPSCs derived from aniridia patients with three different PAX6 nonsense mutations, allowing for a detailed comparison between diseased and healthy control models. These organoids structurally mimicked the human cornea and were used to investigate histologic and metabolomic differences between healthy and aniridia-derived samples. RESULTS Untargeted metabolomic analysis revealed significant metabolic differences between wild-type (WT) and aniridia-associated keratopathy (AAK) hiPSCs. Further metabolomic profiling at different time points demonstrated distinct metabolic shifts, with amino acid metabolism pathways being consistently enriched in AAK organoids. CONCLUSIONS This study emphasizes the profound impact of AAK mutations on metabolism, particularly in amino acid biosynthesis and energy metabolism pathways.
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Affiliation(s)
- Ali Can Koc
- Izmir Biomedicine and Genome Center, 35340, Izmir, Türkiye
- Izmir International Biomedicine and Genome Institute, Dokuz Eylül University, 35340, Izmir, Türkiye
| | - Vedat Sari
- Izmir Biomedicine and Genome Center, 35340, Izmir, Türkiye
- Izmir International Biomedicine and Genome Institute, Dokuz Eylül University, 35340, Izmir, Türkiye
| | - Gamze Kocak
- Izmir Biomedicine and Genome Center, 35340, Izmir, Türkiye
- Izmir International Biomedicine and Genome Institute, Dokuz Eylül University, 35340, Izmir, Türkiye
| | - Tuba Recber
- Department of Analytical Chemistry, Faculty of Pharmacy, Hacettepe University, Sıhhiye, 06100, Ankara, Türkiye
| | - Emirhan Nemutlu
- Department of Analytical Chemistry, Faculty of Pharmacy, Hacettepe University, Sıhhiye, 06100, Ankara, Türkiye
| | - Daniel Aberdam
- INSERM U1138, Centre de Recherche Des Cordeliers, Sorbonne Paris Cité University, Paris, France
| | - Sinan Güven
- Izmir Biomedicine and Genome Center, 35340, Izmir, Türkiye.
- Izmir International Biomedicine and Genome Institute, Dokuz Eylül University, 35340, Izmir, Türkiye.
- Department of Medical Biology and Genetics, Faculty of Medicine, Dokuz Eylül University, 35340, Izmir, Türkiye.
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Gurdal M, Ercan G, Barut Selver O, Aberdam D, Zeugolis DI. Development of Biomimetic Substrates for Limbal Epithelial Stem Cells Using Collagen-Based Films, Hyaluronic Acid, Immortalized Cells, and Macromolecular Crowding. Life (Basel) 2024; 14:1552. [PMID: 39768260 PMCID: PMC11678493 DOI: 10.3390/life14121552] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2024] [Revised: 11/20/2024] [Accepted: 11/22/2024] [Indexed: 01/05/2025] Open
Abstract
Despite the promising potential of cell-based therapies developed using tissue engineering techniques to treat a wide range of diseases, including limbal stem cell deficiency (LSCD), which leads to corneal blindness, their commercialization remains constrained. This is primarily attributable to the limited cell sources, the use of non-standardizable, unscalable, and unsustainable techniques, and the extended manufacturing processes required to produce transplantable tissue-like surrogates. Herein, we present the first demonstration of the potential of a novel approach combining collagen films (CF), hyaluronic acid (HA), human telomerase-immortalized limbal epithelial stem cells (T-LESCs), and macromolecular crowding (MMC) to develop innovative biomimetic substrates for limbal epithelial stem cells (LESCs). The initial step involved the fabrication and characterization of CF and CF enriched with HA (CF-HA). Subsequently, T-LESCs were seeded on CF, CF-HA, and tissue culture plastic (TCP). Thereafter, the effect of these matrices on basic cellular function and tissue-specific extracellular matrix (ECM) deposition with or without MMC was evaluated. The viability and metabolic activity of cells cultured on CF, CF-HA, and TCP were found to be similar, while CF-HA induced the highest (p < 0.05) cell proliferation. It is notable that CF and HA induced cell growth, whereas MMC increased (p < 0.05) the deposition of collagen IV, fibronectin, and laminin in the T-LESC culture. The data highlight the potential of, in particular, immortalized cells and MMC for the development of biomimetic cell culture substrates, which could be utilized in ocular surface reconstruction following further in vitro, in vivo, and clinical validation of the approach.
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Affiliation(s)
- Mehmet Gurdal
- Department of Medical Biochemistry, Faculty of Medicine, Ege University, 35100 Izmir, Türkiye;
- Regenerative, Modular & Developmental Engineering Laboratory (REMODEL) and Science Foundation Ireland (SFI) Centre for Research in Medical Devices (CÚRAM), Biomedical Sciences Building, University of Galway, H91 TK33 Galway, Ireland;
| | - Gulinnaz Ercan
- Department of Medical Biochemistry, Faculty of Medicine, Ege University, 35100 Izmir, Türkiye;
- Department of Stem Cell, Institute of Health Sciences, Ege University, 35100 Izmir, Türkiye;
| | - Ozlem Barut Selver
- Department of Stem Cell, Institute of Health Sciences, Ege University, 35100 Izmir, Türkiye;
- Department of Ophthalmology, Faculty of Medicine, Ege University, 35100 Izmir, Türkiye
| | - Daniel Aberdam
- INSERM U1138, Centre des Cordeliers, Université de Paris, 75006 Paris, France;
| | - Dimitrios I. Zeugolis
- Regenerative, Modular & Developmental Engineering Laboratory (REMODEL) and Science Foundation Ireland (SFI) Centre for Research in Medical Devices (CÚRAM), Biomedical Sciences Building, University of Galway, H91 TK33 Galway, Ireland;
- Regenerative, Modular & Developmental Engineering Laboratory (REMODEL), Charles Institute of Dermatology, Conway Institute of Biomolecular & Biomedical Research and School of Mechanical & Materials Engineering, University College Dublin (UCD), D04 V1W8 Dublin, Ireland
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Di Girolamo N. Biologicals and Biomaterials for Corneal Regeneration and Vision Restoration in Limbal Stem Cell Deficiency. ADVANCED MATERIALS (DEERFIELD BEACH, FLA.) 2024; 36:e2401763. [PMID: 38777343 DOI: 10.1002/adma.202401763] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/01/2024] [Revised: 05/15/2024] [Indexed: 05/25/2024]
Abstract
The mammalian cornea is decorated with stem cells bestowed with the life-long task of renewing the epithelium, provided they remain healthy, functional, and in sufficient numbers. If not, a debilitating disease known as limbal stem cell deficiency (LSCD) can develop causing blindness. Decades after the first stem cell (SC) therapy is devised to treat this condition, patients continue to suffer unacceptable failures. During this time, improvements to therapeutics have included identifying better markers to isolate robust SC populations and nurturing them on crudely modified biological or biomaterial scaffolds including human amniotic membrane, fibrin, and contact lenses, prior to their delivery. Researchers are now gathering information about the biomolecular and biomechanical properties of the corneal SC niche to decipher what biological and/or synthetic materials can be incorporated into these carriers. Advances in biomedical engineering including electrospinning and 3D bioprinting with surface functionalization and micropatterning, and self-assembly models, have generated a wealth of biocompatible, biodegradable, integrating scaffolds to choose from, some of which are being tested for their SC delivery capacity in the hope of improving clinical outcomes for patients with LSCD.
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Affiliation(s)
- Nick Di Girolamo
- Mechanisms of Disease and Translational Research, School of Biomedical Sciences, Faculty of Medicine and Health, University of New South Wales, Sydney, NSW, 2052, Australia
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Suanno G, Genna VG, Maurizi E, Dieh AA, Griffith M, Ferrari G. Cell therapy in the cornea: The emerging role of microenvironment. Prog Retin Eye Res 2024; 102:101275. [PMID: 38797320 DOI: 10.1016/j.preteyeres.2024.101275] [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: 10/11/2023] [Revised: 05/22/2024] [Accepted: 05/23/2024] [Indexed: 05/29/2024]
Abstract
The cornea is an ideal testing field for cell therapies. Its highly ordered structure, where specific cell populations are sequestered in different layers, together with its accessibility, has allowed the development of the first stem cell-based therapy approved by the European Medicine Agency. Today, different techniques have been proposed for autologous and allogeneic limbal and non-limbal cell transplantation. Cell replacement has also been attempted in cases of endothelial cell decompensation as it occurs in Fuchs dystrophy: injection of cultivated allogeneic endothelial cells is now in advanced phases of clinical development. Recently, stromal substitutes have been developed with excellent integration capability and transparency. Finally, cell-derived products, such as exosomes obtained from different sources, have been investigated for the treatment of severe corneal diseases with encouraging results. Optimization of the success rate of cell therapies obviously requires high-quality cultured cells/products, but the role of the surrounding microenvironment is equally important to allow engraftment of transplanted cells, to preserve their functions and, ultimately, lead to restoration of tissue integrity and transparency of the cornea.
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Affiliation(s)
- Giuseppe Suanno
- Vita-Salute San Raffaele University, Milan, Italy; Eye Repair Unit, Division of Neuroscience, IRCCS San Raffaele Scientific Institute, Milan, Italy
| | | | - Eleonora Maurizi
- Centre for Regenerative Medicine ''S. Ferrari'', University of Modena and Reggio Emilia, Modena, Italy
| | - Anas Abu Dieh
- Maisonneuve-Rosemont Hospital Research Centre, Montreal, Quebec, Canada
| | - May Griffith
- Maisonneuve-Rosemont Hospital Research Centre, Montreal, Quebec, Canada.
| | - Giulio Ferrari
- Vita-Salute San Raffaele University, Milan, Italy; Eye Repair Unit, Division of Neuroscience, IRCCS San Raffaele Scientific Institute, Milan, Italy; Ophthalmology Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy.
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Sanie-Jahromi F, Khaki M, Heydari M, Nowroozzadeh MH, Akbarizadeh AR, Daneshamouz S, NejatyJahromy Y, Nejabat M, Mahmoudi A, Zareei A, Nejabat M. Effect of low dose honey on the apoptosis and inflammation gene expression in corneal limbal stem cells and keratocytes and its efficacy as an ophthalmic formulation in the treatment of dry eye: in-vitro and clinical study. Front Med (Lausanne) 2024; 11:1359463. [PMID: 38831993 PMCID: PMC11144896 DOI: 10.3389/fmed.2024.1359463] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2023] [Accepted: 04/29/2024] [Indexed: 06/05/2024] Open
Abstract
Background The use of honey as an eye treatment encounters challenges due to its high osmolarity, low pH, and difficulties in sterilization. This study addresses these issues by employing a low concentration of honey, focusing on both in-vitro experiments and clinical trials for treating dry eye disease in corneal cells. Methods In the in-vitro experiment, we investigated the impact of a 1% honey-supplemented medium (HSM) on limbal stem cells (LSCs) and keratocytes using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and real-time polymerase chain reaction (PCR) for BCL-2, BAX, and IL-1β gene expression. Simultaneously, in the clinical trial, 80 participants were divided into two groups, receiving either a 1% w/v honey ophthalmic formulation or a placebo for 3 months. Study outcomes included subjective improvement in dry eye symptoms, tear break-up time (TBUT), and Schirmer's test results. Results MTT results indicated that 1% HSM did not compromise the survival of corneal cells and significantly reduced the expression of the IL-1β gene. Additionally, participants in the honey group demonstrated a higher rate of improvement in dry eye symptoms and a significant enhancement in TBUT values at the three-month follow-up. However, there was no significant difference between the study groups in terms of Schirmer's test values. No adverse events were observed or reported. Conclusion In conclusion, 1% honey exhibits anti-inflammatory and anti-infective properties, proving effective in ameliorating dry eye symptoms and enhancing tear film stability in patients with dry eye disease.Clinical Trial Registration: https://irct.behdasht.gov.ir/trial/63800.
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Affiliation(s)
- Fatemeh Sanie-Jahromi
- Poostchi Ophthalmology Research Center, Department of Ophthalmology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Mehdi Khaki
- Poostchi Ophthalmology Research Center, Department of Ophthalmology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Mojtaba Heydari
- Poostchi Ophthalmology Research Center, Department of Ophthalmology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Mohammad Hossein Nowroozzadeh
- Poostchi Ophthalmology Research Center, Department of Ophthalmology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Amin Reza Akbarizadeh
- Department of Quality Control, Food and Drug, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Saeid Daneshamouz
- Department of Pharmaceutics, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Yaser NejatyJahromy
- Poostchi Ophthalmology Research Center, Department of Ophthalmology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Maryam Nejabat
- HIV/AIDS Research Center, Institute of Health, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Ahmad Mahmoudi
- Poostchi Ophthalmology Research Center, Department of Ophthalmology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Athar Zareei
- Poostchi Ophthalmology Research Center, Department of Ophthalmology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Mahmood Nejabat
- Poostchi Ophthalmology Research Center, Department of Ophthalmology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
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10
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Verma S, Lin X, Coulson-Thomas VJ. The Potential Reversible Transition between Stem Cells and Transient-Amplifying Cells: The Limbal Epithelial Stem Cell Perspective. Cells 2024; 13:748. [PMID: 38727284 PMCID: PMC11083486 DOI: 10.3390/cells13090748] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2024] [Revised: 04/18/2024] [Accepted: 04/24/2024] [Indexed: 05/13/2024] Open
Abstract
Stem cells (SCs) undergo asymmetric division, producing transit-amplifying cells (TACs) with increased proliferative potential that move into tissues and ultimately differentiate into a specialized cell type. Thus, TACs represent an intermediary state between stem cells and differentiated cells. In the cornea, a population of stem cells resides in the limbal region, named the limbal epithelial stem cells (LESCs). As LESCs proliferate, they generate TACs that move centripetally into the cornea and differentiate into corneal epithelial cells. Upon limbal injury, research suggests a population of progenitor-like cells that exists within the cornea can move centrifugally into the limbus, where they dedifferentiate into LESCs. Herein, we summarize recent advances made in understanding the mechanism that governs the differentiation of LESCs into TACs, and thereafter, into corneal epithelial cells. We also outline the evidence in support of the existence of progenitor-like cells in the cornea and whether TACs could represent a population of cells with progenitor-like capabilities within the cornea. Furthermore, to gain further insights into the dynamics of TACs in the cornea, we outline the most recent findings in other organ systems that support the hypothesis that TACs can dedifferentiate into SCs.
