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Li X, Hayashi R, Imaizumi T, Harrington J, Kudo Y, Takayanagi H, Baba K, Nishida K. Extracellular vesicles from adipose-derived mesenchymal stem cells promote colony formation ability and EMT of corneal limbal epithelial cells. PLoS One 2025; 20:e0321579. [PMID: 40257992 PMCID: PMC12011229 DOI: 10.1371/journal.pone.0321579] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2024] [Accepted: 03/07/2025] [Indexed: 04/23/2025] Open
Abstract
Corneal diseases are a leading cause of visual impairment, and their treatment remains challenging. Corneal epithelial stem cells exist in the limbus, the peripheral region of the cornea, and play an important role in corneal regeneration. Here, we evaluated the effects of extracellular vesicles from human adipose-derived mesenchymal stem cells (AdMSC-EVs) on limbal epithelial cells (LECs). Colony formation assays showed that the colony-forming efficiency of LECs significantly increased in the presence of AdMSC-EVs. We next demonstrated that AdMSC-EVs accelerated the migration of LECs in a scratch assay, whereas the proliferation of LECs was decreased by AdMSC-EVs in the cell proliferation assay. RNA sequencing analysis of LECs indicated that AdMSC-EVs maintained their stem cell properties and improved epithelial-mesenchymal transition (EMT). Furthermore, after identifying the six most abundant microRNAs (miRNAs) in AdMSC-EVs, LEC transfection with miRNA mimics indicated that miR-25, miR-191, and miR-335 were the most probable miRNA factors within AdMSC-EVs at improving colony formation ability and EMT. Taken together, our findings indicated that AdMSC-EVs enhanced the colony formation ability and EMT of LECs, and the effects of AdMSC-EVs were in-part mediated by the miRNAs within the AdMSC-EVs.
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Affiliation(s)
- Xiaoqin Li
- Department of Ophthalmology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan
| | - Ryuhei Hayashi
- Department of Ophthalmology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan
- Department of Stem Cells and Applied Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan
- Institute for Open and Transdisciplinary Research Initiatives, Osaka University, Suita, Osaka, Japan
| | - Tsutomu Imaizumi
- Department of Stem Cells and Applied Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan
- Basic Research Development Division, ROHTO Pharmaceutical, Ikuno-ku, Osaka, Japan
| | - Jodie Harrington
- Department of Stem Cells and Applied Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan
- Faculty of Health, Medicine and Social Care, Medical Technology Research Centre, Anglia Ruskin University, Chelmsford Campus, England, United Kingdom
| | - Yuji Kudo
- Department of Stem Cells and Applied Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan
- Basic Research Development Division, ROHTO Pharmaceutical, Ikuno-ku, Osaka, Japan
| | - Hiroshi Takayanagi
- Department of Ophthalmology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan
- Research, Development and Production Department of RAYMEI Inc, Suita, Osaka, Japan
| | - Koichi Baba
- Department of Ophthalmology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan
- Department of Advanced Device Regenerative Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan
- Visual Regenerative Medicine, Division of Health Sciences, Osaka University Graduate School of Medicine, Suita, Osaka, Japan
| | - Kohji Nishida
- Department of Ophthalmology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan
- Institute for Open and Transdisciplinary Research Initiatives, Osaka University, Suita, Osaka, Japan
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Tran JA, Dohlman TH, Zhang LJ, Lorch A, Elze T, Miller JW, Yin J, Oke I, Dana R. Visual Outcomes of Limbal Stem Cell Transplantation in the IRIS® Registry. Ophthalmology 2025:S0161-6420(25)00236-2. [PMID: 40228697 DOI: 10.1016/j.ophtha.2025.04.009] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2024] [Revised: 04/02/2025] [Accepted: 04/08/2025] [Indexed: 04/16/2025] Open
Affiliation(s)
| | | | - Lyvia J Zhang
- Massachusetts Eye and Ear Eye & Ear, Boston, Massachusetts
| | - Alice Lorch
- Massachusetts Eye and Ear Eye & Ear, Boston, Massachusetts
| | - Tobias Elze
- Massachusetts Eye and Ear Eye & Ear, Boston, Massachusetts
| | - Joan W Miller
- Massachusetts Eye and Ear Eye & Ear, Boston, Massachusetts
| | - Jia Yin
- Massachusetts Eye and Ear Eye & Ear, Boston, Massachusetts
| | - Isdin Oke
- Boston Children's Hospital, Boston, Massachusetts
| | - Reza Dana
- Massachusetts Eye and Ear Eye & Ear, Boston, Massachusetts
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Hernandez BJ, Robertson DM. Exosomes in Corneal Homeostasis and Wound Healing. Curr Eye Res 2025:1-9. [PMID: 39936626 DOI: 10.1080/02713683.2025.2459335] [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: 10/01/2024] [Revised: 01/17/2025] [Accepted: 01/21/2025] [Indexed: 02/13/2025]
Abstract
PURPOSE The cornea is a transparent avascular tissue that serves as the first line of defense against opportunistic pathogens and provides a smooth refractive surface for vision. Due to its external location, the cornea is vulnerable to stress from the outer environment. This can lead to corneal epithelial breakdown and subsequent corneal disease. Extracellular vesicles (EVs) are nano-sized vesicles enclosed within a lipid bilayer that are secreted by all cells in the body and play a key role in cell-to-cell communication. Within the cornea field, EVs and exosomes, the latter of which represents a subpopulation of small EVs, have emerged as potential therapies for treating corneal diseases and have increased our understanding of the mechanisms by which EVs, and more specifically, exosomes released by stressed or unhealthy cells, leads to corneal dysfunction and disease. METHODS We conducted a literature search using PubMed and Google Scholar using keywords relevant to exosomes, extracellular vesicles, and cornea. We reviewed the literature focusing on EV studies on corneal wound healing and therapy. RESULTS This review provides a comprehensive overview of the current state of exosome biology as it relates to corneal disease and wound healing. Studies to date provide compelling data indicating that EVs and exosomes may play an integral role in the maintenance of corneal homeostasis. EVs and exosomes also have exciting potential as therapeutics in corneal wound healing and disease; and their presence in tear fluid may serve as potential diagnostic biomarkers for ocular and systemic diseases. CONCLUSION While corneal exosome biology is still in its infancy state, continued progress in this area will improve our understanding of the functional capacity of these small vesicles in the human cornea and may lead to the development of novel regenerative therapies.
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Affiliation(s)
- Belinda J Hernandez
- Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, TX, USA
| | - Danielle M Robertson
- Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, TX, USA
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Elalfy M, Elsawah K, Maqsood S, Jordan N, Hassan M, Zaki A, Gatzioufas Z, Hamada S, Lake D. Allogenic Cultured Limbal Epithelial Transplantation and Cultivated Oral Mucosal Epithelial Transplantation in Limbal Stem Cells Deficiency: A Comparative Study. Ophthalmol Ther 2025; 14:413-432. [PMID: 39755899 PMCID: PMC11754549 DOI: 10.1007/s40123-024-01083-x] [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: 07/31/2024] [Accepted: 12/04/2024] [Indexed: 01/06/2025] Open
Abstract
INTRODUCTION This study compared the clinical outcomes of allogenic cultured limbal epithelial transplantation (ACLET) and cultivated oral mucosal epithelial transplantation (COMET) in the management of limbal stem cell deficiency (LSCD). METHODS Forty-one COMET procedures in 40 eyes and 69 ACLET procedures in 54 eyes were performed in the Corneoplastic Unit of Queen Victoria Hospital, East Grinstead. Data were examined for demographics, indications, ocular surface stability, absence of epithelial defect, ocular surface inflammation, visual outcomes, and intra- and postoperative complications. RESULTS Kaplan-Meier analysis showed that patients in the ACLET group with longer follow-up had a significantly higher graft survival rate (81.7%, n = 56) than the COMET group (60.7%, n = 25) and the difference was statistically significant (p = 0.01). In the COMET group, there was no statistically significant improvement in the visual acuity (VA) while in the ACLET group there was statistically significant improvement in the final VA. Elevated intraocular pressure (IOP) developed in 9 eyes (22.0%) in the COMET group and in 18 eyes (26.1%) in the ACLET group; infection developed in 4 eyes (9.8%) in the COMET group and in 10 eyes (14.5%) in the ACLET group; and perforation or melting happened in 4 eyes (9.8%) in the COMET group and in 1 eye (1.4%) in the ACLET group. Postoperative immunosuppression complications were noted in 9 eyes (13.0%) in the ACLET group. No graft rejection was observed in either group. CONCLUSION Both ACLET and COMET are effective therapeutic procedures for managing advanced and bilateral cases of LSCD. Although COMET has lower graft survival rate than ACLET, it does not mandate systemic immunosuppression therapy to protect against potential graft rejection.
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Affiliation(s)
- Mohamed Elalfy
- Corneoplastic Unit and Eye Bank, Queen Victoria Hospital NHS Foundation Trust, East Grinstead, UK.
- Cornea Unit, Research Institute of Ophthalmology, Giza, Egypt.
- Department of Ophthalmology, Maidstone and Tunbridge Wells NHS Trust, Maidstone, UK.
| | - Kareem Elsawah
- Cornea Unit, Research Institute of Ophthalmology, Giza, Egypt
| | - Sundas Maqsood
- Department of Ophthalmology, Maidstone and Tunbridge Wells NHS Trust, Maidstone, UK
| | - Nigel Jordan
- Corneoplastic Unit and Eye Bank, Queen Victoria Hospital NHS Foundation Trust, East Grinstead, UK
| | - Mansour Hassan
- Department of Ophthalmology, Beni Suef University, Beni Suef, Egypt
| | - Ahmed Zaki
- Cornea Unit, Research Institute of Ophthalmology, Giza, Egypt
| | - Zisis Gatzioufas
- Department of Ophthalmology, University of Basel, Basel, Switzerland
| | - Samer Hamada
- Corneoplastic Unit and Eye Bank, Queen Victoria Hospital NHS Foundation Trust, East Grinstead, UK
| | - Damian Lake
- Corneoplastic Unit and Eye Bank, Queen Victoria Hospital NHS Foundation Trust, East Grinstead, UK
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Hermo L, Oliveira R, Dufresne J, Gregory M, Cyr DG. Basal and Immune Cells of the Epididymis: An Electron Microscopy View of Their Association. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2025; 1469:67-87. [PMID: 40301253 DOI: 10.1007/978-3-031-82990-1_4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/01/2025]
Abstract
The epididymis is a highly coiled duct divided into the initial segment, caput, corpus, and cauda regions. It is a pseudostratified epithelium consisting of principal, narrow, apical, basal, and clear cells. Circulating halo cells, identified as nonepithelial cells, monocytes/macrophages (M/M) and T-lymphocytes, in addition to dendritic cells and a resident population of M/M cells, also inhabit the epididymal epithelium. Using electron microscopy (EM), we characterized the ultrastructural features of each of these different cell types. Basal cells with stem cell characteristics suggest a role in sustaining the epithelium following injury and inflammation, as well as maintaining the steady state of the epithelium. Interestingly, a close morphological affiliation was noted between circulating M/M cells with basal cells and an intraepithelial resident M/M population of cells, as well as between T-lymphocytes and dendritic cells. The association of all these cell types with one another suggests complex interactions enabling the coordination of their functions related to maturation, protection, survival of sperm, and renewal of the epithelium.
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Affiliation(s)
- Louis Hermo
- Department of Anatomy and Cell Biology, McGill University, Montreal, QC, Canada
| | - Regiana Oliveira
- Department of Anatomy and Cell Biology, McGill University, Montreal, QC, Canada
| | - Julie Dufresne
- Laboratory for Reproductive Toxicology, INRS-Centre Armand-Frappier Santé Biotechnologie, Université du Québec, Laval, QC, Canada
| | - Mary Gregory
- Laboratory for Reproductive Toxicology, INRS-Centre Armand-Frappier Santé Biotechnologie, Université du Québec, Laval, QC, Canada
| | - Daniel G Cyr
- Department of Anatomy and Cell Biology, McGill University, Montreal, QC, Canada.
- Laboratory for Reproductive Toxicology, INRS-Centre Armand-Frappier Santé Biotechnologie, Université du Québec, Laval, QC, Canada.
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Peng H, Kaplan N, Liu M, Jiang H, Lavker RM. Keeping an Eye Out for Autophagy in the Cornea: Sample Preparation for Single-Cell RNA-Sequencing. Methods Mol Biol 2025; 2879:113-122. [PMID: 37930627 PMCID: PMC11162605 DOI: 10.1007/7651_2023_502] [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] [Indexed: 11/07/2023]
Abstract
Single-cell RNA-sequencing (scRNA-seq) is a powerful technique that can barcode individual cells and thus used to obtain a gene expression profile for every individual cell within a tissue. This makes scRNA-seq an excellent method for characterizing rare cell populations such as stem cells. We describe how scRNA-seq can be utilized to examine limbal epithelial stem cell population as well as investigate the contribution of autophagy to the function of limbal epithelial stem cells. To accomplish this, we used the Beclin1 heterozygous (Beclin1 het) mouse, a well-established model of autophagy deficiency. We provide a protocol that we developed for the isolation of viable, single-cell suspensions of limbal/corneal tissues, as well as the analysis of scRNA-seq data.
