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Garg A, Goel K, Gour A, Sapra M, Sangwan VS, Tripathi R, Tiwari A. Unveiling the Molecular Mechanisms Underlying the Success of Simple Limbal Epithelial Transplantation (SLET). Cells 2025; 14:200. [PMID: 39936991 PMCID: PMC11817669 DOI: 10.3390/cells14030200] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/26/2024] [Revised: 01/17/2025] [Accepted: 01/19/2025] [Indexed: 02/13/2025] Open
Abstract
Simple limbal epithelial transplantation (SLET) has emerged as an effective treatment option for limbal stem cell deficiency (LSCD). However, the precise molecular mechanisms underlying its success remain incompletely understood. This review delves into the proposed mechanisms involving the donor limbus, host microenvironment, and the amniotic membrane as a scaffold in SLET. The donor limbus contributes to SLET efficacy through various factors secreted by limbal epithelial stem cells, including hepatocyte growth factor (HGF), soluble Fms-like tyrosine kinase-1 (sFLT-1), and pigment epithelium-derived factor (PEDF), which support corneal healing and transparency. Additionally, the presence of melanocytes, immune cells, limbal fibroblasts, and adhesion molecules within the donor tissue helps preserve the integrity of the limbal niche. The host environment plays a critical role in supporting the transplanted stem cells, with mesenchymal stem cell-secreted factors promoting proliferation and differentiation. Although the amniotic membrane has traditionally been used as a scaffold, emerging evidence suggests that it may not always be necessary. Further studies are needed to validate this scaffold-free approach and to evaluate the vitality and functional contributions of individual components used in SLET. Understanding these complex interactions and molecular mechanisms sheds light on the importance of the donor tissue, host microenvironment, and scaffold in SLET, paving the way for the optimization of this technique for the effective treatment of LSCD.
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Affiliation(s)
- Aastha Garg
- Department of Cornea and Anterior Segment Services, Dr. Shroff’s Charity Eye Hospital, New Delhi 110002, India; (A.G.); (A.G.); (V.S.S.)
- Department of Stem Cells Research, Dr. Shroff’s Charity Eye Hospital, New Delhi 110002, India; (K.G.); (M.S.)
| | - Kartik Goel
- Department of Stem Cells Research, Dr. Shroff’s Charity Eye Hospital, New Delhi 110002, India; (K.G.); (M.S.)
| | - Abha Gour
- Department of Cornea and Anterior Segment Services, Dr. Shroff’s Charity Eye Hospital, New Delhi 110002, India; (A.G.); (A.G.); (V.S.S.)
- Department of Stem Cells Research, Dr. Shroff’s Charity Eye Hospital, New Delhi 110002, India; (K.G.); (M.S.)
| | - Mehak Sapra
- Department of Stem Cells Research, Dr. Shroff’s Charity Eye Hospital, New Delhi 110002, India; (K.G.); (M.S.)
| | - Virender Singh Sangwan
- Department of Cornea and Anterior Segment Services, Dr. Shroff’s Charity Eye Hospital, New Delhi 110002, India; (A.G.); (A.G.); (V.S.S.)
- Department of Stem Cells Research, Dr. Shroff’s Charity Eye Hospital, New Delhi 110002, India; (K.G.); (M.S.)
| | - Ratnakar Tripathi
- Department of Veterinary Medicine, University of Missouri, Columbia, MO 65201, USA
| | - Anil Tiwari
- Department of Stem Cells Research, Dr. Shroff’s Charity Eye Hospital, New Delhi 110002, India; (K.G.); (M.S.)
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Dimmock R, Zhang Y, Butt GF, Rauz S, Huang Z, Yang Y. Characterizing Biomechanics of Limbal Niche Using Vibrational Optical Coherence Elastography. JOURNAL OF BIOPHOTONICS 2024; 17:e202400172. [PMID: 39340174 DOI: 10.1002/jbio.202400172] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/21/2024] [Revised: 08/05/2024] [Accepted: 08/22/2024] [Indexed: 09/30/2024]
Abstract
The limbal niche is an adult source of epithelial stem cells which regenerate the cornea epithelium. The architecture and biomechanical properties of the limbus have previously been demonstrated to change due to aging and disease. This study aims to non-destructively and simultaneously quantify these limbal niche properties, along with their age-related changes. A lab-built vibrational optical coherence elastography (OCE) device consisting of a phase-sensitive optical coherence tomography (OCT) with a vibrational stimulator has been used to collect structural images and the depth-resolved elasticity of human corneoscleral tissues (aged 4-96 years old). The limbal palisades of Vogt (POV) were delineated well in OCT images which were validated by histology. The POVs have been spatially mapped with simultaneous elasticity measurements in cross-sections, showing tissue stiffness distributions across the undulations. A significant influence of age on the dimensions of the POVs was explored. The elastic modulus within the limbal niches for the ≥65-year-old group was significantly higher than that of the <65-year-old group.
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Affiliation(s)
- Ryan Dimmock
- School of Pharmacy and Bioengineering, Keele University, Stoke-on-Trent, UK
| | - Yilong Zhang
- School of Science and Engineering, University of Dundee, Dundee, UK
| | - Gibran F Butt
- Ophthalmology, Institute of Inflammation and Ageing, University of Birmingham, Birmingham, UK
| | - Saaeha Rauz
- Ophthalmology, Institute of Inflammation and Ageing, University of Birmingham, Birmingham, UK
| | - Zhihong Huang
- School of Science and Engineering, University of Dundee, Dundee, UK
| | - Ying Yang
- School of Pharmacy and Bioengineering, Keele University, Stoke-on-Trent, UK
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Verma N, Khare D, Poe AJ, Amador C, Ghiam S, Fealy A, Ebrahimi S, Shadrokh O, Song XY, Santiskulvong C, Mastali M, Parker S, Stotland A, Van Eyk JE, Ljubimov AV, Saghizadeh M. MicroRNA and Protein Cargos of Human Limbal Epithelial Cell-Derived Exosomes and Their Regulatory Roles in Limbal Stromal Cells of Diabetic and Non-Diabetic Corneas. Cells 2023; 12:2524. [PMID: 37947602 PMCID: PMC10649916 DOI: 10.3390/cells12212524] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2023] [Revised: 10/08/2023] [Accepted: 10/18/2023] [Indexed: 11/12/2023] Open
Abstract
Epithelial and stromal/mesenchymal limbal stem cells contribute to corneal homeostasis and cell renewal. Extracellular vesicles (EVs), including exosomes (Exos), can be paracrine mediators of intercellular communication. Previously, we described cargos and regulatory roles of limbal stromal cell (LSC)-derived Exos in non-diabetic (N) and diabetic (DM) limbal epithelial cells (LECs). Presently, we quantify the miRNA and proteome profiles of human LEC-derived Exos and their regulatory roles in N- and DM-LSC. We revealed some miRNA and protein differences in DM vs. N-LEC-derived Exos' cargos, including proteins involved in Exo biogenesis and packaging that may affect Exo production and ultimately cellular crosstalk and corneal function. Treatment by N-Exos, but not by DM-Exos, enhanced wound healing in cultured N-LSCs and increased proliferation rates in N and DM LSCs vs. corresponding untreated (control) cells. N-Exos-treated LSCs reduced the keratocyte markers ALDH3A1 and lumican and increased the MSC markers CD73, CD90, and CD105 vs. control LSCs. These being opposite to the changes quantified in wounded LSCs. Overall, N-LEC Exos have a more pronounced effect on LSC wound healing, proliferation, and stem cell marker expression than DM-LEC Exos. This suggests that regulatory miRNA and protein cargo differences in DM- vs. N-LEC-derived Exos could contribute to the disease state.
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Affiliation(s)
- Nagendra Verma
- Eye Program, Board of Governors Regenerative Medicine Institute, Cedars Sinai Medical Center, 8700 Beverly Boulevard, AHSP-A8104, Los Angeles, CA 90048, USA; (N.V.); (D.K.); (C.A.); (A.F.); (S.E.); (O.S.); (A.V.L.)
- Departments of Biomedical Sciences, Cedars Sinai Medical Center, Los Angeles, CA 90048, USA
| | - Drirh Khare
- Eye Program, Board of Governors Regenerative Medicine Institute, Cedars Sinai Medical Center, 8700 Beverly Boulevard, AHSP-A8104, Los Angeles, CA 90048, USA; (N.V.); (D.K.); (C.A.); (A.F.); (S.E.); (O.S.); (A.V.L.)
- Departments of Biomedical Sciences, Cedars Sinai Medical Center, Los Angeles, CA 90048, USA
- Division of Pediatric Blood and Marrow Transplantation & Cellular Therapy, University of Minnesota, Minneapolis, MN 55455, USA
| | - Adam J. Poe
- Eye Program, Board of Governors Regenerative Medicine Institute, Cedars Sinai Medical Center, 8700 Beverly Boulevard, AHSP-A8104, Los Angeles, CA 90048, USA; (N.V.); (D.K.); (C.A.); (A.F.); (S.E.); (O.S.); (A.V.L.)
- Departments of Biomedical Sciences, Cedars Sinai Medical Center, Los Angeles, CA 90048, USA
| | - Cynthia Amador
- Eye Program, Board of Governors Regenerative Medicine Institute, Cedars Sinai Medical Center, 8700 Beverly Boulevard, AHSP-A8104, Los Angeles, CA 90048, USA; (N.V.); (D.K.); (C.A.); (A.F.); (S.E.); (O.S.); (A.V.L.)
- Departments of Biomedical Sciences, Cedars Sinai Medical Center, Los Angeles, CA 90048, USA
| | - Sean Ghiam
- Eye Program, Board of Governors Regenerative Medicine Institute, Cedars Sinai Medical Center, 8700 Beverly Boulevard, AHSP-A8104, Los Angeles, CA 90048, USA; (N.V.); (D.K.); (C.A.); (A.F.); (S.E.); (O.S.); (A.V.L.)
- Departments of Biomedical Sciences, Cedars Sinai Medical Center, Los Angeles, CA 90048, USA
- Sackler School of Medicine, New York State/American Program of Tel Aviv University, Tel Aviv 6997801, Israel
| | - Andrew Fealy
- Eye Program, Board of Governors Regenerative Medicine Institute, Cedars Sinai Medical Center, 8700 Beverly Boulevard, AHSP-A8104, Los Angeles, CA 90048, USA; (N.V.); (D.K.); (C.A.); (A.F.); (S.E.); (O.S.); (A.V.L.)
- Departments of Biomedical Sciences, Cedars Sinai Medical Center, Los Angeles, CA 90048, USA
| | - Shaghaiegh Ebrahimi
- Eye Program, Board of Governors Regenerative Medicine Institute, Cedars Sinai Medical Center, 8700 Beverly Boulevard, AHSP-A8104, Los Angeles, CA 90048, USA; (N.V.); (D.K.); (C.A.); (A.F.); (S.E.); (O.S.); (A.V.L.)
- Departments of Biomedical Sciences, Cedars Sinai Medical Center, Los Angeles, CA 90048, USA
| | - Odelia Shadrokh
- Eye Program, Board of Governors Regenerative Medicine Institute, Cedars Sinai Medical Center, 8700 Beverly Boulevard, AHSP-A8104, Los Angeles, CA 90048, USA; (N.V.); (D.K.); (C.A.); (A.F.); (S.E.); (O.S.); (A.V.L.)
- Departments of Biomedical Sciences, Cedars Sinai Medical Center, Los Angeles, CA 90048, USA
| | - Xue-Ying Song
- Genomics Core, Cedars Sinai Medical Center, Los Angeles, CA 90048, USA; (X.-Y.S.); (C.S.)
| | - Chintda Santiskulvong
- Genomics Core, Cedars Sinai Medical Center, Los Angeles, CA 90048, USA; (X.-Y.S.); (C.S.)
| | - Mitra Mastali
- Advanced Clinical Biosystems Research Institute, The Smidt Heart Institute, Cedars Sinai Medical Center, Los Angeles, CA 90048, USA; (M.M.); (S.P.); (A.S.); (J.E.V.E.)
| | - Sarah Parker
- Advanced Clinical Biosystems Research Institute, The Smidt Heart Institute, Cedars Sinai Medical Center, Los Angeles, CA 90048, USA; (M.M.); (S.P.); (A.S.); (J.E.V.E.)
| | - Aleksandr Stotland
- Advanced Clinical Biosystems Research Institute, The Smidt Heart Institute, Cedars Sinai Medical Center, Los Angeles, CA 90048, USA; (M.M.); (S.P.); (A.S.); (J.E.V.E.)
| | - Jennifer E. Van Eyk
- Advanced Clinical Biosystems Research Institute, The Smidt Heart Institute, Cedars Sinai Medical Center, Los Angeles, CA 90048, USA; (M.M.); (S.P.); (A.S.); (J.E.V.E.)
| | - Alexander V. Ljubimov
- Eye Program, Board of Governors Regenerative Medicine Institute, Cedars Sinai Medical Center, 8700 Beverly Boulevard, AHSP-A8104, Los Angeles, CA 90048, USA; (N.V.); (D.K.); (C.A.); (A.F.); (S.E.); (O.S.); (A.V.L.)
- Departments of Biomedical Sciences, Cedars Sinai Medical Center, Los Angeles, CA 90048, USA
- Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA 90024, USA
| | - Mehrnoosh Saghizadeh
- Eye Program, Board of Governors Regenerative Medicine Institute, Cedars Sinai Medical Center, 8700 Beverly Boulevard, AHSP-A8104, Los Angeles, CA 90048, USA; (N.V.); (D.K.); (C.A.); (A.F.); (S.E.); (O.S.); (A.V.L.)
- Departments of Biomedical Sciences, Cedars Sinai Medical Center, Los Angeles, CA 90048, USA
- Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA 90024, USA
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Moreno IY, Parsaie A, Gesteira TF, Coulson-Thomas VJ. Characterization of the Limbal Epithelial Stem Cell Niche. Invest Ophthalmol Vis Sci 2023; 64:48. [PMID: 37906057 PMCID: PMC10619699 DOI: 10.1167/iovs.64.13.48] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2023] [Accepted: 10/09/2023] [Indexed: 11/02/2023] Open
Abstract
Purpose Limbal epithelial stem cells (LESCs) reside within a LSC niche (LSCN). We recently identified that hyaluronan (HA) is a major constituent of the LSCN, and that HA is necessary for maintaining LESCs in the "stem cell" state, both in vitro and in vivo. Herein, we characterized the LSCN to identify key components of the HA-specific LSCN. Methods The cornea and limbal rim were dissected from mouse corneas, subjected to mRNA extraction, and sequenced using a NextSeq 500 (Illumina) and data processed using CLC Genomics Workbench 20 (Qiagen) and the STRING database to identify key components of the LSCN. Their expression was confirmed by real-time PCR, Western blotting, and immunohistochemistry. Furthermore, the differential expression of key compounds in different corneal cell types were determined with single-cell RNA sequencing. Results We identified that the hyaladherins inter-alpha-inhibitor (IαI), TSG-6 and versican are highly expressed in the limbus. Specifically, HA/HC complexes are present in the LSCN, in the stroma underlying the limbal epithelium, and surrounding the limbal vasculature. For IαI, heavy chains 5 and 2 (HC5 and HC2) were found to be the most highly expressed HCs in the mouse and human limbus and were associate with HA-forming HA/HC-specific matrices. Conclusions The LSCN contains HA/HC complexes, which have been previously correlated with stem cell niches. The identification of HA/HC complexes in the LSCN could serve as a new therapeutic avenue for treating corneal pathology. Additionally, HA/HC complexes could be used as a substrate for culturing LESCs before LESC transplantation.