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Affiliation(s)
- Sudhir Verma
- College of Optometry, University of Houston, 4901 Calhoun Road, Houston, TX 77204, USA;
- Deen Dayal Upadhyaya College, University of Delhi, Delhi 110078, India
| | - Xiao Lin
- College of Optometry, University of Houston, 4901 Calhoun Road, Houston, TX 77204, USA;
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11
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Kistenmacher S, Schwämmle M, Martin G, Ulrich E, Tholen S, Schilling O, Gießl A, Schlötzer-Schrehardt U, Bucher F, Schlunck G, Nazarenko I, Reinhard T, Polisetti N. Enrichment, Characterization, and Proteomic Profiling of Small Extracellular Vesicles Derived from Human Limbal Mesenchymal Stromal Cells and Melanocytes. Cells 2024; 13:623. [PMID: 38607062 PMCID: PMC11011788 DOI: 10.3390/cells13070623] [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: 03/11/2024] [Revised: 03/27/2024] [Accepted: 03/28/2024] [Indexed: 04/13/2024] Open
Abstract
Limbal epithelial progenitor cells (LEPC) rely on their niche environment for proper functionality and self-renewal. While extracellular vesicles (EV), specifically small EVs (sEV), have been proposed to support LEPC homeostasis, data on sEV derived from limbal niche cells like limbal mesenchymal stromal cells (LMSC) remain limited, and there are no studies on sEVs from limbal melanocytes (LM). In this study, we isolated sEV from conditioned media of LMSC and LM using a combination of tangential flow filtration and size exclusion chromatography and characterized them by nanoparticle tracking analysis, transmission electron microscopy, Western blot, multiplex bead arrays, and quantitative mass spectrometry. The internalization of sEV by LEPC was studied using flow cytometry and confocal microscopy. The isolated sEVs exhibited typical EV characteristics, including cell-specific markers such as CD90 for LMSC-sEV and Melan-A for LM-sEV. Bioinformatics analysis of the proteomic data suggested a significant role of sEVs in extracellular matrix deposition, with LMSC-derived sEV containing proteins involved in collagen remodeling and cell matrix adhesion, whereas LM-sEV proteins were implicated in other cellular bioprocesses such as cellular pigmentation and development. Moreover, fluorescently labeled LMSC-sEV and LM-sEV were taken up by LEPC and localized to their perinuclear compartment. These findings provide valuable insights into the complex role of sEV from niche cells in regulating the human limbal stem cell niche.
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Affiliation(s)
- Sebastian Kistenmacher
- Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106 Freiburg, Germany
| | - Melanie Schwämmle
- Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106 Freiburg, Germany
- Faculty of Biology, University of Freiburg, Schaenzlestrasse 1, D–79104 Freiburg, Germany
| | - Gottfried Martin
- Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106 Freiburg, Germany
| | - Eva Ulrich
- Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106 Freiburg, Germany
| | - Stefan Tholen
- Institute of Surgical Pathology, Faculty of Medicine, Freiburg, Medical Center, University of Freiburg, 79085 Freiburg im Breisgau, Germany
| | - Oliver Schilling
- Institute of Surgical Pathology, Faculty of Medicine, Freiburg, Medical Center, University of Freiburg, 79085 Freiburg im Breisgau, Germany
| | - Andreas Gießl
- Department of Ophthalmology, University Hospital Erlangen, Friedrich-Alexander-University of Erlan-gen-Nürnberg, Schwabachanlage 6, 91054 Erlangen, Germany
| | - Ursula Schlötzer-Schrehardt
- Department of Ophthalmology, University Hospital Erlangen, Friedrich-Alexander-University of Erlan-gen-Nürnberg, Schwabachanlage 6, 91054 Erlangen, Germany
| | - Felicitas Bucher
- Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106 Freiburg, Germany
| | - Günther Schlunck
- Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106 Freiburg, Germany
| | - Irina Nazarenko
- Institute for Infection Prevention and Hospital Epidemiology, Faculty of Medicine, Medical Center, University of Freiburg, 79106 Freiburg, Germany
| | - Thomas Reinhard
- Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106 Freiburg, Germany
| | - Naresh Polisetti
- Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106 Freiburg, Germany
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12
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Altshuler A, Amitai-Lange A, Nasser W, Dimri S, Bhattacharya S, Tiosano B, Barbara R, Aberdam D, Shimmura S, Shalom-Feuerstein R. Eyes open on stem cells. Stem Cell Reports 2023; 18:2313-2327. [PMID: 38039972 PMCID: PMC10724227 DOI: 10.1016/j.stemcr.2023.10.023] [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: 08/02/2023] [Revised: 10/27/2023] [Accepted: 10/30/2023] [Indexed: 12/03/2023] Open
Abstract
Recently, the murine cornea has reemerged as a robust stem cell (SC) model, allowing individual SC tracing in living animals. The cornea has pioneered seminal discoveries in SC biology and regenerative medicine, from the first corneal transplantation in 1905 to the identification of limbal SCs and their transplantation to successfully restore vision in the early 1990s. Recent experiments have exposed unexpected properties attributed to SCs and progenitors and revealed flexibility in the differentiation program and a key role for the SC niche. Here, we discuss the limbal SC model and its broader relevance to other tissues, disease, and therapy.
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Affiliation(s)
- Anna Altshuler
- Department of Genetics & Developmental Biology, The Rappaport Faculty of Medicine & Research Institute, Technion Integrated Cancer Center, Technion - Israel Institute of Technology, Haifa 31096, Israel.
| | - Aya Amitai-Lange
- Department of Genetics & Developmental Biology, The Rappaport Faculty of Medicine & Research Institute, Technion Integrated Cancer Center, Technion - Israel Institute of Technology, Haifa 31096, Israel
| | - Waseem Nasser
- Department of Genetics & Developmental Biology, The Rappaport Faculty of Medicine & Research Institute, Technion Integrated Cancer Center, Technion - Israel Institute of Technology, Haifa 31096, Israel
| | - Shalini Dimri
- Department of Genetics & Developmental Biology, The Rappaport Faculty of Medicine & Research Institute, Technion Integrated Cancer Center, Technion - Israel Institute of Technology, Haifa 31096, Israel
| | - Swarnabh Bhattacharya
- Department of Genetics & Developmental Biology, The Rappaport Faculty of Medicine & Research Institute, Technion Integrated Cancer Center, Technion - Israel Institute of Technology, Haifa 31096, Israel
| | - Beatrice Tiosano
- Department of Ophthalmology, Hillel Yaffe Medical Center, Hadera, Israel
| | - Ramez Barbara
- Department of Ophthalmology, Hillel Yaffe Medical Center, Hadera, Israel
| | - Daniel Aberdam
- Université Paris-Cité, INSERM U1138, Centre des Cordeliers, 75270 Paris, France
| | - Shigeto Shimmura
- Department of Clinical Regenerative Medicine, Fujita Medical Innovation Center, Tokyo, Japan
| | - Ruby Shalom-Feuerstein
- Department of Genetics & Developmental Biology, The Rappaport Faculty of Medicine & Research Institute, Technion Integrated Cancer Center, Technion - Israel Institute of Technology, Haifa 31096, Israel.
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13
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Polisetti N, Martin G, Ulrich E, Glegola M, Schlötzer-Schrehardt U, Schlunck G, Reinhard T. Influence of Organ Culture on the Characteristics of the Human Limbal Stem Cell Niche. Int J Mol Sci 2023; 24:16856. [PMID: 38069177 PMCID: PMC10706739 DOI: 10.3390/ijms242316856] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2023] [Revised: 11/23/2023] [Accepted: 11/24/2023] [Indexed: 12/18/2023] Open
Abstract
Organ culture storage techniques for corneoscleral limbal (CSL) tissue have improved the quality of corneas for transplantation and allow for longer storage times. Cultured limbal tissue has been used for stem cell transplantation to treat limbal stem cell deficiency (LSCD) as well as for research purposes to assess homeostasis mechanisms in the limbal stem cell niche. However, the effects of organ culture storage conditions on the quality of limbal niche components are less well described. Therefore, in this study, the morphological and immunohistochemical characteristics of organ-cultured limbal tissue are investigated and compared to fresh limbal tissues by means of light and electron microscopy. Organ-cultured limbal tissues showed signs of deterioration, such as edema, less pronounced basement membranes, and loss of the most superficial layers of the epithelium. In comparison to the fresh limbal epithelium, organ-cultured limbal epithelium showed signs of ongoing proliferative activity (more Ki-67+ cells) and exhibited an altered limbal epithelial phenotype with a loss of N-cadherin and desmoglein expression as well as a lack of precise staining patterns for cytokeratin ((CK)14, CK17/19, CK15). The analyzed extracellular matrix composition was mainly intact (collagen IV, fibronectin, laminin chains) except for Tenascin-C, whose expression was increased in organ-cultured limbal tissue. Nonetheless, the expression patterns of cell-matrix adhesion proteins varied in organ-cultured limbal tissue compared to fresh limbal tissue. A decrease in the number of melanocytes (Melan-A+ cells) and Langerhans cells (HLA-DR+, CD1a+, CD18+) was observed in the organ-cultured limbal tissue. The organ culture-induced alterations of the limbal epithelial stem cell niche might hamper its use in the treatment of LSCD as well as in research studies. In contrast, reduced numbers of donor-derived Langerhans cells seem associated with better clinical outcomes. However, there is a need to consider the preferential use of fresh CSL for limbal transplants and to look at ways of improving the limbal stem cell properties of stored CSL tissue.
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Affiliation(s)
- Naresh Polisetti
- Eye Center, Medical Center—Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106 Freiburg, Germany
| | - Gottfried Martin
- Eye Center, Medical Center—Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106 Freiburg, Germany
| | - Eva Ulrich
- Eye Center, Medical Center—Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106 Freiburg, Germany
| | - Mateusz Glegola
- Eye Center, Medical Center—Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106 Freiburg, Germany
| | - Ursula Schlötzer-Schrehardt
- Department of Ophthalmology, University Hospital Erlangen, Friedrich-Alexander-University of Erlangen-Nürnberg, Schwabachanlage 6, 91054 Erlangen, Germany
| | - Günther Schlunck
- Eye Center, Medical Center—Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106 Freiburg, Germany
| | - Thomas Reinhard
- Eye Center, Medical Center—Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106 Freiburg, Germany
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14
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Moreno IY, Parsaie A, Gesteira TF, Coulson-Thomas VJ. Characterization of the Limbal Epithelial Stem Cell Niche. Invest Ophthalmol Vis Sci 2023; 64:48. [PMID: 37906057 PMCID: PMC10619699 DOI: 10.1167/iovs.64.13.48] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2023] [Accepted: 10/09/2023] [Indexed: 11/02/2023] Open
Abstract
Purpose Limbal epithelial stem cells (LESCs) reside within a LSC niche (LSCN). We recently identified that hyaluronan (HA) is a major constituent of the LSCN, and that HA is necessary for maintaining LESCs in the "stem cell" state, both in vitro and in vivo. Herein, we characterized the LSCN to identify key components of the HA-specific LSCN. Methods The cornea and limbal rim were dissected from mouse corneas, subjected to mRNA extraction, and sequenced using a NextSeq 500 (Illumina) and data processed using CLC Genomics Workbench 20 (Qiagen) and the STRING database to identify key components of the LSCN. Their expression was confirmed by real-time PCR, Western blotting, and immunohistochemistry. Furthermore, the differential expression of key compounds in different corneal cell types were determined with single-cell RNA sequencing. Results We identified that the hyaladherins inter-alpha-inhibitor (IαI), TSG-6 and versican are highly expressed in the limbus. Specifically, HA/HC complexes are present in the LSCN, in the stroma underlying the limbal epithelium, and surrounding the limbal vasculature. For IαI, heavy chains 5 and 2 (HC5 and HC2) were found to be the most highly expressed HCs in the mouse and human limbus and were associate with HA-forming HA/HC-specific matrices. Conclusions The LSCN contains HA/HC complexes, which have been previously correlated with stem cell niches. The identification of HA/HC complexes in the LSCN could serve as a new therapeutic avenue for treating corneal pathology. Additionally, HA/HC complexes could be used as a substrate for culturing LESCs before LESC transplantation.
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Affiliation(s)
- Isabel Y. Moreno
- College of Optometry, University of Houston, Houston, Texas, United States
| | - Arian Parsaie
- College of Optometry, University of Houston, Houston, Texas, United States
- College of Natural Science and Mathematics, University of Houston, Houston, Texas, United States
| | - Tarsis F. Gesteira
- College of Optometry, University of Houston, Houston, Texas, United States
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15
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Yam GHF, Pi S, Du Y, Mehta JS. Posterior corneoscleral limbus: Architecture, stem cells, and clinical implications. Prog Retin Eye Res 2023; 96:101192. [PMID: 37392960 DOI: 10.1016/j.preteyeres.2023.101192] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2023] [Revised: 06/22/2023] [Accepted: 06/23/2023] [Indexed: 07/03/2023]
Abstract
The limbus is a transition from the cornea to conjunctiva and sclera. In human eyes, this thin strip has a rich variation of tissue structures and composition, typifying a change from scleral irregularity and opacity to corneal regularity and transparency; a variation from richly vascularized conjunctiva and sclera to avascular cornea; the neural passage and drainage of aqueous humor. The limbal stroma is enriched with circular fibres running parallel to the corneal circumference, giving its unique role in absorbing small pressure changes to maintain corneal curvature and refractivity. It contains specific niches housing different types of stem cells for the corneal epithelium, stromal keratocytes, corneal endothelium, and trabecular meshwork. This truly reflects the important roles of the limbus in ocular physiology, and the limbal functionality is crucial for corneal health and the entire visual system. Since the anterior limbus containing epithelial structures and limbal epithelial stem cells has been extensively reviewed, this article is focused on the posterior limbus. We have discussed the structural organization and cellular components of the region beneath the limbal epithelium, the characteristics of stem cell types: namely corneal stromal stem cells, endothelial progenitors and trabecular meshwork stem cells, and recent advances leading to the emergence of potential cell therapy options to replenish their respective mature cell types and to correct defects causing corneal abnormalities. We have reviewed different clinical disorders associated with defects of the posterior limbus and summarized the available preclinical and clinical evidence about the developing topic of cell-based therapy for corneal disorders.