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Affiliation(s)
- Han Peng
- Department of Dermatology, Northwestern University, Chicago, IL, USA
| | - Nihal Kaplan
- Department of Dermatology, Northwestern University, Chicago, IL, USA
| | - Min Liu
- Department of Dermatology, Northwestern University, Chicago, IL, USA
| | - Huimin Jiang
- Department of Dermatology, Northwestern University, Chicago, IL, USA
- Department of Ophthalmology, The Second Hospital of Anhui Medical University, Hefei, China
| | - Robert M Lavker
- Department of Dermatology, Northwestern University, Chicago, IL, USA.
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Li Z, Böhringer D, Stachon T, Nastaranpour M, Fries FN, Seitz B, Ulrich M, Munteanu C, Langenbucher A, Szentmáry N. Culturing Limbal Epithelial Cells of Long-term Stored Corneal Donors (Organ Culture) In Vitro - A Stepwise Linear Regression Algorithm. Klin Monbl Augenheilkd 2024; 241:964-971. [PMID: 37130569 DOI: 10.1055/a-2084-7168] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/04/2023]
Abstract
PURPOSE To assess various potential factors on human limbal epithelial cell (LEC) outgrowth in vitro using corneal donor tissue following long-term storage (organ culture) and a stepwise linear regression algorithm. METHODS Of 215 donors, 304 corneoscleral rings were used for our experiments. For digestion of the limbal tissue and isolation of the limbal epithelial cells, the tissue pieces were incubated with 4.0 mg/mL collagenase A at 37 °C with 95% relative humidity and a 5% CO2 atmosphere overnight. Thereafter, limbal epithelial cells were separated from limbal keratocytes using a 20-µm CellTricks filter. The separated human LECs were cultured in keratinocyte serum-free medium medium, 1% penicillin/streptomycin (P/S), 0.02% epidermal growth factor (EGF), and 0.3% bovine pituitary extract (BPE). The potential effect of donor age (covariate), postmortem time (covariate), medium time (covariate), size of the used corneoscleral ring (360°, 270°180°, 120°, 90°, less than 90°) (covariate), endothelial cell density (ECD) (covariate), gender (factor), number of culture medium changes during organ culture (factor), and origin of the donor (donating institution and storing institution, factor) on the limbal epithelial cell outgrowth was analyzed with a stepwise linear regression algorithm. RESULTS The rate of successful human LEC outgrowth was 37.5%. From the stepwise linear regression algorithm, we found out that the relevant influencing parameters on the LEC growth were intercept (p < 0.001), donor age (p = 0.002), number of culture medium changes during organ culture (p < 0.001), total medium time (p = 0.181), and size of the used corneoscleral ring (p = 0.007), as well as medium time × size of the corneoscleral ring (p = 0.007). CONCLUSIONS The success of LEC outgrowth increases with lower donor age, lower number of organ culture medium changes during storage, shorter medium time in organ culture, and smaller corneoscleral ring size. Our stepwise linear regression algorithm may help us in optimizing LEC cultures in vitro.
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Affiliation(s)
- Zhen Li
- Dr. Rolf M. Schwiete Center for Limbal Stem Cell and Congenital Aniridia Research, Saarland University, Homburg, Germany
| | - Daniel Böhringer
- Department of Ophthalmology, University of Freiburg, Freiburg im Breisgau, Germany
| | - Tanja Stachon
- Dr. Rolf M. Schwiete Center for Limbal Stem Cell and Congenital Aniridia Research, Saarland University, Homburg, Germany
| | - Mahsa Nastaranpour
- Dr. Rolf M. Schwiete Center for Limbal Stem Cell and Congenital Aniridia Research, Saarland University, Homburg, Germany
| | - Fabian Norbert Fries
- Dr. Rolf M. Schwiete Center for Limbal Stem Cell and Congenital Aniridia Research, Saarland University, Homburg, Germany
- Department of Ophthalmology, Saarland University, Homburg, Germany
| | - Berthold Seitz
- Department of Ophthalmology, Saarland University, Homburg, Germany
| | - Myriam Ulrich
- Dr. Rolf M. Schwiete Center for Limbal Stem Cell and Congenital Aniridia Research, Saarland University, Homburg, Germany
| | | | | | - Nóra Szentmáry
- Dr. Rolf M. Schwiete Center for Limbal Stem Cell and Congenital Aniridia Research, Saarland University, Homburg, Germany
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Sahay P, Behbehani M, Filippini P, Bruti G, Townsend M, McKean R, Dua HS. A Biosynthetic Alternative to Human Amniotic Membrane for Use in Ocular Surface Surgery. Transl Vis Sci Technol 2024; 13:3. [PMID: 38696180 PMCID: PMC11077906 DOI: 10.1167/tvst.13.5.3] [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: 12/18/2023] [Accepted: 03/06/2024] [Indexed: 05/12/2024] Open
Abstract
Purpose The biosynthetic Symatix membrane (SM) was developed to replace fresh human amniotic membrane (hAM) in ocular surgical applications. The purpose of this study was to test the biocompatibility of the SM with human limbus-derived epithelial cells with regard to their physical and biological properties. Methods Different physical properties of SM were tested ex vivo by simulation on human corneas. In vitro, primary limbal epithelial cells from limbal explants were used to test biological properties such as cell migration, proliferation, metabolic activity, and limbal epithelial cell markers on the SM, hAM, and freeze-dried amniotic membrane (FDAM). Results The surgical handleability of the SM was equivalent to that of the hAM. Ultrastructural and histological studies demonstrated that epithelial cells on the SM had the typical tightly apposed, polygonal, corneal epithelial cell morphology. The epithelial cells were well stratified on the SM, unlike on the hAM and FDAM. Rapid wound healing occurred on the SM within 3 days. Immunofluorescence studies showed positive expression of CK-19, Col-1, laminin, ZO-1, FN, and p-63 on the SM, plastic, and FDAM compared to positive expression of ZO-1, Col-1, laminin, FN, and p63 and negative expression of CK-19 in the hAM. Conclusions These results indicate that the SM is a better substrate for limbal epithelial cell migration, proliferation, and tight junction formation. Altogether, the SM can provide a suitable alternative to the hAM for surgical application in sight-restoring operations. Translational Relevance The hAM, currently widely used in ocular surface surgery, has numerous variations and limitations. The biocompatibility of corneal epithelial cells with the SM demonstrated in this study suggests that it can be a viable substitute for the hAM.
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Affiliation(s)
- Prity Sahay
- Academic Ophthalmology, Division of Clinical Neuroscience, School of Medicine, University of Nottingham, UK
| | | | - Perla Filippini
- Academic Ophthalmology, Division of Clinical Neuroscience, School of Medicine, University of Nottingham, UK
| | | | | | - Rob McKean
- The Electrospinning Company, Oxfordshire, UK
| | - Harminder S. Dua
- Academic Ophthalmology, Division of Clinical Neuroscience, School of Medicine, University of Nottingham, UK
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Abstract
Regenerative medicine is a highly anticipated field with hopes to provide cures for previously uncurable diseases such as spinal cord injuries and retinal blindness. Most regenerative medical products use either autologous or allogeneic stem cells, which may or may not be genetically modified. The introduction of induced-pluripotent stem cells (iPSCs) has fueled research in the field, and several iPSC-derived cells/tissues are currently undergoing clinical trials. The cornea is one of the pioneering areas of regenerative medicine, and already four cell therapy products are approved for clinical use in Japan. There is one other government-approved cell therapy product approved in Europe, but none are approved in the USA at present. The cornea is transparent and avascular, making it unique as a target for stem cell therapy. This review discusses the unique properties of the cornea and ongoing research in the field.
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Affiliation(s)
- Shigeto Shimmura
- Department of Clinical Regenerative Medicine, Fujita Medical Innovation Center, Tokyo, Japan
- Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan
| | - Emi Inagaki
- Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan
| | - Masatoshi Hirayama
- Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan
| | - Shin Hatou
- Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan
- Cellusion Inc., Tokyo, Japan
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Rowe-Rendleman C, Eveleth D, Goldberg JL, Jurkunas U, Okumura N, Dawson D, Sawant OB. Development of Anterior Segment Focused Biologic Therapies to Regenerate Corneal Tissue for the Treatment of Disease: Drug Development Experience. J Ocul Pharmacol Ther 2023; 39:551-562. [PMID: 37733302 DOI: 10.1089/jop.2023.0044] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/22/2023] Open
Abstract
On February 24-27, 2021, the Association for Ocular Pharmacology and Therapeutics (AOPT) held its 15th biennial scientific meeting online. The meeting was organized by Dr. Sanjoy Bhattacharya of the University of Miami in conjunction with the board of trustees of the AOPT. The 3-day conference was attended by academic scientists, clinicians, and industry and regulatory professionals. The theme of the meeting was Restoring Vision through Regeneration and it was sponsored, in part, by the National Institutes of Health, Bright Focus, Regeneron, and Santen (USA). During the 3 days of the meeting, presentations from several sessions explored different aspects of regenerative medicine in ophthalmology, including optic nerve regeneration, drugs and devices in glaucoma, retinal neuroprotection and plasticity, visual perception, and degeneration of trabecular meshwork. This article summarizes the proceedings of the session on corneal regenerative medicine research and discusses emerging concepts in drug development for corneal epithelial and endothelial regeneration. Since the meeting in 2021, several of these concepts have advanced to clinical-stage therapies, but so far as of 2023, none has been approved by regional regulatory authorities in the United States. One form of corneal endothelial cell therapy has been approved in Japan and only for bullous keratopathy. Ongoing work is proceeding in the United States and other countries. Clinical Registration No: National Clinical Trials 04894110, 04812667; Japan Registry for Clinical Trials a031210199.
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Affiliation(s)
| | | | | | - Ula Jurkunas
- Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts, USA
| | | | | | - Onkar B Sawant
- Center for Vision and Eye Banking Research, Eversight, Cleveland, Ohio, USA
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Swamynathan SK, Swamynathan S. Corneal epithelial development and homeostasis. Differentiation 2023; 132:4-14. [PMID: 36870804 PMCID: PMC10363238 DOI: 10.1016/j.diff.2023.02.002] [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: 10/26/2022] [Revised: 01/27/2023] [Accepted: 02/20/2023] [Indexed: 03/06/2023]
Abstract
The corneal epithelium (CE), the most anterior cellular structure of the eye, is a self-renewing stratified squamous tissue that protects the rest of the eye from external elements. Each cell in this exquisite three-dimensional structure needs to have proper polarity and positional awareness for the CE to serve as a transparent, refractive, and protective tissue. Recent studies have begun to elucidate the molecular and cellular events involved in the embryonic development, post-natal maturation, and homeostasis of the CE, and how they are regulated by a well-coordinated network of transcription factors. This review summarizes the status of related knowledge and aims to provide insight into the pathophysiology of disorders caused by disruption of CE development, and/or homeostasis.
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Affiliation(s)
| | - Sudha Swamynathan
- Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15213, USA
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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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Comparative Histology of the Cornea and Palisades of Vogt in the Different Wild Ruminants (Bovidae, Camelidae, Cervidae, Giraffidae, Tragulidae). Animals (Basel) 2022; 12:ani12223188. [PMID: 36428415 PMCID: PMC9687073 DOI: 10.3390/ani12223188] [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: 10/14/2022] [Revised: 11/14/2022] [Accepted: 11/15/2022] [Indexed: 11/19/2022] Open
Abstract
In the study, we data concerning the histological and morphometrical examination of the cornea and palisades of Vogt in the different species of ruminants from the families Bovidae, Camelidae, Cervidae, Giraffidae and Tragulidae, coming from the Warsaw Zoological Garden, the Wroclaw Zoological Garden and the Division of Animal Anatomy. The following ruminant species were investigated: common wildebeest, Kirk's dik-dik, Natal red duiker, scimitar oryx, sitatunga, Philippine spotted deer, Père David's deer, moose, reindeer, reticulated giraffe, okapi, Balabac mouse-deer and alpaca. The cornea of ruminant species such as the common wildebeest, Kirk's dik-dik, Natal red duiker, scimitar oryx, reindeer and Balabac mouse-deer consisted of four layers (not found in the Bowman's layer): the anterior corneal epithelium, the proper substance of the cornea, the posterior limiting membrane (Descemet's membrane) and the posterior corneal epithelium (endothelium). The anterior corneal epithelium was composed of a multilayer keratinizing squamous epithelium, which was characterized in the studied ruminants with a variable number of cell layers but also with a different thickness both in the central epithelium part and in the peripheral part. Moreover, the proper substance of cornea was thinnest in Balabac mouse-deer, Kirk's dik-dik, Natal red duiker, scimitar oryx, Philippine spotted deer, alpaca, reindeer and sitatunga and was thickest in the reticulated giraffe. The thickest Descemet's membrane was observed in the Père David's deer. The corneal limbus is characterized by a large number of pigment cell clusters in Kirk's dik-dik, scimitar oryx, moose, Balabac mouse-deer and alpaca. In the common wildebeest, Père David's deer, moose, reticulated giraffe, okapi and alpaca, the palisades of Vogt were marked in the form of a crypt-like structure. The corneal limbus epithelium in the examined ruminants was characterized by a variable number of cell layers but also a variable number of melanocytes located in different layers of this epithelium. The detailed knowledge of the corneal structure of domestic and wild animals can contribute to the even better development of methods for treating eye diseases in veterinary medicine.