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Affiliation(s)
- Isabel Y. Moreno
- College of Optometry, University of Houston, Houston, Texas, United States
| | - Arian Parsaie
- College of Optometry, University of Houston, Houston, Texas, United States
- College of Natural Science and Mathematics, University of Houston, Houston, Texas, United States
| | - Tarsis F. Gesteira
- College of Optometry, University of Houston, Houston, Texas, United States
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Bains KK, Young RD, Koudouna E, Lewis PN, Quantock AJ. Cell-Cell and Cell-Matrix Interactions at the Presumptive Stem Cell Niche of the Chick Corneal Limbus. Cells 2023; 12:2334. [PMID: 37830548 PMCID: PMC10571731 DOI: 10.3390/cells12192334] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2023] [Revised: 09/16/2023] [Accepted: 09/19/2023] [Indexed: 10/14/2023] Open
Abstract
(1) Background: Owing to its ready availability and ease of acquisition, developing chick corneal tissue has long been used for research purposes. Here, we seek to ascertain the three-dimensional microanatomy and spatiotemporal interrelationships of the cells (epithelial and stromal), extracellular matrix, and vasculature at the corneo-scleral limbus as the site of the corneal stem cell niche of the chicken eye. (2) Methods: The limbus of developing (i.e., embryonic days (E) 16 and 18, just prior to hatch) and mature chicken eyes was imaged using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and the volume electron microscopy technique, serial-block face SEM (SBF-SEM), the latter technique allowing us to generate three-dimensional reconstructions from data sets of up to 1000 serial images; (3) Results: Data revealed that miniature limbal undulations of the embryonic basement membrane, akin to Palisades of Vogt (PoV), matured into distinct invaginations of epithelial cells that extended proximally into a vascularized limbal stroma. Basal limbal epithelial cells, moreover, occasionally exhibited a high nuclear:cytoplasmic ratio, which is a characteristic feature of stem cells. SBF-SEM identified direct cell-cell associations between corneal epithelial and stromal cells at the base of structures akin to limbal crypts (LCs), with cord-like projections of extracellular matrix extending from the basal epithelial lamina into the subjacent stroma, where they made direct contact with stomal cells in the immature limbus. (4) Conclusion: Similarities with human tissue suggest that the corneal limbus of the mature chicken eye is likely the site of a corneal stem cell niche. The ability to study embryonic corneas pre-hatch, where we see characteristic niche-like features emerge, thus provides an opportunity to chart the development of the limbal stem cell niche of the cornea.
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Affiliation(s)
| | | | | | | | - Andrew J. Quantock
- Structural Biophysics Group, School of Optometry and Vision Sciences, Cardiff University, Maindy Road, Cardiff CF10 3AT, UK; (K.K.B.); (R.D.Y.); (E.K.); (P.N.L.)
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6
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Yam GHF, Pi S, Du Y, Mehta JS. Posterior corneoscleral limbus: Architecture, stem cells, and clinical implications. Prog Retin Eye Res 2023; 96:101192. [PMID: 37392960 DOI: 10.1016/j.preteyeres.2023.101192] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2023] [Revised: 06/22/2023] [Accepted: 06/23/2023] [Indexed: 07/03/2023]
Abstract
The limbus is a transition from the cornea to conjunctiva and sclera. In human eyes, this thin strip has a rich variation of tissue structures and composition, typifying a change from scleral irregularity and opacity to corneal regularity and transparency; a variation from richly vascularized conjunctiva and sclera to avascular cornea; the neural passage and drainage of aqueous humor. The limbal stroma is enriched with circular fibres running parallel to the corneal circumference, giving its unique role in absorbing small pressure changes to maintain corneal curvature and refractivity. It contains specific niches housing different types of stem cells for the corneal epithelium, stromal keratocytes, corneal endothelium, and trabecular meshwork. This truly reflects the important roles of the limbus in ocular physiology, and the limbal functionality is crucial for corneal health and the entire visual system. Since the anterior limbus containing epithelial structures and limbal epithelial stem cells has been extensively reviewed, this article is focused on the posterior limbus. We have discussed the structural organization and cellular components of the region beneath the limbal epithelium, the characteristics of stem cell types: namely corneal stromal stem cells, endothelial progenitors and trabecular meshwork stem cells, and recent advances leading to the emergence of potential cell therapy options to replenish their respective mature cell types and to correct defects causing corneal abnormalities. We have reviewed different clinical disorders associated with defects of the posterior limbus and summarized the available preclinical and clinical evidence about the developing topic of cell-based therapy for corneal disorders.
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Affiliation(s)
- Gary Hin-Fai Yam
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA, USA; Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore; McGowan Institute for Regenerative Medicine, Pittsburgh, PA, USA.
| | - Shaohua Pi
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA, USA
| | - Yiqin Du
- Department of Ophthalmology, University of South Florida, Tampa, FL, USA
| | - Jodhbir S Mehta
- Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore; Department of Cornea and External Eye Disease, Singapore National Eye Centre, Singapore; Ophthalmology and Visual Sciences Academic Clinical Program, Duke-National University of Singapore (NUS) Medical School, Singapore.
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Polisetti N, Reinhard T, Schlunck G. Efficient Isolation and Expansion of Limbal Melanocytes for Tissue Engineering. Int J Mol Sci 2023; 24:ijms24097827. [PMID: 37175529 PMCID: PMC10177947 DOI: 10.3390/ijms24097827] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2023] [Revised: 04/13/2023] [Accepted: 04/21/2023] [Indexed: 05/15/2023] Open
Abstract
Limbal melanocytes (LMs) are found in the corneoscleral limbus basal epithelial layer and interact with neighboring limbal epithelial progenitor cells. The difficulty of isolating and cultivating LMs is due to the small fraction of LMs in the overall limbal population and the frequent contamination of primary cultures by other cell types. This has limited the research on freshly isolated LMs and the investigation of their biological significance in the maintenance of the limbal stem cell niche. Here, we describe an optimized protocol for the efficient isolation and expansion of LMs from cadaveric corneal limbal tissue using CD90 and CD117 as selective markers in fluorescence-activated cell sorting to obtain a pure population of LMs (CD90- CD117+) with self-renewal capacity and sustained melanin production. The isolation of pure LMs from a single preparation enables direct transcriptomic and proteomic analyses, as well as functional studies on freshly isolated LMs, which can be considered the proper counterparts of LMs in vivo and have potential applications in tissue engineering.
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Affiliation(s)
- Naresh Polisetti
- Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany
| | - Thomas Reinhard
- Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany
| | - Günther Schlunck
- Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany
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Bonnet C, Ruiz M, Gonzalez S, Tseng CH, Bourges JL, Behar-Cohen F, Deng SX. Single mRNA detection of Wnt signaling pathway in the human limbus. Exp Eye Res 2023; 229:109337. [PMID: 36702232 DOI: 10.1016/j.exer.2022.109337] [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: 08/16/2022] [Revised: 11/02/2022] [Accepted: 11/23/2022] [Indexed: 01/24/2023]
Abstract
Limbal epithelial stem/progenitor cells (LSCs) are adult stem cells located at the limbus, tightly regulated by their close microenvironment. It has been shown that Wnt signaling pathway is crucial for LSCs regulation. Previous differential gene profiling studies confirmed the preferential expression of specific Wnt ligands (WNT2, WNT6, WNT11, WNT16) and Wnt inhibitors (DKK1, SFRP5, WIF1, FRZB) in the limbal region compared to the cornea. Among all frizzled receptors, frizzled7 (Fzd7) was found to be preferentially expressed in the basal limbal epithelium. However, the exact localization of Wnt signaling molecules-producing cells in the limbus remains unknown. The current study aims to evaluate the in situ spatial expression of these 4 Wnt ligands, 4 Wnt inhibitors, and Fzd7. Wnt ligands, DKK1, and Fzd7 expression were scattered within the limbal epithelium, at a higher abundance in the basal layer than the superficial layer. SFRP5 expression was diffuse among the limbal epithelium, whereas WIF1 and FRZB expression was clustered at the basal limbal epithelial layer corresponding to the areas of high levels of Fzd7 expression. Quantitation of the fluorescence intensity showed that all 4 Wnt ligands, 3 Wnt inhibitors (WIF1, DKK1, FRZB), and Fzd7 were highly expressed at the basal layer of the limbus, then in a decreasing gradient toward the superficial layer (P < 0.05). The expression levels of all 4 Wnt ligands, FRZB, and Fzd7 in the basal epithelial layer were higher in the limbus than the central cornea (P < 0.05). All 4 Wnt ligands, 4 Wnt inhibitors, and Fzd7 were also highly expressed in the limbal stroma immediately below the epithelium but not in the corneal stroma (P < 0.05). In addition, Fzd7 had a preferential expression in the superior limbus compared to other limbal quadrants (P < 0.05). Taken together, the unique expression patterns of the Wnt molecules in the limbus suggests the involvement of both paracrine and autocrine effects in LSCs regulation, and a fine balance between Wnt activators and inhibitors to govern LSC fate.
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Affiliation(s)
- Clémence Bonnet
- Stein Eye Institute, Department of Ophthalmology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA; INSERM, UMRS1138, Team 17, From Physiopathology of Ocular Diseases to Clinical Development, Centre de Recherche des Cordeliers, Paris University, And Cornea Departement, Cochin Hospital, AP-HP, F-75014, Paris, France
| | - Maxime Ruiz
- Stein Eye Institute, Department of Ophthalmology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA
| | - Sheyla Gonzalez
- Stein Eye Institute, Department of Ophthalmology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA
| | - Chi-Hong Tseng
- David Geffen School of Medicine, Division of General Internal Medicine and Health Services Research, University of California, Los Angeles, USA
| | - Jean-Louis Bourges
- INSERM, UMRS1138, Team 17, From Physiopathology of Ocular Diseases to Clinical Development, Centre de Recherche des Cordeliers, Paris University, And Cornea Departement, Cochin Hospital, AP-HP, F-75014, Paris, France
| | - Francine Behar-Cohen
- INSERM, UMRS1138, Team 17, From Physiopathology of Ocular Diseases to Clinical Development, Centre de Recherche des Cordeliers, Paris University, And Cornea Departement, Cochin Hospital, AP-HP, F-75014, Paris, France
| | - Sophie X Deng
- Stein Eye Institute, Department of Ophthalmology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA; Molecular Biology Institute, University of California, Los Angeles, CA, USA.
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Bayati V, Nasrolahi A, Zamani M, Nejaddehbashi F. Evaluation of the effects of ethanol and mitomycin on survival of rat limbal stem cells: an in vitro study. Int Ophthalmol 2023:10.1007/s10792-023-02650-0. [PMID: 36877318 DOI: 10.1007/s10792-023-02650-0] [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: 09/12/2022] [Accepted: 02/19/2023] [Indexed: 03/07/2023]
Abstract
PURPOSE Ethanol and mitomycin C (MMC) are clinically used to treat corneal diseases such as LASEK and LASIK surgery. In this study, we investigated the effects of time-dependent alcohol and MMC in cultured rat limbal stem cells (LSCs) to determine the appropriate time for the use of this compound in the clinical setting. METHODS LSCs (N = 10 eyes) isolated from male Wistar rats were cultured and characterized; then, isolates were divided into three groups. One group was exposed to a 20% concentration of ethanol for 5, 10, 15, 20, 25, and 30 s, and cell viability was assessed one, three, and five days following ethanol exposure using an MTT assay. To investigate the effect of MMC, cells in the second group were treated with 0.02% MMC in various periods (i.e., 15 s, 30 s, 60 s, 90 s, and 120 s) and time-dependent responses of cultured LSCs were recorded. Cells in the third group were co-treated with ethanol and MMC; then, dose and time dependency was evaluated. RESULTS In comparison with the viable cells in the control group, ethanol markedly decreased the viability of cells in a time-dependent manner in days one and three. On day five, the viability of LSCs was improved significantly (p < 0.05) in comparison with day one. The number of viable progenitor cells was significantly decreased after MMC treatment in a time-dependent manner, as determined by the MTT assay (p < 0.001). The use of mitomycin, along with alcohol, decreased cell viability in all groups treated with ethanol + MMC compared to the control on days one, three, and five (p < 0.0001). CONCLUSIONS Our findings suggest that ethanol and MMC reduced cell viability in cultured LSCs in a time-dependent manner. In addition, when LSCs were exposed to alcohol alone, they had a better recovery process within 5 days in comparison to when exposed to mitomycin alone or mitomycin + alcohol.
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Affiliation(s)
- Vahid Bayati
- Cellular and Molecular Research Center, Medical Basic Sciences Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.,Department of Anatomical Sciences, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Ava Nasrolahi
- Infectious Ophthalmologic Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Mitra Zamani
- Infectious Ophthalmologic Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Fereshteh Nejaddehbashi
- Cellular and Molecular Research Center, Medical Basic Sciences Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. .,Infectious Ophthalmologic Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
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10
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In situ transduction of cells in human corneal limbus using adeno-associated viruses: an ex vivo study. Sci Rep 2022; 12:22481. [PMID: 36577775 PMCID: PMC9797548 DOI: 10.1038/s41598-022-26926-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2022] [Accepted: 12/21/2022] [Indexed: 12/30/2022] Open
Abstract
This study aimed to evaluate the efficacy of in situ adeno-associated virus (AAV)-mediated gene delivery into the human corneal limbal region via targeted sub-limbal injection technique. Human cadaveric corneal tissues were fixed on an artificial anterior chamber. Feasibility of sub-limbal injection technique was tested using trypan blue and black India ink. An enhanced green fluorescent protein (eGFP) encoding AAV DJ was injected into sub-limbal region. After AAV injection, corneal tissues were incubated in air-lift culture and prepared for immunohistochemical analysis. Cell survivial and expression of eGFP, stem cell markers (p63α and cytokeratin 19 (KRT19)), and differentiation marker cytokeratin 3 (KRT3) were evaluated using confocal microscopy. Both trypan blue and black India ink stained and were retained sub-limbally establishing specificity of the injection technique. Immunohistochemical analysis of corneas injected with AAV DJ-eGFP indicated that AAV-transduced cells in the limbal region co-express eGFP, p63α, and KRT19 and that these transduced cells were capable of differentiating to KRT3 postitive corneal epithelial cells. Our sub-limbal injection technique can target cells in the human limbus in a reproducible and efficient manner. Thus, we demonstrate that in situ injection of corneal limbus may provide a feasible mode of genetic therapy for corneal disorders with an epithelial etiology.
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11
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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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12
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Puri S, Moreno IY, Sun M, Verma S, Lin X, Gesteira TF, Coulson-Thomas VJ. Hyaluronan supports the limbal stem cell phenotype during ex vivo culture. Stem Cell Res Ther 2022; 13:384. [PMID: 35907870 PMCID: PMC9338506 DOI: 10.1186/s13287-022-03084-8] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2022] [Accepted: 07/21/2022] [Indexed: 11/29/2022] Open
Abstract
BACKGROUND Hyaluronan (HA) has previously been identified as an integral component of the limbal stem cell niche in vivo. In this study, we investigated whether a similar HA matrix is also expressed in vitro providing a niche supporting limbal epithelial stem cells (LESCs) during ex vivo expansion. We also investigated whether providing exogenous HA in vitro is beneficial to LESCs during ex vivo expansion. METHOD Human LESCs (hLESCs) were isolated from donor corneas and a mouse corneal epithelial progenitor cell line (TKE2) was obtained. The HA matrix was identified surrounding LESCs in vitro using immunocytochemistry, flow cytometry and red blood exclusion assay. Thereafter, LESCs were maintained on HA coated dishes or in the presence of HA supplemented in the media, and viability, proliferation, cell size, colony formation capabilities and expression of putative stem cell markers were compared with cells maintained on commonly used coated dishes. RESULTS hLESCs and TKE2 cells express an HA-rich matrix in vitro, and this matrix is essential for maintaining LESCs. Further supplying exogenous HA, as a substrate and supplemented to the media, increases LESC proliferation, colony formation capabilities and the expression levels of putative limbal stem cell markers. CONCLUSION Our data show that both exogenous and endogenous HA help to maintain the LESC phenotype. Exogenous HA provides improved culture conditions for LESC during ex vivo expansion. Thus, HA forms a favorable microenvironment for LESCs during ex vivo expansion and, therefore, could be considered as an easy and cost-effective substrate and/or supplement for culturing LESCs in the clinic.
