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Wang B, Zhao JL, Cai WY, Wang GY, Li YZ, Wang JS, Xie HT, Zhang MC. Progress in Transdifferentiation of Autologous Alternative Cell Sources into Corneal Epithelial Cells. Stem Cell Rev Rep 2025; 21:226-235. [PMID: 39480612 PMCID: PMC11762461 DOI: 10.1007/s12015-024-10808-1] [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] [Accepted: 10/20/2024] [Indexed: 11/02/2024]
Abstract
Corneal limbal epithelial stem cells (LESCs) play a crucial role in corneal epithelium regeneration. Severe damage to these cells can result in limbal stem cell deficiency (LSCD), characterized by repeated corneal conjunctivalization, leading to corneal turbidity and scar formation. Restoring functional LESCs and their ecological location are essential for treating LSCD. The goal of this review is to provide researchers and clinicians with key insights into LESCs biology and to conclude the current cell-based therapies advancement in LSCD treatments. Therapeutic cell resources mainly include mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), skin keratinocyte stem cells (SKCs), and oral mucosal epithelial cells (OMECs).
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Affiliation(s)
- Bei Wang
- Department of Ophthalmology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China
| | - Jiang-Lan Zhao
- Department of Ophthalmology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China
| | - Wan-Ying Cai
- Department of Ophthalmology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China
| | - Gong-Yue Wang
- Department of Ophthalmology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China
| | - Yu-Zhi Li
- Department of Ophthalmology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China
| | - Jia-Song Wang
- Department of Ophthalmology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China
| | - Hua-Tao Xie
- Department of Ophthalmology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
| | - Ming-Chang Zhang
- Department of Ophthalmology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
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2
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Davidson KC, Sung M, Brown KD, Contet J, Belluschi S, Hamel R, Moreno-Moral A, Dos Santos RL, Gough J, Polo JM, Daniell M, Parfitt GJ. Single nuclei transcriptomics of the in situ human limbal stem cell niche. Sci Rep 2024; 14:6749. [PMID: 38514716 PMCID: PMC10957941 DOI: 10.1038/s41598-024-57242-4] [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: 09/06/2023] [Accepted: 03/15/2024] [Indexed: 03/23/2024] Open
Abstract
The corneal epithelium acts as a barrier to pathogens entering the eye; corneal epithelial cells are continuously renewed by uni-potent, quiescent limbal stem cells (LSCs) located at the limbus, where the cornea transitions to conjunctiva. There has yet to be a consensus on LSC markers and their transcriptome profile is not fully understood, which may be due to using cadaveric tissue without an intact stem cell niche for transcriptomics. In this study, we addressed this problem by using single nuclei RNA sequencing (snRNAseq) on healthy human limbal tissue that was immediately snap-frozen after excision from patients undergoing cataract surgery. We identified the quiescent LSCs as a sub-population of corneal epithelial cells with a low level of total transcript counts. Moreover, TP63, KRT15, CXCL14, and ITGβ4 were found to be highly expressed in LSCs and transiently amplifying cells (TACs), which constitute the corneal epithelial progenitor populations at the limbus. The surface markers SLC6A6 and ITGβ4 could be used to enrich human corneal epithelial cell progenitors, which were also found to specifically express the putative limbal progenitor cell markers MMP10 and AC093496.1.
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Affiliation(s)
- Kathryn C Davidson
- Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC, Australia
| | | | - Karl D Brown
- Centre for Eye Research Australia (CERA), Melbourne, Australia
| | - Julian Contet
- Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC, Australia
| | | | | | | | | | - Julian Gough
- Mogrify Limited, Cambridge, England, UK
- MRC Laboratory of Molecular Biology, Cambridge, England, UK
| | - Jose M Polo
- Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC, Australia.
- Mogrify Limited, Cambridge, England, UK.
- Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Clayton, VIC, Australia.
- Australian Regenerative Medicine Institute, Monash University, Clayton, VIC, Australia.
- Adelaide Centre for Epigenetics, Faculty of Medicine Nursing and Medical Sciences, The University of Adelaide, Adelaide, Australia.
- The South Australian Immunogenomics Cancer Institute, Faculty of Medicine Nursing and Medical Sciences, The University of Adelaide, Adelaide, Australia.
| | - Mark Daniell
- Centre for Eye Research Australia (CERA), Melbourne, Australia.
| | - Geraint J Parfitt
- Mogrify Limited, Cambridge, England, UK.