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14
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Sonam S, Bangru S, Perry KJ, Chembazhi UV, Kalsotra A, Henry JJ. Cellular and molecular profiles of larval and adult Xenopus corneal epithelia resolved at the single-cell level. Dev Biol 2022; 491:13-30. [PMID: 36049533 PMCID: PMC10241109 DOI: 10.1016/j.ydbio.2022.08.007] [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: 01/20/2022] [Revised: 08/22/2022] [Accepted: 08/23/2022] [Indexed: 11/24/2022]
Abstract
Corneal Epithelial Stem Cells (CESCs) and their proliferative progeny, the Transit Amplifying Cells (TACs), are responsible for homeostasis and maintaining corneal transparency. Owing to our limited knowledge of cell fates and gene activity within the cornea, the search for unique markers to identify and isolate these cells remains crucial for ocular surface reconstruction. We performed single-cell RNA sequencing of corneal cells from larval and adult stages of Xenopus. Our results indicate that as the cornea develops and matures, there is an increase in cellular diversity, which is accompanied by a substantial shift in transcriptional profile, gene regulatory network and cell-cell communication dynamics. Our data also reveals several novel genes expressed in corneal cells and changes in gene expression during corneal differentiation at both developmental time-points. Importantly, we identify specific basal cell clusters in both the larval and adult cornea that comprise a relatively undifferentiated cell type and express distinct stem cell markers, which we propose are the putative larval and adult CESCs, respectively. This study offers a detailed atlas of single-cell transcriptomes in the frog cornea. In the future, this work will be useful to elucidate the function of novel genes in corneal epithelial homeostasis, wound healing and regeneration.
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Affiliation(s)
- Surabhi Sonam
- Department of Cell and Developmental Biology, University of Illinois, Urbana-Champaign, IL, USA
| | - Sushant Bangru
- Department of Biochemistry, University of Illinois, Urbana-Champaign, IL, USA; Cancer Center@Illinois, University of Illinois, Urbana-Champaign, IL, USA
| | - Kimberly J Perry
- Department of Cell and Developmental Biology, University of Illinois, Urbana-Champaign, IL, USA
| | - Ullas V Chembazhi
- Department of Biochemistry, University of Illinois, Urbana-Champaign, IL, USA
| | - Auinash Kalsotra
- Department of Biochemistry, University of Illinois, Urbana-Champaign, IL, USA; Carl R. Woese Institute for Genomic Biology, University of Illinois, Urbana-Champaign, IL, USA; Cancer Center@Illinois, University of Illinois, Urbana-Champaign, IL, USA.
| | - Jonathan J Henry
- Department of Cell and Developmental Biology, University of Illinois, Urbana-Champaign, IL, USA.
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15
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Sugihara K, Kaidzu S, Sasaki M, Ichioka S, Takayanagi Y, Shimizu H, Sano I, Hara K, Tanito M. One-Year Ocular Safety Observation of Workers and Estimations of Microorganism Inactivation Efficacy in the Room Irradiated with 222-Nm far Ultraviolet-C Lamps. Photochem Photobiol 2022; 99:967-974. [PMID: 36081379 DOI: 10.1111/php.13710] [Citation(s) in RCA: 20] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2022] [Accepted: 09/05/2022] [Indexed: 11/30/2022]
Abstract
Two krypton-chloride germicidal excimer lamp units (Care222 TRT-104C11-UI-U3, USHIO Inc.) were installed in the examination room of an ophthalmology department. The irradiation dose was set not to exceed the former (i.e., before 2022) threshold limit value (TLV) (22 mJ/cm2 /8 hours) recommended by the ACGIH. Section 1: The eyes and lids of the 6 ophthalmologists (5 wore glasses for myopic correction) who worked in the room for a mean stay of 6.7 hours/week were prospectively observed for 12 months. Slitlamp examinations revealed neither acute adverse events such as corneal erosion, conjunctival hyperemia, lid skin erythema nor chronic adverse events such as pterygium, cataract, or lid tumor. The visual acuity, refractive error, corneal endothelial cell density remained unchanged during the study. Section 2: The irradiation of samples placed on the table or floor using the same fixtures in the room (5-7.5 mJ/cm2 ) was associated with >99% inhibition of φX174 phage and >90% inhibition of S. aureus. In conclusion, no acute or chronic health effects in human participants was observed in a clinical setting of full-room ultraviolet germicidal irradiation by 222-nm lamp units and high efficacy in deactivation of microorganisms was determined in the same setting.
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Affiliation(s)
- Kazunobu Sugihara
- Department of Ophthalmology, Shimane University Faculty of Medicine, Izumo, Japan
| | - Sachiko Kaidzu
- Department of Ophthalmology, Shimane University Faculty of Medicine, Izumo, Japan
| | | | - Sho Ichioka
- Department of Ophthalmology, Shimane University Faculty of Medicine, Izumo, Japan
| | - Yuji Takayanagi
- Department of Ophthalmology, Shimane University Faculty of Medicine, Izumo, Japan
| | - Hiroshi Shimizu
- Department of Ophthalmology, Shimane University Faculty of Medicine, Izumo, Japan
| | - Ichiya Sano
- Department of Ophthalmology, Shimane University Faculty of Medicine, Izumo, Japan
| | - Katsunori Hara
- Department of Ophthalmology, Shimane University Faculty of Medicine, Izumo, Japan
| | - Masaki Tanito
- Department of Ophthalmology, Shimane University Faculty of Medicine, Izumo, Japan
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16
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Ghavami Shahri SH, Balali-Mood M, Heidarzadeh HR, Abrishami M. Ophthalmic Complications and Managements of Sulfur Mustard Exposure: A Narrative Review. ARCHIVES OF IRANIAN MEDICINE 2022; 25:647-657. [PMID: 37543890 PMCID: PMC10685765 DOI: 10.34172/aim.2022.100] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/30/2022] [Accepted: 05/23/2022] [Indexed: 08/07/2023]
Abstract
Sulfur mustard (SM) is a lethal chemical agent that affects many organs, particularly the eyes, respiratory system and skin. Even asymptomatic patients with documented SM vapor exposure may develop organ disorder many years later. Patients with even minor signs in the acute stage may experience late complications that necessitate surgery. Early decontamination and conservative measures could help the patients and decrease the complications. Despite decades of research, there is still no effective treatment for either acute or long-term SM-induced ocular complications. Even after multiple medications and surgical procedures, the majority of patients continue to have symptoms. For dry eye, punctual occlusion, autologous eye drops, and aggressive lubrication are used; for persistent epithelial defects (PED), tarsorrhaphy, amniotic membrane transplant, and stem cell transplantation are used; for total limbal stem cell deficiency (LSCD), living-related conjunctivolimbal allograft and keratolimbal allograft are used; for corneal vascularization, steroids, non-steroidal anti-inflammatory drugs, and anti-vascular endothelial growth factor prescribed; and for corneal opacities, corneal transplantation is done. Platelet rich plasma and topical drops containing stem cell transplantation for LSCD, photodynamic therapy paired with subconjunctival or topical anti-vascular endothelial growth factors for corneal vascularization, topical curcumin and topical ciclosporin-A for dry eye, and orbital fat-derived stem cells for PED are all alternative treatments that can be suggested. Despite the experimental and clinical research on the complications of SM exposure over the past decades, there is still no effective treatment for eye complications. However, supportive medical and surgical management has been applied with relatively good outcome.
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Affiliation(s)
| | - Mahdi Balali-Mood
- Medical Toxicology and Drug Abuse Research Center, Birjand University of Medical Sciences, Birjand, Iran
| | | | - Mojtaba Abrishami
- Eye Research Center, Mashhad University of Medical Sciences, Birjand, Iran
- Ocular Oncology Service, Department of Ophthalmology and Visual Sciences, University of Toronto, Toronto, Canada
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17
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Abdellah N, Desoky SMME. Novel detection of stem cell niche within the stroma of limbus in the rabbit during postnatal development. Sci Rep 2022; 12:13711. [PMID: 35962026 PMCID: PMC9374725 DOI: 10.1038/s41598-022-18090-2] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2022] [Accepted: 08/04/2022] [Indexed: 11/16/2022] Open
Abstract
Identifying and locating stem cell populations in the limbus may lead to developing a cell-based strategy for treating the corneal injury. Therefore, this study was the first to design a follow-up on the microscopical and histomorphometric changes in the rabbit limbus and to localize and demonstrate the limbal stem cell niche during postnatal development. The paraffin sections from the eyes of different postnatal-developmental stages were stained and examined using light microscopy. Furthermore, sections were immunohistologically stained for the epithelial stem cell differentiation marker, cytokeratin-14. Moreover, semithin and ultrathin sections were applied for ultrastructural demonstration of the stem cell niche. This study revealed that the number and thickness of limbal epithelial layers increased with age, whereas the thickness of limbal stroma decreased. Additionally, the immunohistochemical data showed that ck14 staining intensity increased in the limbal region where limbal stem cells reside. The semithin and ultrastructure investigation revealed stem cell clusters within the limbus's underlying stroma close to the blood and nerve supply and surrounded by telocytes. Conclusively, isolated clusters of limbal epithelial stem cells combined with blood vessels, nerve fibers, and telocytes propose a harmonious microenvironment of a stem cell niche.
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Affiliation(s)
- Nada Abdellah
- Department of Histology, Faculty of Veterinary Medicine, Sohag University, Sohag, 82524, Egypt.
| | - Sara M M El- Desoky
- Department of Anatomy and Embryology, Faculty of Veterinary Medicine, Assiut University, Asyut, 71526, Egypt
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18
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Guo L, Wang Z, Li J, Li J, Cui L, Dong J, Meng X, Qian C, Wang H. Immortalization effect of SV40T lentiviral vectors on canine corneal epithelial cells. BMC Vet Res 2022; 18:181. [PMID: 35578336 PMCID: PMC9109393 DOI: 10.1186/s12917-022-03288-3] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2021] [Accepted: 05/10/2022] [Indexed: 11/10/2022] Open
Abstract
Background Primary canine corneal epithelial cells (CCECs) easily become senescent, and cell proliferation is limited. Therefore, sampling for experimentation requires a large number of animals, which is problematic in terms of animal welfare and fails to maintain the stability of the cells for in vitro analyses. Results In this study, CCECs were separated and purified by trypsin and dispase II enzymatic analysis. Next, the cells were immortalized by transfection with a lentiviral vector expressing Simian vacuolating virus 40 large T (SV40T). The immortalized canine corneal epithelial cell line (CCEC-SV40T) was established by serial passages and monoclonal selection. The biological characteristics of CCEC-SV40T cells were evaluated based on the cell proliferation rate, cell cycle pattern, serum dependence, karyotype, and cytokeratin 12 immunofluorescence detection. In addition, we infected CCEC-SV40T cells with Staphylococcus pseudintermedius (S. pseudintermedius) and detected the inflammatory response of the cells. After the CCEC-SV40T cells were passaged continuously for 40 generations, the cells grew in a cobblestone pattern, which was similar to CCECs. The SV40T gene and cytokeratin 12 can be detected in each generation. CCEC-SV40T cells were observed to have a stronger proliferation capacity than CCECs. CCEC-SV40T cells maintained the same diploid karyotype and serum-dependent ability as CCECs. After CCEC-SV40T cells were infected with S. pseudintermedius, the mRNA expression levels of NLRP3, Caspase-1 and proinflammatory cytokines, including IL-1β, IL-6, IL-8 and TNF-α, were upregulated, and the protein levels of MyD88, NLRP3 and the phosphorylation of Iκbα and p65 were upregulated. Conclusions In conclusion, the CCEC-SV40T line was successfully established and can be used for in vitro studies, such as research on corneal diseases or drug screening. Supplementary Information The online version contains supplementary material available at 10.1186/s12917-022-03288-3.
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Affiliation(s)
- Long Guo
- College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, Jiangsu, China.,Joint International Research Laboratory of Agriculture and Agri-Product Safety of the Ministry of Education, Yangzhou, 225009, Jiangsu, China
| | - Zhihao Wang
- College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, Jiangsu, China.,Joint International Research Laboratory of Agriculture and Agri-Product Safety of the Ministry of Education, Yangzhou, 225009, Jiangsu, China
| | - Jun Li
- College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, Jiangsu, China.,Joint International Research Laboratory of Agriculture and Agri-Product Safety of the Ministry of Education, Yangzhou, 225009, Jiangsu, China
| | - Jianji Li
- College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, Jiangsu, China.,Joint International Research Laboratory of Agriculture and Agri-Product Safety of the Ministry of Education, Yangzhou, 225009, Jiangsu, China
| | - Luying Cui
- College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, Jiangsu, China.,Joint International Research Laboratory of Agriculture and Agri-Product Safety of the Ministry of Education, Yangzhou, 225009, Jiangsu, China
| | - Junsheng Dong
- College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, Jiangsu, China.,Joint International Research Laboratory of Agriculture and Agri-Product Safety of the Ministry of Education, Yangzhou, 225009, Jiangsu, China
| | - Xia Meng
- College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, Jiangsu, China.,Joint International Research Laboratory of Agriculture and Agri-Product Safety of the Ministry of Education, Yangzhou, 225009, Jiangsu, China
| | - Chen Qian
- College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, Jiangsu, China.,Joint International Research Laboratory of Agriculture and Agri-Product Safety of the Ministry of Education, Yangzhou, 225009, Jiangsu, China
| | - Heng Wang
- College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, Jiangsu, China. .,Joint International Research Laboratory of Agriculture and Agri-Product Safety of the Ministry of Education, Yangzhou, 225009, Jiangsu, China.