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Affiliation(s)
- Sudan Puri
- College of Optometry, University of Houston, Houston, TX, USA
| | - Isabel Y Moreno
- College of Optometry, University of Houston, Houston, TX, USA
| | - Mingxia Sun
- College of Optometry, University of Houston, Houston, TX, USA
| | - Sudhir Verma
- College of Optometry, University of Houston, Houston, TX, USA
- Department of Zoology, Deen Dayal Upadhyaya College, University of Delhi, New Delhi, India
| | - Xiao Lin
- College of Optometry, University of Houston, Houston, TX, USA
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13
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Tavakkoli F, Damala M, Koduri MA, Gangadharan A, Rai AK, Dash D, Basu S, Singh V. Transcriptomic Profiling of Human Limbus-Derived Stromal/Mesenchymal Stem Cells-Novel Mechanistic Insights into the Pathways Involved in Corneal Wound Healing. Int J Mol Sci 2022; 23:ijms23158226. [PMID: 35897793 PMCID: PMC9368612 DOI: 10.3390/ijms23158226] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2022] [Revised: 07/16/2022] [Accepted: 07/16/2022] [Indexed: 01/27/2023] Open
Abstract
Limbus-derived stromal/mesenchymal stem cells (LMSCs) are vital for corneal homeostasis and wound healing. However, despite multiple pre-clinical and clinical studies reporting the potency of LMSCs in avoiding inflammation and scarring during corneal wound healing, the molecular basis for the ability of LMSCs remains unknown. This study aimed to uncover the factors and pathways involved in LMSC-mediated corneal wound healing by employing RNA-Sequencing (RNA-Seq) in human LMSCs for the first time. We characterized the cultured LMSCs at the stages of initiation (LMSC−P0) and pure population (LMSC−P3) and subjected them to RNA-Seq to identify the differentially expressed genes (DEGs) in comparison to native limbus and cornea, and scleral tissues. Of the 28,000 genes detected, 7800 DEGs were subjected to pathway-specific enrichment Gene Ontology (GO) analysis. These DEGs were involved in Wnt, TGF-β signaling pathways, and 16 other biological processes, including apoptosis, cell motility, tissue remodeling, and stem cell maintenance, etc. Two hundred fifty-four genes were related to wound healing pathways. COL5A1 (11.81 ± 0.48) and TIMP1 (20.44 ± 0.94) genes were exclusively up-regulated in LMSC−P3. Our findings provide new insights involved in LMSC-mediated corneal wound healing.
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Affiliation(s)
- Fatemeh Tavakkoli
- Prof. Brien Holden Eye Research Center, LV Prasad Eye Institute, Hyderabad 500034, India; (F.T.); (M.D.); (M.A.K.); (S.B.)
- Center for Genetic Disorders, Banaras Hindu University, Varanasi 221005, India;
| | - Mukesh Damala
- Prof. Brien Holden Eye Research Center, LV Prasad Eye Institute, Hyderabad 500034, India; (F.T.); (M.D.); (M.A.K.); (S.B.)
- School of Life Sciences, University of Hyderabad, Hyderabad 500046, India
| | - Madhuri Amulya Koduri
- Prof. Brien Holden Eye Research Center, LV Prasad Eye Institute, Hyderabad 500034, India; (F.T.); (M.D.); (M.A.K.); (S.B.)
- Manipal Academy of Higher Education, Manipal 576104, India
| | - Abhilash Gangadharan
- CSIR-Institute of Genomics and Integrative Biology, Mathura Road Campus, New Delhi 110025, India; (A.G.); (D.D.)
| | - Amit K. Rai
- Center for Genetic Disorders, Banaras Hindu University, Varanasi 221005, India;
| | - Debasis Dash
- CSIR-Institute of Genomics and Integrative Biology, Mathura Road Campus, New Delhi 110025, India; (A.G.); (D.D.)
| | - Sayan Basu
- Prof. Brien Holden Eye Research Center, LV Prasad Eye Institute, Hyderabad 500034, India; (F.T.); (M.D.); (M.A.K.); (S.B.)
- Center for Ocular Regeneration (CORE), Prof. Brien Holden Eye Research Center, LV Prasad Eye Institute, Hyderabad 500034, India
| | - Vivek Singh
- Prof. Brien Holden Eye Research Center, LV Prasad Eye Institute, Hyderabad 500034, India; (F.T.); (M.D.); (M.A.K.); (S.B.)
- Center for Ocular Regeneration (CORE), Prof. Brien Holden Eye Research Center, LV Prasad Eye Institute, Hyderabad 500034, India
- Correspondence: ; Tel.: +91-40-6810-2286
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14
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Polisetti N, Sharaf L, Reinhard T, Schlunck G. Isolation and ex vivo Expansion of Limbal Mesenchymal Stromal Cells. Bio Protoc 2022; 12:e4471. [PMID: 35978577 PMCID: PMC9350925 DOI: 10.21769/bioprotoc.4471] [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: 04/04/2022] [Revised: 06/08/2022] [Accepted: 06/09/2022] [Indexed: 12/29/2022] Open
Abstract
Limbal mesenchymal stromal cells (LMSC), a cellular component of the limbal stem cell niche, have the capability of determining the fate of limbal epithelial progenitor cells (LEPC), which are responsible for the homeostasis of corneal epithelium. However, the isolation of these LMSC has proven to be difficult due to the small fraction of LMSC in the total limbal population, and primary cultures are always hampered by contamination with other cell types. We recently published the efficient isolation and functional characterization of LMSC from the human corneal limbus using CD90 as a selective marker. We observed that flow sorting yielded a pure population of LMSC with superior self-renewal capacity and transdifferentiation potential, and supported the maintenance of the LEPC phenotype. Here, we describe an optimized protocol for the isolation of LMSC from cadaveric corneal limbal tissue by combined collagenase digestion and flow sorting with expansion of LMSC on plastic. Graphical abstract.
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Affiliation(s)
- Naresh Polisetti
- Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany
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*For correspondence:
| | - Lyne Sharaf
- Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Thomas Reinhard
- Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Günther Schlunck
- Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany
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15
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Efficient Isolation and Functional Characterization of Niche Cells from Human Corneal Limbus. Int J Mol Sci 2022; 23:ijms23052750. [PMID: 35269891 PMCID: PMC8911296 DOI: 10.3390/ijms23052750] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2022] [Revised: 02/23/2022] [Accepted: 02/28/2022] [Indexed: 12/13/2022] Open
Abstract
The fate decision of limbal epithelial progenitor cells (LEPC) at the human corneal limbus is determined by the surrounding microenvironment with limbal niche cells (LNC) as one of its essential components. Research on freshly isolated LNC which mainly include limbal mesenchymal stromal cells (LMSC) and limbal melanocytes (LM) has been hampered by a lack of efficient protocols to isolate and purify these cells. We devised a protocol for rapid retrieval of pure LMSC, LM and LEPC populations by collagenase digestion of limbal tissue and subsequent fluorescence-activated cell sorting (FACS) using antibodies against CD90 and CD117. The sorted cells were characterized by immunophenotyping and functional assays. The effects of LMSC and LM on LEPC were studied in 3D co-cultures and LEPC differentiation status was assessed by immunohistochemistry. Enzymatic digestion and flow sorting yielded pure populations of LMSC (CD117−CD90+), LM (CD117+CD90−), and LEPC (CD117−CD90−). The LMSC exhibited self-renewal capacity (55.0 ± 4.6 population doublings), expressed mesenchymal stem cell markers (CD73, CD90, CD105, and CD44), and transdifferentiated to adipocytes, osteocytes, or chondrocytes. The LM exhibited self-renewal capacity and sustained melanin production. The sorted LEPC expressed epithelial progenitor markers (CK14, CK19, and CK15) and showed a colony-forming ability. Co-cultivation of LMSC and LM with LEPC resulted in a 4–5-layered stratified epithelium and supported the preservation of a LEPC phenotype, as reflected by increased p63+ and Ki67+ cells and decreased CK12+ cells compared with LEPC monocultures. A highly efficient isolation of pure LM, LMSC, and LEPC populations from a single preparation may allow for direct transcriptomic and proteomic profiling as well as functional studies on native unpassaged LNC, which can be considered as proper equivalents of LNC in vivo. The developed biomimetic 3D co-culture method could provide an experimental model for investigating the functional role of LNC in the limbal stem cell niche.
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16
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Elhusseiny AM, Soleimani M, Eleiwa TK, ElSheikh RH, Frank CR, Naderan M, Yazdanpanah G, Rosenblatt MI, Djalilian AR. OUP accepted manuscript. Stem Cells Transl Med 2022; 11:259-268. [PMID: 35303110 PMCID: PMC8968724 DOI: 10.1093/stcltm/szab028] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2021] [Accepted: 09/06/2021] [Indexed: 11/24/2022] Open
Abstract
The corneal epithelium serves to protect the underlying cornea from the external environment and is essential for corneal transparency and optimal visual function. Regeneration of this epithelium is dependent on a population of stem cells residing in the basal layer of the limbus, the junction between the cornea and the sclera. The limbus provides the limbal epithelial stem cells (LESCs) with an optimal microenvironment, the limbal niche, which strictly regulates their proliferation and differentiation. Disturbances to the LESCs and/or their niche can lead to the pathologic condition known as limbal stem cell deficiency (LSCD) whereby the corneal epithelium is not generated effectively. This has deleterious effects on the corneal and visual function, due to impaired healing and secondary corneal opacification. In this concise review, we summarize the characteristics of LESCs and their niche, and present the current and future perspectives in the management of LSCD with an emphasis on restoring the function of the limbal niche.
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Affiliation(s)
- Abdelrahman M Elhusseiny
- Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, IL, USA
- Department of Ophthalmology, Harvey and Bernice Jones Eye Institute, University of Arkansas for Medical Sciences, Little Rock, AR, USA
| | - Mohammad Soleimani
- Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, IL, USA
| | - Taher K Eleiwa
- Department of Ophthalmology, Faculty of Medicine, Benha University, Benha, Egypt
| | - Reem H ElSheikh
- Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, IL, USA
| | - Charles R Frank
- Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, IL, USA
| | - Morteza Naderan
- Department of Ophthalmology, Farabi Eye Hospital, Tehran University of Medical Sciences, Tehran, Iran
| | - Ghasem Yazdanpanah
- Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, IL, USA
| | - Mark I Rosenblatt
- Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, IL, USA
| | - Ali R Djalilian
- Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, IL, USA
- Corresponding author: Ali R. Djalilian, Cornea Service, Stem Cell Therapy and Corneal Tissue Engineering Laboratory, Illinois Eye and Ear Infirmary, 1855 W. Taylor Street, M/C 648, Chicago, IL 60612, USA.
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17
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Bonnet C, González S, Roberts JS, Robertson SYT, Ruiz M, Zheng J, Deng SX. Human limbal epithelial stem cell regulation, bioengineering and function. Prog Retin Eye Res 2021; 85:100956. [PMID: 33676006 PMCID: PMC8428188 DOI: 10.1016/j.preteyeres.2021.100956] [Citation(s) in RCA: 47] [Impact Index Per Article: 11.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2020] [Revised: 02/21/2021] [Accepted: 02/26/2021] [Indexed: 12/13/2022]
Abstract
The corneal epithelium is continuously renewed by limbal stem/progenitor cells (LSCs), a cell population harbored in a highly regulated niche located at the limbus. Dysfunction and/or loss of LSCs and their niche cause limbal stem cell deficiency (LSCD), a disease that is marked by invasion of conjunctival epithelium into the cornea and results in failure of epithelial wound healing. Corneal opacity, pain, loss of vision, and blindness are the consequences of LSCD. Successful treatment of LSCD depends on accurate diagnosis and staging of the disease and requires restoration of functional LSCs and their niche. This review highlights the major advances in the identification of potential LSC biomarkers and components of the LSC niche, understanding of LSC regulation, methods and regulatory standards in bioengineering of LSCs, and diagnosis and staging of LSCD. Overall, this review presents key points for researchers and clinicians alike to consider in deepening the understanding of LSC biology and improving LSCD therapies.
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Affiliation(s)
- Clémence Bonnet
- Cornea Division, Stein Eye Institute, University of California, Los Angeles, CA, 90095, USA; Cornea Department, Paris University, Cochin Hospital, AP-HP, F-75014, Paris, France
| | - Sheyla González
- Cornea Division, Stein Eye Institute, University of California, Los Angeles, CA, 90095, USA
| | - JoAnn S Roberts
- Cornea Division, Stein Eye Institute, University of California, Los Angeles, CA, 90095, USA
| | - Sarah Y T Robertson
- Cornea Division, Stein Eye Institute, University of California, Los Angeles, CA, 90095, USA
| | - Maxime Ruiz
- Cornea Division, Stein Eye Institute, University of California, Los Angeles, CA, 90095, USA
| | - Jie Zheng
- Basic Science Division, Stein Eye Institute, University of California, Los Angeles, CA, 90095, USA
| | - Sophie X Deng
- Cornea Division, Stein Eye Institute, University of California, Los Angeles, CA, 90095, USA.
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18
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Hidalgo-Alvarez V, Dhowre HS, Kingston OA, Sheridan CM, Levis HJ. Biofabrication of Artificial Stem Cell Niches in the Anterior Ocular Segment. Bioengineering (Basel) 2021; 8:135. [PMID: 34677208 PMCID: PMC8533470 DOI: 10.3390/bioengineering8100135] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2021] [Revised: 09/27/2021] [Accepted: 09/27/2021] [Indexed: 11/16/2022] Open
Abstract
The anterior segment of the eye is a complex set of structures that collectively act to maintain the integrity of the globe and direct light towards the posteriorly located retina. The eye is exposed to numerous physical and environmental insults such as infection, UV radiation, physical or chemical injuries. Loss of transparency to the cornea or lens (cataract) and dysfunctional regulation of intra ocular pressure (glaucoma) are leading causes of worldwide blindness. Whilst traditional therapeutic approaches can improve vision, their effect often fails to control the multiple pathological events that lead to long-term vision loss. Regenerative medicine approaches in the eye have already had success with ocular stem cell therapy and ex vivo production of cornea and conjunctival tissue for transplant recovering patients' vision. However, advancements are required to increase the efficacy of these as well as develop other ocular cell therapies. One of the most important challenges that determines the success of regenerative approaches is the preservation of the stem cell properties during expansion culture in vitro. To achieve this, the environment must provide the physical, chemical and biological factors that ensure the maintenance of their undifferentiated state, as well as their proliferative capacity. This is likely to be accomplished by replicating the natural stem cell niche in vitro. Due to the complex nature of the cell microenvironment, the creation of such artificial niches requires the use of bioengineering techniques which can replicate the physico-chemical properties and the dynamic cell-extracellular matrix interactions that maintain the stem cell phenotype. This review discusses the progress made in the replication of stem cell niches from the anterior ocular segment by using bioengineering approaches and their therapeutic implications.
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Affiliation(s)
- Veronica Hidalgo-Alvarez
- Institute of Biological Chemistry, Biophysics and Bioengineering, School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh EH14 4AS, UK
| | - Hala S. Dhowre
- Department of Eye and Vision Science, Institute of Life Course and Medical Sciences, University of Liverpool, Liverpool L7 8TX, UK; (H.S.D.); (O.A.K.)
| | - Olivia A. Kingston
- Department of Eye and Vision Science, Institute of Life Course and Medical Sciences, University of Liverpool, Liverpool L7 8TX, UK; (H.S.D.); (O.A.K.)
| | - Carl M. Sheridan
- Department of Eye and Vision Science, Institute of Life Course and Medical Sciences, University of Liverpool, Liverpool L7 8TX, UK; (H.S.D.); (O.A.K.)
| | - Hannah J. Levis
- Department of Eye and Vision Science, Institute of Life Course and Medical Sciences, University of Liverpool, Liverpool L7 8TX, UK; (H.S.D.); (O.A.K.)