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Affiliation(s)
- Gary Hin-Fai Yam
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA, USA; Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore; McGowan Institute for Regenerative Medicine, Pittsburgh, PA, USA.
| | - Shaohua Pi
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA, USA
| | - Yiqin Du
- Department of Ophthalmology, University of South Florida, Tampa, FL, USA
| | - Jodhbir S Mehta
- Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore; Department of Cornea and External Eye Disease, Singapore National Eye Centre, Singapore; Ophthalmology and Visual Sciences Academic Clinical Program, Duke-National University of Singapore (NUS) Medical School, Singapore.
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16
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Clahsen T, Hadrian K, Notara M, Schlereth SL, Howaldt A, Prokosch V, Volatier T, Hos D, Schroedl F, Kaser-Eichberger A, Heindl LM, Steven P, Bosch JJ, Steinkasserer A, Rokohl AC, Liu H, Mestanoglu M, Kashkar H, Schumacher B, Kiefer F, Schulte-Merker S, Matthaei M, Hou Y, Fassbender S, Jantsch J, Zhang W, Enders P, Bachmann B, Bock F, Cursiefen C. The novel role of lymphatic vessels in the pathogenesis of ocular diseases. Prog Retin Eye Res 2023; 96:101157. [PMID: 36759312 DOI: 10.1016/j.preteyeres.2022.101157] [Citation(s) in RCA: 20] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2022] [Revised: 12/13/2022] [Accepted: 12/17/2022] [Indexed: 02/10/2023]
Abstract
Historically, the eye has been considered as an organ free of lymphatic vessels. In recent years, however, it became evident, that lymphatic vessels or lymphatic-like vessels contribute to several ocular pathologies at various peri- and intraocular locations. The aim of this review is to outline the pathogenetic role of ocular lymphatics, the respective molecular mechanisms and to discuss current and future therapeutic options based thereon. We will give an overview on the vascular anatomy of the healthy ocular surface and the molecular mechanisms contributing to corneal (lymph)angiogenic privilege. In addition, we present (i) current insights into the cellular and molecular mechanisms occurring during pathological neovascularization of the cornea triggered e.g. by inflammation or trauma, (ii) the role of lymphatic vessels in different ocular surface pathologies such as dry eye disease, corneal graft rejection, ocular graft versus host disease, allergy, and pterygium, (iii) the involvement of lymphatic vessels in ocular tumors and metastasis, and (iv) the novel role of the lymphatic-like structure of Schlemm's canal in glaucoma. Identification of the underlying molecular mechanisms and of novel modulators of lymphangiogenesis will contribute to the development of new therapeutic targets for the treatment of ocular diseases associated with pathological lymphangiogenesis in the future. The preclinical data presented here outline novel therapeutic concepts for promoting transplant survival, inhibiting metastasis of ocular tumors, reducing inflammation of the ocular surface, and treating glaucoma. Initial data from clinical trials suggest first success of novel treatment strategies to promote transplant survival based on pretransplant corneal lymphangioregression.
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Affiliation(s)
- Thomas Clahsen
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany; Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany
| | - Karina Hadrian
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany; Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany
| | - Maria Notara
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany; Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany
| | - Simona L Schlereth
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany; Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany
| | - Antonia Howaldt
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
| | - Verena Prokosch
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
| | - Thomas Volatier
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
| | - Deniz Hos
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany; Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany
| | - Falk Schroedl
- Center for Anatomy and Cell Biology, Institute of Anatomy and Cell Biology - Salzburg, Paracelsus Medical University, Salzburg, Austria
| | - Alexandra Kaser-Eichberger
- Center for Anatomy and Cell Biology, Institute of Anatomy and Cell Biology - Salzburg, Paracelsus Medical University, Salzburg, Austria
| | - Ludwig M Heindl
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
| | - Philipp Steven
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany; Cluster of Excellence: Cellular Stress Responses in Ageing-Associated Diseases, CECAD, University of Cologne, Cologne, Germany
| | - Jacobus J Bosch
- Centre for Human Drug Research and Leiden University Medical Center, Leiden, the Netherlands
| | | | - Alexander C Rokohl
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
| | - Hanhan Liu
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
| | - Mert Mestanoglu
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
| | - Hamid Kashkar
- Institute for Molecular Immunology, Center for Molecular Medicine Cologne (CMMC), CECAD Research Center, Faculty of Medicine and University Hospital of Cologne, University of Cologne, Cologne, Germany
| | - Björn Schumacher
- Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany; Cluster of Excellence: Cellular Stress Responses in Ageing-Associated Diseases, CECAD, University of Cologne, Cologne, Germany
| | - Friedemann Kiefer
- European Institute for Molecular Imaging (EIMI), University of Münster, 48149, Münster, Germany
| | - Stefan Schulte-Merker
- Institute for Cardiovascular Organogenesis and Regeneration, Faculty of Medicine, WWU Münster, Münster, Germany
| | - Mario Matthaei
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
| | - Yanhong Hou
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany; Eye Institute and Department of Ophthalmology, Eye & ENT Hospital, Fudan University, 83 Fenyang Road, Xuhui District, Shanghai, China
| | - Sonja Fassbender
- IUF‒Leibniz Research Institute for Environmental Medicine, Duesseldorf, Germany; Immunology and Environment, Life & Medical Sciences (LIMES) Institute, University of Bonn, Bonn, Germany
| | - Jonathan Jantsch
- Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
| | - Wei Zhang
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
| | - Philip Enders
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
| | - Björn Bachmann
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
| | - Felix Bock
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany; Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany
| | - Claus Cursiefen
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany; Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany; Cluster of Excellence: Cellular Stress Responses in Ageing-Associated Diseases, CECAD, University of Cologne, Cologne, Germany.
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17
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Meshko B, Volatier TLA, Hadrian K, Deng S, Hou Y, Kluth MA, Ganss C, Frank MH, Frank NY, Ksander B, Cursiefen C, Notara M. ABCB5+ Limbal Epithelial Stem Cells Inhibit Developmental but Promote Inflammatory (Lymph) Angiogenesis While Preventing Corneal Inflammation. Cells 2023; 12:1731. [PMID: 37443766 PMCID: PMC10341195 DOI: 10.3390/cells12131731] [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: 05/20/2023] [Revised: 06/16/2023] [Accepted: 06/20/2023] [Indexed: 07/15/2023] Open
Abstract
The limbus, the vascularized junction between the cornea and conjunctiva, is thought to function as a barrier against corneal neovascularization. However, the exact mechanisms regulating this remain unknown. In this study, the limbal epithelial stem cell (LESC) marker ABCB5 was used to investigate the role of LESCs in corneal neovascularization. In an ABCB5KO model, a mild but significant increase of limbal lymphatic and blood vascular network complexity was observed in developing mice (4 weeks) but not in adult mice. Conversely, when using a cornea suture model, the WT animals exhibited a mild but significant increase in the number of lymphatic vessel sprouts compared to the ABCB5KO, suggesting a contextual anti-lymphangiogenic effect of ABCB5 on the limbal vasculature during development, but a pro-lymphangiogenic effect under inflammatory challenge in adulthood. In addition, conditioned media from ABCB5-positive cultured human limbal epithelial cells (ABCB5+) stimulated human blood and lymphatic endothelial cell proliferation and migration. Finally, a proteomic analysis demonstrated ABCB5+ cells have a pro(lymph)angiogenic as well as an anti-inflammatory profile. These data suggest a novel dual, context-dependent role of ABCB5+ LESCs, inhibiting developmental but promoting inflammatory (lymph)angiogenesis in adulthood and exerting anti-inflammatory effects. These findings are of high clinical relevance in relation to LESC therapy against blindness.
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Affiliation(s)
- Berbang Meshko
- Department of Ophthalmology, University of Cologne, 50937 Cologne, Germany; (B.M.); (T.L.A.V.); (Y.H.)
| | - Thomas L. A. Volatier
- Department of Ophthalmology, University of Cologne, 50937 Cologne, Germany; (B.M.); (T.L.A.V.); (Y.H.)
| | - Karina Hadrian
- Department of Ophthalmology, University of Cologne, 50937 Cologne, Germany; (B.M.); (T.L.A.V.); (Y.H.)
| | - Shuya Deng
- Department of Ophthalmology, University of Cologne, 50937 Cologne, Germany; (B.M.); (T.L.A.V.); (Y.H.)
| | - Yanhong Hou
- Department of Ophthalmology, University of Cologne, 50937 Cologne, Germany; (B.M.); (T.L.A.V.); (Y.H.)
| | - Mark Andreas Kluth
- TICEBA GmbH, Im Neuenheimer Feld 517, 69120 Heidelberg, Germany; (M.A.K.); (C.G.)
- RHEACELL GmbH & Co. KG, Im Neuenheimer Feld 517, 69120 Heidelberg, Germany
| | - Christoph Ganss
- TICEBA GmbH, Im Neuenheimer Feld 517, 69120 Heidelberg, Germany; (M.A.K.); (C.G.)
- RHEACELL GmbH & Co. KG, Im Neuenheimer Feld 517, 69120 Heidelberg, Germany
| | - Markus H. Frank
- Transplant Research Program, Boston Children’s Hospital, Boston, MA 02115, USA
- Harvard Skin Disease Research Center, Department of Dermatology, Brigham and Women’s Hospital, Boston, MA 02115, USA
- Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA;
- School of Medical and Health Sciences, Edith Cowan University, Perth, WA 6027, Australia
| | - Natasha Y. Frank
- Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA;
- Department of Medicine, VA Boston Healthcare System, Boston, MA 02132, USA
- Division of Genetics, Brigham and Women’s Hospital, Boston, MA 02115, USA
| | - Bruce Ksander
- Massachusetts Eye & Ear Infirmary, Schepens Eye Research Institute, Boston, MA 02114, USA;
| | - Claus Cursiefen
- Department of Ophthalmology, University of Cologne, 50937 Cologne, Germany; (B.M.); (T.L.A.V.); (Y.H.)
- Institute for Genome Stability in Ageing and Disease, CECAD Research Center, Joseph-Stelzmann-Str. 26, 50931 Cologne, Germany
| | - Maria Notara
- Department of Ophthalmology, University of Cologne, 50937 Cologne, Germany; (B.M.); (T.L.A.V.); (Y.H.)
- Center for Molecular Medicine Cologne (CMMC), University of Cologne, 50937 Cologne, Germany
- Institute for Genome Stability in Ageing and Disease, CECAD Research Center, Joseph-Stelzmann-Str. 26, 50931 Cologne, Germany
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18
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Bisevac J, Katta K, Petrovski G, Moe MC, Noer A. Wnt/β-Catenin Signaling Activation Induces Differentiation in Human Limbal Epithelial Stem Cells Cultured Ex Vivo. Biomedicines 2023; 11:1829. [PMID: 37509479 PMCID: PMC10377110 DOI: 10.3390/biomedicines11071829] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2023] [Revised: 06/05/2023] [Accepted: 06/13/2023] [Indexed: 07/30/2023] Open
Abstract
Human limbal epithelial stem cells (hLESCs) continuously replenish lost or damaged human corneal epithelial cells. The percentage of stem/progenitor cells in autologous ex vivo expanded tissue is essential for the long-term success of transplantation in patients with limbal epithelial stem cell deficiency. However, the molecular processes governing the stemness and differentiation state of hLESCs remain uncertain. Therefore, we sought to explore the impact of canonical Wnt/β-catenin signaling activation on hLESCs by treating ex vivo expanded hLESC cultures with GSK-3 inhibitor LY2090314. Real-time qRT-PCR and microarray data reveal the downregulation of stemness (TP63), progenitor (SOX9), quiescence (CEBPD), and proliferation (MKI67, PCNA) genes and the upregulation of genes for differentiation (CX43, KRT3) in treated- compared to non-treated samples. The pathway activation was shown by AXIN2 upregulation and enhanced levels of accumulated β-catenin. Immunocytochemistry and Western blot confirmed the findings for most of the above-mentioned markers. The Wnt/β-catenin signaling profile demonstrated an upregulation of WNT1, WNT3, WNT5A, WNT6, and WNT11 gene expression and a downregulation for WNT7A and DKK1 in the treated samples. No significant differences were found for WNT2, WNT16B, WIF1, and DKK2 gene expression. Overall, our results demonstrate that activation of Wnt/β-catenin signaling in ex vivo expanded hLESCs governs the cells towards differentiation and reduces proliferation and stem cell maintenance capability.