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19
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Kaplan N, Liu M, Wang J, Yang W, Fiolek E, Peng H, Lavker RM. Eph signaling is regulated by miRNA-210: Implications for corneal epithelial repair. FASEB J 2022; 36:e22076. [PMID: 34856019 PMCID: PMC8647904 DOI: 10.1096/fj.202101423r] [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: 09/13/2021] [Revised: 10/25/2021] [Accepted: 11/17/2021] [Indexed: 01/03/2023]
Abstract
A distinct boundary exists between the progenitor cells in the basal limbal epithelium and the more differentiated corneal epithelial basal cells. We have shown that reciprocal expression patterns of EphA2 and Ephrin-A1 are likely to contribute to normal limbal-corneal epithelial compartmentalization as well as play a role in response to injury. How this signaling axis is regulated remains unclear. We have demonstrated that microRNAs (miRNAs) play critical roles in corneal epithelial wound healing and several miRNAs (e.g. miR-210) have been predicted to target ephrins. Previous expression profiling experiments demonstrated that miR-210 is prominently expressed in corneal epithelial cells. RNA-seq data acquired from miR-210-depleted HCECs showed up-regulation of genes involved in cellular migration. In addition, miR-210 is decreased after corneal injury while EphA2 is increased. Moreover, antago-210-treated HCECs markedly enhanced wound closure in a scratch wound assay. Antago-210 treatment resulted in increased EphA2 protein levels as well as pS897-EphA2, the pro-migratory form of EphA2. As expected, Ephrin-A1 levels were reduced, while levels of a well-known target of miR-210, Ephrin-A3, were increased by antago-210 treatment. The increase in migration with antago-210 could be inhibited by Ephrin-A1 overexpression, Ephrin-A1-Fc treatment or siRNA depletion of EphA2. However, depletion of Ephrin-A3 did not have effects on the antago-210-induced increase in migration. In addition, Ephrin-A1 overexpression and siEphA2 dampened EGFR signaling, which is increased by antago-210. Our data clearly demonstrate a link between miR-210 and EphA2/Ephrin-A1 signaling that regulates, in part, corneal epithelial migration. This interaction might potentially control the limbal-corneal epithelial boundary.
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Affiliation(s)
- Nihal Kaplan
- Department of Dermatology, Northwestern University, Chicago, IL, 60611, USA
| | - Min Liu
- Department of Dermatology, Northwestern University, Chicago, IL, 60611, USA
| | - Junyi Wang
- Department of Dermatology, Northwestern University, Chicago, IL, 60611, USA,Department of Ophthalmology, Ophthalmology and Visual Science Key Lab of PLA, Chinese PLA General Hospital, Beijing, China
| | - Wending Yang
- Department of Dermatology, Northwestern University, Chicago, IL, 60611, USA
| | - Elaina Fiolek
- Department of Dermatology, Northwestern University, Chicago, IL, 60611, USA
| | - Han Peng
- Department of Dermatology, Northwestern University, Chicago, IL, 60611, USA,Correspondence: Robert M. Lavker, Ph.D., Department of Dermatology, Northwestern University, 303 East Chicago Avenue, Ward 9-124, Chicago, IL 60611, USA;
| | - Robert M. Lavker
- Department of Dermatology, Northwestern University, Chicago, IL, 60611, USA,Correspondence: Han Peng, Ph.D., Department of Dermatology, Northwestern University, 303 East Chicago Avenue, Ward 9-120, Chicago, IL 60611, USA;
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20
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Song Z, Tsai CH, Mei H. Comparison of different methods to isolate mouse limbal epithelial cells. Exp Eye Res 2021; 212:108767. [PMID: 34534542 DOI: 10.1016/j.exer.2021.108767] [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: 03/29/2021] [Revised: 08/30/2021] [Accepted: 09/12/2021] [Indexed: 01/18/2023]
Abstract
Limbal stem cells (LSCs) are the stem cell reservoir for corneal epithelium. The protocol to isolate LSCs from human cornea has been examined and optimized. However, the isolation protocol has not been optimized for mouse cornea, which is crucial for the downstream cell analysis. Here we compared four different isolation methods evolved from the previous reports to obtain mouse limbal epithelial cells which are heterogeneous and contain LSCs in a single-cell suspension: (1) the dissected limbal rim was cut into pieces and digested by 10-cycle incubation in trypsin; (2) after the removal of corneal epithelium by a rotating bur, the remaining eyeball was incubated in dispase at 4 °C for overnight to obtain limbal epithelial sheet, followed by trypsin digestion into a single-cell suspension; (3) same as method 2 except that the incubation was in dispase at 37 °C for 2h and an additional collagenase incubation at 37 °C for 20 min; (4) same as method 3 except that the corneal epithelium was punctured by a 1.5 mm trephine instead of being removed by a rotating bur. Method 1 showed the lowest cell yield, the lowest percentage of single cells, and the lowest number of limbal epithelial stem/progenitor cells in the harvested cells among the four methods, thus not a recommended protocol. Method 2, 3, and 4 isolated a comparable number of K14+ and p63α-bright stem/progenitor cells per eye. The remaining eye globe after cell collection in the three methods showed a complete removal of limbal epithelium albeit different extent of corneal and limbal stromal digestion. Among the three methods, method 2 showed a higher cell viability than method 4; method 3 yielded the lowest cell number; method 4 led to the highest percentage of single cells in cell suspension. Results suggest that method 2, 3, and 4 are preferred methods to isolate heterogeneous-LSCs from mouse corneas.
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Affiliation(s)
- Zhenwei Song
- Department of Ophthalmology, School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA; School of Medicine, Hunan Normal University, 371 Tongzipo Road, Chang Sha, 410003, China.
| | - Chi-Hao Tsai
- Department of Ophthalmology, School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA.
| | - Hua Mei
- Department of Ophthalmology, School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA; Department of Cell Biology and Physiology, School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA.
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21
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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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22
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Farrelly O, Suzuki-Horiuchi Y, Brewster M, Kuri P, Huang S, Rice G, Bae H, Xu J, Dentchev T, Lee V, Rompolas P. Two-photon live imaging of single corneal stem cells reveals compartmentalized organization of the limbal niche. Cell Stem Cell 2021; 28:1233-1247.e4. [PMID: 33984283 DOI: 10.1016/j.stem.2021.02.022] [Citation(s) in RCA: 34] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2020] [Revised: 11/18/2020] [Accepted: 02/16/2021] [Indexed: 12/11/2022]
Abstract
The functional heterogeneity of resident stem cells that support adult organs is incompletely understood. Here, we directly visualize the corneal limbus in the eyes of live mice and identify discrete stem cell niche compartments. By recording the life cycle of individual stem cells and their progeny, we directly analyze their fates and show that their location within the tissue can predict their differentiation status. Stem cells in the inner limbus undergo mostly symmetric divisions and are required to sustain the population of transient progenitors that support corneal homeostasis. Using in situ photolabeling, we captured their progeny exiting the niche before moving centripetally in unison. The long-implicated slow-cycling stem cells are functionally distinct and display local clonal dynamics during homeostasis but can contribute to corneal regeneration after injury. This study demonstrates how the compartmentalized organization of functionally diverse stem cell populations supports the maintenance and regeneration of an adult organ.
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Affiliation(s)
- Olivia Farrelly
- Department of Dermatology, Department of Cell and Developmental Biology, Institute for Regenerative Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA
| | - Yoko Suzuki-Horiuchi
- Department of Dermatology, Department of Cell and Developmental Biology, Institute for Regenerative Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA
| | - Megan Brewster
- Department of Dermatology, Department of Cell and Developmental Biology, Institute for Regenerative Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA
| | - Paola Kuri
- Department of Dermatology, Department of Cell and Developmental Biology, Institute for Regenerative Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA
| | - Sixia Huang
- Department of Dermatology, Department of Cell and Developmental Biology, Institute for Regenerative Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA
| | - Gabriella Rice
- Department of Dermatology, Department of Cell and Developmental Biology, Institute for Regenerative Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA
| | - Hyunjin Bae
- Department of Dermatology, Department of Cell and Developmental Biology, Institute for Regenerative Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA
| | - Jianming Xu
- Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA
| | - Tzvete Dentchev
- Department of Dermatology, Department of Cell and Developmental Biology, Institute for Regenerative Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA
| | - Vivian Lee
- Department of Ophthalmology, Scheie Eye Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA
| | - Panteleimon Rompolas
- Department of Dermatology, Department of Cell and Developmental Biology, Institute for Regenerative Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA; Department of Ophthalmology, Scheie Eye Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
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Xiao YT, Qu JY, Xie HT, Zhang MC, Zhao XY. A Comparison of Methods for Isolation of Limbal Niche Cells: Maintenance of Limbal Epithelial Stem/Progenitor Cells. Invest Ophthalmol Vis Sci 2021; 61:16. [PMID: 33320169 PMCID: PMC7745628 DOI: 10.1167/iovs.61.14.16] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Purpose Limbal niche cells (LNCs) play a vital role in the maintenance of limbal epithelial stem/progenitor cells (LESCs). Four methods have been reported to isolate and expand LNCs: digestion by collagenase alone (C-LNC), collagenase following dispase removal of the limbal epithelium (DC-LNC), dissection of dispase-isolated limbal epithelial sheets (D-LNC), and explant cultures of limbal stromal tissues (Ex-LNC). This study aimed to isolate LNCs using those four methods and to compare their capacity to maintain LESCs. Methods LNCs were isolated from the rat corneal limbus by the following methods: C-LNC, DC-LNC, D-LNC, and Ex-LNC. Quantitative real-time PCR and immunofluorescence staining were used to analyze the expression of embryonic stem cell (ESC) markers. The ability to maintain LESCs was assessed on the basis of colony-forming capacity and the expression of progenitor, proliferation, and differentiation markers in three-dimensional (3D) Matrigel and Transwell systems. Notch signaling of LESCs supported by different LNCs in Transwell inserts was analyzed by quantitative real-time PCR. Results DC-LNCs exhibited lower expression of CK12 during isolation and expansion. Among P4-expanded LNCs, DC-LNCs expressed significantly higher levels of Sox2, Oct4, Nanog, and N-cadherin than C-LNCs, D-LNCs, and Ex-LNCs. Compared with other LNCs, DC-LNCs were more effective in maintaining LESCs with higher holoclone-forming efficiency, greater expression of ΔNp63α and Ki67, and lower expression of CK12. DC-LNCs were also more capable of downregulating Notch signaling of LESCs. Conclusions DC-LNCs were more effective in expressing ESC markers and maintaining LESCs compared to other LNCs. This study identifies an optimal method for the isolation of LNCs in tissue engineering and ocular surface reconstruction.
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Affiliation(s)
- Yu-Ting Xiao
- Department of Ophthalmology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, People's Republic of China
| | - Jing-Yu Qu
- Department of Ophthalmology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, People's Republic of China
| | - Hua-Tao Xie
- Department of Ophthalmology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, People's Republic of China
| | - Ming-Chang Zhang
- Department of Ophthalmology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, People's Republic of China
| | - Xin-Yue Zhao
- Department of Ophthalmology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, People's Republic of China
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Dou S, Wang Q, Qi X, Zhang B, Jiang H, Chen S, Duan H, Lu Y, Dong J, Cao Y, Xie L, Zhou Q, Shi W. Molecular identity of human limbal heterogeneity involved in corneal homeostasis and privilege. Ocul Surf 2021; 21:206-220. [PMID: 33964410 DOI: 10.1016/j.jtos.2021.04.010] [Citation(s) in RCA: 45] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2021] [Revised: 04/10/2021] [Accepted: 04/24/2021] [Indexed: 12/20/2022]
Abstract
PURPOSE The corneal limbus maintains the homeostasis, immune and angiogenic privilege of cornea. This study aimed to depict the landscape of human limbal tissues by single-cell RNA sequencing (scRNA-seq). METHODS Single cells of human limbus collected from donor corneas were subjected to 10x scRNA-seq, followed by clustering cell types through the t-distributed stochastic neighbor embedding (t-SNE) and unbiased computational informatic analysis. Immunofluorescent staining was performed using human corneas to validate the analysis results. RESULTS 47,627 cells acquired from six human limbal tissues were collected and subjected to scRNA-seq. 14 distinct clusters were identified and 8 cell types were annotated with representative markers. In-depth dissection revealed three limbal epithelial cell subtypes and refined the X-Y-Z hypothesis of corneal epithelial maintenance. We further unveiled two cell states with higher stemness (TP63+ and CCL20+ cells), and two other differentiated cell states (GPHA2+ and KRT6B + cells) in homeostatic limbal stem/progenitor cells (LSPCs) that differ in transcriptional profiles. Cell-cell communication analysis revealed the central role of LSPCs and their bidirectional regulation with various niche cells. Moreover, comparative analysis between limbus and skin deciphered the pivotal contribution of limbal immune cells, vascular and lymphatic endothelial cells to corneal immune and angiogenic privilege. CONCLUSIONS The human limbus atlas provided valuable resources and foundations for understanding corneal biology, disease and potential interventions.