- Ophthalmology Discovery Research, AbbVie, Irvine, CA, USA.
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3
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Xiao Y, McGhee CNJ, Zhang J. Adult stem cells in the eye: Identification, characterisation, and therapeutic application in ocular regeneration - A review. Clin Exp Ophthalmol 2024; 52:148-166. [PMID: 38214071 DOI: 10.1111/ceo.14309] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2023] [Accepted: 09/25/2023] [Indexed: 01/13/2024]
Abstract
Adult stem cells, present in various parts of the human body, are undifferentiated cells that can proliferate and differentiate to replace dying cells within tissues. Stem cells have specifically been identified in the cornea, trabecular meshwork, crystalline lens, iris, ciliary body, retina, choroid, sclera, conjunctiva, eyelid, lacrimal gland, and orbital fat. The identification of ocular stem cells broadens the potential therapeutic strategies for untreatable eye diseases. Currently, stem cell transplantation for corneal and conjunctival diseases remains the most common stem cell-based therapy in ocular clinical management. Lens epithelial stem cells have been applied in the treatment of paediatric cataracts. Several early-phase clinical trials for corneal and retinal regeneration using ocular stem cells are also underway. Extensive preclinical studies using ocular stem cells have been conducted, showing encouraging outcomes. Ocular stem cells currently demonstrate great promise in potential treatments of eye diseases. In this review, we focus on the identification, characterisation, and therapeutic application of adult stem cells in the eye.
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Affiliation(s)
- Yuting Xiao
- Department of Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand
| | - Charles N J McGhee
- Department of Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand
| | - Jie Zhang
- Department of Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand
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4
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Tonti E, Manco GA, Spadea L, Zeppieri M. Focus on limbal stem cell deficiency and limbal cell transplantation. World J Transplant 2023; 13:321-330. [PMID: 38174150 PMCID: PMC10758683 DOI: 10.5500/wjt.v13.i6.321] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/19/2023] [Revised: 10/10/2023] [Accepted: 11/02/2023] [Indexed: 12/15/2023] Open
Abstract
Limbal stem cell deficiency (LSCD) causes severe vision impairment and can lead to blindness, representing one of the most challenging ocular surface disorders. Stem cell deficiency can be congenital or, more often, acquired. The categorization of ocular surface transplantation techniques is crucial to achieving treatment homogeneity and quality of care, according to the anatomic source of the tissue being transplanted, genetic source, autologous or allogenic transplantation (to reflect histocompatibility in the latter group), and cell culture and tissue engi neering techniques. The aim of this minireview is to provide a summary of the management of LSCD, from clinical characteristics and therapeutic outcomes to the development of novel therapeutic approaches. The manuscript also briefly summarizes recent findings in the current literature and outlines the future challenges to overcome in the management of the major types of ocular surface failure.
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Affiliation(s)
- Emanuele Tonti
- Eye Clinic, Policlinico Umberto I, "Sapienza" University of Rome, Rome 00142, Italy
| | | | - Leopoldo Spadea
- Eye Clinic, Policlinico Umberto I, "Sapienza" University of Rome, Rome 00142, Italy
| | - Marco Zeppieri
- Department of Ophthalmology, University Hospital of Udine, Udine 33100, Italy
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Yao G, Mo X, Liu S, Wang Q, Xie M, Lou W, Chen S, Pan T, Chen K, Yao D, Lin Y. Snowflake-inspired and blink-driven flexible piezoelectric contact lenses for effective corneal injury repair. Nat Commun 2023; 14:3604. [PMID: 37330515 PMCID: PMC10276863 DOI: 10.1038/s41467-023-39315-6] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2022] [Accepted: 06/06/2023] [Indexed: 06/19/2023] Open
Abstract
The cornea is a tissue susceptible to various injuries and traumas with a complicated cascade repair process, in which conserving its integrity and clarity is critical to restoring visual function. Enhancing the endogenous electric field is recognized as an effective method of accelerating corneal injury repair. However, current equipment limitations and implementation complexities hinder its widespread adoption. Here, we propose a snowflake-inspired, blink-driven flexible piezoelectric contact lens that can convert mechanical blink motions into a unidirectional pulsed electric field for direct application to moderate corneal injury repair. The device is validated on mouse and rabbit models with different relative corneal alkali burn ratios to modulate the microenvironment, alleviate stromal fibrosis, promote orderly epithelial arrangement and differentiation, and restore corneal clarity. Within an 8-day intervention, the corneal clarity of mice and rabbits improves by more than 50%, and the repair rate of mouse and rabbit corneas increases by over 52%. Mechanistically, the device intervention is advantageous in blocking growth factors' signaling pathways specifically involved in stromal fibrosis whilst preserving and harnessing the signaling pathways required for indispensable epithelial metabolism. This work put forward an efficient and orderly corneal therapeutic technology utilizing artificial endogenous-strengthened signals generated by spontaneous body activities.