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Affiliation(s)
- Jovana Bisevac
- Center for Eye Research and Innovative Diagnostics, Department of Ophthalmology, Oslo University Hospital, P.O. Box 4956 Nydalen, 0424 Oslo, Norway
- Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, 0318 Oslo, Norway
| | - Kirankumar Katta
- Department of Immunology, Oslo University Hospital, Hf Rikshospitalet, 0424 Oslo, Norway
| | - Goran Petrovski
- Center for Eye Research and Innovative Diagnostics, Department of Ophthalmology, Oslo University Hospital, P.O. Box 4956 Nydalen, 0424 Oslo, Norway
- Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, 0318 Oslo, Norway
| | - Morten Carstens Moe
- Center for Eye Research and Innovative Diagnostics, Department of Ophthalmology, Oslo University Hospital, P.O. Box 4956 Nydalen, 0424 Oslo, Norway
- Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, 0318 Oslo, Norway
| | - Agate Noer
- Center for Eye Research and Innovative Diagnostics, Department of Ophthalmology, Oslo University Hospital, P.O. Box 4956 Nydalen, 0424 Oslo, Norway
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19
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Polisetti N, Martin G, Cristina Schmitz HR, Schlötzer-Schrehardt U, Schlunck G, Reinhard T. Characterization of Porcine Ocular Surface Epithelial Microenvironment. Int J Mol Sci 2023; 24:ijms24087543. [PMID: 37108705 PMCID: PMC10145510 DOI: 10.3390/ijms24087543] [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: 02/28/2023] [Revised: 04/13/2023] [Accepted: 04/14/2023] [Indexed: 04/29/2023] Open
Abstract
The porcine ocular surface is used as a model of the human ocular surface; however, a detailed characterization of the porcine ocular surface has not been documented. This is due, in part, to the scarcity of antibodies produced specifically against the porcine ocular surface cell types or structures. We performed a histological and immunohistochemical investigation on frozen and formalin-fixed, paraffin-embedded ocular surface tissue from domestic pigs using a panel of 41 different antibodies related to epithelial progenitor/differentiation phenotypes, extracellular matrix and associated molecules, and various niche cell types. Our observations suggested that the Bowman's layer is not evident in the cornea; the deep invaginations of the limbal epithelium in the limbal zone are analogous to the limbal interpalisade crypts of human limbal tissue; and the presence of goblet cells in the bulbar conjunctiva. Immunohistochemistry analysis revealed that the epithelial progenitor markers cytokeratin (CK)15, CK14, p63α, and P-cadherin were expressed in both the limbal and conjunctival basal epithelium, whereas the basal cells of the limbal and conjunctival epithelium did not stain for CK3, CK12, E-cadherin, and CK13. Antibodies detecting marker proteins related to the extracellular matrix (collagen IV, Tenascin-C), cell-matrix adhesion (β-dystroglycan, integrin α3 and α6), mesenchymal cells (vimentin, CD90, CD44), neurons (neurofilament), immune cells (HLA-ABC; HLA-DR, CD1, CD4, CD14), vasculature (von Willebrand factor), and melanocytes (SRY-homeobox-10, human melanoma black-45, Tyrosinase) on the normal human ocular surface demonstrated similar immunoreactivity on the normal porcine ocular surface. Only a few antibodies (directed against N-cadherin, fibronectin, agrin, laminin α3 and α5, melan-A) appeared unreactive on porcine tissues. Our findings characterize the main immunohistochemical properties of the porcine ocular surface and provide a morphological and immunohistochemical basis useful to research using porcine models. Furthermore, the analyzed porcine ocular structures are similar to those of humans, confirming the potential usefulness of pig eyes to study ocular surface physiology and pathophysiology.
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Affiliation(s)
- Naresh Polisetti
- Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106 Freiburg, Germany
| | - Gottfried Martin
- Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106 Freiburg, Germany
| | - Heidi R Cristina Schmitz
- CEMT-Freiburg, Experimental Surgery, Hospital-Medical Center, Faculty of Medicine, University of Freiburg, Breisacher Str. 66, 79106 Freiburg, Germany
| | - Ursula Schlötzer-Schrehardt
- Department of Ophthalmology, University Hospital Erlangen, Friedrich-Alexander-University of Erlangen-Nürnberg, Schwabachanlage 6, 91054 Erlangen, Germany
| | - Günther Schlunck
- Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106 Freiburg, Germany
| | - Thomas Reinhard
- Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106 Freiburg, Germany
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20
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Makuloluwa AK, Hamill KJ, Rauz S, Bosworth L, Haneef A, Romano V, Williams RL, Dartt DA, Kaye SB. The conjunctival extracellular matrix, related disorders and development of substrates for conjunctival restoration. Ocul Surf 2023; 28:322-335. [PMID: 34102309 DOI: 10.1016/j.jtos.2021.05.011] [Citation(s) in RCA: 12] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2021] [Revised: 05/05/2021] [Accepted: 05/26/2021] [Indexed: 12/15/2022]
Abstract
The conjunctiva can be damaged by numerous diseases with scarring, loss of tissue and dysfunction. Depending on extent of damage, restoration of function may require a conjunctival graft. A wide variety of biological and synthetic substrates have been tested in the search for optimal conditions for ex vivo culture of conjunctival epithelial cells as a route toward tissue grafts. Each substrate has specific advantages but also disadvantages related to their unique physical and biological characteristics, and identification and development of an improved substrate remains a priority. To achieve the goal of mimicking and restoring a biological material, requires information from the material. Specifically, extracellular matrix (ECM) derived from conjunctival tissue. Knowledge of the composition and structure of native ECM and identifying contributions of individual components to its function would enable using or mimicking those components to develop improved biological substrates. ECM is comprised of two components: basement membrane secreted predominantly by epithelial cells containing laminins and type IV collagens, which directly support epithelial and goblet cell adhesion differentiation and growth and, interstitial matrix secreted by fibroblasts in lamina propria, which provides mechanical and structural support. This review presents current knowledge on anatomy, composition of conjunctival ECM and related conjunctival disorders. Requirements of potential substrates for conjunctival tissue engineering and transplantation are discussed. Biological and synthetic substrates and their components are described in an accompanying review.
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Affiliation(s)
- Aruni K Makuloluwa
- Department of Eye and Vision Science, University of Liverpool, William Duncan Building, 6 West Derby Street, Liverpool, L7 8TX, UK
| | - Kevin J Hamill
- Department of Eye and Vision Science, University of Liverpool, William Duncan Building, 6 West Derby Street, Liverpool, L7 8TX, UK
| | - Saaeha Rauz
- Academic Unit of Ophthalmology, Institute of Inflammation and Ageing, University of Birmingham and Birmingham and Midland Eye Centre, Dudley Road Birmingham, B18 7QU, UK
| | - Lucy Bosworth
- Department of Eye and Vision Science, University of Liverpool, William Duncan Building, 6 West Derby Street, Liverpool, L7 8TX, UK
| | - Atikah Haneef
- Department of Eye and Vision Science, University of Liverpool, William Duncan Building, 6 West Derby Street, Liverpool, L7 8TX, UK
| | - Vito Romano
- Department of Eye and Vision Science, University of Liverpool, William Duncan Building, 6 West Derby Street, Liverpool, L7 8TX, UK
| | - Rachel L Williams
- Department of Eye and Vision Science, University of Liverpool, William Duncan Building, 6 West Derby Street, Liverpool, L7 8TX, UK
| | - Darlene A Dartt
- Schepens Eye Research Institute, Mass Eye and Ear Infirmary, Harvard Medical School, 20 Staniford St. Boston, MA, 02114, USA
| | - Stephen B Kaye
- Department of Eye and Vision Science, University of Liverpool, William Duncan Building, 6 West Derby Street, Liverpool, L7 8TX, UK.
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21
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Soleimani M, Cheraqpour K, Koganti R, Baharnoori SM, Djalilian AR. Concise Review: Bioengineering of Limbal Stem Cell Niche. Bioengineering (Basel) 2023; 10:111. [PMID: 36671683 PMCID: PMC9855097 DOI: 10.3390/bioengineering10010111] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2022] [Revised: 01/01/2023] [Accepted: 01/10/2023] [Indexed: 01/15/2023] Open
Abstract
The corneal epithelium is composed of nonkeratinized stratified squamous cells and has a significant turnover rate. Limbal integrity is vital to maintain the clarity and avascularity of the cornea as well as regeneration of the corneal epithelium. Limbal epithelial stem cells (LESCs) are located in the basal epithelial layer of the limbus and preserve this homeostasis. Proper functioning of LESCs is dependent on a specific microenvironment, known as the limbal stem cell niche (LSCN). This structure is made up of various cells, an extracellular matrix (ECM), and signaling molecules. Different etiologies may damage the LSCN, leading to limbal stem cell deficiency (LSCD), which is characterized by conjunctivalization of the cornea. In this review, we first summarize the basics of the LSCN and then focus on current and emerging bioengineering strategies for LSCN restoration to combat LSCD.
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Affiliation(s)
- Mohammad Soleimani
- Eye Research Center, Farabi Eye Hospital, Tehran University of Medical Sciences, Tehran 1336616351, Iran
- Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, IL 60612, USA
| | - Kasra Cheraqpour
- Eye Research Center, Farabi Eye Hospital, Tehran University of Medical Sciences, Tehran 1336616351, Iran
| | - Raghuram Koganti
- Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, IL 60612, USA
| | - Seyed Mahbod Baharnoori
- Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, IL 60612, USA
| | - Ali R. Djalilian
- Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, IL 60612, USA
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22
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The progress in techniques for culturing human limbal epithelial stem cells. Hum Cell 2023; 36:1-14. [PMID: 36181663 DOI: 10.1007/s13577-022-00794-2] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2022] [Accepted: 09/11/2022] [Indexed: 01/07/2023]
Abstract
In vitro culture of human limbal epithelial stem cells (hLESCs) is crucial to cell therapy in the treatment of limbal stem cell deficiency, a potentially vision-threatening disease that is characterized by persistent corneal epithelial defects and corneal epithelium conjunctivalization. Traditionally, hLESCs are cultivated based on either limbal tissue explants or single-cell suspensions in culture media containing xenogenous components, such as fetal bovine serum and murine 3T3 feeder cells. Plastic culture dishes and human amniotic membranes are classical growth substrates used in conventional hLESC culture systems. The past few decades have witnessed considerable progress and innovations in hLESC culture techniques to ensure a higher level of biosafety and lower immunogenicity for further cell treatment, including complete removal of xenogenous components from culture media, the application of human-derived feeder cells, and the development of novel scaffolds. Three-dimensional artificial niches and three-dimensional culture techniques have also been established to simulate the real microenvironment of limbal crypts for better cell outgrowth and proliferation. All these progresses ensure that in vitro cultured hLESCs are more adaptable to translational stem cell therapy for limbal stem cell deficiency.
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23
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Cárdenas-León CG, Mäemets-Allas K, Klaas M, Lagus H, Kankuri E, Jaks V. Matricellular proteins in cutaneous wound healing. Front Cell Dev Biol 2022; 10:1073320. [PMID: 36506087 PMCID: PMC9730256 DOI: 10.3389/fcell.2022.1073320] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2022] [Accepted: 11/14/2022] [Indexed: 11/25/2022] Open
Abstract
Cutaneous wound healing is a complex process that encompasses alterations in all aspects of the skin including the extracellular matrix (ECM). ECM consist of large structural proteins such as collagens and elastin as well as smaller proteins with mainly regulative properties called matricellular proteins. Matricellular proteins bind to structural proteins and their functions include but are not limited to interaction with cell surface receptors, cytokines, or protease and evoking a cellular response. The signaling initiated by matricellular proteins modulates differentiation and proliferation of cells having an impact on the tissue regeneration. In this review we give an overview of the matricellular proteins that have been found to be involved in cutaneous wound healing and summarize the information known to date about their functions in this process.
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Affiliation(s)
| | - Kristina Mäemets-Allas
- Department of Cell Biology, Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia
| | - Mariliis Klaas
- Department of Cell Biology, Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia
| | - Heli Lagus
- Department of Plastic Surgery and Wound Healing Centre, Helsinki University Hospital, University of Helsinki, Helsinki, Finland
| | - Esko Kankuri
- Department of Pharmacology, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | - Viljar Jaks
- Department of Cell Biology, Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia,Dermatology Clinic, Tartu University Clinics, Tartu, Estonia,*Correspondence: Viljar Jaks,
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24
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Ramos T, Parekh M, Meleady P, O’Sullivan F, Stewart RMK, Kaye SB, Hamill K, Ahmad S. Specific decellularized extracellular matrix promotes the plasticity of human ocular surface epithelial cells. Front Med (Lausanne) 2022; 9:974212. [PMID: 36457571 PMCID: PMC9705355 DOI: 10.3389/fmed.2022.974212] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2022] [Accepted: 10/21/2022] [Indexed: 11/25/2023] Open
Abstract
The ocular surface is composed of two phenotypically and functionally different epithelial cell types: corneal and the conjunctival epithelium. Upon injury or disease, ocular surface homeostasis is impaired resulting in migration of conjunctival epithelium on to the corneal surface. This can lead to incomplete transdifferentiation toward corneal epithelial-like cells in response to corneal basement membrane cues. We show that corneal extracellular matrix (ECM) proteins induce conjunctival epithelial cells to express corneal associated markers losing their conjunctival associated phenotype at both, mRNA and protein level. Corneal epithelial cells behave the same in the presence of conjunctival ECM proteins, expressing markers associated with conjunctival epithelium. This process of differentiation is accompanied by an intermediate step of cell de-differentiation as an up-regulation in the expression of epithelial stem cell markers is observed. In addition, analysis of ECM proteins by laminin screening assays showed that epithelial cell response is laminin-type dependent, and cells cultured on laminin-511 showed lower levels of lineage commitment. The phosphorylation and proteolysis levels of proteins mainly involved in cell growth and differentiation showed lower modifications in cells with lower lineage commitment. These observations showed that the ECM proteins may serve as tools to induce cell differentiation, which may have potential applications for the treatment of ocular surface injuries.
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Affiliation(s)
- Tiago Ramos
- Department of Eye and Vision Science, University of Liverpool, Liverpool, United Kingdom
- Faculty of Brain Sciences, Institute of Ophthalmology, University College London, London, United Kingdom
| | - Mohit Parekh
- Faculty of Brain Sciences, Institute of Ophthalmology, University College London, London, United Kingdom
| | - Paula Meleady
- Primary Department, National Institute for Cellular Biotechnology, Dublin City University, Dublin, Ireland
| | - Finbarr O’Sullivan
- Primary Department, National Institute for Cellular Biotechnology, Dublin City University, Dublin, Ireland
| | - Rosalind M. K. Stewart
- Department of Eye and Vision Science, University of Liverpool, Liverpool, United Kingdom
- St Paul’s Eye Unit, Royal Liverpool University Hospital, Liverpool, United Kingdom
- Department of Ophthalmology, Aberdeen Royal Infirmary, Aberdeen, United Kingdom
| | - Stephen B. Kaye
- Department of Eye and Vision Science, University of Liverpool, Liverpool, United Kingdom
- St Paul’s Eye Unit, Royal Liverpool University Hospital, Liverpool, United Kingdom
| | - Kevin Hamill
- Department of Eye and Vision Science, University of Liverpool, Liverpool, United Kingdom
| | - Sajjad Ahmad
- Department of Eye and Vision Science, University of Liverpool, Liverpool, United Kingdom
- Faculty of Brain Sciences, Institute of Ophthalmology, University College London, London, United Kingdom
- St Paul’s Eye Unit, Royal Liverpool University Hospital, Liverpool, United Kingdom
- External Eye Disease Service, Moorfields Eye Hospital, London, United Kingdom
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25
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Di Girolamo N, Park M. Cell identity changes in ocular surface Epithelia. Prog Retin Eye Res 2022:101148. [DOI: 10.1016/j.preteyeres.2022.101148] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2022] [Revised: 10/13/2022] [Accepted: 11/09/2022] [Indexed: 11/21/2022]
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26
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Masood F, Chang JH, Akbar A, Song A, Hu WY, Azar DT, Rosenblatt MI. Therapeutic Strategies for Restoring Perturbed Corneal Epithelial Homeostasis in Limbal Stem Cell Deficiency: Current Trends and Future Directions. Cells 2022; 11:3247. [PMID: 36291115 PMCID: PMC9600167 DOI: 10.3390/cells11203247] [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: 09/19/2022] [Revised: 10/11/2022] [Accepted: 10/12/2022] [Indexed: 02/03/2023] Open
Abstract
Limbal stem cells constitute an important cell population required for regeneration of the corneal epithelium. If insults to limbal stem cells or their niche are sufficiently severe, a disease known as limbal stem cell deficiency occurs. In the absence of functioning limbal stem cells, vision-compromising conjunctivalization of the corneal epithelium occurs, leading to opacification, inflammation, neovascularization, and chronic scarring. Limbal stem cell transplantation is the standard treatment for unilateral cases of limbal stem cell deficiency, but bilateral cases require allogeneic transplantation. Herein we review the current therapeutic utilization of limbal stem cells. We also describe several limbal stem cell markers that impact their phenotype and function and discuss the possibility of modulating limbal stem cells and other sources of stem cells to facilitate the development of novel therapeutic interventions. We finally consider several hurdles for widespread adoption of these proposed methodologies and discuss how they can be overcome to realize vision-restoring interventions.