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Affiliation(s)
- Shengqian Dou
- State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Shandong Eye Institute, Shandong First Medical University & Shandong Academy of Medical Sciences, Qingdao, China; Qingdao Eye Hospital of Shandong First Medical University, Qingdao, China
| | - Qun Wang
- State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Shandong Eye Institute, Shandong First Medical University & Shandong Academy of Medical Sciences, Qingdao, China; Qingdao Eye Hospital of Shandong First Medical University, Qingdao, China
| | - Xia Qi
- State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Shandong Eye Institute, Shandong First Medical University & Shandong Academy of Medical Sciences, Qingdao, China; Qingdao Eye Hospital of Shandong First Medical University, Qingdao, China
| | - Bin Zhang
- State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Shandong Eye Institute, Shandong First Medical University & Shandong Academy of Medical Sciences, Qingdao, China; Qingdao Eye Hospital of Shandong First Medical University, Qingdao, China
| | - Hui Jiang
- State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Shandong Eye Institute, Shandong First Medical University & Shandong Academy of Medical Sciences, Qingdao, China; Qingdao Eye Hospital of Shandong First Medical University, Qingdao, China
| | - Shengwen Chen
- State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Shandong Eye Institute, Shandong First Medical University & Shandong Academy of Medical Sciences, Qingdao, China; Qingdao Eye Hospital of Shandong First Medical University, Qingdao, China
| | - Haoyun Duan
- State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Shandong Eye Institute, Shandong First Medical University & Shandong Academy of Medical Sciences, Qingdao, China; Qingdao Eye Hospital of Shandong First Medical University, Qingdao, China
| | - Yao Lu
- OE Biotech Co., Ltd, Shanghai, Shanghai, China
| | | | - Yihai Cao
- Department of Microbiology, Tumor, and Cell Biology, Karolinska Institute, Stockholm, Sweden
| | - Lixin Xie
- State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Shandong Eye Institute, Shandong First Medical University & Shandong Academy of Medical Sciences, Qingdao, China; Qingdao Eye Hospital of Shandong First Medical University, Qingdao, China
| | - Qingjun Zhou
- State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Shandong Eye Institute, Shandong First Medical University & Shandong Academy of Medical Sciences, Qingdao, China; Qingdao Eye Hospital of Shandong First Medical University, Qingdao, China.
| | - Weiyun Shi
- State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Shandong Eye Institute, Shandong First Medical University & Shandong Academy of Medical Sciences, Qingdao, China; Qingdao Eye Hospital of Shandong First Medical University, Qingdao, China; Eye Hospital of Shandong First Medical University, Jinan, China.
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Kaczmarek R, Zimmer K, Gajdzis P, Gajdzis M. The Role of Eph Receptors and Ephrins in Corneal Physiology and Diseases. Int J Mol Sci 2021; 22:ijms22094567. [PMID: 33925443 PMCID: PMC8123804 DOI: 10.3390/ijms22094567] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2021] [Revised: 04/21/2021] [Accepted: 04/26/2021] [Indexed: 11/29/2022] Open
Abstract
The cornea, while appearing to be simple tissue, is actually an extremely complex structure. In order for it to retain its biomechanical and optical properties, perfect organization of its cells is essential. Proper regeneration is especially important after injuries and in the course of various diseases. Eph receptors and ephrin are mainly responsible for the proper organization of tissues as well as cell migration and communication. In this review, we present the current state of knowledge on the role of Eph and ephrins in corneal physiology and diseases, in particular, we focused on the functions of the epithelium and endothelium. Since the role of Eph and ephrins in the angiogenesis process has been well established, we also analyzed their influence on conditions with corneal neovascularization.
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Affiliation(s)
- Radoslaw Kaczmarek
- Department of Ophthalmology, Wroclaw Medical University, 50-556 Wroclaw, Poland; (R.K.); (K.Z.)
| | - Katarzyna Zimmer
- Department of Ophthalmology, Wroclaw Medical University, 50-556 Wroclaw, Poland; (R.K.); (K.Z.)
| | - Pawel Gajdzis
- Department of Pathomorphology and Oncological Cytology, Wroclaw Medical University, 50-556 Wroclaw, Poland;
| | - Malgorzata Gajdzis
- Department of Ophthalmology, Wroclaw Medical University, 50-556 Wroclaw, Poland; (R.K.); (K.Z.)
- Correspondence: ; Tel.: +48-71-736-43-00
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26
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Sliney DH, Stuck BE. A Need to Revise Human Exposure Limits for Ultraviolet UV-C Radiation †. Photochem Photobiol 2021; 97:485-492. [PMID: 33590879 PMCID: PMC8252557 DOI: 10.1111/php.13402] [Citation(s) in RCA: 27] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2020] [Revised: 01/28/2021] [Accepted: 02/09/2021] [Indexed: 01/03/2023]
Abstract
The COVID‐19 pandemic has greatly heightened interest in ultraviolet germicidal irradiation (UVGI) as an important intervention strategy to disinfect air in medical treatment facilities and public indoor spaces. However, a major drawback of UVGI is the challenge posed by assuring safe installation of potentially hazardous short‐wavelength (UV‐C) ultraviolet lamps. Questions have arisen regarding what appear to be unusually conservative exposure limit values in the UV‐C spectral band between 180 and 280 nm. We review the bases for the current limits and proposes some adjustments that would provide separate limits for the eye and the skin at wavelengths less than 300 nm and to increase both skin and eye limits in the UV‐C below 250 nm.
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Affiliation(s)
- David H Sliney
- Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD, USA
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27
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Liu XN, Mi SL, Chen Y, Wang Y. Corneal stromal mesenchymal stem cells: reconstructing a bioactive cornea and repairing the corneal limbus and stromal microenvironment. Int J Ophthalmol 2021; 14:448-455. [PMID: 33747824 DOI: 10.18240/ijo.2021.03.19] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2020] [Accepted: 11/17/2020] [Indexed: 02/07/2023] Open
Abstract
Corneal stroma-derived mesenchymal stem cells (CS-MSCs) are mainly distributed in the anterior part of the corneal stroma near the corneal limbal stem cells (LSCs). CS-MSCs are stem cells with self-renewal and multidirectional differentiation potential. A large amount of data confirmed that CS-MSCs can be induced to differentiate into functional keratocytes in vitro, which is the motive force for maintaining corneal transparency and producing a normal corneal stroma. CS-MSCs are also an important component of the limbal microenvironment. Furthermore, they are of great significance in the reconstruction of ocular surface tissue and tissue engineering for active biocornea construction. In this paper, the localization and biological characteristics of CS-MSCs, the use of CS-MSCs to reconstruct a tissue-engineered active biocornea, and the repair of the limbal and matrix microenvironment by CS-MSCs are reviewed, and their application prospects are discussed.
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Affiliation(s)
- Xian-Ning Liu
- Department of Ophthalmology, First Hospital of Xi'an; Shaanxi Institute of Ophthalmology, Shaanxi Provincial Key Lab of Ophthalmology, Clinical Research Center for Ophthalmology Diseases of Shaanxi Province, the First Affiliated Hospital of Northwest University, Xi'an 710002, Shaanxi Province, China
| | - Sheng-Li Mi
- Open FIESTA Center, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, Guangdong Province, China.,Biomanufacturing Engineering Laboratory, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, Guangdong Province, China
| | - Yun Chen
- Open FIESTA Center, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, Guangdong Province, China
| | - Yao Wang
- Department of Ophthalmology, First Hospital of Xi'an; Shaanxi Institute of Ophthalmology, Shaanxi Provincial Key Lab of Ophthalmology, Clinical Research Center for Ophthalmology Diseases of Shaanxi Province, the First Affiliated Hospital of Northwest University, Xi'an 710002, Shaanxi Province, China
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28
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Sharma A, Riaz KM, Gill MS, Patnaik A, Ulahannan SV, Wang JS, Gombos DS, Ang Q, Cicic D, Bergonio GR, Zhang C, Wirostko BM. Reversible HER2 antibody-drug conjugate-induced ocular toxicity. Can J Ophthalmol 2021; 57:118-126. [PMID: 33727105 DOI: 10.1016/j.jcjo.2021.02.028] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2020] [Revised: 01/23/2021] [Accepted: 02/16/2021] [Indexed: 12/01/2022]
Abstract
PURPOSE To report 3 cases of reversible epitheliopathy induced by A166-a human epidermal growth factor receptor (HER2)-targeted antibody-drug conjugate (ADC) therapy for resistant HER2 tumours. METHODS Advanced HER2 tumour patients were enrolled in A166 phase I/II clinical trial using Bayesian logistic regression model dose escalation. Key exclusion criteria were ≥grade 2 (G2) corneal pathology, severe organ disease, and other cancer therapy within 4 weeks. Eye exams were performed at baseline, regularly scheduled intervals, and additionally upon A166-induced ocular symptoms. Topical therapy with autologous serum tears (ASTs) was implemented based on visual acuity, symptoms, and slit lamp exam. A166 was withheld if ≥G2 ocular toxicity developed; if status improved to ≤G1, A166 therapy was resumed. Visual acuity, corneal exam, and subjective comfort were recorded. RESULTS After ≥2 cycles of A166, 6 eyes of 3/23 enrolled patients developed whorl pattern epitheliopathy suggestive of limbal stem cell (LSC) dysfunction requiring cessation of A166 despite positive tumour response. Patients 1 and 3 received 3.6 mg/kg A166 dose, and patient 2 received 3.0 mg/kg. Topical steroids (2/4 eyes) failed to improve epitheliopathy. Adding ASTs improved vision, ocular comfort, and whorl pattern epitheliopathy in 6/6 eyes within 3 weeks. Patient 1 continues to improve on ASTs; patient 2 withdrew from the study; and patient 3 resumed A166 therapy. CONCLUSION A166 precipitates LSC dysfunction-like epitheliopathy. Combination therapy including aggressive lubrication, withholding drug, and ASTs help reverse toxicity. Recognizing that ADC-induced epitheliopathy can respond to ocular management may enable cancer patients to continue lifesaving therapy.
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Affiliation(s)
| | - Kamran M Riaz
- Dean McGee Eye Institute, University of Oklahoma, Oklahoma City, Okla..
| | - Mohsain S Gill
- University of Oklahoma Medical School, Oklahoma City, Okla
| | - Amita Patnaik
- South Texas Accelerated Research Therapeutics, San Antonio, Tex
| | - Susanna V Ulahannan
- The University of Oklahoma Health Sciences Center/Sarah Cannon Research Institute, Oklahoma City, Okla
| | - Judy S Wang
- Florida Cancer Specialists/Sarah Cannon Research Institute, Sarasota, Fla
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29
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Adil MT, Henry JJ. Understanding cornea epithelial stem cells and stem cell deficiency: Lessons learned using vertebrate model systems. Genesis 2021; 59:e23411. [PMID: 33576188 DOI: 10.1002/dvg.23411] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2020] [Revised: 01/08/2021] [Accepted: 01/09/2021] [Indexed: 12/13/2022]
Abstract
Animal models have contributed greatly to our understanding of human diseases. Here, we focus on cornea epithelial stem cell (CESC) deficiency (commonly called limbal stem cell deficiency, LSCD). Corneal development, homeostasis and wound healing are supported by specific stem cells, that include the CESCs. Damage to or loss of these cells results in blindness and other debilitating ocular conditions. Here we describe the contributions from several vertebrate models toward understanding CESCs and LSCD treatments. These include both mammalian models, as well as two aquatic models, Zebrafish and the amphibian, Xenopus. Pioneering developments have been made using stem cell transplants to restore normal vision in patients with LSCD, but questions still remain about the basic biology of CESCs, including their precise cell lineages and behavior in the cornea. We describe various cell lineage tracing studies to follow their patterns of division, and the fates of their progeny during development, homeostasis, and wound healing. In addition, we present some preliminary results using the Xenopus model system. Ultimately, a more thorough understanding of these cornea cells will advance our knowledge of stem cell biology and lead to better cornea disease therapeutics.
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Affiliation(s)
- Mohd Tayyab Adil
- Department of Cell & Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA
| | - Jonathan J Henry
- Department of Cell & Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA
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30
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Strinkovsky L, Havkin E, Shalom-Feuerstein R, Savir Y. Spatial correlations constrain cellular lifespan and pattern formation in corneal epithelium homeostasis. eLife 2021; 10:56404. [PMID: 33433326 PMCID: PMC7803374 DOI: 10.7554/elife.56404] [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: 02/26/2020] [Accepted: 12/08/2020] [Indexed: 02/06/2023] Open
Abstract
Homeostasis in adult tissues relies on the replication dynamics of stem cells, their progenitors and the spatial balance between them. This spatial and kinetic coordination is crucial to the successful maintenance of tissue size and its replenishment with new cells. However, our understanding of the role of cellular replicative lifespan and spatial correlation between cells in shaping tissue integrity is still lacking. We developed a mathematical model for the stochastic spatial dynamics that underlie the rejuvenation of corneal epithelium. Our model takes into account different spatial correlations between cell replication and cell removal. We derive the tradeoffs between replicative lifespan, spatial correlation length, and tissue rejuvenation dynamics. We determine the conditions that allow homeostasis and are consistent with biological timescales, pattern formation, and mutants phenotypes. Our results can be extended to any cellular system in which spatial homeostasis is maintained through cell replication.