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Affiliation(s)
- Guang Yao
- School of Materials and Energy, University of Electronic Science and Technology of China, Chengdu, 610054, Sichuan, China.
- State Key Laboratory of Electronic Thin films and Integrated Devices, University of Electronic Science and Technology of China, Chengdu, 610054, Sichuan, China.
- Shenzhen Institute for Advanced Study, University of Electronic Science and Technology of China, Shenzhen, 518110, China.
| | - Xiaoyi Mo
- School of Materials and Energy, University of Electronic Science and Technology of China, Chengdu, 610054, Sichuan, China
| | - Shanshan Liu
- MOE Key Laboratory for Neuroinformation, The Clinical Hospital of Chengdu Brain Sciences Institute, University of Electronic Science and Technology of China, Chengdu, 610054, Sichuan, China
| | - Qian Wang
- School of Materials and Energy, University of Electronic Science and Technology of China, Chengdu, 610054, Sichuan, China
| | - Maowen Xie
- School of Materials and Energy, University of Electronic Science and Technology of China, Chengdu, 610054, Sichuan, China
| | - Wenhao Lou
- School of Materials and Energy, University of Electronic Science and Technology of China, Chengdu, 610054, Sichuan, China
| | - Shiyan Chen
- Department of Ophthalmology, Sichuan Academy of Medical Sciences and Sichuan Provincial People's Hospital, Medical School, University of Electronic Science and Technology of China, Chengdu, 610054, Sichuan, China
| | - Taisong Pan
- School of Materials and Energy, University of Electronic Science and Technology of China, Chengdu, 610054, Sichuan, China
| | - Ke Chen
- MOE Key Laboratory for Neuroinformation, The Clinical Hospital of Chengdu Brain Sciences Institute, University of Electronic Science and Technology of China, Chengdu, 610054, Sichuan, China.
- Department of Ophthalmology, Sichuan Academy of Medical Sciences and Sichuan Provincial People's Hospital, Medical School, University of Electronic Science and Technology of China, Chengdu, 610054, Sichuan, China.
| | - Dezhong Yao
- MOE Key Laboratory for Neuroinformation, The Clinical Hospital of Chengdu Brain Sciences Institute, University of Electronic Science and Technology of China, Chengdu, 610054, Sichuan, China
| | - Yuan Lin
- School of Materials and Energy, University of Electronic Science and Technology of China, Chengdu, 610054, Sichuan, China.
- State Key Laboratory of Electronic Thin films and Integrated Devices, University of Electronic Science and Technology of China, Chengdu, 610054, Sichuan, China.
- Medico-Engineering Cooperation on Applied Medicine Research Center, University of Electronic Science and Technology of China, Chengdu, 610054, Sichuan, China.