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Affiliation(s)
- Faisal Masood
- Department of Ophthalmology and Visual Sciences, Illinois Eye and Ear Infirmary, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA
| | - Jin-Hong Chang
- Department of Ophthalmology and Visual Sciences, Illinois Eye and Ear Infirmary, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA
| | - Anosh Akbar
- Department of Ophthalmology and Visual Sciences, Illinois Eye and Ear Infirmary, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA
| | - Amy Song
- Department of Ophthalmology and Visual Sciences, Illinois Eye and Ear Infirmary, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA
| | - Wen-Yang Hu
- Department of Urology, University of Illinois at Chicago, Chicago, IL 60612, USA
| | - Dimitri T. Azar
- Department of Ophthalmology and Visual Sciences, Illinois Eye and Ear Infirmary, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA
| | - Mark I. Rosenblatt
- Department of Ophthalmology and Visual Sciences, Illinois Eye and Ear Infirmary, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA
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27
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Menzel-Severing J, Spaniol K, Groeber-Becker F, Geerling G. [Regenerative medicine for the corneal epithelium : Cell therapy from bench to bedside]. DIE OPHTHALMOLOGIE 2022; 119:891-901. [PMID: 35925345 DOI: 10.1007/s00347-022-01674-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Accepted: 05/31/2022] [Indexed: 06/15/2023]
Abstract
In the case of thermal or caustic burns of the ocular surface, loss of limbal epithelial stem cells leads to compromised self-renewal of the corneal epithelium. This results in permanent loss of vision. In these situations, transplantation of cultured limbal epithelial cells on an amniotic membrane or fibrin gel as substrate (Holoclar®) can help to regenerate the corneal surface. The required cells are obtained from the healthy partner eye, if available. Adult stem cells from other parts of the body potentially serve as alternative cell sources: hair follicles, oral mucosa, mesenchymal stromal cells, or induced pluripotent stem cells (originally, e.g., skin fibroblasts). The reprogramming of such cells can be achieved with the help of transcription factors. In addition, work is being done on biosynthetic or synthetic matrices, which not only serve as substrate material for the transplantation but also support the functional properties of these cells (self-renewal, corneal epithelial-typical phenotype).
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Affiliation(s)
- Johannes Menzel-Severing
- Klinik für Augenheilkunde, Universitätsklinikum Düsseldorf, Moorenstr. 5, 40225, Düsseldorf, Deutschland.
| | - Kristina Spaniol
- Klinik für Augenheilkunde, Universitätsklinikum Düsseldorf, Moorenstr. 5, 40225, Düsseldorf, Deutschland
| | - Florian Groeber-Becker
- Translationszentrum Regenerative Therapien | TLZ-RT, Leitung In-vitro-Testsysteme, Fraunhofer-Institut für Silicatforschung ISC, Würzburg, Deutschland
| | - Gerd Geerling
- Klinik für Augenheilkunde, Universitätsklinikum Düsseldorf, Moorenstr. 5, 40225, Düsseldorf, Deutschland
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28
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Puri S, Moreno IY, Sun M, Verma S, Lin X, Gesteira TF, Coulson-Thomas VJ. Hyaluronan supports the limbal stem cell phenotype during ex vivo culture. Stem Cell Res Ther 2022; 13:384. [PMID: 35907870 PMCID: PMC9338506 DOI: 10.1186/s13287-022-03084-8] [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: 05/19/2022] [Accepted: 07/21/2022] [Indexed: 11/29/2022] Open
Abstract
BACKGROUND Hyaluronan (HA) has previously been identified as an integral component of the limbal stem cell niche in vivo. In this study, we investigated whether a similar HA matrix is also expressed in vitro providing a niche supporting limbal epithelial stem cells (LESCs) during ex vivo expansion. We also investigated whether providing exogenous HA in vitro is beneficial to LESCs during ex vivo expansion. METHOD Human LESCs (hLESCs) were isolated from donor corneas and a mouse corneal epithelial progenitor cell line (TKE2) was obtained. The HA matrix was identified surrounding LESCs in vitro using immunocytochemistry, flow cytometry and red blood exclusion assay. Thereafter, LESCs were maintained on HA coated dishes or in the presence of HA supplemented in the media, and viability, proliferation, cell size, colony formation capabilities and expression of putative stem cell markers were compared with cells maintained on commonly used coated dishes. RESULTS hLESCs and TKE2 cells express an HA-rich matrix in vitro, and this matrix is essential for maintaining LESCs. Further supplying exogenous HA, as a substrate and supplemented to the media, increases LESC proliferation, colony formation capabilities and the expression levels of putative limbal stem cell markers. CONCLUSION Our data show that both exogenous and endogenous HA help to maintain the LESC phenotype. Exogenous HA provides improved culture conditions for LESC during ex vivo expansion. Thus, HA forms a favorable microenvironment for LESCs during ex vivo expansion and, therefore, could be considered as an easy and cost-effective substrate and/or supplement for culturing LESCs in the clinic.
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Affiliation(s)
- Sudan Puri
- College of Optometry, University of Houston, Houston, TX, USA
| | - Isabel Y Moreno
- College of Optometry, University of Houston, Houston, TX, USA
| | - Mingxia Sun
- College of Optometry, University of Houston, Houston, TX, USA
| | - Sudhir Verma
- College of Optometry, University of Houston, Houston, TX, USA
- Department of Zoology, Deen Dayal Upadhyaya College, University of Delhi, New Delhi, India
| | - Xiao Lin
- College of Optometry, University of Houston, Houston, TX, USA
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29
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Ramos T, Parekh M, Kaye SB, Ahmad S. Epithelial Cell-Derived Extracellular Vesicles Trigger the Differentiation of Two Epithelial Cell Lines. Int J Mol Sci 2022; 23:1718. [PMID: 35163646 PMCID: PMC8836104 DOI: 10.3390/ijms23031718] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2022] [Revised: 01/26/2022] [Accepted: 01/26/2022] [Indexed: 12/13/2022] Open
Abstract
Extracellular vesicles (EVs), specifically exosomes, carry a cell-type dependent cargo that is transported to the recipient cell and translated in the presence of a required machinery. Differences in the cargo carried by the corneal and conjunctival-derived EVs could be the agent that triggers the transdifferentiation of these two cell populations. Therefore, this study investigates the role of EVs in triggering the plasticity of corneal and conjunctival epithelial cells and identifies prospective miRNA and genes responsible for maintaining ocular surface homeostasis. The EVs were extracted from the conditioned media (after starving) of corneal epithelial (hTCEpi) and conjunctival (HCjE-Gi) cell lines using ultracentrifugation. HCjE-Gi cells were cultured with hTCEpi-derived EVs and vice-versa. The EVs were characterized as exosomes using Nanosight and Flow cytometry. KRT3 and KRT12 were used as associated corneal markers, whereas KRT7 and KRT13 were used as associated conjunctival markers with ΔNp63 as a differentiation marker. Shift of these markers was an indication of transdifferentiation. The cargo of the extracted exosomes from both the cell types was explored using next-generation sequencing. The hTCEpi-derived EVs induced conjunctival epithelial cells to express the corneal-associated markers KRT3 and KRT12, losing their conjunctival phenotype at both the mRNA and protein level. Simultaneously, HCjE-Gi-derived EVs induced corneal epithelial cells to express the conjunctival associated markers KRT7 and KRT13, losing their corneal phenotype. This process of differentiation was accompanied by an intermediate step of cell de-differentiation showed by up-regulation in the expression of epithelial stem cell marker ΔNp63, also shown on the ex vivo human cadaveric donor corneas. miRNA molecules (total of 11 including precursor and mature) with significant differences in their relative abundance between the two populations (p < 0.05) were found and investigated. miR-9-5p expression was higher in HCjE-Gi cells and HCjE-Gi-derived EVs when compared to hTCEpi cells and hTCEPi-derived EVs (p < 0.001). The results suggest that EVs released by the two cell types have the ability to influence the transdifferentiation of human conjunctival and corneal epithelial cells. miR-9-5p could have a role in stem cell homeostasis and cell differentiation via HES-1 gene.
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Affiliation(s)
- Tiago Ramos
- Department of Eye and Vision Science, University of Liverpool, Liverpool L7 8TX, UK; (T.R.); (S.B.K.)
- Institute of Ophthalmology, Faculty of Brain Sciences, University College London, London EC1V 9EL, UK;
| | - Mohit Parekh
- Institute of Ophthalmology, Faculty of Brain Sciences, University College London, London EC1V 9EL, UK;
| | - Stephen B. Kaye
- Department of Eye and Vision Science, University of Liverpool, Liverpool L7 8TX, UK; (T.R.); (S.B.K.)
- St Paul’s Eye Unit, Royal Liverpool University Hospital, Liverpool L7 8XP, UK
| | - Sajjad Ahmad
- Department of Eye and Vision Science, University of Liverpool, Liverpool L7 8TX, UK; (T.R.); (S.B.K.)
- Institute of Ophthalmology, Faculty of Brain Sciences, University College London, London EC1V 9EL, UK;
- St Paul’s Eye Unit, Royal Liverpool University Hospital, Liverpool L7 8XP, UK
- Moorfields Eye Hospital NHS Foundation Trust, London EC1V 2PD, UK
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Elhusseiny AM, Soleimani M, Eleiwa TK, ElSheikh RH, Frank CR, Naderan M, Yazdanpanah G, Rosenblatt MI, Djalilian AR. OUP accepted manuscript. Stem Cells Transl Med 2022; 11:259-268. [PMID: 35303110 PMCID: PMC8968724 DOI: 10.1093/stcltm/szab028] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2021] [Accepted: 09/06/2021] [Indexed: 11/24/2022] Open
Abstract
The corneal epithelium serves to protect the underlying cornea from the external environment and is essential for corneal transparency and optimal visual function. Regeneration of this epithelium is dependent on a population of stem cells residing in the basal layer of the limbus, the junction between the cornea and the sclera. The limbus provides the limbal epithelial stem cells (LESCs) with an optimal microenvironment, the limbal niche, which strictly regulates their proliferation and differentiation. Disturbances to the LESCs and/or their niche can lead to the pathologic condition known as limbal stem cell deficiency (LSCD) whereby the corneal epithelium is not generated effectively. This has deleterious effects on the corneal and visual function, due to impaired healing and secondary corneal opacification. In this concise review, we summarize the characteristics of LESCs and their niche, and present the current and future perspectives in the management of LSCD with an emphasis on restoring the function of the limbal niche.
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Affiliation(s)
- Abdelrahman M Elhusseiny
- Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, IL, USA
- Department of Ophthalmology, Harvey and Bernice Jones Eye Institute, University of Arkansas for Medical Sciences, Little Rock, AR, USA
| | - Mohammad Soleimani
- Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, IL, USA
| | - Taher K Eleiwa
- Department of Ophthalmology, Faculty of Medicine, Benha University, Benha, Egypt
| | - Reem H ElSheikh
- Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, IL, USA
| | - Charles R Frank
- Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, IL, USA
| | - Morteza Naderan
- Department of Ophthalmology, Farabi Eye Hospital, Tehran University of Medical Sciences, Tehran, Iran
| | - Ghasem Yazdanpanah
- Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, IL, USA
| | - Mark I Rosenblatt
- Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, IL, USA
| | - Ali R Djalilian
- Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, IL, USA
- Corresponding author: Ali R. Djalilian, Cornea Service, Stem Cell Therapy and Corneal Tissue Engineering Laboratory, Illinois Eye and Ear Infirmary, 1855 W. Taylor Street, M/C 648, Chicago, IL 60612, USA.
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Robertson SYT, Roberts JS, Deng SX. Regulation of Limbal Epithelial Stem Cells: Importance of the Niche. Int J Mol Sci 2021; 22:11975. [PMID: 34769405 PMCID: PMC8584795 DOI: 10.3390/ijms222111975] [Citation(s) in RCA: 12] [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: 10/12/2021] [Revised: 10/29/2021] [Accepted: 11/03/2021] [Indexed: 12/11/2022] Open
Abstract
Limbal epithelial stem/progenitor cells (LSCs) reside in a niche that contains finely tuned balances of various signaling pathways including Wnt, Notch, BMP, Shh, YAP, and TGFβ. The activation or inhibition of these pathways is frequently dependent on the interactions of LSCs with various niche cell types and extracellular substrates. In addition to receiving molecular signals from growth factors, cytokines, and other soluble molecules, LSCs also respond to their surrounding physical structure via mechanotransduction, interaction with the ECM, and interactions with other cell types. Damage to LSCs or their niche leads to limbal stem cell deficiency (LSCD). The field of LSCD treatment would greatly benefit from an understanding of the molecular regulation of LSCs in vitro and in vivo. This review synthesizes current literature around the niche factors and signaling pathways that influence LSC function. Future development of LSCD therapies should consider all these niche factors to achieve improved long-term restoration of the LSC population.