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Affiliation(s)
- Lior Strinkovsky
- Department of Physiology, Biophysics and System Biology, Faculty of Medicine, Technion, Haifa, Israel
| | - Evgeny Havkin
- Department of Physiology, Biophysics and System Biology, Faculty of Medicine, Technion, Haifa, Israel
| | - Ruby Shalom-Feuerstein
- Department of Genetics & Developmental Biology, Faculty of Medicine, Technion, Haifa, Israel.,The Rappaport Family Institute for Research in the Medical Sciences, Technion, Haifa, Israel
| | - Yonatan Savir
- Department of Physiology, Biophysics and System Biology, Faculty of Medicine, Technion, Haifa, Israel.,The Rappaport Family Institute for Research in the Medical Sciences, Technion, Haifa, Israel
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31
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Single-cell transcriptomics identifies limbal stem cell population and cell types mapping its differentiation trajectory in limbal basal epithelium of human cornea. Ocul Surf 2021; 20:20-32. [PMID: 33388438 DOI: 10.1016/j.jtos.2020.12.004] [Citation(s) in RCA: 70] [Impact Index Per Article: 17.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2020] [Revised: 12/17/2020] [Accepted: 12/27/2020] [Indexed: 12/15/2022]
Abstract
PURPOSE This study aimed to uncover novel cell types in heterogenous basal limbus of human cornea for identifying LSC at single cell resolution. METHODS Single cells of human limbal basal epithelium were isolated from young donor corneas. Single-cell RNA-Sequencing was performed using 10x Genomics platform, followed by clustering cell types through the graph-based visualization method UMAP and unbiased computational informatic analysis. Tissue RNA in situ hybridization with RNAscope, immunofluorescent staining and multiple functional assays were performed using human corneas and limbal epithelial culture models. RESULTS Single-cell transcriptomics of 16,360 limbal basal cells revealed 12 cell clusters belonging to three lineages. A smallest cluster (0.4% of total cells) was identified as LSCs based on their quiescent and undifferentiated states with enriched marker genes for putative epithelial stem cells. TSPAN7 and SOX17 are discovered and validated as new LSC markers based on their exclusive expression pattern and spatial localization in limbal basal epithelium by RNAscope and immunostaining, and functional role in cell growth and tissue regeneration models with RNA interference in cultures. Interestingly, five cell types/states mapping a developmental trajectory of LSC from quiescence to proliferation and differentiation are uncovered by Monocle3 and CytoTRACE pseudotime analysis. The transcription factor networks linking novel signaling pathways are revealed to maintain LSC stemness. CONCLUSIONS This human corneal scRNA-Seq identifies the LSC population and uncovers novel cell types mapping the differentiation trajectory in heterogenous limbal basal epithelium. The findings provide insight into LSC concept and lay the foundation for understanding the corneal homeostasis and diseases.
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Ishii R, Yanagisawa H, Sada A. Defining compartmentalized stem cell populations with distinct cell division dynamics in the ocular surface epithelium. Development 2020; 147:dev197590. [PMID: 33199446 PMCID: PMC7758628 DOI: 10.1242/dev.197590] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2020] [Accepted: 11/04/2020] [Indexed: 12/11/2022]
Abstract
Adult tissues contain label-retaining cells (LRCs), which are relatively slow-cycling and considered to represent a property of tissue stem cells (SCs). In the ocular surface epithelium, LRCs are present in the limbus and conjunctival fornix; however, the character of these LRCs remains unclear, owing to lack of appropriate molecular markers. Using three CreER transgenic mouse lines, we demonstrate that the ocular surface epithelium accommodates spatially distinct populations with different cell division dynamics. In the limbus, long-lived Slc1a3CreER-labeled SCs either migrate centripetally toward the central cornea or slowly expand their clones laterally within the limbal region. In the central cornea, non-LRCs labeled with Dlx1CreER and K14CreER behave as short-lived progenitor cells. The conjunctival epithelium in the bulbar, fornix and palpebral compartment is regenerated by regionally unique SC populations. Severe damage to the cornea leads to the cancellation of SC compartments and conjunctivalization, whereas milder limbal injury induces a rapid increase of laterally expanding clones in the limbus. Taken together, our work defines compartmentalized multiple SC/progenitor populations of the mouse eye in homeostasis and their behavioral changes in response to injury.
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Affiliation(s)
- Ryutaro Ishii
- Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Tsukuba 305-8577, Japan
- Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba 305-8577, Japan
| | - Hiromi Yanagisawa
- Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Tsukuba 305-8577, Japan
- Faculty of Medicine, University of Tsukuba, Tsukuba 305-8577, Japan
| | - Aiko Sada
- Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Tsukuba 305-8577, Japan
- International Research Center for Medical Sciences (IRCMS), Kumamoto University, Kumamoto 860-0811, Japan
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Ortiz-Melo MT, Garcia-Murillo MJ, Salazar-Rojas VM, Campos JE, Castro-Muñozledo F. Transcriptional profiles along cell programming into corneal epithelial differentiation. Exp Eye Res 2020; 202:108302. [PMID: 33098888 DOI: 10.1016/j.exer.2020.108302] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2020] [Revised: 09/23/2020] [Accepted: 10/12/2020] [Indexed: 12/15/2022]
Abstract
Using the rabbit corneal epithelial cell line RCE1(5T5) as a model, we analyzed three differentiation stages, distinguished on basis to the growth state of cultured cells and after studying the expression of transcription factors such as Oct4, Pax6 and ΔNp63α, selected differentiation markers, and signaling or epigenetic markers such as Notch receptors and Prdm3. Namely, proliferative non-differentiated cells, committed cells, and cells that constitute a stratified epithelium with a limbal epithelial-like structure. RNAseq based transcriptome analysis showed that 4891 genes were differentially expressed among these stages displaying distinctive gene signatures: proliferative cells had 1278 genes as gene signature, and seem to be early epithelial progenitors with an Oct4+, KLF4+, Myc+, ΔNp63α+, ABCG2+, Vimentin+, Zeb1+, VANGL1+, Krt3-, Krt12- phenotype. Committed cells had a gene signature with 417 genes and displayed markers indicative of the beginning of corneal differentiation, and genes characteristic of proliferative cells; we found the possible participation of Six3 and Six4 transcription factors along this stage. The third stage matches with a stratified corneal epithelium (gene signature comprising 979 genes) and is typified by an increase in the expression of WNT10A and NOTCH 2 and 3 signaling and Cux1 transcription factor, besides Pax6, KLF4 or Sox9. The differentiated cells express about 50% of the genes that belong to the Epidermal Differentiation Complex (EDC). Analysis of the differences between corneal epithelium and epidermis could be crucial to understand the regulatory mechanisms that lead to the expression of the differentiated phenotype.
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Affiliation(s)
- María Teresa Ortiz-Melo
- Department of Cell Biology, Centro de Investigación y de Estudios Avanzados del IPN, Apdo. Postal 14-740. México City, 07000, Mexico; Unidad de Biotecnología y Prototipos, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Ap. Postal 314, 54000, Tlalnepantla, Edo. de México, Mexico; Posgrado en Ciencias Biológicas, Universidad Nacional Autónoma de México. Unidad de Posgrado, Edificio A, 1° Piso, Circuito de Posgrados, Ciudad Universitaria, Coyoacán, C.P. 04510, Ciudad de México, Mexico
| | - Maria Jimena Garcia-Murillo
- Department of Cell Biology, Centro de Investigación y de Estudios Avanzados del IPN, Apdo. Postal 14-740. México City, 07000, Mexico
| | - Víctor Manuel Salazar-Rojas
- Unidad de Biotecnología y Prototipos, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Ap. Postal 314, 54000, Tlalnepantla, Edo. de México, Mexico
| | - Jorge E Campos
- Unidad de Biotecnología y Prototipos, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Ap. Postal 314, 54000, Tlalnepantla, Edo. de México, Mexico
| | - Federico Castro-Muñozledo
- Department of Cell Biology, Centro de Investigación y de Estudios Avanzados del IPN, Apdo. Postal 14-740. México City, 07000, Mexico.
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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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Lavker RM, Kaplan N, Wang J, Peng H. Corneal epithelial biology: Lessons stemming from old to new. Exp Eye Res 2020; 198:108094. [PMID: 32697979 DOI: 10.1016/j.exer.2020.108094] [Citation(s) in RCA: 29] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2020] [Revised: 05/27/2020] [Accepted: 05/29/2020] [Indexed: 12/13/2022]
Abstract
The anterior surface of the eye functions as a barrier to the external environment and protects the delicate underlying tissues from injury. Central to this protection are the corneal, limbal and conjunctival epithelia. The corneal epithelium is a self-renewing stratified squamous epithelium that protects the underlying delicate structures of the eye, supports a tear film and maintains transparency so that light can be transmitted to the interior of the eye (Basu et al., 2014; Cotsarelis et al., 1989; Funderburgh et al., 2016; Lehrer et al., 1998; Pajoohesh-Ganji and Stepp, 2005; Parfitt et al., 2015; Peng et al., 2012b; Stepp and Zieske, 2005). In this review, dedicated to James Funderburgh and his contributions to visual science, in particular the limbal niche, corneal stroma and corneal stromal stem cells, we will focus on recent data on the identification of novel regulators in corneal epithelial cell biology, their roles in stem cell homeostasis, wound healing, limbal/corneal boundary maintenance and the utility of single cell RNA sequencing (scRNA-seq) in vision biology studies.
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Affiliation(s)
- Robert M Lavker
- Departments of Dermatology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA.
| | - Nihal Kaplan
- Departments of Dermatology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA
| | - Junyi Wang
- Departments of Dermatology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA; Department of Ophthalmology, The First Center of the PLA General Hospital, Haidian District, Beijing, China
| | - Han Peng
- Departments of Dermatology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA
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Micera A, Jirsova K, Esposito G, Balzamino BO, Di Zazzo A, Bonini S. Mast Cells Populate the Corneoscleral Limbus: New Insights for Our Understanding of Limbal Microenvironment. Invest Ophthalmol Vis Sci 2020; 61:43. [PMID: 32207813 PMCID: PMC7401584 DOI: 10.1167/iovs.61.3.43] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Purpose Although stem cell activity represents a crucial feature in corneal and ocular surface homeostasis, other cells populating this region and the neighboring zones might participate and influence local microenvironment. Mast cells, the long-lived and tissue-sited immune cells, have been previously reported in corneoscleral specimens. Herein, mast cells were investigated in corneoscleral tissues and related to microenvironment protein expression. Methods Twenty-six (14 male/12 female; older than 60 years) human corneoscleral specimens were sectioned for light and fluorescent immunostaining (CD45, p63, Ck-3/7/12/19, tryptase/AA1, and chymase/CC1). Corneal, limbal, and conjunctival squares were produced for molecular and biochemical analysis. Statistical comparisons were carried out by ANOVA. Results Toluidine blue staining identified metachromatic intact or degranulated mast cells in the area below the palisades' Vogt (Ck-3/12-positive epithelium and underneath p63 immunoreactivity). Tryptase immunoreactivity was observed close to palisades' Vogt, whereas no specific signal was detected for chymase. Tryptase/AA1 transcripts were quantified in limbal and conjunctival RNA extracts, whereas no specific amplification was detected in corneal ones. Few mediators were overexpressed in limbal extracts with respect to corneal (Neural cell adhesion molecule (NCAM), Intercellular adhesion molecule 3 (ICAM3), Brain-derived Neurotrophic factor (BDNF), and neurotrophin 3 (NT3); P < 0.00083) and conjunctival (NCAM, ICAM3, and NT3; P < 0.05) protein extracts. A trend to an increase was observed for Nerve Growth Factor (NGF) in limbal extracts (P > 0.05). Conclusions The specific observation of tryptase phenotype and the interesting protein signature of microenvironment (adhesion molecules, growth factors, and neurotrophins), known to partake mast cell behavior, at least in other areas, would provide additional information to better understand this crucial zone in the framework of ocular surface healthiness.
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Short- and Long-Term Results of Xenogeneic-Free Cultivated Autologous and Allogeneic Limbal Epithelial Stem Cell Transplantations. Cornea 2020; 38:1543-1549. [PMID: 31569145 PMCID: PMC6830964 DOI: 10.1097/ico.0000000000002153] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
To evaluate the short- and long-term success rates of xenogeneic-free cultivated limbal epithelial stem cell transplantation (CLET) for the treatment of limbal stem cell deficiency (LSCD).
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Foster JW, Wahlin KJ, Chakravarti S. A Guide to the Development of Human CorneaOrganoids from Induced Pluripotent Stem Cells in Culture. Methods Mol Biol 2020; 2145:51-58. [PMID: 32542600 PMCID: PMC7565088 DOI: 10.1007/978-1-0716-0599-8_5] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
The cornea is the outermost transparent and refractive barrier surface of the eye necessary for vision. Development of the cornea involves the coordinated production of extracellular matrix, epithelial differentiation, and endothelial cell expansion to produce a highly transparent tissue. Here we describe the production of multilayered three-dimensional organoids from human-induced pluripotent stem cells. These organoids have the potential for multiple downstream applications which are currently unattainable using traditional in vitro techniques.
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Affiliation(s)
- James W Foster
- The Wilmer Eye Institute, Johns Hopkins University, Baltimore, MD, USA
| | - Karl J Wahlin
- Shiley Eye Institute, UC San Diego, La Jolla, CA, USA.
| | - Shukti Chakravarti
- Department of Ophthalmology and Pathology, NYU Langone Health, Alexandria Life Sciences Center, New York, NY, USA.