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19
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Latta L, Figueiredo FC, Ashery-Padan R, Collinson JM, Daniels J, Ferrari S, Szentmáry N, Solá S, Shalom-Feuerstein R, Lako M, Xapelli S, Aberdam D, Lagali N. Pathophysiology of aniridia-associated keratopathy: Developmental aspects and unanswered questions. Ocul Surf 2021; 22:245-266. [PMID: 34520870 DOI: 10.1016/j.jtos.2021.09.001] [Citation(s) in RCA: 29] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2021] [Revised: 07/19/2021] [Accepted: 09/08/2021] [Indexed: 12/13/2022]
Abstract
Aniridia, a rare congenital disease, is often characterized by a progressive, pronounced limbal insufficiency and ocular surface pathology termed aniridia-associated keratopathy (AAK). Due to the characteristics of AAK and its bilateral nature, clinical management is challenging and complicated by the multiple coexisting ocular and systemic morbidities in aniridia. Although it is primarily assumed that AAK originates from a congenital limbal stem cell deficiency, in recent years AAK and its pathogenesis has been questioned in the light of new evidence and a refined understanding of ocular development and the biology of limbal stem cells (LSCs) and their niche. Here, by consolidating and comparing the latest clinical and preclinical evidence, we discuss key unanswered questions regarding ocular developmental aspects crucial to AAK. We also highlight hypotheses on the potential role of LSCs and the ocular surface microenvironment in AAK. The insights thus gained lead to a greater appreciation for the role of developmental and cellular processes in the emergence of AAK. They also highlight areas for future research to enable a deeper understanding of aniridia, and thereby the potential to develop new treatments for this rare but blinding ocular surface disease.
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Affiliation(s)
- L Latta
- Dr. Rolf. M. Schwiete Center for Limbal Stem Cell and Aniridia Research, Saarland University, Homburg, Saar, Germany; Department of Ophthalmology, Saarland University Medical Center, Homburg, Saar, Germany.
| | - F C Figueiredo
- Department of Ophthalmology, Royal Victoria Infirmary, Newcastle Upon Tyne, United Kingdom
| | - R Ashery-Padan
- Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, 69978, Israel
| | - J M Collinson
- School of Medicine, Medical Sciences and Nutrition, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen, AB25 2ZD, United Kingdom
| | - J Daniels
- Cells for Sight, UCL Institute of Ophthalmology, University College London, London, EC1V 9EL, UK
| | - S Ferrari
- The Veneto Eye Bank Foundation, Venice, Italy
| | - N Szentmáry
- Dr. Rolf. M. Schwiete Center for Limbal Stem Cell and Aniridia Research, Saarland University, Homburg, Saar, Germany
| | - S Solá
- Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon, Portugal
| | - R Shalom-Feuerstein
- Department of Genetics and Developmental Biology, The Rappaport Faculty of Medicine and Research Institute, Technion - Israel Institute of Technology, Haifa, Israel
| | - M Lako
- Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle Upon Tyne, UK
| | - S Xapelli
- Instituto Farmacologia e Neurociências, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal; Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal
| | - D Aberdam
- Centre de Recherche des Cordeliers, INSERM U1138, Team 17, France; Université de Paris, 75006, Paris, France.
| | - N Lagali
- Department of Biomedical and Clinical Sciences, Linköping University, Linköping, Sweden; Department of Ophthalmology, Sørlandet Hospital Arendal, Arendal, Norway.
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20
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Polisetti N, Gießl A, Zenkel M, Heger L, Dudziak D, Naschberger E, Stich L, Steinkasserer A, Kruse FE, Schlötzer-Schrehardt U. Melanocytes as emerging key players in niche regulation of limbal epithelial stem cells. Ocul Surf 2021; 22:172-189. [PMID: 34425298 DOI: 10.1016/j.jtos.2021.08.006] [Citation(s) in RCA: 32] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2021] [Revised: 08/05/2021] [Accepted: 08/14/2021] [Indexed: 02/09/2023]
Abstract
PURPOSE Limbal melanocytes (LMel) represent essential components of the corneal epithelial stem cell niche and are known to protect limbal epithelial stem/progenitor cells (LEPCs) from UV damage by transfer of melanosomes. Here, we explored additional functional roles for LMel in niche homeostasis, immune regulation and angiostasis. METHODS Human corneoscleral tissues were morphologically analyzed in normal, inflammatory and wound healing conditions. The effects of LMel on LEPCs were analyzed in direct and indirect co-culture models using electron microscopy, immunocytochemistry, qRT-PCR, Western blotting and functional assays; limbal mesenchymal stromal cells and murine embryonic 3T3 fibroblasts served as controls. The immunophenotype of LMel was assessed by flow cytometry before and after interferon-γ stimulation, and their immunomodulatory properties were analyzed by mixed lymphocytes reaction, monocyte adhesion assays and cytometric bead arrays. Their angiostatic effects on human umbilical cord endothelial cells (HUVECs) were evaluated by proliferation, migration, and tube formation assays. RESULTS LMel and LEPCs formed structural units in the human limbal stem cell niche in situ, which could be functionally replicated, including melanosome transfer, by co-cultivation in vitro. LMel supported LEPCs during clonal expansion and during epithelial wound healing by stimulating proliferation and migration, and suppressed their differentiation through direct contact and paracrine effects. Under inflammatory conditions, LMel were increased in numbers and upregulated expression of ICAM-1 and MHC II molecules (HLA-DR), but lacked expression of HLA-G, -DP, -DQ and costimulatory molecules CD80 and CD86. They were also found to be potent suppressors of alloreactive T- cell proliferation and cytokine secretion, which largely depended on direct cell-cell interaction. Moreover, the LMel secretome exerted angiostatic activity by inhibiting vascular endothelial cell proliferation and capillary network formation. CONCLUSION These findings suggest that LMel are not only professional melanin-producing cells, but exert various non-canonical functions in limbal niche homeostasis by regulating LEPC maintenance, immune responses, and angiostasis. Their potent regulatory, immunomodulatory and anti-angiogenic properties may have important implications for future regenerative cell therapies.
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Affiliation(s)
- Naresh Polisetti
- Department of Ophthalmology, University Hospital Erlangen, Friedrich-Alexander-University of Erlangen-Nürnberg, Erlangen, Germany; Eye Center, Medical Center - Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Andreas Gießl
- Department of Ophthalmology, University Hospital Erlangen, Friedrich-Alexander-University of Erlangen-Nürnberg, Erlangen, Germany
| | - Matthias Zenkel
- Department of Ophthalmology, University Hospital Erlangen, Friedrich-Alexander-University of Erlangen-Nürnberg, Erlangen, Germany
| | - Lukas Heger
- Laboratory of Dendritic Cell Biology, Department of Dermatology, University Hospital Erlangen, Friedrich-Alexander-University of Erlangen-Nürnberg, Erlangen, Germany
| | - Diana Dudziak
- Laboratory of Dendritic Cell Biology, Department of Dermatology, University Hospital Erlangen, Friedrich-Alexander-University of Erlangen-Nürnberg, Erlangen, Germany; Deutsches Zentrum Immuntherapie (DZI), Erlangen, Germany; Comprehensive Cancer Center Erlangen-EMN (CCC ER-EMN), Erlangen, Germany; Medical Immunology Campus Erlangen, Erlangen, Germany
| | - Elisabeth Naschberger
- Division of Molecular and Experimental Surgery, Translational Research Center, Department of Surgery, University Hospital Erlangen, Friedrich-Alexander-University of Erlangen-Nürnberg, Erlangen, Germany
| | - Lena Stich
- Department of Immune Modulation, University Hospital Erlangen, Friedrich-Alexander-University of Erlangen-Nürnberg, Erlangen, Germany
| | - Alexander Steinkasserer
- Deutsches Zentrum Immuntherapie (DZI), Erlangen, Germany; Medical Immunology Campus Erlangen, Erlangen, Germany; Department of Immune Modulation, University Hospital Erlangen, Friedrich-Alexander-University of Erlangen-Nürnberg, Erlangen, Germany
| | - Friedrich E Kruse
- Department of Ophthalmology, University Hospital Erlangen, Friedrich-Alexander-University of Erlangen-Nürnberg, Erlangen, Germany
| | - Ursula Schlötzer-Schrehardt
- Department of Ophthalmology, University Hospital Erlangen, Friedrich-Alexander-University of Erlangen-Nürnberg, Erlangen, Germany.
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21
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Romo-Valera C, Pérez-Garrastachu M, Hernáez-Moya R, Rodriguez-Astigarraga M, Romano-Ruiz P, Etxebarria J, Arluzea J, Andollo N. Characterisation of corneas following different time and storage methods for their use as a source of stem-like limbal epithelial cells. Exp Eye Res 2021; 211:108720. [PMID: 34389315 DOI: 10.1016/j.exer.2021.108720] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2021] [Revised: 07/21/2021] [Accepted: 08/05/2021] [Indexed: 12/13/2022]
Abstract
The transplantation of expansions of limbal epithelial stem cells (LESC) remains one of the most efficient therapies for the treatment of limbal stem cell deficiency (LSCD) to date. However, the available donor corneas are scarce, and the corneas conserved for long time, under hypothermic conditions (after 7 days) or in culture (more than 28 days), are usually discarded due to poor viability of the endothelial cells. To establish an objective criterion for the utilisation or discarding of corneas as a source of LESC, we characterized, by immunohistochemistry analysis, donor corneas conserved in different conditions and for different periods of time. We also studied the potency of LESCs isolated from these corneas and maintained in culture up to 3 cell passages. We hoped that the study of markers of LESCs present in both the corneoscleral histological sections and the cell cultures would show the adequacy of the methods used for cell isolation and how fit the LESC enrichment of the obtained cell populations to be expanded was. Thus, the expressions of markers of the cells residing in the human limbal and corneal epithelium (cytokeratin CK15 and CK12, vimentin, Collagen VII, p63α, ABCG2, Ki67, Integrin β4, ZO1, and melan A) were analysed in sections of corneoscleral tissues conserved in hypothermic conditions for 2-9 days with post-mortem time (pmt) < 8 h or for 1 day with pmt > 16 h, and in sclerocorneal rims maintained in an organ culture medium for 29 days. Cell populations isolated from donor corneoscleral tissues were also assessed based on these markers to verify the adequacy of isolation methods and the potential of expanding LESCs from these tissues. Positivity for several putative stem cell markers such as CK15 and p63α was detected in all corneoscleral tissues, although a decrease was recorded in the ones conserved for longer times. The barrier function and the ability to adhere to the extracellular matrix were maintained in all the analysed tissues. In limbal epithelial cell cultures, a simultaneous decrease in the melan A melanocyte marker and the putative stem cell markers was detected, suggesting a close relationship between the melanocytes and the limbal stem cells of the niche. Holoclones stained with putative stem cell markers were obtained from long-term, hypothermic, stored sclerocorneal rims. The results showed that the remaining sclerocorneal rims after corneal transplantation, which were conserved under hypothermic conditions for up to 7 days and would have been discarded at a first glance, still maintained their potential as a source of LESC cultures.
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Affiliation(s)
- Cristina Romo-Valera
- Department of Cell Biology and Histology, School of Medicine and Nursing, University of the Basque Country, Sarriena, S/N, 48940, Leioa, Spain
| | - Miguel Pérez-Garrastachu
- Department of Cell Biology and Histology, School of Medicine and Nursing, University of the Basque Country, Sarriena, S/N, 48940, Leioa, Spain
| | - Raquel Hernáez-Moya
- Department of Cell Biology and Histology, School of Medicine and Nursing, University of the Basque Country, Sarriena, S/N, 48940, Leioa, Spain
| | - Maddalen Rodriguez-Astigarraga
- Department of Cell Biology and Histology, School of Medicine and Nursing, University of the Basque Country, Sarriena, S/N, 48940, Leioa, Spain
| | - Paula Romano-Ruiz
- Department of Cell Biology and Histology, School of Medicine and Nursing, University of the Basque Country, Sarriena, S/N, 48940, Leioa, Spain
| | - Jaime Etxebarria
- Department of Cell Biology and Histology, School of Medicine and Nursing, University of the Basque Country, Sarriena, S/N, 48940, Leioa, Spain; Department of Ophthalmology, University Hospital of Cruces, Cruces Plaza S/N, 48903, Barakaldo, Spain; BioCruces Bizkaia Health Research Institute, Begiker, Cruces Plaza S/N, 48903, Barakaldo, Spain
| | - Jon Arluzea
- Department of Cell Biology and Histology, School of Medicine and Nursing, University of the Basque Country, Sarriena, S/N, 48940, Leioa, Spain
| | - Noelia Andollo
- Department of Cell Biology and Histology, School of Medicine and Nursing, University of the Basque Country, Sarriena, S/N, 48940, Leioa, Spain; BioCruces Bizkaia Health Research Institute, Begiker, Cruces Plaza S/N, 48903, Barakaldo, Spain.
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22
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Abdul-Al M, Kyeremeh GK, Saeinasab M, Heidari Keshel S, Sefat F. Stem Cell Niche Microenvironment: Review. Bioengineering (Basel) 2021; 8:bioengineering8080108. [PMID: 34436111 PMCID: PMC8389324 DOI: 10.3390/bioengineering8080108] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2021] [Revised: 07/14/2021] [Accepted: 07/16/2021] [Indexed: 12/13/2022] Open
Abstract
The cornea comprises a pool of self-regenerating epithelial cells that are crucial to preserving clarity and visibility. Limbal epithelial stem cells (LESCs), which live in a specialized stem cell niche (SCN), are crucial for the survival of the human corneal epithelium. They live at the bottom of the limbal crypts, in a physically enclosed microenvironment with a number of neighboring niche cells. Scientists also simplified features of these diverse microenvironments for more analysis in situ by designing and recreating features of different SCNs. Recent methods for regenerating the corneal epithelium after serious trauma, including burns and allergic assaults, focus mainly on regenerating the LESCs. Mesenchymal stem cells, which can transform into self-renewing and skeletal tissues, hold immense interest for tissue engineering and innovative medicinal exploration. This review summarizes all types of LESCs, identity and location of the human epithelial stem cells (HESCs), reconstruction of LSCN and artificial stem cells for self-renewal.
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Affiliation(s)
- Mohamed Abdul-Al
- Department of Biomedical and Electronics Engineering, School of Engineering, University of Bradford, Bradford BD71DP, UK; (M.A.-A.); (G.K.K.)
| | - George Kumi Kyeremeh
- Department of Biomedical and Electronics Engineering, School of Engineering, University of Bradford, Bradford BD71DP, UK; (M.A.-A.); (G.K.K.)
| | - Morvarid Saeinasab
- Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad 91779 48974, Iran;
| | - Saeed Heidari Keshel
- Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran 19839 69411, Iran;
| | - Farshid Sefat
- Department of Biomedical and Electronics Engineering, School of Engineering, University of Bradford, Bradford BD71DP, UK; (M.A.-A.); (G.K.K.)
- Interdisciplinary Research Centre in Polymer Science & Technology (Polymer IRC), University of Bradford, Bradford BD71DP, UK
- Correspondence:
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23
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Bonnet C, Roberts JS, Deng SX. Limbal stem cell diseases. Exp Eye Res 2021; 205:108437. [PMID: 33571530 PMCID: PMC8044031 DOI: 10.1016/j.exer.2021.108437] [Citation(s) in RCA: 28] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2020] [Revised: 12/14/2020] [Accepted: 01/02/2021] [Indexed: 12/13/2022]
Abstract
The function of limbal stem/progenitor cells (LSCs) is critical to maintain corneal epithelial homeostasis. Many external insults and intrinsic defects can be deleterious to LSCs and their niche microenvironment, resulting in limbal stem cell dysfunction or deficiency (LSCD). Ocular comorbidities, frequent in eyes with LSCD, can exacerbate the dysfunction of residual LSCs, and limit the survival of transplanted LSCs. Clinical presentation and disease evolution vary among different etiologies of LSCD. New ocular imaging modalities and molecular markers are now available to standardize the diagnosis criteria and stage the severity of the disease. Medical therapies may be sufficient to reverse the disease if residual LSCs are present. A stepwise approach should be followed to optimize the ocular surface, eliminate the causative factors and treat comorbid conditions, before considering surgical interventions. Furthermore, surgical options are selected depending on the severity and laterality of the disease. The standardized diagnostic criteria to stage the disease is necessary to objectively evaluate and compare the efficacy of the emerging customized therapies.