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6
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Verta R, Saccu G, Tanzi A, Grange C, Buono L, Fagoonee S, Deregibus MC, Camussi G, Scalabrin S, Nuzzi R, Bussolati B. Phenotypic and functional characterization of aqueous humor derived extracellular vesicles. Exp Eye Res 2023; 228:109393. [PMID: 36709863 DOI: 10.1016/j.exer.2023.109393] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2022] [Revised: 12/16/2022] [Accepted: 01/25/2023] [Indexed: 01/27/2023]
Abstract
Extracellular vesicles (EVs) are double membrane vesicles, abundant in all biological fluids. However, the characterization of EVs in aqueous humor (AH) is still limited. The aim of the present work was to characterize EVs isolated from AH (AH-EVs) in terms of surface markers of cellular origin and functional properties. We obtained AHs from patients with cataract undergoing surgical phacoemulsification and insertion of intraocular lenses (n = 10). Nanoparticle tracking analysis, electron microscopy, super resolution microscopy and bead-based cytofluorimetry were used to characterize EVs from AH. Subsequently, we investigated the effects of AH-EVs on viability, proliferation and wound healing of human immortalized keratinocyte (HaCaT) cells in vitro in comparison with the effect of mesenchymal stromal cell-EVs (MSC-EVs). AH-EVs had a mean size of around 100 nm and expressed the classical tetraspanins (CD9, CD63 and CD81). Super resolution microscopy revealed co-expression of CD9, CD63 and CD81. Moreover, cytofluorimetric analysis highlighted the expression of mesenchymal, stem, epithelial and endothelial markers. In the in vitro wound healing assay on HaCaT cells, AH-EVs induced a significantly faster wound repair, comparable to the effects of MSC-EVs, and promoted HaCaT cell viability and proliferation. We provide evidence, herein, of the possible AH-EV origin from stromal cells, limbal epithelial/stem cells, ciliary epithelium and corneal endothelium. In addition, we showed their in vitro proliferative and regenerative capacities.
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Affiliation(s)
- Roberta Verta
- Department of Molecular Biotechnology and Health Sciences, University of Torino, Italy
| | - Gabriele Saccu
- Department of Medical Sciences, University of Torino, Torino, Italy
| | - Adele Tanzi
- Department of Molecular Biotechnology and Health Sciences, University of Torino, Italy
| | - Cristina Grange
- Department of Medical Sciences, University of Torino, Torino, Italy
| | - Lola Buono
- Department of Molecular Biotechnology and Health Sciences, University of Torino, Italy
| | - Sharmila Fagoonee
- Institute of Biostructure and Bioimaging, National Research Council, Molecular Biotechnology Center, Turin, Italy
| | | | - Giovanni Camussi
- Department of Medical Sciences, University of Torino, Torino, Italy
| | | | - Raffaele Nuzzi
- Department of Surgical Sciences, University of Torino, Italy
| | - Benedetta Bussolati
- Department of Molecular Biotechnology and Health Sciences, University of Torino, Italy.
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7
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Blanco-Elices C, Morales-Álvarez C, Chato-Astrain J, González-Gallardo C, Ávila-Fernández P, Campos F, Carmona R, Martín-Piedra MÁ, Garzón I, Alaminos M. Development of stromal differentiation patterns in heterotypical models of artificial corneas generated by tissue engineering. Front Bioeng Biotechnol 2023; 11:1124995. [PMID: 37034263 PMCID: PMC10076743 DOI: 10.3389/fbioe.2023.1124995] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2022] [Accepted: 03/16/2023] [Indexed: 04/11/2023] Open
Abstract
Purpose: We carried out a histological characterization analysis of the stromal layer of human heterotypic cornea substitutes generated with extra-corneal cells to determine their putative usefulness in tissue engineering. Methods: Human bioartificial corneas were generated using nanostructured fibrin-agarose biomaterials with corneal stromal cells immersed within. To generate heterotypical corneas, umbilical cord Wharton's jelly stem cells (HWJSC) were cultured on the surface of the stromal substitutes to obtain an epithelial-like layer. These bioartificial corneas were compared with control native human corneas and with orthotypical corneas generated with human corneal epithelial cells on top of the stromal substitute. Both the corneal stroma and the basement membrane were analyzed using histological, histochemical and immunohistochemical methods in samples kept in culture and grafted in vivo for 12 months in the rabbit cornea. Results: Our results showed that the stroma of the bioartificial corneas kept ex vivo showed very low levels of fibrillar and non-fibrillar components of the tissue extracellular matrix. However, in vivo implantation resulted in a significant increase of the contents of collagen, proteoglycans, decorin, keratocan and lumican in the corneal stroma, showing higher levels of maturation and spatial organization of these components. Heterotypical corneas grafted in vivo for 12 months showed significantly higher contents of collagen fibers, proteoglycans and keratocan. When the basement membrane was analyzed, we found that all corneas grafted in vivo showed intense PAS signal and higher contents of nidogen-1, although the levels found in human native corneas was not reached, and a rudimentary basement membrane was observed using transmission electron microscopy. At the epithelial level, HWJSC used to generate an epithelial-like layer in ex vivo corneas were mostly negative for p63, whereas orthotypical corneas and heterotypical corneas grafted in vivo were positive. Conclusion: These results support the possibility of generating bioengineered artificial corneas using non-corneal HWJSC. Although heterotypical corneas were not completely biomimetic to the native human corneas, especially ex vivo, in vivo grafted corneas demonstrated to be highly biocompatible, and the animal cornea became properly differentiated at the stroma and basement membrane compartments. These findings open the door to the future clinical use of these bioartificial corneas.