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Kaplan N, Wang J, Wray B, Patel P, Yang W, Peng H, Lavker RM. Single-Cell RNA Transcriptome Helps Define the Limbal/Corneal Epithelial Stem/Early Transit Amplifying Cells and How Autophagy Affects This Population. Invest Ophthalmol Vis Sci 2019; 60:3570-3583. [PMID: 31419300 PMCID: PMC6701873 DOI: 10.1167/iovs.19-27656] [Citation(s) in RCA: 82] [Impact Index Per Article: 13.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Purpose Single-cell RNA-sequencing (scRNA-seq) was used to interrogate the relatively rare stem (SC) and early transit amplifying (TA) cell populations in limbal/corneal epithelia from wild-type and autophagy-compromised mice. Methods We conducted scRNA-seq on ocular anterior segmental tissue from wild-type and beclin 1–deficient (beclin1+/−) mice, using a 10X Gemomics pipeline. Cell populations were distinguished by t-distributed stochastic neighbor embedding. Seurat analysis was conducted to compare gene expression profiles between these two groups of mice. Differential protein expression patterns were validated by immunofluorescence staining and immunoblotting. Results Unbiased clustering detected 10 distinct populations: three clusters of mesenchymal and seven clusters of epithelial cells, based on their unique molecular signatures. A discrete group of mesenchymal cells expressed genes associated with corneal stromal SCs. We identified three limbal/corneal epithelial cell subpopulations designated as stem/early TA, mature TA, and differentiated corneal epithelial cells. Thioredoxin-interacting protein and PDZ-binding kinase (PBK) were identified as novel regulators of stem/early TA cell quiescence. PBK arrested corneal epithelial cells in G2/M phase of the cell cycle. Beclin1+/− mice displayed a decrease in proliferation-associated (Ki67, Lrig1) and stress-response (H2ax) genes. The most increased gene in beclin1+/− mice was transcription factor ATF3, which negatively regulates limbal epithelial cell proliferation. Conclusions Establishment of a comprehensive atlas of genes expressed by stromal and epithelial cells from limbus and cornea forms the foundation for unraveling regulatory networks among these distinct tissues. Similarly, scRNA-seq profiling of the anterior segmental epithelia from wild-type and autophagy-deficient mice provides new insights into how autophagy influences proliferation in these tissues.
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Affiliation(s)
- Nihal Kaplan
- Department of Dermatology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, United States
| | - Junyi Wang
- Department of Dermatology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, United States.,Department of Ophthalmology, Ophthalmology and Visual Science Key Lab of PLA, Chinese PLA General Hospital, Beijing, China
| | - Brian Wray
- Center for Genetic Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, United States
| | - Priyam Patel
- Center for Genetic Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, United States
| | - Wending Yang
- Department of Dermatology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, United States
| | - Han Peng
- Department of Dermatology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, United States
| | - Robert M Lavker
- Department of Dermatology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, United States
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Sakimoto T, Sakimoto A, Yamagami S. Autologous transplantation of conjunctiva by modifying simple limbal epithelial transplantation for limbal stem cell deficiency. Jpn J Ophthalmol 2019; 64:54-61. [PMID: 31760516 DOI: 10.1007/s10384-019-00701-7] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2019] [Accepted: 10/31/2019] [Indexed: 11/28/2022]
Abstract
PURPOSE As a treatment to replace regenerative medicine to treat limbal stem cell deficiency (LSCD), we performed 4 consecutive cases of autologous transplantation of conjunctival explants by modifying simple limbal epithelial transplantation (SLET). STUDY DESIGN Single-center case series. METHODS Four patients with LSCD were enrolled in this study. After resection of scar tissue with neovascularization from the ocular surface, human amniotic membrane (AM) was placed over the bare ocular surface. The bulbar conjunctiva of the operated eye was dissected at the temporal superior fornix, divided into small pieces, and transplanted onto AM with fibrin glue. Keratoplasty was performed simultaneously or few months after surgery. RESULTS Epithelialization was achieved in all patients. Best-corrected visual acuity was improved in all patients. CONCLUSION This is the first report of ocular surface reconstruction using autologous conjunctival epithelial transplants from the affected eye. Transplantation by modifying SLET effectively restored a clear corneal surface with minimal neovascularization in 4 patients with LSCD. Autologous conjunctival transplants combined with AM transplantation could be a practical option for treating bilateral LSCD in patients without symblepharon or severe keratinization.
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Affiliation(s)
- Tohru Sakimoto
- Division of Ophthalmology, Department of Visual Sciences, Nihon University School of Medicine, 30-1 Oyaguchi Kamimachi, Itabashi-ku, Tokyo, 173-8610, Japan
| | - Akira Sakimoto
- Division of Ophthalmology, Department of Visual Sciences, Nihon University School of Medicine, 30-1 Oyaguchi Kamimachi, Itabashi-ku, Tokyo, 173-8610, Japan
| | - Satoru Yamagami
- Division of Ophthalmology, Department of Visual Sciences, Nihon University School of Medicine, 30-1 Oyaguchi Kamimachi, Itabashi-ku, Tokyo, 173-8610, Japan.
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Son YJ, Tse JW, Zhou Y, Mao W, Yim EKF, Yoo HS. Biomaterials and controlled release strategy for epithelial wound healing. Biomater Sci 2019; 7:4444-4471. [PMID: 31436261 DOI: 10.1039/c9bm00456d] [Citation(s) in RCA: 49] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
The skin and cornea are tissues that provide protective functions. Trauma and other environmental threats often cause injuries, infections and damage to these tissues, where the degree of injury is directly correlated to the recovery time. For example, a superficial skin or corneal wound may recover within days; however, more severe injuries can last up to several months and may leave scarring. Thus, therapeutic strategies have been introduced to enhance the wound healing efficiency and quality. Although the skin and cornea share similar anatomic structures and wound healing process, therapeutic agents and formulations for skin and cornea wound healing differ in accordance with the tissue and wound type. In this review, we describe the anatomy and epithelial wound healing processes of the skin and cornea, and summarize the therapeutic molecules that are beneficial to the respective regeneration process. In addition, biomaterial scaffolds that inherently possess bioactive properties or modified with therapeutic molecules for topical controlled release and enhanced wound healing efficiency are also discussed.
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Affiliation(s)
- Young Ju Son
- Department of Biomedical Materials Engineering, Kangwon National University, Chuncheon, 24341, Republic of Korea.
| | - John W Tse
- Department of Chemical Engineering, University of Waterloo, Waterloo, ON, Canada N2L 3G1.
| | - Yiran Zhou
- Department of Chemical Engineering, University of Waterloo, Waterloo, ON, Canada N2L 3G1.
| | - Wei Mao
- Department of Biomedical Materials Engineering, Kangwon National University, Chuncheon, 24341, Republic of Korea.
| | - Evelyn K F Yim
- Department of Chemical Engineering, University of Waterloo, Waterloo, ON, Canada N2L 3G1.
| | - Hyuk Sang Yoo
- Department of Biomedical Materials Engineering, Kangwon National University, Chuncheon, 24341, Republic of Korea. and Institute of Bioscience and Biotechnology, Kangwon National University, Republic of Korea
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The Neural Differentiation Potential of Limbal Stem Cells: A Model for Studying the Multipotency of Limbal Stem Cells. Cornea 2019; 38 Suppl 1:S4-S10. [PMID: 31397733 DOI: 10.1097/ico.0000000000002084] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
PURPOSE To investigate the multipotency, especially the neural differentiation potential, of limbal stem cells (LSCs) using a coculture system and to provide a relevant literature review. METHODS Limbal tissue was harvested from adult New Zealand white rabbits and treated with collagenase A. Small pieces of the resulting limbal epithelial sheets were cocultured with a neuroblastoma cell line (Neuro-2A) in transwells. Morphological observation of the growing epithelial sheets was accomplished by microscopy, and marker expression was detected by immunocytochemistry for ßIII-tubulin and microtubule-associated protein 2. A literature review of associated studies was performed. RESULTS In the coculture group, directly adherent colonies of neuron-like (DACN) cells were observed among the growing limbal epithelial sheets from day 3. The DACN cells exhibited neuron-like morphology. The control group comprising limbal cell sheets cultured alone showed a very small number of DACN cells at the end of the culture period (day 14). Immunocytochemical staining revealed that the DACN cells were positive for ßIII-tubulin and microtubule-associated protein 2, confirming the neuronal phenotype of the neuron-like cells. By contrast, the DACN cells in the control group produced negative results. In previous reports, LSCs and niches exhibited neural potential, but most differentiated neural cells were observed as floating spheres, in contrast to the DACN cells observed in the present study. CONCLUSIONS We developed a coculture system of LSCs and Neuro-2A neuroblastoma cells and obtained DACN cells with neural differentiation potential. Our findings confirm the neural potential of LSCs, consistent with previous reports, but in a form other than floating spheres.
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Chen SY, Cheng AMS, Zhang Y, Zhu YT, He H, Mahabole M, Tseng SCG. Pax 6 Controls Neural Crest Potential of Limbal Niche Cells to Support Self-Renewal of Limbal Epithelial Stem Cells. Sci Rep 2019; 9:9763. [PMID: 31278274 PMCID: PMC6611810 DOI: 10.1038/s41598-019-45100-7] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2018] [Accepted: 05/23/2019] [Indexed: 12/31/2022] Open
Abstract
On ocular surface, corneal epithelial stem cells (SC) reside in limbus between cornea and conjunctiva. Pax6, an evolutionally conserved transcription factor essential for eye development, is expressed in post-natal corneal and limbal epithelia progenitors (LEPC) but not in underlying stroma. Because Pax6 is transiently expressed in developing corneal stroma and a subset of limbal and corneal stromal progenitors, we examined the role of Pax6 in limbal niche cells (LNC) in maintaining the phenotype of neural crest (NC) progenitors to support LEPC. Our results showed that nuclear Pax6 staining was found in freshly isolated LNC but not corneal stromal cells. Serial passaged LNC resulted in gradual loss of nuclear Pax6 (46 kDa) staining and neural crest progenitor status defined by the expression of embryonic SCs and NC markers, neurosphere formation, and differentiation into neurons, oligodendrocytes and astrocytes. Gain of function of 46 kDa Pax6 in late-passaged LNC resulted in nuclear Pax6 staining and promotion of the aforementioned NC progenitor status. In an in vitro reunion assay, early passaged LNC and late passaged LNC with overexpression of Pax6 inhibited the expression of corneal epithelial differentiation marker and promoted holoclone by LEPC. Therefore, expression of nuclear 46 kDa Pax6 in LNC plays an important developmental role in maintaining NC progenitor status to support self-renewal of corneal epithelial SCs in the limbal niche.
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Affiliation(s)
- Szu-Yu Chen
- R&D Department, Tissue Tech, Inc., Miami, FL, 33126, USA.,Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, FL33136, USA
| | - Anny M S Cheng
- Department of Ophthalmology, Florida International University, Herbert Wertheim College of Medicine, Miami, FL33199, USA.,Ocular Surface Center, and Ocular Surface Research & Education Foundation, Miami, FL, 33126, USA
| | - Yuan Zhang
- R&D Department, Tissue Tech, Inc., Miami, FL, 33126, USA
| | - Ying-Ting Zhu
- R&D Department, Tissue Tech, Inc., Miami, FL, 33126, USA
| | - Hua He
- R&D Department, Tissue Tech, Inc., Miami, FL, 33126, USA
| | - Megha Mahabole
- R&D Department, Tissue Tech, Inc., Miami, FL, 33126, USA
| | - Scheffer C G Tseng
- R&D Department, Tissue Tech, Inc., Miami, FL, 33126, USA. .,Ocular Surface Center, and Ocular Surface Research & Education Foundation, Miami, FL, 33126, USA.
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Zhu L, Titone R, Robertson DM. The impact of hyperglycemia on the corneal epithelium: Molecular mechanisms and insight. Ocul Surf 2019; 17:644-654. [PMID: 31238114 DOI: 10.1016/j.jtos.2019.06.007] [Citation(s) in RCA: 48] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2018] [Revised: 06/11/2019] [Accepted: 06/21/2019] [Indexed: 12/15/2022]
Abstract
Type 2 Diabetes Mellitus (T2DM) is reaching epidemic levels worldwide and with it, there is a significant increase in complications associated with the disease. T2DM affects virtually all organ systems including the eye. While frequently overlooked, diabetic keratopathy is the most common ocular complication of diabetes and can manifest in mild to severe forms, the latter of which poses a major threat to vision. As the initial barrier between the environment and the eye, the corneal epithelium functions in innate immune defense. Compromise of this barrier may predispose the cornea to infection and can hinder the refractive capabilities of the eye. The clinical burden in patients with diabetic keratopathy lies primarily in the inability of the corneal epithelium to repair damage and maintain its tight barrier function. Current therapies for diabetic keratopathy are supportive, centering on the prevention of infection and promotion of an optimal healing environment. With no clear disease-modifying agent identified as of yet, a thorough understanding of the pathophysiology that underlies the development of diabetic keratopathy at the cellular level is critical to identify and develop potential therapeutic agents capable of promoting corneal re-epithelialization to accelerate the wound healing process. The focus of this review is to examine what is known regarding the cellular and molecular mechanisms needed to maintain epithelial homeostasis and how it goes awry in diabetes.