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Affiliation(s)
- Clémence Bonnet
- Stein Eye Institute, Department of Ophthalmology, David Geffen School of Medicine, University of California, Los Angeles, CA, 90095, USA; Cornea Department, Paris University, Cochin Hospital, AP-HP, F-75014, Paris, France.
| | - JoAnn S Roberts
- Stein Eye Institute, Department of Ophthalmology, David Geffen School of Medicine, University of California, Los Angeles, CA, 90095, USA.
| | - Sophie X Deng
- Stein Eye Institute, Department of Ophthalmology, David Geffen School of Medicine, University of California, Los Angeles, CA, 90095, USA.
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24
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Galindo S, de la Mata A, López-Paniagua M, Herreras JM, Pérez I, Calonge M, Nieto-Miguel T. Subconjunctival injection of mesenchymal stem cells for corneal failure due to limbal stem cell deficiency: state of the art. Stem Cell Res Ther 2021; 12:60. [PMID: 33441175 PMCID: PMC7805216 DOI: 10.1186/s13287-020-02129-0] [Citation(s) in RCA: 40] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2020] [Accepted: 12/28/2020] [Indexed: 12/13/2022] Open
Abstract
Mesenchymal stem cells (MSCs) have unique and beneficial properties and are currently used to treat a broad variety of diseases. These properties include the potential for differentiation into other cell types, secretion of different trophic factors that promote a regenerative microenvironment, anti-inflammatory actions, selective migration to damaged tissues, and non-immunogenicity. MSCs are effective for the treatment of ocular surface diseases such as dry eye, corneal burns, and limbal stem cell deficiency (LSCD), both in experimental models and in humans. LSCD is a pathological condition in which damage occurs to the limbal epithelial stem cells, or their niche, that are responsible for the continuous regeneration of the corneal epithelium. If LSCD is extensive and/or severe, it usually causes corneal epithelial defects, ulceration, and conjunctival overgrowth of the cornea. These changes can result in neovascularization and corneal opacity, severe inflammation, pain, and visual loss. The effectiveness of MSCs to reduce corneal opacity, neovascularization, and inflammation has been widely studied in different experimental models of LSCD and in some clinical trials; however, the methodological disparity used in the different studies makes it hard to compare outcomes among them. In this regard, the MSC route of administration used to treat LSCD and other ocular surface diseases is an important factor. It should be efficient, minimally invasive, and safe. So far, intravenous and intraperitoneal injections, topical administration, and MSC transplantation using carrier substrata like amniotic membrane (AM), fibrin, or synthetic biopolymers have been the most commonly used administration routes in experimental models. However, systemic administration carries the risk of potential side effects and transplantation requires surgical procedures that could complicate the process. Alternatively, subconjunctival injection is a minimally invasive and straightforward technique frequently used in ophthalmology. It enables performance of local treatments using high cell doses. In this review, we provide an overview of the current status of MSC administration by subconjunctival injection, analyzing the convenience, safety, and efficacy for treatment of corneal failure due to LSCD in different experimental models. We also provide a summary of the clinical trials that have been completed, are in progress, or being planned.
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Affiliation(s)
- Sara Galindo
- Instituto de Oftalmobiología Aplicada (IOBA), Universidad de Valladolid, Edificio IOBA, Campus Miguel Delibes, Paseo de Belén 17, 47011, Valladolid, Spain.,Centro de Investigación Biomédica en Red de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Instituto de Salud Carlos III, Madrid, Spain.,Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, Valladolid, Spain
| | - Ana de la Mata
- Instituto de Oftalmobiología Aplicada (IOBA), Universidad de Valladolid, Edificio IOBA, Campus Miguel Delibes, Paseo de Belén 17, 47011, Valladolid, Spain. .,Centro de Investigación Biomédica en Red de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Instituto de Salud Carlos III, Madrid, Spain. .,Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, Valladolid, Spain.
| | - Marina López-Paniagua
- Instituto de Oftalmobiología Aplicada (IOBA), Universidad de Valladolid, Edificio IOBA, Campus Miguel Delibes, Paseo de Belén 17, 47011, Valladolid, Spain.,Centro de Investigación Biomédica en Red de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Instituto de Salud Carlos III, Madrid, Spain.,Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, Valladolid, Spain
| | - Jose M Herreras
- Instituto de Oftalmobiología Aplicada (IOBA), Universidad de Valladolid, Edificio IOBA, Campus Miguel Delibes, Paseo de Belén 17, 47011, Valladolid, Spain.,Centro de Investigación Biomédica en Red de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Instituto de Salud Carlos III, Madrid, Spain.,Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, Valladolid, Spain
| | - Inmaculada Pérez
- Instituto de Oftalmobiología Aplicada (IOBA), Universidad de Valladolid, Edificio IOBA, Campus Miguel Delibes, Paseo de Belén 17, 47011, Valladolid, Spain.,Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, Valladolid, Spain
| | - Margarita Calonge
- Instituto de Oftalmobiología Aplicada (IOBA), Universidad de Valladolid, Edificio IOBA, Campus Miguel Delibes, Paseo de Belén 17, 47011, Valladolid, Spain.,Centro de Investigación Biomédica en Red de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Instituto de Salud Carlos III, Madrid, Spain.,Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, Valladolid, Spain
| | - Teresa Nieto-Miguel
- Instituto de Oftalmobiología Aplicada (IOBA), Universidad de Valladolid, Edificio IOBA, Campus Miguel Delibes, Paseo de Belén 17, 47011, Valladolid, Spain. .,Centro de Investigación Biomédica en Red de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Instituto de Salud Carlos III, Madrid, Spain. .,Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, Valladolid, Spain.
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25
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Ashworth S, Harrington J, Hammond GM, Bains KK, Koudouna E, Hayes AJ, Ralphs JR, Regini JW, Young RD, Hayashi R, Nishida K, Hughes CE, Quantock AJ. Chondroitin Sulfate as a Potential Modulator of the Stem Cell Niche in Cornea. Front Cell Dev Biol 2021; 8:567358. [PMID: 33511110 PMCID: PMC7835413 DOI: 10.3389/fcell.2020.567358] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2020] [Accepted: 12/07/2020] [Indexed: 12/13/2022] Open
Abstract
Chondroitin sulfate (CS) is an important component of the extracellular matrix in multiple biological tissues. In cornea, the CS glycosaminoglycan (GAG) exists in hybrid form, whereby some of the repeating disaccharides are dermatan sulfate (DS). These CS/DS GAGs in cornea, through their presence on the proteoglycans, decorin and biglycan, help control collagen fibrillogenesis and organization. CS also acts as a regulatory ligand for a spectrum of signaling molecules, including morphogens, cytokines, chemokines, and enzymes during corneal growth and development. There is a growing body of evidence that precise expression of CS or CS/DS with specific sulfation motifs helps define the local extracellular compartment that contributes to maintenance of the stem cell phenotype. Indeed, recent evidence shows that CS sulfation motifs recognized by antibodies 4C3, 7D4, and 3B3 identify stem cell populations and their niches, along with activated progenitor cells and transitional areas of tissue development in the fetal human elbow. Various sulfation motifs identified by some CS antibodies are also specifically located in the limbal region at the edge of the mature cornea, which is widely accepted to represent the corneal epithelial stem cell niche. Emerging data also implicate developmental changes in the distribution of CS during corneal morphogenesis. This article will reflect upon the potential roles of CS and CS/DS in maintenance of the stem cell niche in cornea, and will contemplate the possible involvement of CS in the generation of eye-like tissues from human iPS (induced pluripotent stem) cells.
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Affiliation(s)
- Sean Ashworth
- Structural Biophysics Group, School of Optometry and Vision Sciences, College of Biomedical and Life Sciences, Cardiff University, Cardiff, United Kingdom.,School of Biosciences, College of Biomedical and Life Sciences, Cardiff University, Cardiff, United Kingdom
| | - Jodie Harrington
- Structural Biophysics Group, School of Optometry and Vision Sciences, College of Biomedical and Life Sciences, Cardiff University, Cardiff, United Kingdom.,Department of Stem Cells and Applied Medicine, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Greg M Hammond
- Structural Biophysics Group, School of Optometry and Vision Sciences, College of Biomedical and Life Sciences, Cardiff University, Cardiff, United Kingdom
| | - Kiranjit K Bains
- Structural Biophysics Group, School of Optometry and Vision Sciences, College of Biomedical and Life Sciences, Cardiff University, Cardiff, United Kingdom
| | - Elena Koudouna
- Structural Biophysics Group, School of Optometry and Vision Sciences, College of Biomedical and Life Sciences, Cardiff University, Cardiff, United Kingdom
| | - Anthony J Hayes
- School of Biosciences, College of Biomedical and Life Sciences, Cardiff University, Cardiff, United Kingdom
| | - James R Ralphs
- School of Biosciences, College of Biomedical and Life Sciences, Cardiff University, Cardiff, United Kingdom
| | - Justyn W Regini
- Structural Biophysics Group, School of Optometry and Vision Sciences, College of Biomedical and Life Sciences, Cardiff University, Cardiff, United Kingdom
| | - Robert D Young
- Structural Biophysics Group, School of Optometry and Vision Sciences, College of Biomedical and Life Sciences, Cardiff University, Cardiff, United Kingdom
| | - Ryuhei Hayashi
- Department of Stem Cells and Applied Medicine, Osaka University Graduate School of Medicine, Osaka, Japan.,Department of Ophthalmology, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Kohji Nishida
- Department of Ophthalmology, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Clare E Hughes
- School of Biosciences, College of Biomedical and Life Sciences, Cardiff University, Cardiff, United Kingdom
| | - Andrew J Quantock
- Structural Biophysics Group, School of Optometry and Vision Sciences, College of Biomedical and Life Sciences, Cardiff University, Cardiff, United Kingdom
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26
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Laminin-511-E8 promotes efficient in vitro expansion of human limbal melanocytes. Sci Rep 2020; 10:11074. [PMID: 32632213 PMCID: PMC7338389 DOI: 10.1038/s41598-020-68120-0] [Citation(s) in RCA: 21] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2020] [Accepted: 06/18/2020] [Indexed: 01/08/2023] Open
Abstract
Limbal melanocytes, located in the basal epithelial layer of the corneoscleral limbus, represent essential components of the corneal epithelial stem cell niche, but, due to difficulties in their isolation and cultivation, their biological roles and potential for stem cell-based tissue engineering approaches have not been comprehensively studied. Here, we established a protocol for the efficient isolation and cultivation of pure populations of human limbal melanocytes, which could be expanded at high yield by using recombinant laminin (LN)-511-E8 as culture substrate. Co-cultivation of limbal melanocytes with limbal epithelial stem/progenitor cells on fibrin hydrogels pre-incubated with LN-511-E8 resulted in multilayered stratified epithelial constructs within ten days. By reproducing physiological cell–cell and cell–matrix interactions of the native niche environment, these biomimetic co-culture systems provide a promising experimental model for investigating the functional roles of melanocytes in the limbal stem cell niche and their suitability for developing advanced epithelial grafts for ocular surface surface reconstruction.
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27
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O'Callaghan AR, Dziasko MA, Sheth-Shah R, Lewis MP, Daniels JT. Oral Mucosa Tissue Equivalents for the Treatment of Limbal Stem Cell Deficiency. ACTA ACUST UNITED AC 2020; 4:e1900265. [PMID: 32515079 DOI: 10.1002/adbi.201900265] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2019] [Revised: 05/07/2020] [Indexed: 12/13/2022]
Abstract
Cultured limbal and oral epithelial cells have been successfully used to treat patients with limbal stem cell deficiency (LSCD). The most common culture method for these cell therapies utilizes amniotic membrane as a cell support and/or murine 3T3s as feeder fibroblasts. The aim of this study is to refine the production of autologous oral mucosal cell therapy for the treatment of LSCD. Real architecture for 3D tissue (RAFT) is used as an alternative cell culture support. In addition, oral mucosal cells (epithelial and fibroblast) are used as autologous alternatives to donor human limbal epithelial cells (HLE) and murine 3T3s. The following tissue equivalents are produced and characterized: first, for patients with bilateral LSCD, an oral mucosa tissue equivalent consisting of human oral mucosal epithelial cells on RAFT supported by human oral mucosal fibroblasts (HOMF). Second, for patients with unilateral LSCD, HLE on RAFT supported by HOMF. For both tissue equivalent types, features of the cornea are observed including a multi-layered epithelium with small cells with a stem cell like phenotype in the basal layer and squamous cells in the top layers, and p63α and PAX6 expression. These tissue equivalents may therefore be useful in the treatment of LSCD.
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Affiliation(s)
- Anna R O'Callaghan
- Cells for Sight, UCL Institute of Ophthalmology, University College London, London, EC1V 9EL, UK
| | - Marc A Dziasko
- Cells for Sight, UCL Institute of Ophthalmology, University College London, London, EC1V 9EL, UK
| | - Radhika Sheth-Shah
- Cells for Sight, UCL Institute of Ophthalmology, University College London, London, EC1V 9EL, UK
| | - Mark P Lewis
- National Centre for Sport and Exercise Medicine (NCSEM), School of Sport, Exercise and Health Sciences, Loughborough University, Leicestershire, LE11 3TU, UK
| | - Julie T Daniels
- Cells for Sight, UCL Institute of Ophthalmology, University College London, London, EC1V 9EL, UK
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28
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Seyed-Safi AG, Daniels JT. The limbus: Structure and function. Exp Eye Res 2020; 197:108074. [PMID: 32502532 DOI: 10.1016/j.exer.2020.108074] [Citation(s) in RCA: 26] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2020] [Revised: 05/14/2020] [Accepted: 05/17/2020] [Indexed: 12/12/2022]
Abstract
Limbal function is a key determinant of corneal epithelial integrity. Lineage tracing studies in mice have highlighted that the centripetal movement of epithelial progenitors from the limbus drives both the steady-state maintenance of the corneal epithelium and its regeneration following injury. It is well established that this is facilitated by a population of limbal epithelial stem cells within the limbus. It is becoming increasingly apparent that the behaviour of these stem cells and their ability to respond to the needs of the tissue are closely linked to their immediate microenvironment - the stem cell niche. Increasing understanding of the structural features of this niche and the signalling networks that they coordinate is required to enhance the therapeutic application of these cells in the treatment of limbal stem cell deficiency. Importantly, an improved characterisation of the hierarchy of limbal epithelial progenitors using both new and old putative markers will enable a greater appreciation for the effects of many of these limbal niche factors on stem cell fate.
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Seyed-Safi AG, Daniels JT. A validated porcine corneal organ culture model to study the limbal response to corneal epithelial injury. Exp Eye Res 2020; 197:108063. [PMID: 32417262 DOI: 10.1016/j.exer.2020.108063] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2020] [Revised: 04/03/2020] [Accepted: 05/05/2020] [Indexed: 12/13/2022]
Abstract
Limbal epithelial stem cells are required for the maintenance and repair of the corneal epithelial surface. The difficulty in obtaining human corneal tissue for research purposes means that animal models for studying the corneal and limbal epithelium are extremely useful. Porcine corneal tissue represents an attractive experimental model, however, functional analysis of the limbal epithelial cell population is needed to validate the use of this tissue. Single cell clonal analysis revealed that holoclone-generating cells were enriched in the limbus as compared with the central cornea (38.3% vs 8.3%) and that label-retaining cells were also enriched in the limbus and compared with the central cornea (44.7 ± 6.4 vs 4.7 ± 1.5). Furthermore, it was demonstrated that in a 3D-printed organ culture system, porcine tissue was capable of maintaining and healing the corneal epithelium. Ki67 staining of corneal sections revealed that in response to central epithelial wounding, a greater proportion of progenitors in the basal limbal epithelium enter an actively dividing state. The authors present a comprehensively validated model system for studying the interactions between limbal niche factors and limbal epithelial stem cell fate.