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Affiliation(s)
- Cristina Blanco-Elices
- Tissue Engineering Group, Department of Histology, Faculty of Medicine, Universidad de Granada, Granada, Spain
- Instituto de Investigación Biosanitaria ibs.GRANADA, Granada, Spain
| | - Carmen Morales-Álvarez
- GENYO, Centre for Genomics and Oncological Research: Pfizer, University of Granada, Andalusian Regional Government, PTS Granada, Granada, Spain
- Department of Biochemistry and Molecular Biology III, Faculty of Medicine, University of Granada, Granada, Spain
| | - Jesús Chato-Astrain
- Tissue Engineering Group, Department of Histology, Faculty of Medicine, Universidad de Granada, Granada, Spain
- Instituto de Investigación Biosanitaria ibs.GRANADA, Granada, Spain
| | | | - Paula Ávila-Fernández
- Tissue Engineering Group, Department of Histology, Faculty of Medicine, Universidad de Granada, Granada, Spain
| | - Fernando Campos
- Tissue Engineering Group, Department of Histology, Faculty of Medicine, Universidad de Granada, Granada, Spain
- Instituto de Investigación Biosanitaria ibs.GRANADA, Granada, Spain
| | - Ramón Carmona
- Department of Cell Biology, Faculty of Sciences, University of Granada, Granada, Spain
| | - Miguel Ángel Martín-Piedra
- Tissue Engineering Group, Department of Histology, Faculty of Medicine, Universidad de Granada, Granada, Spain
- Instituto de Investigación Biosanitaria ibs.GRANADA, Granada, Spain
- *Correspondence: Miguel Ángel Martín-Piedra, ; Ingrid Garzón,
| | - Ingrid Garzón
- Tissue Engineering Group, Department of Histology, Faculty of Medicine, Universidad de Granada, Granada, Spain
- Instituto de Investigación Biosanitaria ibs.GRANADA, Granada, Spain
- *Correspondence: Miguel Ángel Martín-Piedra, ; Ingrid Garzón,
| | - Miguel Alaminos
- Tissue Engineering Group, Department of Histology, Faculty of Medicine, Universidad de Granada, Granada, Spain
- Instituto de Investigación Biosanitaria ibs.GRANADA, Granada, Spain
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8
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Fortingo N, Melnyk S, Sutton SH, Watsky MA, Bollag WB. Innate Immune System Activation, Inflammation and Corneal Wound Healing. Int J Mol Sci 2022; 23:14933. [PMID: 36499260 PMCID: PMC9740891 DOI: 10.3390/ijms232314933] [Citation(s) in RCA: 41] [Impact Index Per Article: 13.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2022] [Revised: 11/09/2022] [Accepted: 11/14/2022] [Indexed: 12/05/2022] Open
Abstract
Corneal wounds resulting from injury, surgeries, or other intrusions not only cause pain, but also can predispose an individual to infection. While some inflammation may be beneficial to protect against microbial infection of wounds, the inflammatory process, if excessive, may delay corneal wound healing. An examination of the literature on the effect of inflammation on corneal wound healing suggests that manipulations that result in reductions in severe or chronic inflammation lead to better outcomes in terms of corneal clarity, thickness, and healing. However, some acute inflammation is necessary to allow efficient bacterial and fungal clearance and prevent corneal infection. This inflammation can be triggered by microbial components that activate the innate immune system through toll-like receptor (TLR) pathways. In particular, TLR2 and TLR4 activation leads to pro-inflammatory nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) activation. Similarly, endogenous molecules released from disrupted cells, known as damage-associated molecular patterns (DAMPs), can also activate TLR2, TLR4 and NFκB, with the resultant inflammation worsening the outcome of corneal wound healing. In sterile keratitis without infection, inflammation can occur though TLRs to impact corneal wound healing and reduce corneal transparency. This review demonstrates the need for acute inflammation to prevent pathogenic infiltration, while supporting the idea that a reduction in chronic and/or excessive inflammation will allow for improved wound healing.