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Affiliation(s)
| | | | - Sophie X. Deng
- Jules Stein Eye Institute, University of California, Los Angeles, CA 94143, USA; (S.Y.T.R.); (J.S.R.)
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Bonnet C, González S, Roberts JS, Robertson SYT, Ruiz M, Zheng J, Deng SX. Human limbal epithelial stem cell regulation, bioengineering and function. Prog Retin Eye Res 2021; 85:100956. [PMID: 33676006 PMCID: PMC8428188 DOI: 10.1016/j.preteyeres.2021.100956] [Citation(s) in RCA: 47] [Impact Index Per Article: 11.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2020] [Revised: 02/21/2021] [Accepted: 02/26/2021] [Indexed: 12/13/2022]
Abstract
The corneal epithelium is continuously renewed by limbal stem/progenitor cells (LSCs), a cell population harbored in a highly regulated niche located at the limbus. Dysfunction and/or loss of LSCs and their niche cause limbal stem cell deficiency (LSCD), a disease that is marked by invasion of conjunctival epithelium into the cornea and results in failure of epithelial wound healing. Corneal opacity, pain, loss of vision, and blindness are the consequences of LSCD. Successful treatment of LSCD depends on accurate diagnosis and staging of the disease and requires restoration of functional LSCs and their niche. This review highlights the major advances in the identification of potential LSC biomarkers and components of the LSC niche, understanding of LSC regulation, methods and regulatory standards in bioengineering of LSCs, and diagnosis and staging of LSCD. Overall, this review presents key points for researchers and clinicians alike to consider in deepening the understanding of LSC biology and improving LSCD therapies.
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Affiliation(s)
- Clémence Bonnet
- Cornea Division, Stein Eye Institute, University of California, Los Angeles, CA, 90095, USA; Cornea Department, Paris University, Cochin Hospital, AP-HP, F-75014, Paris, France
| | - Sheyla González
- Cornea Division, Stein Eye Institute, University of California, Los Angeles, CA, 90095, USA
| | - JoAnn S Roberts
- Cornea Division, Stein Eye Institute, University of California, Los Angeles, CA, 90095, USA
| | - Sarah Y T Robertson
- Cornea Division, Stein Eye Institute, University of California, Los Angeles, CA, 90095, USA
| | - Maxime Ruiz
- Cornea Division, Stein Eye Institute, University of California, Los Angeles, CA, 90095, USA
| | - Jie Zheng
- Basic Science Division, Stein Eye Institute, University of California, Los Angeles, CA, 90095, USA
| | - Sophie X Deng
- Cornea Division, Stein Eye Institute, University of California, Los Angeles, CA, 90095, USA.
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A Decellularized Human Limbal Scaffold for Limbal Stem Cell Niche Reconstruction. Int J Mol Sci 2021; 22:ijms221810067. [PMID: 34576227 PMCID: PMC8471675 DOI: 10.3390/ijms221810067] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2021] [Revised: 09/13/2021] [Accepted: 09/14/2021] [Indexed: 12/13/2022] Open
Abstract
The transplantation of ex vivo expanded limbal epithelial progenitor cells (LEPCs) on amniotic membrane or fibrin gel is an established therapeutic strategy to regenerate the damaged corneal surface in patients with limbal stem cell deficiency (LSCD), but the long-term success rate is restricted. A scaffold with niche-specific structure and extracellular matrix (ECM) composition might have the advantage to improve long-term clinical outcomes, in particular for patients with severe damage or complete loss of the limbal niche tissue structure. Therefore, we evaluated the decellularized human limbus (DHL) as a biomimetic scaffold for the transplantation of LEPCs. Corneoscleral tissue was decellularized by sodium deoxycholate and deoxyribonuclease I in the presence or absence of dextran. We evaluated the efficiency of decellularization and its effects on the ultrastructure and ECM composition of the human corneal limbus. The recellularization of these scaffolds was studied by plating cultured LEPCs and limbal melanocytes (LMs) or by allowing cells to migrate from the host tissue following a lamellar transplantation ex vivo. Our decellularization protocol rapidly and effectively removed cellular and nuclear material while preserving the native ECM composition. In vitro recellularization by LEPCs and LMs demonstrated the good biocompatibility of the DHL and intrastromal invasion of LEPCs. Ex vivo transplantation of DHL revealed complete epithelialization as well as melanocytic and stromal repopulation from the host tissue. Thus, the generated DHL scaffold could be a promising biological material as a carrier for the transplantation of LEPCs to treat LSCD.
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Abdul-Al M, Kyeremeh GK, Saeinasab M, Heidari Keshel S, Sefat F. Stem Cell Niche Microenvironment: Review. Bioengineering (Basel) 2021; 8:bioengineering8080108. [PMID: 34436111 PMCID: PMC8389324 DOI: 10.3390/bioengineering8080108] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2021] [Revised: 07/14/2021] [Accepted: 07/16/2021] [Indexed: 12/13/2022] Open
Abstract
The cornea comprises a pool of self-regenerating epithelial cells that are crucial to preserving clarity and visibility. Limbal epithelial stem cells (LESCs), which live in a specialized stem cell niche (SCN), are crucial for the survival of the human corneal epithelium. They live at the bottom of the limbal crypts, in a physically enclosed microenvironment with a number of neighboring niche cells. Scientists also simplified features of these diverse microenvironments for more analysis in situ by designing and recreating features of different SCNs. Recent methods for regenerating the corneal epithelium after serious trauma, including burns and allergic assaults, focus mainly on regenerating the LESCs. Mesenchymal stem cells, which can transform into self-renewing and skeletal tissues, hold immense interest for tissue engineering and innovative medicinal exploration. This review summarizes all types of LESCs, identity and location of the human epithelial stem cells (HESCs), reconstruction of LSCN and artificial stem cells for self-renewal.
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Affiliation(s)
- Mohamed Abdul-Al
- Department of Biomedical and Electronics Engineering, School of Engineering, University of Bradford, Bradford BD71DP, UK; (M.A.-A.); (G.K.K.)
| | - George Kumi Kyeremeh
- Department of Biomedical and Electronics Engineering, School of Engineering, University of Bradford, Bradford BD71DP, UK; (M.A.-A.); (G.K.K.)
| | - Morvarid Saeinasab
- Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad 91779 48974, Iran;
| | - Saeed Heidari Keshel
- Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran 19839 69411, Iran;
| | - Farshid Sefat
- Department of Biomedical and Electronics Engineering, School of Engineering, University of Bradford, Bradford BD71DP, UK; (M.A.-A.); (G.K.K.)
- Interdisciplinary Research Centre in Polymer Science & Technology (Polymer IRC), University of Bradford, Bradford BD71DP, UK
- Correspondence:
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Dysfunction of the limbal epithelial stem cell niche in aniridia-associated keratopathy. Ocul Surf 2021; 21:160-173. [PMID: 34102310 DOI: 10.1016/j.jtos.2021.06.002] [Citation(s) in RCA: 25] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2021] [Revised: 04/22/2021] [Accepted: 06/01/2021] [Indexed: 02/06/2023]
Abstract
PURPOSE Abnormalities in the limbal niche microenvironment have been suggested to be causally involved in aniridia-associated keratopathy (AAK), but histological analyses on the limbal structure and composition in AAK are lacking. Here, we investigated morphologic and molecular alterations of the limbal epithelial stem cell niche in human congenital aniridia. METHODS The blind, buphthalmic and painful left eye of a 16-year old girl with congenital aniridia and juvenile glaucoma had to be enucleated because of uncontrolled intraocular pressure. The diagnosis of AAK was based on classical clinical features and partial limbal stem cell deficiency in the superior half. Genetic analysis identified a large heterozygous PAX6 gene deletion encompassing exons 11-15 as well as exon 9 of the neighboring ELP4 gene. Three limbal biopsies were taken from the superior, nasal and temporal regions to isolate and cultivate limbal epithelial progenitor cells and subject them to mRNA expression analyses. The globe was vertically bisected and processed for light and transmission electron microscopy and immunohistochemistry. RESULTS Comparative analysis of the superior and inferior limbal zones showed a gradual degradation of palisade structures associated with the transition from a hyperplastic to an attenuated corneal epithelium, inflammatory cell infiltrations and basement membrane irregularities. The clinically unaffected inferior part revealed no distinct stem cell clusters in the preserved palisade region, but a uniform population of hyperproliferative, undifferentiated progenitor cells in the basal/suprabasal layers of limbal and corneal epithelia, which gave rise to maldifferentiated epithelial cells exhibiting a conjunctival/epidermal phenotype and nuclear-to-cytoplasmic translocation of Pax6. The structure of the limbal niche was fundamentally perturbed, showing marked alterations in extracellular matrix composition, dislocation of atypical melanocytes lacking melanosomes and melanin, aberrant Wnt/β-catenin and retinoic acid signaling, and massive immune cell infiltration. CONCLUSIONS Considering the limitations of a single Case study, the findings suggest that ocular surface alterations in AAK are caused by a primary dysfunction and gradual breakdown of the limbal stem cell niche through Pax6-related effects on both melanogenesis and epithelial differentiation.
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Mutoji KN, Sun M, Elliott G, Moreno IY, Hughes C, Gesteira TF, Coulson-Thomas VJ. Extracellular Matrix Deposition and Remodeling after Corneal Alkali Burn in Mice. Int J Mol Sci 2021; 22:5708. [PMID: 34071909 PMCID: PMC8199272 DOI: 10.3390/ijms22115708] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2021] [Revised: 05/24/2021] [Accepted: 05/25/2021] [Indexed: 12/13/2022] Open
Abstract
Corneal transparency relies on the precise arrangement and orientation of collagen fibrils, made of mostly Type I and V collagen fibrils and proteoglycans (PGs). PGs are essential for correct collagen fibrillogenesis and maintaining corneal homeostasis. We investigated the spatial and temporal distribution of glycosaminoglycans (GAGs) and PGs after a chemical injury. The chemical composition of chondroitin sulfate (CS)/dermatan sulfate (DS) and heparan sulfate (HS) were characterized in mouse corneas 5 and 14 days after alkali burn (AB), and compared to uninjured corneas. The expression profile and corneal distribution of CS/DSPGs and keratan sulfate (KS) PGs were also analyzed. We found a significant overall increase in CS after AB, with an increase in sulfated forms of CS and a decrease in lesser sulfated forms of CS. Expression of the CSPGs biglycan and versican was increased after AB, while decorin expression was decreased. We also found an increase in KS expression 14 days after AB, with an increase in lumican and mimecan expression, and a decrease in keratocan expression. No significant changes in HS composition were noted after AB. Taken together, our study reveals significant changes in the composition of the extracellular matrix following a corneal chemical injury.
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Affiliation(s)
- Kazadi N. Mutoji
- College of Optometry, University of Houston, Houston, TX 77204, USA; (K.N.M.); (M.S.); (G.E.); (I.Y.M.); (T.F.G.)
| | - Mingxia Sun
- College of Optometry, University of Houston, Houston, TX 77204, USA; (K.N.M.); (M.S.); (G.E.); (I.Y.M.); (T.F.G.)
| | - Garrett Elliott
- College of Optometry, University of Houston, Houston, TX 77204, USA; (K.N.M.); (M.S.); (G.E.); (I.Y.M.); (T.F.G.)
| | - Isabel Y. Moreno
- College of Optometry, University of Houston, Houston, TX 77204, USA; (K.N.M.); (M.S.); (G.E.); (I.Y.M.); (T.F.G.)
| | - Clare Hughes
- School of Biosciences, Cardiff University, Cardiff CF10 3AT, UK;
| | - Tarsis F. Gesteira
- College of Optometry, University of Houston, Houston, TX 77204, USA; (K.N.M.); (M.S.); (G.E.); (I.Y.M.); (T.F.G.)
- Optimvia, Batavia, OH 45103, USA
| | - Vivien J. Coulson-Thomas
- College of Optometry, University of Houston, Houston, TX 77204, USA; (K.N.M.); (M.S.); (G.E.); (I.Y.M.); (T.F.G.)
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Bonnet C, Roberts JS, Deng SX. Limbal stem cell diseases. Exp Eye Res 2021; 205:108437. [PMID: 33571530 PMCID: PMC8044031 DOI: 10.1016/j.exer.2021.108437] [Citation(s) in RCA: 28] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2020] [Revised: 12/14/2020] [Accepted: 01/02/2021] [Indexed: 12/13/2022]
Abstract
The function of limbal stem/progenitor cells (LSCs) is critical to maintain corneal epithelial homeostasis. Many external insults and intrinsic defects can be deleterious to LSCs and their niche microenvironment, resulting in limbal stem cell dysfunction or deficiency (LSCD). Ocular comorbidities, frequent in eyes with LSCD, can exacerbate the dysfunction of residual LSCs, and limit the survival of transplanted LSCs. Clinical presentation and disease evolution vary among different etiologies of LSCD. New ocular imaging modalities and molecular markers are now available to standardize the diagnosis criteria and stage the severity of the disease. Medical therapies may be sufficient to reverse the disease if residual LSCs are present. A stepwise approach should be followed to optimize the ocular surface, eliminate the causative factors and treat comorbid conditions, before considering surgical interventions. Furthermore, surgical options are selected depending on the severity and laterality of the disease. The standardized diagnostic criteria to stage the disease is necessary to objectively evaluate and compare the efficacy of the emerging customized therapies.
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Affiliation(s)
- Clémence Bonnet
- Stein Eye Institute, Department of Ophthalmology, David Geffen School of Medicine, University of California, Los Angeles, CA, 90095, USA; Cornea Department, Paris University, Cochin Hospital, AP-HP, F-75014, Paris, France.
| | - JoAnn S Roberts
- Stein Eye Institute, Department of Ophthalmology, David Geffen School of Medicine, University of California, Los Angeles, CA, 90095, USA.
| | - Sophie X Deng
- Stein Eye Institute, Department of Ophthalmology, David Geffen School of Medicine, University of California, Los Angeles, CA, 90095, USA.