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Gupta N, Farooqui JH, Dziasko MA, Daniels JT, Mathur U, Sangwan VS. Reappearance of limbal pigmentation post-simple limbal epithelial transplant. Indian J Ophthalmol 2020; 68:927-929. [PMID: 32317494 PMCID: PMC7350484 DOI: 10.4103/ijo.ijo_155_19] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2019] [Revised: 10/28/2019] [Accepted: 10/28/2019] [Indexed: 11/17/2022] Open
Abstract
We report the repigmentation at the limbus in patients who underwent simple limbal epithelial transplant (SLET) for uniocular chemical injury. The first case is of an 8-year-old child who presented with grade 4 chemical injury, with limbal stem cell deficiency (LSCD) corresponding to 6 o' clock till 11 o' clock. He was managed by amniotic membrane graft in the acute stage and SLET after 6 months of the initial injury. The second case is of a 15-year-old female who presented with lime injury, which had resulted in 6 o' clock of limbal involvement (10 o' clock till 4 o' clock). The patient was managed on similar lines with amniotic membrane graft (AMG) in the acute phase and SLET after 6 months of injury. The ocular surface was stable in both the patients post-SLET. The effected limbus showed pigmentation at 8 months of follow-up which eventually became distinct and remained stable. We speculate that the pigmentation at limbus could be attributed to proliferation and movement of melanocytes from limbal biopsy in SLET. These may be capable of supporting the proliferation of limbal epithelial cells and modulation of corneal wound healing.
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Affiliation(s)
- Nidhi Gupta
- Department of Cornea, Refractive Surgery and Ocular Surface Disorders, Dr. Shroff's Charity Eye Hospital, New Delhi, India
| | - Javed H Farooqui
- Department of Cornea, Refractive Surgery and Ocular Surface Disorders, Dr. Shroff's Charity Eye Hospital, New Delhi, India
| | | | | | - Umang Mathur
- Department of Cornea, Refractive Surgery and Ocular Surface Disorders, Dr. Shroff's Charity Eye Hospital, New Delhi, India
| | - Virender S Sangwan
- Department of Cornea, Refractive Surgery and Ocular Surface Disorders, Dr. Shroff's Charity Eye Hospital, New Delhi, India
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31
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Kafarnik C, McClellan A, Dziasko M, Daniels JT, Guest DJ. Canine Corneal Stromal Cells Have Multipotent Mesenchymal Stromal Cell Properties In Vitro. Stem Cells Dev 2020; 29:425-439. [PMID: 31973649 DOI: 10.1089/scd.2019.0163] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
The objective of this study was to determine whether corneal stromal cells (CSCs) from the limbal and central corneal stroma in dogs have multipotent mesenchymal stem/stromal cell (MSC) properties, and whether this cell population can be differentiated into keratocyte-like cells (KDCs). Normal, donated, mesocephalic dog corneas were used to isolate CSC in vitro. Immunohistochemistry demonstrated a distinct population of CD90 expressing cells in the anterior stroma throughout the limbal and central cornea. CSC could be cultured from both the limbal and central cornea and the culture kinetics showed a progenitor cell profile. The CSC expressed stem cell markers CD90, CD73, CD105, N-cadherin, and Pax6, while CD34 was negative. Limbal and central CSC differentiated into osteoblasts, chondrocytes, and adipocytes confirming their multipotency. Coculturing allogeneic peripheral blood mononuclear cells (PBMCs) with limbal CSCs did not affect baseline PBMC proliferation indicating a degree of innate immune privilege. Limbal CSC could be differentiated into KDCs that expressed Keratocan, Lumican, and ALDH1A3 and downregulated Pax6 and N-cadherin. In conclusion, canine CSCs have multipotent MSC properties similarly described in humans and could serve as a source of cells for cell therapy and studying corneal diseases.
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Affiliation(s)
- Christiane Kafarnik
- Centre for Preventive Medicine, Animal Health Trust, Newmarket, United Kingdom.,Rescue, Repair and Regeneration Theme, Institute of Ophthalmology, University College London, London, United Kingdom
| | - Alyce McClellan
- Centre for Preventive Medicine, Animal Health Trust, Newmarket, United Kingdom
| | - Marc Dziasko
- Rescue, Repair and Regeneration Theme, Institute of Ophthalmology, University College London, London, United Kingdom
| | - Julie T Daniels
- Rescue, Repair and Regeneration Theme, Institute of Ophthalmology, University College London, London, United Kingdom
| | - Deborah J Guest
- Centre for Preventive Medicine, Animal Health Trust, Newmarket, United Kingdom
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32
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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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Current Trends and Future Perspective of Mesenchymal Stem Cells and Exosomes in Corneal Diseases. Int J Mol Sci 2019; 20:ijms20122853. [PMID: 31212734 PMCID: PMC6627168 DOI: 10.3390/ijms20122853] [Citation(s) in RCA: 79] [Impact Index Per Article: 13.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2019] [Revised: 06/01/2019] [Accepted: 06/10/2019] [Indexed: 02/07/2023] Open
Abstract
The corneal functions (transparency, refractivity and mechanical strength) deteriorate in many corneal diseases but can be restored after corneal transplantation (penetrating and lamellar keratoplasties). However, the global shortage of transplantable donor corneas remains significant and patients are subject to life-long risk of immune response and graft rejection. Various studies have shown the differentiation of multipotent mesenchymal stem cells (MSCs) into various corneal cell types. With the unique properties of immunomodulation, anti-angiogenesis and anti-inflammation, they offer the advantages in corneal reconstruction. These effects are widely mediated by MSC differentiation and paracrine signaling via exosomes. Besides the cell-free nature of exosomes in circumventing the problems of cell-fate control and tumorigenesis, the vesicle content can be genetically modified for optimal therapeutic affinity. The pharmacology and toxicology, xeno-free processing with sustained delivery, scale-up production in compliant to Good Manufacturing Practice regulations, and cost-effectiveness are the current foci of research. Routes of administration via injection, topical and/or engineered bioscaffolds are also explored for its applicability in treating corneal diseases.
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Calonge M, Pérez I, Galindo S, Nieto-Miguel T, López-Paniagua M, Fernández I, Alberca M, García-Sancho J, Sánchez A, Herreras JM. A proof-of-concept clinical trial using mesenchymal stem cells for the treatment of corneal epithelial stem cell deficiency. Transl Res 2019; 206:18-40. [PMID: 30578758 DOI: 10.1016/j.trsl.2018.11.003] [Citation(s) in RCA: 85] [Impact Index Per Article: 14.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/17/2018] [Revised: 10/18/2018] [Accepted: 11/18/2018] [Indexed: 01/02/2023]
Abstract
Ocular stem cell transplantation derived from either autologous or allogeneic donor corneoscleral junction is a functional cell therapy to manage extensive and/or severe limbal stem cell deficiencies that lead to corneal epithelial failure. Mesenchymal stem cells have been properly tested in animal models of this ophthalmic pathology, but never in human eyes despite their potential advantages. We conducted a 6- to 12-month proof-of-concept, randomized, and double-masked pilot trial to test whether allogeneic bone marrow-derived mesenchymal stem cell transplantation (MSCT], n = 17) was as safe and as equally efficient as allogeneic cultivated limbal epithelial transplantation (CLET), (n = 11) to improve corneal epithelial damage due to limbal stem cell deficiency. Primary endpoints demanded combination of symptoms, signs, and the objective improvement of the epithelial phenotype in central cornea by in vivo confocal microscopy. This proof-of-concept trial showed that MSCT was as safe and efficacious as CLET. Global success at 6-12 months was 72.7%-77.8% for CLET cases and 76.5%-85.7% for MSCT cases (not significant differences). Central corneal epithelial phenotype improved in 71.4% and 66.7% of MSCT and CLET cases, respectively at 12 months (P = 1.000). There were no adverse events related to cell products. This trial suggests first evidence that MSCT facilitated improvement of a diseased corneal epithelium due to lack of its stem cells as efficiently as CLET. Consequently, not only CLET but also MSCT deserves more preclinical investigational resources before the favorable results of this proof-of-concept trial could be transformed into the larger numbers of the multicenter trials that would provide stronger evidence. (ClinicalTrials.gov number, NCT01562002.).
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Affiliation(s)
- Margarita Calonge
- IOBA (Institute of Applied Ophthalmobiology), University of Valladolid, Valladolid, Spain; CIBER-BBN (Biomedical Research Networking Centre in Bioengineering, Biomaterials, and Nanomedicine), Carlos III National Institute of Health, Valladolid, Spain.
| | - Inmaculada Pérez
- IOBA (Institute of Applied Ophthalmobiology), University of Valladolid, Valladolid, Spain.
| | - Sara Galindo
- IOBA (Institute of Applied Ophthalmobiology), University of Valladolid, Valladolid, Spain; CIBER-BBN (Biomedical Research Networking Centre in Bioengineering, Biomaterials, and Nanomedicine), Carlos III National Institute of Health, Valladolid, Spain.
| | - Teresa Nieto-Miguel
- IOBA (Institute of Applied Ophthalmobiology), University of Valladolid, Valladolid, Spain; CIBER-BBN (Biomedical Research Networking Centre in Bioengineering, Biomaterials, and Nanomedicine), Carlos III National Institute of Health, Valladolid, Spain.
| | - Marina López-Paniagua
- IOBA (Institute of Applied Ophthalmobiology), University of Valladolid, Valladolid, Spain; CIBER-BBN (Biomedical Research Networking Centre in Bioengineering, Biomaterials, and Nanomedicine), Carlos III National Institute of Health, Valladolid, Spain.
| | - Itziar Fernández
- IOBA (Institute of Applied Ophthalmobiology), University of Valladolid, Valladolid, Spain; CIBER-BBN (Biomedical Research Networking Centre in Bioengineering, Biomaterials, and Nanomedicine), Carlos III National Institute of Health, Valladolid, Spain.
| | - Mercedes Alberca
- IBGM (Institute of Molecular Biology and Genetics), University of Valladolid and National Research Council (CSIC), and University Scientific Park, Valladolid, Spain.
| | - Javier García-Sancho
- IBGM (Institute of Molecular Biology and Genetics), University of Valladolid and National Research Council (CSIC), and University Scientific Park, Valladolid, Spain.
| | - Ana Sánchez
- IBGM (Institute of Molecular Biology and Genetics), University of Valladolid and National Research Council (CSIC), and University Scientific Park, Valladolid, Spain.
| | - José M Herreras
- IOBA (Institute of Applied Ophthalmobiology), University of Valladolid, Valladolid, Spain; CIBER-BBN (Biomedical Research Networking Centre in Bioengineering, Biomaterials, and Nanomedicine), Carlos III National Institute of Health, Valladolid, Spain.
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35
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Strategies for reconstructing the limbal stem cell niche. Ocul Surf 2019; 17:230-240. [PMID: 30633966 DOI: 10.1016/j.jtos.2019.01.002] [Citation(s) in RCA: 64] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2018] [Revised: 10/21/2018] [Accepted: 01/07/2019] [Indexed: 12/19/2022]
Abstract
The epithelial cell layer that covers the surface of the cornea provides a protective barrier while maintaining corneal transparency. The rapid and effective turnover of these epithelial cells depends, in part, on the limbal epithelial stem cells (LESCs) located in a specialized microenvironment known as the limbal niche. Many disorders affecting the regeneration of the corneal epithelium are related to deficiency and/or dysfunction of LESCs and the limbal niche. Current approaches for regenerating the corneal epithelium following significant injuries such as burns and inflammatory attacks are primarily aimed at repopulating the LESCs. This review summarizes and assesses the clinical feasibility and efficacy of current and emerging approaches for reconstruction of the limbal niche. In particular, the application of mesenchymal stem cells along with appropriate biological scaffolds appear to be promising strategies for long-term revitalization of the limbal niche.
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36
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Masterton S, Ahearne M. Mechanobiology of the corneal epithelium. Exp Eye Res 2018; 177:122-129. [PMID: 30086260 PMCID: PMC6280025 DOI: 10.1016/j.exer.2018.08.001] [Citation(s) in RCA: 56] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2018] [Revised: 07/16/2018] [Accepted: 08/03/2018] [Indexed: 12/16/2022]
Abstract
There has been a drive to develop new cell based therapies to treat corneal blindness, one of the most common causes of blindness worldwide. Mechanical and physical cues are known to regulate the behavior of many cell types, however studies examining these effects on corneal epithelial cells have been limited in number and their findings have not previously been amalgamated and contrasted. Here, we provide an overview of the different types of mechanical stimuli to which the corneal epithelium is exposed and the influence that these have on the cells. Shear stress from the tear film motion and blinking, extracellular matrix stiffness and external physical forces such as eye rubbing and contact lens wear are among some of the forms of mechanical stimuli that the epithelium experiences. In vivo and in vitro studies examining the mechanobiology on corneal epithelial cells under differing mechanical environments are explored. A greater understanding of the mechanobiology of the corneal epithelium has the potential to lead to improved tissue engineering and cell based therapies to repair and regenerate damaged cornea.
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Affiliation(s)
- Sophia Masterton
- Dept of Mechanical and Manufacturing Engineering, School of Engineering, Trinity College Dublin, University of Dublin, Ireland; Trinity Centre for Bioengineering, Trinity Biomedical Science Institute, Trinity College Dublin, University of Dublin, Ireland
| | - Mark Ahearne
- Dept of Mechanical and Manufacturing Engineering, School of Engineering, Trinity College Dublin, University of Dublin, Ireland; Trinity Centre for Bioengineering, Trinity Biomedical Science Institute, Trinity College Dublin, University of Dublin, Ireland.
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37
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Limbal Stem Cell Transplantation: Clinical Results, Limits, and Perspectives. Stem Cells Int 2018; 2018:8086269. [PMID: 30405723 PMCID: PMC6201383 DOI: 10.1155/2018/8086269] [Citation(s) in RCA: 57] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2018] [Revised: 07/10/2018] [Accepted: 08/27/2018] [Indexed: 12/13/2022] Open
Abstract
Limbal stem cell deficiency (LSCD) is a clinical condition characterized by damage of cornea limbal stem cells, which results in an impairment of corneal epithelium turnover and in an invasion of the cornea by the conjunctival epithelium. In these patients, the conjunctivalization of the cornea is associated with visual impairment and cornea transplantation has poor prognosis for recurrence of the conjunctivalization. Current treatments of LSCD are aimed at replacing the damaged corneal stem cells in order to restore a healthy corneal epithelium. The autotransplantation of limbal tissue from the healthy, fellow eye is effective in unilateral LSCD but leads to depauperation of the stem cell reservoir. In the last decades, novel techniques such as cultivated limbal epithelial transplantation (CLET) have been proposed in order to reduce the damage of the healthy fellow eye. Clinical and experimental evidence showed that CLET is effective in inducing long-term regeneration of a healthy corneal epithelium in patients with LSCD with a success rate of 70%–80%. Current limitations for the treatment of LSCD are represented by the lack of a marker able to unequivocally identify limbal stem cells and the treatment of total, bilateral LSCD which requires other sources of stem cells for ocular surface reconstruction.
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38
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Exosomes from normal and diabetic human corneolimbal keratocytes differentially regulate migration, proliferation and marker expression of limbal epithelial cells. Sci Rep 2018; 8:15173. [PMID: 30310159 PMCID: PMC6182003 DOI: 10.1038/s41598-018-33169-5] [Citation(s) in RCA: 54] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2018] [Accepted: 09/19/2018] [Indexed: 12/13/2022] Open
Abstract
Limbal epithelial stem cells (LESC) maintenance requires communication between stem cells and neighboring stromal keratocytes. Extracellular vesicles (EVs) are important for intercellular communication in various stem cell niches. We explored the regulatory roles of limbal stromal cell (LSC)-derived exosomes (Exos), an EV sub-population, in limbal epithelial cells (LEC) in normal and diabetic limbal niche and determined differences in Exo cargos from normal and diabetic LSC. Wound healing and proliferation rates in primary normal LEC were significantly enhanced upon treatment by normal Exos (N-Exos), but not by diabetic Exos (DM-Exos). Western analysis showed increased Akt phosphorylation in wounded LECs and organ-cultured corneas treated with N-Exos, compared to untreated wounded cells and DM-Exos treated fellow corneas, respectively. N-Exos treated organ-cultured corneas showed upregulation of putative LESC markers, keratin 15 (K15) and Frizzled-7, compared to the DM-Exos treated fellow corneas. By next generation sequencing, we identified differentially expressed small RNAs including microRNAs in DM-Exos vs. N-Exos. Overall, N-Exos have greater effect on LEC proliferation and wound healing than DM-Exos, likely by activating Akt signaling. The small RNA differences in Exos from diabetic vs. normal LSC could contribute to the disease state. Our study suggests that exosomes may serve as novel therapeutic tools for diabetic cornea.