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Affiliation(s)
- Nyemkuna Fortingo
- Department of Physiology, Medical College of Georgia, Augusta University, Augusta, GA 30907, USA
| | - Samuel Melnyk
- Department of Physiology, Medical College of Georgia, Augusta University, Augusta, GA 30907, USA
- James and Jean Culver Vision Discovery Institute, Medical College of Georgia, Augusta University, Augusta, GA 30907, USA
| | - Sarah H. Sutton
- Department of Medical Illustration, Augusta University, Augusta, GA 30907, USA
| | - Mitchell A. Watsky
- James and Jean Culver Vision Discovery Institute, Medical College of Georgia, Augusta University, Augusta, GA 30907, USA
- Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta University, Augusta, GA 30907, USA
| | - Wendy B. Bollag
- Department of Physiology, Medical College of Georgia, Augusta University, Augusta, GA 30907, USA
- James and Jean Culver Vision Discovery Institute, Medical College of Georgia, Augusta University, Augusta, GA 30907, USA
- Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta University, Augusta, GA 30907, USA
- Charlie Norwood VA Medical Center, Augusta, GA 30904, USA
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Masood F, Chang JH, Akbar A, Song A, Hu WY, Azar DT, Rosenblatt MI. Therapeutic Strategies for Restoring Perturbed Corneal Epithelial Homeostasis in Limbal Stem Cell Deficiency: Current Trends and Future Directions. Cells 2022; 11:3247. [PMID: 36291115 PMCID: PMC9600167 DOI: 10.3390/cells11203247] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2022] [Revised: 10/11/2022] [Accepted: 10/12/2022] [Indexed: 02/03/2023] Open
Abstract
Limbal stem cells constitute an important cell population required for regeneration of the corneal epithelium. If insults to limbal stem cells or their niche are sufficiently severe, a disease known as limbal stem cell deficiency occurs. In the absence of functioning limbal stem cells, vision-compromising conjunctivalization of the corneal epithelium occurs, leading to opacification, inflammation, neovascularization, and chronic scarring. Limbal stem cell transplantation is the standard treatment for unilateral cases of limbal stem cell deficiency, but bilateral cases require allogeneic transplantation. Herein we review the current therapeutic utilization of limbal stem cells. We also describe several limbal stem cell markers that impact their phenotype and function and discuss the possibility of modulating limbal stem cells and other sources of stem cells to facilitate the development of novel therapeutic interventions. We finally consider several hurdles for widespread adoption of these proposed methodologies and discuss how they can be overcome to realize vision-restoring interventions.
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Affiliation(s)
- Faisal Masood
- Department of Ophthalmology and Visual Sciences, Illinois Eye and Ear Infirmary, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA
| | - Jin-Hong Chang
- Department of Ophthalmology and Visual Sciences, Illinois Eye and Ear Infirmary, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA
| | - Anosh Akbar
- Department of Ophthalmology and Visual Sciences, Illinois Eye and Ear Infirmary, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA
| | - Amy Song
- Department of Ophthalmology and Visual Sciences, Illinois Eye and Ear Infirmary, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA
| | - Wen-Yang Hu
- Department of Urology, University of Illinois at Chicago, Chicago, IL 60612, USA
| | - Dimitri T. Azar
- Department of Ophthalmology and Visual Sciences, Illinois Eye and Ear Infirmary, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA
| | - Mark I. Rosenblatt
- Department of Ophthalmology and Visual Sciences, Illinois Eye and Ear Infirmary, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA
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10
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Menzel-Severing J, Spaniol K, Groeber-Becker F, Geerling G. [Regenerative medicine for the corneal epithelium : Cell therapy from bench to bedside]. DIE OPHTHALMOLOGIE 2022; 119:891-901. [PMID: 35925345 DOI: 10.1007/s00347-022-01674-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Accepted: 05/31/2022] [Indexed: 06/15/2023]
Abstract
In the case of thermal or caustic burns of the ocular surface, loss of limbal epithelial stem cells leads to compromised self-renewal of the corneal epithelium. This results in permanent loss of vision. In these situations, transplantation of cultured limbal epithelial cells on an amniotic membrane or fibrin gel as substrate (Holoclar®) can help to regenerate the corneal surface. The required cells are obtained from the healthy partner eye, if available. Adult stem cells from other parts of the body potentially serve as alternative cell sources: hair follicles, oral mucosa, mesenchymal stromal cells, or induced pluripotent stem cells (originally, e.g., skin fibroblasts). The reprogramming of such cells can be achieved with the help of transcription factors. In addition, work is being done on biosynthetic or synthetic matrices, which not only serve as substrate material for the transplantation but also support the functional properties of these cells (self-renewal, corneal epithelial-typical phenotype).