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Affiliation(s)
- Luke Zhu
- Department of Ophthalmology, University of Texas Southwestern Medical Center, United States
| | - Rossella Titone
- Department of Ophthalmology, University of Texas Southwestern Medical Center, United States
| | - Danielle M Robertson
- Department of Ophthalmology, University of Texas Southwestern Medical Center, United States.
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Systematic review and meta-analysis investigating autograft versus allograft cultivated limbal epithelial transplantation in limbal stem cell deficiency. Int Ophthalmol 2019; 39:2685-2696. [PMID: 30826943 DOI: 10.1007/s10792-019-01092-x] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2018] [Accepted: 02/23/2019] [Indexed: 12/13/2022]
Abstract
PURPOSE Currently, regenerative medicine has attracted much attention among researchers investigating new methods to treat ocular surface diseases. Based on this new concept, cultivated limbal epithelial transplantation (CLET), whether in the form of autograft or allograft, has emerged as a promising surgical procedure for treating limbal stem cell deficiency (LSCD). Given that there is no updated comparison between autograft and allograft CLETs, the present review and meta-analysis aims to compare and determine the efficacy of two different CLET techniques, autologous versus allogeneic, based on a literature review of relevant studies. METHODS A comprehensive search of electronic databases, including PubMed, Web of Science, Cochrane Library, Embase and Scopus, for related articles was performed in March 2018 to obtain relevant articles and to conduct a meta-analysis investigating the success rate of ocular surface regeneration and two-line improvement in best-corrected visual acuity (BCVA) using autograft versus allograft transplantations. RESULTS A total of 30 studies, including 1306 eyes from 1288 patients with LSCD, with a sample size ranging from 6 to 200 and follow-up period of 0.6-156 months, were reviewed. Of 1306 eyes, 982 (75.2%) underwent autograft and 324 (24.8%) received allografts from living or deceased donors. Meta-analysis revealed that there was no significant difference between autograft and allograft CLETs in terms of success rate and two-line BCVA improvement. The prospective studies showed a zero difference between the two groups; only two retrospective studies included in the analysis pulled the autografts up to 1.82 and 1.2 times more than allografts in terms of success rate and two-line BCVA improvement, respectively [pooled OR 1.82 (95% CI 0.80-4.11); pooled OR 1.2 (95% CI 0.54-2.65)]. There was no statistically significant evidence of bias in the meta-analysis in terms of success rates and two-line BCVA improvement. CONCLUSIONS The present analysis revealed no significant differences in success rates or visual improvement between autograft and allograft surgical techniques.
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El-Hofi AH, Helaly HA. Evaluation of limbal transplantation in eyes with bilateral severe ocular surface damage secondary to chemical injury. Clin Ophthalmol 2019; 13:383-390. [PMID: 30858688 PMCID: PMC6387598 DOI: 10.2147/opth.s192316] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
Abstract
PURPOSE To evaluate the outcome of limbal transplantation in eyes with bilateral severe ocular surface damage secondary to chemical injury. PATIENTS AND METHODS This was a retrospective case series that included 20 patients who had undergone living related limbal transplantation due to the presence of bilateral severe stem cell deficiency resulting from chemical injury. Medical records of the selected patients were reviewed. The following data were recorded and analyzed: gender; age at the time of the surgery; duration of follow-up; corrected distance visual acuity (CDVA); symptoms; intraocular pressure (IOP); any complications; postoperative treatment; and other surgical procedures needed. RESULTS The mean age of the included patients was 27.4±9.3 years. The causative agent was an alkali in 14 patients. Fifteen patients (75%) had a stable ocular surface (ie, complete corneal re-epithelization and resolution of postoperative inflammation) after the first limbal transplantation, while the other five patients (25%) needed regrafting. As regards the IOP; five patients (25%) needed Ahmed's valve implantation to control the IOP. Other surgical procedures needed were penetrating keratoplasty (PKP) in three patients (15%), cataract surgery in six patients (30%), and lid surgery in one patient (5%). CONCLUSION In conclusion, living related conjunctival limbal allograft transplantation, especially when combined with amniotic membrane transplantation, yielded good results in the management of cases with bilateral severe chemical eye injuries.
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Affiliation(s)
- Abdel Hamid El-Hofi
- Department of Ophthalmology, Faculty of Medicine, Alexandria University, Alexandria, Egypt,
| | - Hany Ahmed Helaly
- Department of Ophthalmology, Faculty of Medicine, Alexandria University, Alexandria, Egypt,
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Stadnikova A, Trosan P, Skalicka P, Utheim TP, Jirsova K. Interleukin-13 maintains the stemness of conjunctival epithelial cell cultures prepared from human limbal explants. PLoS One 2019; 14:e0211861. [PMID: 30742646 PMCID: PMC6370187 DOI: 10.1371/journal.pone.0211861] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2018] [Accepted: 01/23/2019] [Indexed: 11/18/2022] Open
Abstract
To use human limbal explants as an alternative source for generating conjunctival epithelium and to determine the effect of interleukin-13 (IL-13) on goblet cell number, mucin expression, and stemness. Human limbal explants prepared from 17 corneoscleral rims were cultured with or without IL-13 (IL-13+ and IL-13-, respectively) and followed up to passage 2 (primary culture [P0]-P2). Cells were characterized by alcian blue/periodic acid-Schiff (AB/PAS) staining (goblet cells); immunofluorescent staining for p63α (progenitor cells), Ki-67 (proliferation), MUC5AC (mucin, goblet cells), and keratin 7 (K7, conjunctival epithelial and goblet cells); and by quantitative real-time polymerase chain reaction for expression of the p63α (TP63), MUC5AC, MUC4 (conjunctival mucins), K3, K12 (corneal epithelial cells), and K7 genes. Clonogenic ability was determined by colony-forming efficiency (CFE) assay. Using limbal explants, we generated epithelium with conjunctival phenotype and high viability in P0, P1, and P2 cultures under IL-13+ and IL-13- conditions, i.e., epithelium with strong K7 positivity, high K7 and MUC4 expression and the presence of goblet cells (AB/PAS and MUC5AC positivity; MUC5AC expression). p63α positivity was similar in IL-13+ and IL-13- cultures and was decreased in P2 cultures; however, there was increased TP63 expression in the presence of IL-13 (especially in the P1 cultures). Similarly, IL-13 increased proliferative activity in P1 cultures and significantly promoted P0 and P1 culture CFE. IL-13 did not increase goblet cell number in the P0-P2 cultures, nor did it influence MUC5AC and MUC4 expression. By harvesting unattached cells on day 1 of P1 we obtained goblet cell rich subpopulation showing AB/PAS, MUC5AC, and K7 positivity, but with no growth potential. In conclusion, limbal explants were successfully used to develop conjunctival epithelium with the presence of putative stem and goblet cells and with the ability to preserve the stemness of P0 and P1 cultures under IL-13 influence.
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Affiliation(s)
- Andrea Stadnikova
- Laboratory of the Biology and Pathology of the Eye, Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic
| | - Peter Trosan
- Laboratory of the Biology and Pathology of the Eye, Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic
| | - Pavlina Skalicka
- Department of Ophthalmology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic
| | - Tor Paaske Utheim
- Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway
- Department of Plastic and Reconstructive Surgery, Oslo University Hospital, Oslo, Norway
| | - Katerina Jirsova
- Laboratory of the Biology and Pathology of the Eye, Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic
- * E-mail:
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Abstract
PURPOSE To grade the severity of limbal stem cell deficiency (LSCD) based on the extent of clinical presentation and central corneal basal epithelial cell density (BCD). METHODS This is a retrospective observational comparative study of 48 eyes of 35 patients with LSCD and 9 eyes of 7 normal subjects (controls). Confocal images of the central cornea were acquired. A clinical scoring system was created based on the extent of limbal and corneal surface involvement. LSCD was graded as mild, moderate, and severe stages based on the clinical scores. The degree of BCD reduction was given a score of 0 to 3. RESULTS Compared with BCD in controls, BCD decreased by 23.0%, 40.4%, and 69.5% in the mild, moderate, and severe stages of LSCD classified by the clinical scoring system, respectively. The degree of BCD reduction was positively correlated with larger limbal and corneal surface involvement and when the central visual axis was affected (all P ≤ 0.0005). Mean corrected distance visual acuity logarithm of the minimum angle of resolution was 0.0 ± 0.0 in control eyes, 0.2 ± 0.5 in mild LSCD, 0.6 ± 0.4 in moderate LSCD, and 1.6 ± 1.1 in severe LSCD (P < 0.0001). There was a significant correlation between a higher clinical score and corrected distance visual acuity logarithm of the minimum angle of resolution (rho = 0.82; P < 0.0001) and a greater decrease in BCD (rho = -0.78; P < 0.0001). CONCLUSIONS A clinical scoring system was developed to assess the extent of clinical presentation of LSCD. A classification system to grade the severity of LSCD can be established by combining the BCD score with the clinical score.
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Bussan KA, Robertson DM. Contact lens wear and the diabetic corneal epithelium: A happy or disastrous marriage? J Diabetes Complications 2019; 33:75-83. [PMID: 30391097 PMCID: PMC7364814 DOI: 10.1016/j.jdiacomp.2018.09.015] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/25/2018] [Revised: 09/10/2018] [Accepted: 09/23/2018] [Indexed: 12/11/2022]
Abstract
Diabetes mellitus is an epidemic in the US and abroad. With the advent of new contact lens technology, the use of contact lenses as glucose sensors in lieu of the traditional finger stick is quickly becoming realized. This has the potential to rapidly expand the contact lens market into this growing patient population. The independent cellular and physiological effects of contact lens wear and diabetes on the corneal epithelium have been described. However, little evidence exists to date to support whether there is increased risk associated with contact lens wear in diabetes. The focus of this review is to discuss what is known about the cellular effects of contact lenses on the corneal epithelium, the pathophysiological changes in the corneal epithelium that occur in diabetes, and whether an increased risk for corneal epithelial damage and/or infection may negatively impact safety in diabetic contact lens wearers. Available data indicates that there are inherent risks associated with contact lens wear in diabetics. Importantly, eye care practitioners fitting contact lenses in the diabetic patient need to carefully consider the duration of disease, the level of glycemic control, the presence of retinopathy, and the patient's overall health.
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Affiliation(s)
- Katherine A Bussan
- The Department of Ophthalmology, The University of Texas Southwestern Medical Center, United States of America
| | - Danielle M Robertson
- The Department of Ophthalmology, The University of Texas Southwestern Medical Center, United States of America.
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Sagga N, Kuffová L, Vargesson N, Erskine L, Collinson JM. Limbal epithelial stem cell activity and corneal epithelial cell cycle parameters in adult and aging mice. Stem Cell Res 2018; 33:185-198. [PMID: 30439642 PMCID: PMC6288239 DOI: 10.1016/j.scr.2018.11.001] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/18/2018] [Revised: 10/04/2018] [Accepted: 11/04/2018] [Indexed: 12/15/2022] Open
Abstract
Limbal epithelial stem cells (LESCs) are believed to be responsible for corneal epithelial maintenance and repair after injury, but their activity has never been properly quantified in aging or wounded eyes. In this study, labelling with thymidine analogues, 5-iodo-2′-deoxyuridine (IdU), 5-chloro-2′-deoxyuridine (CldU) and 5-ethynyl-2′-deoxyuridine (EdU), was used to estimate cell-cycle time of the corneal and limbal epithelia in wild-type eyes, comparing aging (12 months) and young adult (8 week) mice. In C57BL/6 mice, cells cycled significantly faster in the central corneal epithelium of aging eyes (3.24 ± 0.2 days) compared to 10 week old mice (4.97 ± 0.5 days). Long-term labelling with IdU was used to detect slow-cycling stem cells, followed by CldU or EdU labelling to quantify the proliferative dynamics of LESCs during corneal wound healing. In unwounded eyes, 4.52 ± 1.4% of LESCs were shown to enter S phase in a 24 h period and were estimated to divide every 2–3 weeks. Within 24 h of corneal injury this rose significantly to 32.8 ± 10.0% of stem cells indicating a seven-fold increase in activation. In contrast, no comparable increase in LESC activation was observed in aging mice after wounding. In the 24–48 h period after wounding in young adults, LESC activation continued to increase (86.5 ± 8.2% of label-retaining cells in wounded eye were in S-phase) but surprisingly, 46.0 ± 9.4% of LESCs were observed to reenter S-phase in the contralateral unwounded eye. These data imply an unsuspected systemic effect of corneal wounding on LESC activation suggesting that injury to one eye elicits a regenerative response in both.
Corneal wounding causes a seven-fold increase in the number of limbal epithelial stem cells in mitosis, 24 h after injury. This stem cell response to injury does not occur in aging animals. 24–48 h after wounding, nearly 90% of limbal epithelial stem cells are in mitosis. Wounding to one cornea elicits a systemic stem cell response in the other cornea after 24 h.
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Affiliation(s)
- Nada Sagga
- School of Medicine, Medical Sciences and Nutrition, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, United Kingdom
| | - Lucia Kuffová
- School of Medicine, Medical Sciences and Nutrition, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, United Kingdom; Department of Ophthalmology, NHS Grampian, Aberdeen, UK
| | - Neil Vargesson
- School of Medicine, Medical Sciences and Nutrition, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, United Kingdom
| | - Lynda Erskine
- School of Medicine, Medical Sciences and Nutrition, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, United Kingdom
| | - J Martin Collinson
- School of Medicine, Medical Sciences and Nutrition, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, United Kingdom.
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