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39
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Bojic S, Hallam D, Alcada N, Ghareeb A, Queen R, Pervinder S, Buck H, Amitai Lange A, Figueiredo G, Rooney P, Stojkovic M, Shortt A, Figueiredo FC, Lako M. CD200 Expression Marks a Population of Quiescent Limbal Epithelial Stem Cells with Holoclone Forming Ability. Stem Cells 2018; 36:1723-1735. [PMID: 30157305 DOI: 10.1002/stem.2903] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2018] [Revised: 08/02/2018] [Accepted: 08/09/2018] [Indexed: 12/12/2022]
Abstract
One of the main challenges in limbal stem cell (LSC) biology and transplantation is the lack of definitive cell surface markers which can be used to identify and enrich viable LSCs. In this study, expression of 361 cell surface proteins was assessed in ex vivo expanded limbal epithelial cells. One marker, CD200 was selected for further characterization based on expression in a small subset of limbal epithelial cells (2.25% ± 0.69%) and reduced expression through consecutive passaging and calcium induced differentiation. CD200 was localized to a small population of cells at the basal layer of the human and mouse limbal epithelium. CD200+ cells were slow cycling and contained the majority of side population (SP) and all the holoclone forming progenitors. CD200+ cells displayed higher expression of LSCs markers including PAX6, WNT7A, CDH3, CK14, CK15, and ABCB5 and lower expression of Ki67 when compared to CD200- . Downregulation of CD200 abrogated the ability of limbal epithelial cells to form holoclones, suggesting an important function for CD200 in the maintenance and/or self-renewal of LSCs. A second marker, CD109, which was expressed in 56.29% ± 13.96% of limbal epithelial cells, was also found to co-localize with ΔNp63 in both human and mouse cornea, albeit more abundantly than CD200. CD109 expression decreased slowly through calcium induced cell differentiation and CD109+ cells were characterized by higher expression of Ki67, when compared to CD109- subpopulation. Together our data suggest that CD200 expression marks a quiescent population of LSCs with holoclone forming potential, while CD109 expression is associated with a proliferative progenitor phenotype. Stem Cells 2018;36:1723-1735.
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Affiliation(s)
- Sanja Bojic
- Institute of Genetic Medicine, Newcastle University, Newcastle, United Kingdom
| | - Dean Hallam
- Institute of Genetic Medicine, Newcastle University, Newcastle, United Kingdom
| | - Nuno Alcada
- Institute of Genetic Medicine, Newcastle University, Newcastle, United Kingdom
| | - Ali Ghareeb
- Institute of Genetic Medicine, Newcastle University, Newcastle, United Kingdom
| | - Rachel Queen
- Institute of Genetic Medicine, Newcastle University, Newcastle, United Kingdom
| | - Sagoo Pervinder
- UCL Institute of Immunology and Transplantation, London, United Kingdom
| | - Harley Buck
- UCL Institute of Immunology and Transplantation, London, United Kingdom
| | - Aya Amitai Lange
- Department of Genetics and Developmental Biology, The Ruth and Bruce Rappaport Faculty of Medicine, Technion - Israel Institute of Technology, Haifa, Israel
| | - Gustavo Figueiredo
- Institute of Genetic Medicine, Newcastle University, Newcastle, United Kingdom
| | - Paul Rooney
- Tissue Services, NHS Blood and Transplant, Liverpool, United Kingdom
| | - Miodrag Stojkovic
- Faculty of Medical Sciences, Department of Genetics, University of Kragujevac, Serbia.,SPEBO Medical, Leskovac, Kragujevac, Serbia
| | - Alex Shortt
- UCL Institute of Immunology and Transplantation, London, United Kingdom
| | - Francisco C Figueiredo
- Institute of Genetic Medicine, Newcastle University, Newcastle, United Kingdom.,Department of Ophthalmology, Royal Victoria Infirmary, Newcastle University, Newcastle, United Kingdom
| | - Majlinda Lako
- Institute of Genetic Medicine, Newcastle University, Newcastle, United Kingdom
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40
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Liu L, Nielsen FM, Emmersen J, Bath C, Østergaard Hjortdal J, Riis S, Fink T, Pennisi CP, Zachar V. Pigmentation Is Associated with Stemness Hierarchy of Progenitor Cells Within Cultured Limbal Epithelial Cells. Stem Cells 2018; 36:1411-1420. [PMID: 29781179 DOI: 10.1002/stem.2857] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2018] [Revised: 04/26/2018] [Accepted: 05/07/2018] [Indexed: 12/15/2022]
Abstract
Ex vivo cultured human limbal epithelial stem/progenitor cells (hLESCs) are the main source for regenerative therapy of limbal stem cell deficiency (LSCD), which is worldwide one of the major causes of corneal blindness. Despite many stemness-associated markers have been identified within the limbal niche, the phenotype of the earliest hLESCs has not been hitherto identified. We sought to confirm or refute the use of tumor protein p63 (p63) and ATP binding cassette subfamily B member 5 (ABCB5) as surrogate markers for hLESCs early within the limbal differentiation hierarchy. Based on a robust fluorescence-activated cell sorting and subsequent RNA isolation protocol, a comprehensive transcriptomic profile was obtained from four subpopulations of cultured hLESCs. The subpopulations were defined by co-expression of two putative stem/progenitor markers, the p63 and ABCB5, and the corneal differentiation marker cytokeratin 3. A comparative transcriptomic analysis yielded novel data that indicated association between pigmentation and differentiation, with the p63 positive populations being the most pigmented and immature of the progenitors. In contrast, ABCB5, either alone or in co-expression patterns, identified more committed progenitor cells with less pigmentation. In conclusion, p63 is superior to ABCB5 as a marker for stemness. Stem Cells 2018;36:1411-1420.
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Affiliation(s)
- Lei Liu
- Laboratory for Stem Cell Research, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark.,Department of Pediatric Surgery, First Hospital of Jilin University, Changchun, Jilin, People's Republic of China
| | - Frederik Mølgaard Nielsen
- Laboratory for Stem Cell Research, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark
| | - Jeppe Emmersen
- Laboratory for Stem Cell Research, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark
| | - Chris Bath
- Department of Ophthalmology, Aalborg University Hospital, Aalborg, Denmark
| | | | - Simone Riis
- Laboratory for Stem Cell Research, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark
| | - Trine Fink
- Laboratory for Stem Cell Research, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark
| | - Cristian Pablo Pennisi
- Laboratory for Stem Cell Research, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark
| | - Vladimir Zachar
- Laboratory for Stem Cell Research, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark
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Brejchova K, Trosan P, Studeny P, Skalicka P, Utheim TP, Bednar J, Jirsova K. Characterization and comparison of human limbal explant cultures grown under defined and xeno-free conditions. Exp Eye Res 2018; 176:20-28. [PMID: 29928900 DOI: 10.1016/j.exer.2018.06.019] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2018] [Revised: 06/04/2018] [Accepted: 06/16/2018] [Indexed: 01/19/2023]
Abstract
Human limbal epithelial cells (LECs) intended for treatment of limbal stem cell deficiency are commonly cultivated on a 3T3 feeder layer with complex culture medium supplemented with fetal bovine serum (FBS). However, FBS is a xenogeneic component containing poorly characterised constituents and exhibits quantitative and qualitative lot-to-lot variations. Human limbal explants were plated on untreated or fibrin coated plastic plates and cultured in two non-xenogeneic media (supplemented with either human serum or platelet lysate only). Our aim was to find out whether the characteristics of harvested LEC cultures are comparable to those of LEC cultivated in the gold standard - FBS-supplemented complex medium. The growth kinetics, cell proliferation, differentiation, stemness maintenance, apoptosis and contamination by other cell types were evaluated and compared among these conditions. In all of them LECs were successfully cultivated. Stemness was preserved in both xeno-free media. However, cells cultured with human serum on the fibrin-coated plates had the highest growth rate and cell proliferation and very low fibroblast-like cell contamination. These data suggest that xeno-free cell culture conditions can replace the traditional FBS-supplemented medium and thereby provide a safer protocol for ex vivo cultured limbal stem cell transplants.
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Affiliation(s)
- Kristyna Brejchova
- Research Unit for Rare Diseases, Clinic of Paediatrics and Adolescent Medicine, 1st Faculty of Medicine, Charles University, Ke Karlovu 2, 128 08 Prague 2, Czech Republic; Laboratory of the Biology and Pathology of the Eye, Institute of Biology and Medical Genetics, 1st Faculty of Medicine, Charles University and General University Hospital in Prague, Albertov 4, 128 00 Prague 2, Czech Republic.
| | - Peter Trosan
- Laboratory of the Biology and Pathology of the Eye, Institute of Biology and Medical Genetics, 1st Faculty of Medicine, Charles University and General University Hospital in Prague, Albertov 4, 128 00 Prague 2, Czech Republic
| | - Pavel Studeny
- Ophthalmology Department of 3rd Medical Faculty and University Hospital Kralovske Vinohrady, Šrobárova 1150/50, 100 34 Prague 10, Czech Republic
| | - Pavlina Skalicka
- Research Unit for Rare Diseases, Clinic of Paediatrics and Adolescent Medicine, 1st Faculty of Medicine, Charles University, Ke Karlovu 2, 128 08 Prague 2, Czech Republic; Department of Ophthalmology, 1st Faculty of Medicine, Charles University and General University Hospital in Prague, U nemocnice 499/2, 128 08 Prague 2, Czech Republic
| | - Tor Paaske Utheim
- Department of Medical Biochemistry, Oslo University Hospital, Kirkeveien 166, 0407 Oslo, Norway; Department of Plastic and Reconstructive Surgery, Oslo University Hospital, Kirkeveien 166, 0407 Oslo, Norway
| | - Jan Bednar
- Laboratory of the Biology and Pathology of the Eye, Institute of Biology and Medical Genetics, 1st Faculty of Medicine, Charles University and General University Hospital in Prague, Albertov 4, 128 00 Prague 2, Czech Republic
| | - Katerina Jirsova
- Laboratory of the Biology and Pathology of the Eye, Institute of Biology and Medical Genetics, 1st Faculty of Medicine, Charles University and General University Hospital in Prague, Albertov 4, 128 00 Prague 2, Czech Republic; Ophthalmology Department of 3rd Medical Faculty and University Hospital Kralovske Vinohrady, Šrobárova 1150/50, 100 34 Prague 10, Czech Republic
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Stern JH, Tian Y, Funderburgh J, Pellegrini G, Zhang K, Goldberg JL, Ali RR, Young M, Xie Y, Temple S. Regenerating Eye Tissues to Preserve and Restore Vision. Cell Stem Cell 2018; 22:834-849. [PMID: 29859174 PMCID: PMC6492284 DOI: 10.1016/j.stem.2018.05.013] [Citation(s) in RCA: 117] [Impact Index Per Article: 16.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Ocular regenerative therapies are on track to revolutionize treatment of numerous blinding disorders, including corneal disease, cataract, glaucoma, retinitis pigmentosa, and age-related macular degeneration. A variety of transplantable products, delivered as cell suspensions or as preformed 3D structures combining cells and natural or artificial substrates, are in the pipeline. Here we review the status of clinical and preclinical studies for stem cell-based repair, covering key eye tissues from front to back, from cornea to retina, and including bioengineering approaches that advance cell product manufacturing. While recognizing the challenges, we look forward to a deep portfolio of sight-restoring, stem cell-based medicine. VIDEO ABSTRACT.
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Affiliation(s)
- Jeffrey H Stern
- Neural Stem Cell Institute, Rensselaer, NY 12144, USA; Kellogg Eye Center, University of Michigan, Ann Arbor, MI 48105, USA
| | - Yangzi Tian
- Colleges of Nanoscale Science and Engineering, SUNY Polytechnic Institute, 257 Fuller Road, Albany, NY 12203, USA
| | - James Funderburgh
- Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA
| | - Graziella Pellegrini
- Centre for Regenerative Medicine, University of Modena and Reggio Emilia, via G.Gottardi 100, 41125 Modena, Italy
| | - Kang Zhang
- Shiley Eye Institute and Institute for Engineering in Medicine, University of California, San Diego, La Jolla, CA 92093, USA; State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University and Guangzhou Regenerative Medicine and Health Laboratory, Guangzhou 510060, China
| | - Jeffrey L Goldberg
- Byers Eye Institute at Stanford University, 2452 Watson Court, Palo Alto, CA 94303, USA
| | - Robin R Ali
- Department of Genetics, University College London Institute of Ophthalmology, 11-43 Bath Street, London EC1V 9EL, UK; NIHR Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology, City Road, London EC1V 2PD, UK; Kellogg Eye Center, University of Michigan, Ann Arbor, MI 48105, USA
| | - Michael Young
- The Schepens Eye Research Institute, Massachusetts Eye and Ear, an affiliate of Harvard Medical School, Boston, MA 02114, USA
| | - Yubing Xie
- Colleges of Nanoscale Science and Engineering, SUNY Polytechnic Institute, 257 Fuller Road, Albany, NY 12203, USA
| | - Sally Temple
- Neural Stem Cell Institute, Rensselaer, NY 12144, USA; Kellogg Eye Center, University of Michigan, Ann Arbor, MI 48105, USA.
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The Role of Limbal Epithelial Stem Cells in Regulating Corneal (Lymph)angiogenic Privilege and the Micromilieu of the Limbal Niche following UV Exposure. Stem Cells Int 2018; 2018:8620172. [PMID: 29853920 PMCID: PMC5964490 DOI: 10.1155/2018/8620172] [Citation(s) in RCA: 54] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2017] [Accepted: 04/18/2018] [Indexed: 12/02/2022] Open
Abstract
The cornea is a clear structure, void of blood, and lymphatic vessels, functioning as our window to the world. Limbal epithelial stem cells, occupying the area between avascular cornea and vascularized conjunctiva, have been implicated in tissue border maintenance, preventing conjunctivalisation and propagation of blood and lymphatic vessels into the cornea. Defects in limbal epithelial stem cells are linked to corneal neovascularisation, including lymphangiogenesis, chronic inflammation, conjunctivalisation, epithelial abnormalities including the presence of goblet cells, breaks in Bowman's membrane, persistent epithelial defects and ulceration, ocular surface squamous neoplasia, lipid keratopathy, pain, discomfort, and compromised vision. It has been postulated that pterygium is an example of focal limbal deficiency. Previous reports showing changes occurring in limbal epithelium during pterygium pathogenesis suggest that there is a link to stem cell damage. In this light, pterygium can serve as a model disease of UV-induced stem cell damage also characterised by corneal blood and lymphangiogenesis. This review focuses on the role of corneal and limbal epithelial cells and the stem cell niche in maintaining corneal avascularity and corneal immune privilege and how this may be deregulated following UV exposure. We present an overview of the PUBMED literature in the field as well as recent work from our laboratories.
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Le Q, Xu J, Deng SX. The diagnosis of limbal stem cell deficiency. Ocul Surf 2018; 16:58-69. [PMID: 29113917 PMCID: PMC5844504 DOI: 10.1016/j.jtos.2017.11.002] [Citation(s) in RCA: 125] [Impact Index Per Article: 17.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2017] [Revised: 08/24/2017] [Accepted: 11/03/2017] [Indexed: 12/15/2022]
Abstract
Limbal stem cells (LSCs) maintain the normal homeostasis and wound healing of corneal epithelium. Limbal stem cell deficiency (LSCD) is a pathologic condition that results from the dysfunction and/or an insufficient quantity of LSCs. The diagnosis of LSCD has been made mainly based on medical history and clinical signs, which often are not specific to LSCD. Methods to stage the severity of LSCD have been lacking. With the application of newly developed ocular imaging modalities and molecular methods as diagnostic tools, standardized quantitative criteria for the staging of LSCD can be established. Because of these recent advancements, effective patient-specific therapy for different stages of LSCD may be feasible.