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Wang LY, Zhang YT, Du LQ, Wu XY, Zhu J. The Effect of SPARC on the Proliferation and Migration of Limbal Epithelial Stem Cells During the Corneal Epithelial Wound Healing. Stem Cells Dev 2021; 30:301-308. [PMID: 33487117 DOI: 10.1089/scd.2020.0196] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Secreted protein acidic and rich in cysteine (SPARC) shows a specific colocalization with limbal epithelial stem cells (LESCs) in vivo; however, the inherent relationship between SPARC and LESCs is still unclear. This study investigated the effects of SPARC on the maintenance of LESC stemness and corneal wound healing. To test the influence of different concentration of exogenous SPARC on the proliferation of LESCs, cell counting kit-8 assay and 5-ethynyl-2'-deoxyuridine staining were performed and the results indicated that 1 μg/mL SPARC was the optimum concentration for enhanced LESC proliferation. Compared with a control group, SPARC-treated group showed a higher expression of LESC-positive markers p63α, ABCG-2, and Bmi-1, and a lower level of differentiation marker cytokeratin-3 (CK3), thereby suggesting that SPARC could maintain LESC characteristic phenotype and suppress spontaneous epithelial differentiation in vitro. In vivo, exogenous SPARC accelerated the wound-healing process by both the enhancement of LESC proliferation and promoting the migration of the proliferating cells. However, the intact epithelium impaired this function of SPARC by contact inhibition.
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Affiliation(s)
- Le-Yi Wang
- Department of Ophthalmology, Cheeloo College of Medicine, Qilu Hospital, Shandong University, Jinan, P.R. China
| | - Yu-Ting Zhang
- Department of Ophthalmology, Cheeloo College of Medicine, Qilu Hospital, Shandong University, Jinan, P.R. China
| | - Li-Qun Du
- Department of Ophthalmology, Cheeloo College of Medicine, Qilu Hospital, Shandong University, Jinan, P.R. China
| | - Xin-Yi Wu
- Department of Ophthalmology, Cheeloo College of Medicine, Qilu Hospital, Shandong University, Jinan, P.R. China
| | - Jing Zhu
- Department of Ophthalmology, Cheeloo College of Medicine, Qilu Hospital, Shandong University, Jinan, P.R. China.,The Key Laboratory of Cardiovascular Remodeling and Function Research, The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Chinese Ministry of Education, Chinese National Health Commission and Chinese Academy of Medical Sciences, Qilu Hospital, Shandong University, Jinan, China
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Material Characterisation and Stratification of Conjunctival Epithelial Cells on Electrospun Poly(ε-Caprolactone) Fibres Loaded with Decellularised Tissue Matrices. Pharmaceutics 2021; 13:pharmaceutics13030318. [PMID: 33671006 PMCID: PMC7997349 DOI: 10.3390/pharmaceutics13030318] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2021] [Revised: 02/17/2021] [Accepted: 02/24/2021] [Indexed: 12/16/2022] Open
Abstract
The conjunctiva, an under-researched yet incredibly important tissue, plays key roles in providing protection to the eye and maintaining homeostasis of its ocular surface. Multiple diseases can impair conjunctival function leading to severe consequences that require surgical intervention. Small conjunctival defects can be repaired relatively easily, but larger defects rely on tissue grafts which generally do not provide adequate healing. A tissue engineering approach involving a biomaterial substrate capable of supporting a stratified epithelium with embedded, mucin-secreting goblet cells offers a potential solution. As a first step, this study aimed to induce stratification of human conjunctival epithelial cells cultured on electrospun scaffolds composed from poly(ε-caprolactone) (PCL) and decellularised tissue matrix (small intestinal submucosa (SIS) or urinary bladder matrix (UBM)) and held at the air/liquid interface. Stratification, up to 5 cell layers, occurred more frequently on scaffolds containing PCL + UBM. Incorporation of these decellularised tissue matrices also impacted material properties, with significant changes occurring to their fibre diameter, tensile properties, and chemical composition throughout the scaffold structure compared to PCL alone. These matrix containing scaffolds warrant further long-term investigation as a potential advanced therapy medicinal product for conjunctiva repair and regeneration.
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40
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Polisetti N, Schmid A, Schlötzer-Schrehardt U, Maier P, Lang SJ, Steinberg T, Schlunck G, Reinhard T. A decellularized human corneal scaffold for anterior corneal surface reconstruction. Sci Rep 2021; 11:2992. [PMID: 33542377 PMCID: PMC7862698 DOI: 10.1038/s41598-021-82678-3] [Citation(s) in RCA: 34] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2020] [Accepted: 01/14/2021] [Indexed: 02/07/2023] Open
Abstract
Allogenic transplants of the cornea are prone to rejection, especially in repetitive transplantation and in scarred or highly vascularized recipient sites. Patients with these ailments would particularly benefit from the possibility to use non-immunogenic decellularized tissue scaffolds for transplantation, which may be repopulated by host cells in situ or in vitro. So, the aim of this study was to develop a fast and efficient decellularization method for creating a human corneal extracellular matrix scaffold suitable for repopulation with human cells from the corneal limbus. To decellularize human donor corneas, sodium deoxycholate, deoxyribonuclease I, and dextran were assessed to remove cells and nuclei and to control tissue swelling, respectively. We evaluated the decellularization effects on the ultrastructure, optical, mechanical, and biological properties of the human cornea. Scaffold recellularization was studied using primary human limbal epithelial cells, stromal cells, and melanocytes in vitro and a lamellar transplantation approach ex vivo. Our data strongly suggest that this approach allowed the effective removal of cellular and nuclear material in a very short period of time while preserving extracellular matrix proteins, glycosaminoglycans, tissue structure, and optical transmission properties. In vitro recellularization demonstrated good biocompatibility of the decellularized human cornea and ex vivo transplantation revealed complete epithelialization and stromal repopulation from the host tissue. Thus, the generated decellularized human corneal scaffold could be a promising biological material for anterior corneal reconstruction in the treatment of corneal defects.
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Affiliation(s)
- Naresh Polisetti
- Eye Center, Medical Center - Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106, Freiburg, Germany.
| | - Anke Schmid
- Eye Center, Medical Center - Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106, Freiburg, Germany
| | - Ursula Schlötzer-Schrehardt
- Department of Ophthalmology, University Hospital Erlangen, Friedrich-Alexander-University of Erlangen-Nürnberg, Schwabachanlage 6, 91054, Erlangen, Germany
| | - Philip Maier
- Eye Center, Medical Center - Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106, Freiburg, Germany
| | - Stefan J Lang
- Eye Center, Medical Center - Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106, Freiburg, Germany
| | - Thorsten Steinberg
- Department of Operative Dentistry and Periodontology, Division of Oral Biotechnology, University of Freiburg, Hugstetter Strasse 55, 79106, Freiburg, Germany
| | - Günther Schlunck
- Eye Center, Medical Center - Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106, Freiburg, Germany.
| | - Thomas Reinhard
- Eye Center, Medical Center - Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106, Freiburg, Germany
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Guo ZH, Jia YYS, Zeng YM, Li ZF, Lin JS. Transcriptome analysis identifies the differentially expressed genes related to the stemness of limbal stem cells in mice. Gene 2021; 775:145447. [PMID: 33482278 DOI: 10.1016/j.gene.2021.145447] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2020] [Revised: 12/28/2020] [Accepted: 01/13/2021] [Indexed: 10/22/2022]
Abstract
Limbal stem cells (LSCs) reside in the basal layer of limbal epithelial cells (LECs). They are crucial for maintenance of corneal epithelium homeostasis and corneal wound healing. Their stemness is determined by their gene expression pattern. Despite of several positive identifiers have been reported, the unique biomarker for LSCs still remain elusive. Differentially expressed genes (DEGs) between stem cells and differentiated cells affect the fate of stem cells via specific signaling pathway. In order to understand the DEGs in the LSCs, RNA-seq was firstly conducted using a mouse model. A total of 1907 up-regulated DEGs and 395 down-regulated DEGs were identified in the limbus (L) compared to central cornea (CC) and conjunctiva (Cj). Reliability of the expression of genes from RNA-seq analysis was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and immunofluorescence staining. The expression pattern of putative biomarkers was considered to be age-related. In up-regulated DEGs GO analysis, 570 gene ontology (GO) terms were significantly enriched. Five groups of genes related with biological processes from these significantly enriched GO terms comprised ionic transport, regulation of tissue development, muscle contraction, visual perception, and cell adhesion, which were clustered as a weighted similar network. Whereas, in down-regulated DEGs GO analysis, 61 GO terms were significantly enriched and only one group of ATP biosynthesis and metabolic process were clustered. Furthermore, we identified 55 signaling pathways by the Kyoto Encyclopedia of Genes and Genomes (KEGG) database based on up-regulated genes and 14 KEGG pathways based on down-regulated genes. In this study, we provide a landscape of the expression of putative LSCs biomarkers and stemness-related signaling pathways in a mouse model. Our findings could aid in the identification of LSC niche factors that may be related to the stemness of the LSCs.
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Affiliation(s)
- Zhi Hou Guo
- School of Medicine, Huaqiao University, Quanzhou, Fujian 362021, China; Stem Cell Laboratory, The Second Affiliated Hospital of Fujian Medical University, China
| | | | - Yi Ming Zeng
- The Second Affiliated Hospital of Fujian Medical University, China
| | - Zhao Fa Li
- School of Medicine, Huaqiao University, Quanzhou, Fujian 362021, China
| | - Jun Sheng Lin
- School of Medicine, Huaqiao University, Quanzhou, Fujian 362021, China.
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Ashworth S, Harrington J, Hammond GM, Bains KK, Koudouna E, Hayes AJ, Ralphs JR, Regini JW, Young RD, Hayashi R, Nishida K, Hughes CE, Quantock AJ. Chondroitin Sulfate as a Potential Modulator of the Stem Cell Niche in Cornea. Front Cell Dev Biol 2021; 8:567358. [PMID: 33511110 PMCID: PMC7835413 DOI: 10.3389/fcell.2020.567358] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2020] [Accepted: 12/07/2020] [Indexed: 12/13/2022] Open
Abstract
Chondroitin sulfate (CS) is an important component of the extracellular matrix in multiple biological tissues. In cornea, the CS glycosaminoglycan (GAG) exists in hybrid form, whereby some of the repeating disaccharides are dermatan sulfate (DS). These CS/DS GAGs in cornea, through their presence on the proteoglycans, decorin and biglycan, help control collagen fibrillogenesis and organization. CS also acts as a regulatory ligand for a spectrum of signaling molecules, including morphogens, cytokines, chemokines, and enzymes during corneal growth and development. There is a growing body of evidence that precise expression of CS or CS/DS with specific sulfation motifs helps define the local extracellular compartment that contributes to maintenance of the stem cell phenotype. Indeed, recent evidence shows that CS sulfation motifs recognized by antibodies 4C3, 7D4, and 3B3 identify stem cell populations and their niches, along with activated progenitor cells and transitional areas of tissue development in the fetal human elbow. Various sulfation motifs identified by some CS antibodies are also specifically located in the limbal region at the edge of the mature cornea, which is widely accepted to represent the corneal epithelial stem cell niche. Emerging data also implicate developmental changes in the distribution of CS during corneal morphogenesis. This article will reflect upon the potential roles of CS and CS/DS in maintenance of the stem cell niche in cornea, and will contemplate the possible involvement of CS in the generation of eye-like tissues from human iPS (induced pluripotent stem) cells.
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Affiliation(s)
- Sean Ashworth
- Structural Biophysics Group, School of Optometry and Vision Sciences, College of Biomedical and Life Sciences, Cardiff University, Cardiff, United Kingdom.,School of Biosciences, College of Biomedical and Life Sciences, Cardiff University, Cardiff, United Kingdom
| | - Jodie Harrington
- Structural Biophysics Group, School of Optometry and Vision Sciences, College of Biomedical and Life Sciences, Cardiff University, Cardiff, United Kingdom.,Department of Stem Cells and Applied Medicine, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Greg M Hammond
- Structural Biophysics Group, School of Optometry and Vision Sciences, College of Biomedical and Life Sciences, Cardiff University, Cardiff, United Kingdom
| | - Kiranjit K Bains
- Structural Biophysics Group, School of Optometry and Vision Sciences, College of Biomedical and Life Sciences, Cardiff University, Cardiff, United Kingdom
| | - Elena Koudouna
- Structural Biophysics Group, School of Optometry and Vision Sciences, College of Biomedical and Life Sciences, Cardiff University, Cardiff, United Kingdom
| | - Anthony J Hayes
- School of Biosciences, College of Biomedical and Life Sciences, Cardiff University, Cardiff, United Kingdom
| | - James R Ralphs
- School of Biosciences, College of Biomedical and Life Sciences, Cardiff University, Cardiff, United Kingdom
| | - Justyn W Regini
- Structural Biophysics Group, School of Optometry and Vision Sciences, College of Biomedical and Life Sciences, Cardiff University, Cardiff, United Kingdom
| | - Robert D Young
- Structural Biophysics Group, School of Optometry and Vision Sciences, College of Biomedical and Life Sciences, Cardiff University, Cardiff, United Kingdom
| | - Ryuhei Hayashi
- Department of Stem Cells and Applied Medicine, Osaka University Graduate School of Medicine, Osaka, Japan.,Department of Ophthalmology, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Kohji Nishida
- Department of Ophthalmology, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Clare E Hughes
- School of Biosciences, College of Biomedical and Life Sciences, Cardiff University, Cardiff, United Kingdom
| | - Andrew J Quantock
- Structural Biophysics Group, School of Optometry and Vision Sciences, College of Biomedical and Life Sciences, Cardiff University, Cardiff, United Kingdom
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43
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Markers of Stem Cells. Stem Cells 2021. [DOI: 10.1007/978-981-16-1638-9_6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022]
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Wang Y, Hu G, Hill RC, Dzieciatkowska M, Hansen KC, Zhang XB, Yan Z, Pei M. Matrix reverses immortalization-mediated stem cell fate determination. Biomaterials 2021; 265:120387. [PMID: 32987274 PMCID: PMC7944411 DOI: 10.1016/j.biomaterials.2020.120387] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2020] [Revised: 08/24/2020] [Accepted: 09/14/2020] [Indexed: 12/15/2022]
Abstract
Primary cell culture in vitro suffers from cellular senescence. We hypothesized that expansion on decellularized extracellular matrix (dECM) deposited by simian virus 40 large T antigen (SV40LT) transduced autologous infrapatellar fat pad stem cells (IPFSCs) could rejuvenate high-passage IPFSCs in both proliferation and chondrogenic differentiation. In the study, we found that SV40LT transduced IPFSCs exhibited increased proliferation and adipogenic potential but decreased chondrogenic potential. Expansion on dECMs deposited by passage 5 IPFSCs yielded IPFSCs with dramatically increased proliferation and chondrogenic differentiation capacity; however, this enhanced capacity diminished if IPFSCs were grown on dECM deposited by passage 15 IPFSCs. Interestingly, expansion on dECM deposited by SV40LT transduced IPFSCs yielded IPFSCs with enhanced proliferation and chondrogenic capacity but decreased adipogenic potential, particularly for the dECM group derived from SV40LT transduced passage 15 cells. Our immunofluorescence staining and proteomics data identify matrix components such as basement membrane proteins as top candidates for matrix mediated IPFSC rejuvenation. Both cell proliferation and differentiation were endorsed by transcripts measured by RNASeq during the process. This study provides a promising model for in-depth investigation of the matrix protein influence on surrounding stem cell differentiation.