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Affiliation(s)
- Qihua Le
- Stein Eye Institute, Cornea Division, David Geffen School of Medicine, University of California, Los Angeles, USA; Department of Ophthalmology, Eye & ENT Hospital of Fudan University, Shanghai 200031, China
| | - Jianjiang Xu
- Department of Ophthalmology, Eye & ENT Hospital of Fudan University, Shanghai 200031, China
| | - Sophie X Deng
- Stein Eye Institute, Cornea Division, David Geffen School of Medicine, University of California, Los Angeles, USA.
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Gesteira TF, Sun M, Coulson-Thomas YM, Yamaguchi Y, Yeh LK, Hascall V, Coulson-Thomas VJ. Hyaluronan Rich Microenvironment in the Limbal Stem Cell Niche Regulates Limbal Stem Cell Differentiation. Invest Ophthalmol Vis Sci 2017; 58:4407-4421. [PMID: 28863216 PMCID: PMC5584473 DOI: 10.1167/iovs.17-22326] [Citation(s) in RCA: 56] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2017] [Accepted: 07/31/2017] [Indexed: 02/07/2023] Open
Abstract
Purpose Limbal epithelial stem cells (LSCs), located in the basal layer of the corneal epithelium in the corneal limbus, are vital for maintaining the corneal epithelium. LSCs have a high capacity of self-renewal with increased potential for error-free proliferation and poor differentiation. To date, limited research has focused on unveiling the composition of the limbal stem cell niche, and, more important, on the role the specific stem cell niche may have in LSC differentiation and function. Our work investigates the composition of the extracellular matrix in the LSC niche and how it regulates LSC differentiation and function. Methods Hyaluronan (HA) is naturally synthesized by hyaluronan synthases (HASs), and vertebrates have the following three types: HAS1, HAS2, and HAS3. Wild-type and HAS and TSG-6 knockout mice-HAS1-/-;HAS3-/-, HAS2Δ/ΔCorEpi, TSG-6-/--were used to determine the importance of the HA niche in LSC differentiation and specification. Results Our data demonstrate that the LSC niche is composed of a HA rich extracellular matrix. HAS1-/-;HAS3-/-, HAS2Δ/ΔCorEpi, and TSG-6-/- mice have delayed wound healing and increased inflammation after injury. Interestingly, upon insult the HAS knock-out mice up-regulate HA throughout the cornea through a compensatory mechanism, and in turn this alters LSC and epithelial cell specification. Conclusions The LSC niche is composed of a specialized HA matrix that differs from that present in the rest of the corneal epithelium, and the disruption of this specific HA matrix within the LSC niche leads to compromised corneal epithelial regeneration. Finally, our findings suggest that HA has a major role in maintaining the LSC phenotype.
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MESH Headings
- Animals
- Burns, Chemical/metabolism
- Cell Differentiation/physiology
- Cellular Microenvironment/physiology
- Disease Models, Animal
- Epithelium, Corneal/metabolism
- Eye Burns/chemically induced
- Glucuronosyltransferase/metabolism
- Hyaluronan Synthases
- Hyaluronic Acid/genetics
- Hyaluronic Acid/metabolism
- Immunohistochemistry
- Limbus Corneae/cytology
- Mice
- Mice, Inbred C57BL
- Mice, Knockout
- Mice, Transgenic
- Microscopy, Confocal
- Microscopy, Electron, Transmission
- RNA, Messenger/genetics
- Real-Time Polymerase Chain Reaction
- Sodium Hydroxide
- Stem Cell Niche/physiology
- Stem Cells/metabolism
- Wound Healing/physiology
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Affiliation(s)
| | - Mingxia Sun
- College of Optometry, University of Houston, Houston, Texas, United States
| | | | - Yu Yamaguchi
- Sanford Children's Health Research Center, Sanford-Burnham Medical Research Institute, La Jolla, California, United States
| | - Lung-Kun Yeh
- Department of Ophthalmology, Chang-Gung Memorial Hospital, Chang-Gung University College of Medicine, Linko, Taiwan
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Abstract
PURPOSE OF REVIEW The aim of this review is to describe the underlying mechanisms of corneal epithelial homeostasis in addition to illustrating the vital role of the limbal epithelial stem cells (LESCs) and the limbal niche in epithelial regeneration and wound healing. RECENT FINDINGS The shedded corneal epithelial cells are constantly replenished by the LESCs which give rise to epithelial cells that proliferate, differentiate, and migrate centripetally. While some recent studies have proposed that epithelial stem cells may also be present in the central cornea, the predominant location for the stem cells is the limbus. The limbal niche is the specialized microenvironment consisting of cells, extracellular matrix, and signaling molecules that are essential for the function of LESCs. Disturbances to limbal niche can result in LESC dysfunction; therefore, limbal stem cell deficiency should also be considered a limbal niche deficiency. Current and in-development therapeutic strategies are aimed at restoring the limbal niche, by medical and/or surgical treatments, administration of trophic factors, and cell based therapies. SUMMARY The corneal epithelium is constantly replenished by LESCs that are housed within the limbal niche. The limbal niche is the primary determinant of the LESC function and novel therapeutic approaches should be focused on regeneration of this microenvironment.
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Haagdorens M, Behaegel J, Rozema J, Van Gerwen V, Michiels S, Ní Dhubhghaill S, Tassignon MJ, Zakaria N. A method for quantifying limbal stem cell niches using OCT imaging. Br J Ophthalmol 2017; 101:1250-1255. [PMID: 28228408 PMCID: PMC5574400 DOI: 10.1136/bjophthalmol-2016-309549] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2016] [Revised: 12/16/2016] [Accepted: 12/31/2016] [Indexed: 12/13/2022]
Abstract
Aims To evaluate the efficacy of Fourier domain-optical coherence tomography (FD-OCT) in imaging and quantifying the limbal palisades of Vogt and to correlate these images with histological findings. Methods The superior and inferior limbal region of both eyes of 50 healthy volunteers were imaged by FD-OCT. Images were processed and analysed using Matlab software. In vitro immunofluorescent staining of a cadaveric donor limbus was analysed to correlate the presence of stem cells in the visualised structures. Results FD-OCT could successfully visualise limbal crypts and the palisades of Vogt in the limbus region. Fluorescent labelling confirmed the presence of stem cells in these structures. The mean palisade ridge width (ΔPR) and the mean interpalisade epithelial rete peg width (ΔERP) were both of the order of 72 μm, leading to a palisade density (PD) of about 7.4 palisades/mm. A significant difference in ΔPR, ΔERP and PD was seen between the inferior and superior sides of the right eye and the superior sides of the left and right eye(p<0.05.). A significant influence of iris colour on parameters ΔPR, ΔERP and PD was found, and of age on PD and ΔERP (p<0.05). Conclusions In vivo OCT imaging is a safe and effective modality to image the limbus and can be used to visualise the palisades of Vogt. Image processing using Matlab software enabled quantification and density calculation of imaged limbal palisades of Vogt. This technique may enhance targeted limbal biopsies for transplantation.
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Affiliation(s)
- Michel Haagdorens
- Faculty of Medicine and Health Sciences, Department of Ophthalmology, Visual Optics and Visual Rehabilitation, Antwerp University, Antwerp, Wilrijk, Belgium.,Department of Ophthalmology, Antwerp University Hospital, Edegem, Belgium
| | - Joséphine Behaegel
- Faculty of Medicine and Health Sciences, Department of Ophthalmology, Visual Optics and Visual Rehabilitation, Antwerp University, Antwerp, Wilrijk, Belgium.,Department of Ophthalmology, University Hospital Brussels, Brussels, Belgium
| | - Jos Rozema
- Faculty of Medicine and Health Sciences, Department of Ophthalmology, Visual Optics and Visual Rehabilitation, Antwerp University, Antwerp, Wilrijk, Belgium.,Department of Ophthalmology, Antwerp University Hospital, Edegem, Belgium
| | - Veerle Van Gerwen
- Faculty of Medicine and Health Sciences, Department of Ophthalmology, Visual Optics and Visual Rehabilitation, Antwerp University, Antwerp, Wilrijk, Belgium
| | - Sofie Michiels
- Department of Ophthalmology, Antwerp University Hospital, Edegem, Belgium
| | - Sorcha Ní Dhubhghaill
- Faculty of Medicine and Health Sciences, Department of Ophthalmology, Visual Optics and Visual Rehabilitation, Antwerp University, Antwerp, Wilrijk, Belgium.,Department of Ophthalmology, Antwerp University Hospital, Edegem, Belgium
| | - Marie-José Tassignon
- Faculty of Medicine and Health Sciences, Department of Ophthalmology, Visual Optics and Visual Rehabilitation, Antwerp University, Antwerp, Wilrijk, Belgium.,Department of Ophthalmology, Antwerp University Hospital, Edegem, Belgium
| | - Nadia Zakaria
- Faculty of Medicine and Health Sciences, Department of Ophthalmology, Visual Optics and Visual Rehabilitation, Antwerp University, Antwerp, Wilrijk, Belgium.,Department of Ophthalmology, Antwerp University Hospital, Edegem, Belgium.,Center for Cell Therapy and Regenerative Medicine, Antwerp University Hospital, Edegem, Belgium
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Lužnik Z, Hawlina M, Ferrari S, Ponzin D, Schollmayer P. Ocular surface reconstruction in limbal stem cell deficiency: current treatment options and perspectives. EXPERT REVIEW OF OPHTHALMOLOGY 2017. [DOI: 10.1080/17469899.2017.1263568] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 10/20/2022]
Affiliation(s)
- Zala Lužnik
- Eye Hospital, University Medical Centre, Ljubljana, Slovenia
| | - Marko Hawlina
- Eye Hospital, University Medical Centre, Ljubljana, Slovenia
| | - Stefano Ferrari
- Fondazione Banca degli Occhi del Veneto Onlus, Via Paccagnella 11, Venezia Zelarino, Italy
| | - Diego Ponzin
- Fondazione Banca degli Occhi del Veneto Onlus, Via Paccagnella 11, Venezia Zelarino, Italy
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Kasinathan JR, Namperumalsamy VP, Veerappan M, Chidambaranathan GP. A novel method for a high enrichment of human corneal epithelial stem cells for genomic analysis. Microsc Res Tech 2016; 79:1165-1172. [PMID: 27862636 DOI: 10.1002/jemt.22771] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2016] [Revised: 08/11/2016] [Accepted: 08/18/2016] [Indexed: 12/13/2022]
Abstract
Understanding the molecular mechanisms that regulate the corneal epithelial stem cells (CESCs) in maintaining corneal homeostasis remains elusive largely due to the lack of a specific marker for their isolation. This study aims to enrich CESCs from human donor limbal epithelium and to evaluate the level of enrichment based on expression of ΔNp63α, a putative CESC marker. A two-stage enrichment of CESCs was carried out. (a) The limbal basal epithelial cells were isolated by differential enzymatic treatment and five-fold enrichment was achieved from 2% of CESCs present in the total limbal epithelium. The CESCs were quantified on the basis of two parameters-high expression of p63/ABCG2 and nucleus to cytoplasmic (N/C) ratio ≥0.7. (b) Cytospin smears of isolated basal cells were Giemsa stained and cells with N/C ratio ≥0.7 were separated by laser capture microdissection. This strategy resulted in an enrichment of CESCs to 78.57% based on two-parameter analysis using p63 and 76.66% using ABCG2. RT-PCR was carried out for ΔNp63 isoforms (α, β, and γ) and connexin-43, with GAPDH for normalization. The expression of ΔNp63α was restricted to the enriched population of CESCs in contrast to its absence in limbal basal cells with N/C ratio <0.7 and CCECs. The unique expression of ΔNp63α and 5.9-fold reduced connexin-43 expression in the enriched population of CESCs indicates its high purity. Further analysis of these cells will help in elucidating the molecular mechanisms associated with stemness and also in identifying a specific marker for CESCs.
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Affiliation(s)
- Jhansi Rani Kasinathan
- Department of Immunology and Stem Cell Biology, Aravind Medical Research Foundation, Dr. G. Venkataswamy Eye Research Institute, Madurai, Tamil Nadu, India
| | | | - Muthukkaruppan Veerappan
- Department of Immunology and Stem Cell Biology, Aravind Medical Research Foundation, Dr. G. Venkataswamy Eye Research Institute, Madurai, Tamil Nadu, India
| | - Gowri Priya Chidambaranathan
- Department of Immunology and Stem Cell Biology, Aravind Medical Research Foundation, Dr. G. Venkataswamy Eye Research Institute, Madurai, Tamil Nadu, India
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Lužnik Z, Hawlina M, Maličev E, Bertolin M, Kopitar AN, Ihan A, Ferrari S, Schollmayer P. Effect of Cryopreserved Amniotic Membrane Orientation on the Expression of Limbal Mesenchymal and Epithelial Stem Cell Markers in Prolonged Limbal Explant Cultures. PLoS One 2016; 11:e0164408. [PMID: 27723792 PMCID: PMC5056676 DOI: 10.1371/journal.pone.0164408] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2016] [Accepted: 09/23/2016] [Indexed: 01/20/2023] Open
Abstract
PURPOSE To evaluate the effect of prolonged limbal explants cultured without any scaffolds or on amniotic membrane (AM) on the viability, proliferation and differentiation potential of putative phenotypically defined cultured limbal mesenchymal (LMSC) and epithelial stem cells (LESC). METHODS Limbal explants were cultivated on cryopreserved intact AM or plastic plates using medium supplemented with only human serum. AM was positioned with either the epithelial or stromal side up. The outgrowing cells were immunophenotyped for the co-expression of mesenchymal stem cell markers (CD73/CD90/CD105 positive and CD45 negative), proliferation and putative progenitor markers (CXCR4, CD117), epithelial markers and antigen presenting cell markers (CD80, CD83, CD86) by flow cytometry. Immunohistochemistry on limbal cultures cultivated on AM was carried out with antibodies against pan-cytokeratin, p63, Ki67. RESULTS Morphological and immunostaining analyses revealed two distinct stem cell population types, which could be identified over prolonged culturing time periods. Expression of LMSC markers and CXCR4 was significantly higher (p < 0.05) in cultures cultivated without AM. However, no statistically significant difference was observed in CD117 expression. The cells cultivated on AM retained an epithelial cell structure, which was further confirmed by histology examination. Histology revealed limbal epithelial growth and p63, Ki67 positive cells on both sides of AM. CONCLUSION Limbal cells cultivated on AM exhibited a lower expression profile of LMSC and CXCR4 markers as limbal cells cultivated on plastic culture plates. However, CD117 expression was similar. Histology confirmed limbal epithelial cell growth on both sides of AM, with no morphological differences, or positivity of cells for p63 and Ki67.
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Affiliation(s)
- Zala Lužnik
- Eye Hospital, University Medical Centre, Ljubljana, Slovenia
| | - Marko Hawlina
- Eye Hospital, University Medical Centre, Ljubljana, Slovenia
| | - Elvira Maličev
- Blood Transfusion Centre of Slovenia, Ljubljana. Slovenia
| | - Marina Bertolin
- The Veneto Eye Bank Foundation (Fondazione Banca degli Occhi del Veneto), Zelarino-Venice, Italy
| | - Andreja Nataša Kopitar
- Medical Faculty Ljubljana, Institute of Microbiology and Immunology, Ljubljana, Slovenia
| | - Alojz Ihan
- Medical Faculty Ljubljana, Institute of Microbiology and Immunology, Ljubljana, Slovenia
| | - Stefano Ferrari
- The Veneto Eye Bank Foundation (Fondazione Banca degli Occhi del Veneto), Zelarino-Venice, Italy
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