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Affiliation(s)
- Yiming Wang
- Stem Cell and Tissue Engineering Laboratory, Department of Orthopaedics, West Virginia University, Morgantown, WV, USA; Department of Orthopaedics, Zhongshan Hospital, Fudan University, Shanghai, China
| | - Gangqing Hu
- Department of Microbiology, Immunology, and Cell Biology, West Virginia University, Morgantown, WV, USA; Bioinformatics Core, West Virginia University, Morgantown, WV, USA
| | - Ryan C Hill
- Department of Biochemistry & Molecular Genetics, University of Colorado Denver, Aurora, CO, USA
| | - Monika Dzieciatkowska
- Department of Biochemistry & Molecular Genetics, University of Colorado Denver, Aurora, CO, USA
| | - Kirk C Hansen
- Department of Biochemistry & Molecular Genetics, University of Colorado Denver, Aurora, CO, USA
| | - Xiao-Bing Zhang
- State Key Laboratory of Experimental Hematology, Tianjin, China; Department of Medicine, Loma Linda University, Loma Linda, CA, USA.
| | - Zuoqin Yan
- Department of Orthopaedics, Zhongshan Hospital, Fudan University, Shanghai, China.
| | - Ming Pei
- Stem Cell and Tissue Engineering Laboratory, Department of Orthopaedics, West Virginia University, Morgantown, WV, USA; WVU Cancer Institute, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, WV, USA.
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45
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Isolation and enrichment of melanocytes from human corneal limbus using CD117 (c-Kit) as selection marker. Sci Rep 2020; 10:17588. [PMID: 33067486 PMCID: PMC7567782 DOI: 10.1038/s41598-020-74869-1] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2020] [Accepted: 10/07/2020] [Indexed: 12/13/2022] Open
Abstract
Limbal melanocytes (LM) are located in the basal epithelial layer of the corneoscleral limbus and interact with adjacent limbal epithelial progenitor cells. The exploration of their biological role in the maintenance of the limbal stem cell niche has been limited by the difficulty of LM isolation and cultivation. Here, we report on a facile protocol for the efficient isolation and enrichment of pure populations of human LMs by fluorescence-activated cell sorting (FACS) using antibodies raised against the cell surface marker CD117 (c-Kit). The enriched LMs retain self-renewal capacity and sustained melanin production, and are suitable to study the potential of LMs in stem cell-based corneal tissue engineering.
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Hou L, Fu W, Liu Y, Wang Q, Wang L, Huang Y. Agrin Promotes Limbal Stem Cell Proliferation and Corneal Wound Healing Through Hippo-Yap Signaling Pathway. Invest Ophthalmol Vis Sci 2020; 61:7. [PMID: 32392315 PMCID: PMC7405682 DOI: 10.1167/iovs.61.5.7] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Purpose To investigate the effect and mechanism of Agrin on limbal stem cell proliferation and corneal wound healing. Methods Limbal stem cells were isolated and treated with different concentrations of Agrin. CCK-8 and cell proliferation markers (Ki67 and pH3) were detected to evaluate cell numbers or proliferative potential of limbal stem cells. The corneal epithelium wound model was induced by debridement of central corneal epithelial, and the effects of Agrin on limbal stem cell proliferation and corneal epithelial wound healing rate were determined. Results Agrin promoted the proliferation of cultured limbal stem cells in vitro and increased the expression level of p63α rather than keratin 12. Furthermore, Agrin accelerated the wound healing rate of corneal epithelium through activating limbal stem cell proliferation in vivo. In terms of mechanism, Agrin could facilitate the dephosphorylation of Yap1, which contributed to the nuclear translocation of Yap1 and expression of Cyclin D1, and subsequently promoted proliferation of limbal stem cells. Conclusions Agrin promotes the proliferation of limbal stem cells and accelerates the healing rate of corneal wound through Hippo-Yap signaling pathway.
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Polisetti N, Schlunck G, Reinhard T, Kruse FE, Schlötzer-Schrehardt U. Isolation and ex vivo Expansion of Human Limbal Epithelial Progenitor Cells. Bio Protoc 2020; 10:e3754. [PMID: 33659413 DOI: 10.21769/bioprotoc.3754] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2020] [Revised: 07/28/2020] [Accepted: 07/29/2020] [Indexed: 11/02/2022] Open
Abstract
Limbal stem cell transplantation has been used successfully to treat patients with limbal stem cell deficiency all over the world. However, long term clinical results often proved less satisfactory due to the low quality of the graft or inadequate properties of transplanted cells. To enhance the ex vivo expansion of human limbal epithelial stem or progenitor cells (LEPC) by preserving stem cell phenotype and to improve subsequent transplantation efficiency, cell-matrix interactions ex vivo should mimic the condition in vivo. The laminin isoforms preferentially expressed in the limbal niche can be used as a culture matrix for epithelial tissue engineering. We recently published the expansion of LEPC on various laminin isoforms and observed that laminin alpha 5-derived matrices support the efficient expansion of LEPC compared to tissue culture plates and other laminin isoforms by preserving stem/progenitor cell phenotype. Here, we describe an optimized protocol for the isolation of LEPC from cadaveric corneal limbal tissue by collagenase digestion and efficient expansion of LEPC using recombinant human laminin-511 E8 fragment (LN-511E8) as culture substrate.
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Affiliation(s)
- Naresh Polisetti
- Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Günther Schlunck
- Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Thomas Reinhard
- Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Friedrich E Kruse
- Department of Ophthalmology, University of Erlangen-Nürnberg, Erlangen, Germany
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Al-Jaibaji O, Swioklo S, Shortt A, Figueiredo FC, Connon CJ. Hypothermically Stored Adipose-Derived Mesenchymal Stromal Cell Alginate Bandages Facilitate Use of Paracrine Molecules for Corneal Wound Healing. Int J Mol Sci 2020; 21:ijms21165849. [PMID: 32823996 PMCID: PMC7461547 DOI: 10.3390/ijms21165849] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2020] [Revised: 08/05/2020] [Accepted: 08/05/2020] [Indexed: 12/13/2022] Open
Abstract
Adipose-derived mesenchymal stromal cells (Ad-MSCs) may alleviate corneal injury through the secretion of therapeutic factors delivered at the injury site. We aimed to investigate the therapeutic factors secreted from hypothermically stored, alginate-encapsulated Ad-MSCs’ bandages in in vitro and in vivo corneal wounds. Ad-MSCs were encapsulated in 1.2% w/v alginate gels to form bandages and stored at 15 °C for 72 h before assessing cell viability and co-culture with corneal scratch wounds. Genes of interest, including HGF, TSG-6, and IGF were identified by qPCR and a human cytokine array kit used to profile the therapeutic factors secreted. In vivo, bandages were applied to adult male mice corneas following epithelial debridement. Bandages were shown to maintain Ad-MSCs viability during storage and able to indirectly improve corneal wound healing in vivo. Soluble protein concentration and paracrine factors such as TSG-6, HGF, IL-8, and MCP-1 release were greatest following hypothermic storage. In vivo, Ad-MSCs bandages-treated groups reduced immune cell infiltration when compared to untreated groups. In conclusion, bandages were shown to maintain Ad-MSCs ability to produce a cocktail of key therapeutic factors following storage and that these soluble factors can improve in vitro and in vivo corneal wound healing.
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Affiliation(s)
- Olla Al-Jaibaji
- Biosciences Institute, Newcastle University, International Centre for Life, Central Parkway, Newcastle upon Tyne NE1 3BZ, UK; (O.A.-J.); (S.S.); (F.C.F.)
| | - Stephen Swioklo
- Biosciences Institute, Newcastle University, International Centre for Life, Central Parkway, Newcastle upon Tyne NE1 3BZ, UK; (O.A.-J.); (S.S.); (F.C.F.)
- Atelerix Ltd., The Biosphere, Newcastle upon Tyne NE4 5BX, UK
| | - Alex Shortt
- UCL Institute of Ophthalmology, London EC1V 9EL, UK;
| | - Francisco C. Figueiredo
- Biosciences Institute, Newcastle University, International Centre for Life, Central Parkway, Newcastle upon Tyne NE1 3BZ, UK; (O.A.-J.); (S.S.); (F.C.F.)
- Department of Ophthalmology, Royal Victoria Infirmary & Newcastle University, Newcastle upon Tyne NE1 4LP, UK
| | - Che J. Connon
- Biosciences Institute, Newcastle University, International Centre for Life, Central Parkway, Newcastle upon Tyne NE1 3BZ, UK; (O.A.-J.); (S.S.); (F.C.F.)
- Correspondence: ; Tel.: +44-(0)-191-241-8623
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Tseng SCG, Chen SY, Mead OG, Tighe S. Niche regulation of limbal epithelial stem cells: HC-HA/PTX3 as surrogate matrix niche. Exp Eye Res 2020; 199:108181. [PMID: 32795525 DOI: 10.1016/j.exer.2020.108181] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2020] [Revised: 06/15/2020] [Accepted: 07/31/2020] [Indexed: 12/13/2022]
Abstract
Homeostasis of the corneal epithelium is ultimately maintained by stem cells that reside in a specialized microenvironment within the corneal limbus termed palisades of Vogt. This limbal niche nourishes, protects, and regulates quiescence, self-renewal, and fate decision of limbal epithelial stem/progenitor cells (LEPCs) toward corneal epithelial differentiation. This review focuses on our current understanding of the mechanism by which limbal (stromal) niche cells (LNCs) regulate the aforementioned functions of LEPCs. Based on our discovery and characterization of a unique extracellular matrix termed HC-HA/PTX3 (Heavy chain (HC1)-hyaluronan (HA)/pentraxin 3 (PTX3) complex, "-" denotes covalent linkage; "/" denotes non-covalent binding) in the birth tissue, i.e., amniotic membrane and umbilical cord, we put forth a new paradigm that HC-HA/PTX3 serves as a surrogate matrix niche by maintaining the in vivo nuclear Pax6+ neural crest progenitor phenotype to support quiescence and self-renewal but prevent corneal fate decision of LEPCs. This new paradigm helps explain how limbal stem cell deficiency (LSCD) develops in aniridia due to Pax6-haplotype deficiency and further explains why transplantation of HC-HA/PTX3-containing amniotic membrane prevents LSCD in acute chemical burns and Stevens Johnson syndrome, augments the success of autologous LEPCs transplantation in patients suffering from partial or total LSCD, and assists ex vivo expansion (engineering) of a graft containing LEPCs. We thus envisage that this new paradigm based on regenerative matrix HC-HA/PTX3 as a surrogate niche can set a new standard for regenerative medicine in and beyond ophthalmology.
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Affiliation(s)
- Scheffer C G Tseng
- Research & Development Department, TissueTech, Inc., Miami, FL, 33126, USA; Ocular Surface Center and Ocular Surface Research & Education Foundation, Miami, FL, 33126, USA.
| | - Szu-Yu Chen
- Research & Development Department, TissueTech, Inc., Miami, FL, 33126, USA
| | - Olivia G Mead
- Research & Development Department, TissueTech, Inc., Miami, FL, 33126, USA
| | - Sean Tighe
- Research & Development Department, TissueTech, Inc., Miami, FL, 33126, USA; Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, FL, 33136, USA; Department of Ophthalmology, Herbert Wertheim College of Medicine, Florida International University, Miami, FL, 33199, USA
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Puri S, Coulson-Thomas YM, Gesteira TF, Coulson-Thomas VJ. Distribution and Function of Glycosaminoglycans and Proteoglycans in the Development, Homeostasis and Pathology of the Ocular Surface. Front Cell Dev Biol 2020; 8:731. [PMID: 32903857 PMCID: PMC7438910 DOI: 10.3389/fcell.2020.00731] [Citation(s) in RCA: 58] [Impact Index Per Article: 11.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2020] [Accepted: 07/15/2020] [Indexed: 12/20/2022] Open
Abstract
The ocular surface, which forms the interface between the eye and the external environment, includes the cornea, corneoscleral limbus, the conjunctiva and the accessory glands that produce the tear film. Glycosaminoglycans (GAGs) and proteoglycans (PGs) have been shown to play important roles in the development, hemostasis and pathology of the ocular surface. Herein we review the current literature related to the distribution and function of GAGs and PGs within the ocular surface, with focus on the cornea. The unique organization of ECM components within the cornea is essential for the maintenance of corneal transparency and function. Many studies have described the importance of GAGs within the epithelial and stromal compartment, while very few studies have analyzed the ECM of the endothelial layer. Importantly, GAGs have been shown to be essential for maintaining corneal homeostasis, epithelial cell differentiation and wound healing, and, more recently, a role has been suggested for the ECM in regulating limbal stem cells, corneal innervation, corneal inflammation, corneal angiogenesis and lymphangiogenesis. Reports have also associated genetic defects of the ECM to corneal pathologies. Thus, we also highlight the role of different GAGs and PGs in ocular surface homeostasis, as well as in pathology.
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Affiliation(s)
- Sudan Puri
- College of Optometry, University of Houston, Houston, TX, United States
| | - Yvette M Coulson-Thomas
- Molecular Biology Section, Department of Biochemistry, Universidade Federal de São Paulo, São Paulo, Brazil
| | - Tarsis F Gesteira
- College of Optometry, University of Houston, Houston, TX, United States.,Optimvia, LLC, Batavia, OH, United States
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