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Affiliation(s)
- Johannes Menzel-Severing
- Klinik für Augenheilkunde, Universitätsklinikum Düsseldorf, Moorenstr. 5, 40225, Düsseldorf, Deutschland.
| | - Kristina Spaniol
- Klinik für Augenheilkunde, Universitätsklinikum Düsseldorf, Moorenstr. 5, 40225, Düsseldorf, Deutschland
| | - Florian Groeber-Becker
- Translationszentrum Regenerative Therapien | TLZ-RT, Leitung In-vitro-Testsysteme, Fraunhofer-Institut für Silicatforschung ISC, Würzburg, Deutschland
| | - Gerd Geerling
- Klinik für Augenheilkunde, Universitätsklinikum Düsseldorf, Moorenstr. 5, 40225, Düsseldorf, Deutschland
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Bone Morphogenetic Protein 4 (BMP4) Enhances the Differentiation of Human Induced Pluripotent Stem Cells into Limbal Progenitor Cells. Curr Issues Mol Biol 2021; 43:2124-2134. [PMID: 34940121 PMCID: PMC8929048 DOI: 10.3390/cimb43030147] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2021] [Revised: 11/14/2021] [Accepted: 11/23/2021] [Indexed: 12/21/2022] Open
Abstract
Corneal epithelium maintains visual acuity and is regenerated by the proliferation and differentiation of limbal progenitor cells. Transplantation of human limbal progenitor cells could restore the integrity and functionality of the corneal surface in patients with limbal stem cell deficiency. However, multiple protocols are employed to differentiate human induced pluripotent stem (iPS) cells into corneal epithelium or limbal progenitor cells. The aim of this study was to optimize a protocol that uses bone morphogenetic protein 4 (BMP4) and limbal cell-specific medium. Human dermal fibroblast-derived iPS cells were differentiated into limbal progenitor cells using limbal cell-specific (PI) medium and varying doses (1, 10, and 50 ng/mL) and durations (1, 3, and 10 days) of BMP4 treatment. Differentiated human iPS cells were analyzed by real-time polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemical studies at 2 or 4 weeks after BMP4 treatment. Culturing human dermal fibroblast-derived iPS cells in limbal cell-specific medium and BMP4 gave rise to limbal progenitor and corneal epithelial-like cells. The optimal protocol of 10 ng/mL and three days of BMP4 treatment elicited significantly higher limbal progenitor marker (ABCG2, ∆Np63α) expression and less corneal epithelial cell marker (CK3, CK12) expression than the other combinations of BMP4 dose and duration. In conclusion, this study identified a successful reprogramming strategy to induce limbal progenitor cells from human iPS cells using limbal cell-specific medium and BMP4. Additionally, our experiments indicate that the optimal BMP4 dose and duration favor limbal progenitor cell differentiation over corneal epithelial cells and maintain the phenotype of limbal stem cells. These findings contribute to the development of therapies for limbal stem cell deficiency disorders.
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12
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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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13
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Promoting limbal stem cells proliferation and maintenance using post-thaw human amniotic membranes fortified by platelet lysate. GENE REPORTS 2021. [DOI: 10.1016/j.genrep.2021.101084] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
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14
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Ortiz-Melo MT, Garcia-Murillo MJ, Salazar-Rojas VM, Campos JE, Castro-Muñozledo F. Transcriptional profiles along cell programming into corneal epithelial differentiation. Exp Eye Res 2020; 202:108302. [PMID: 33098888 DOI: 10.1016/j.exer.2020.108302] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2020] [Revised: 09/23/2020] [Accepted: 10/12/2020] [Indexed: 12/15/2022]
Abstract
Using the rabbit corneal epithelial cell line RCE1(5T5) as a model, we analyzed three differentiation stages, distinguished on basis to the growth state of cultured cells and after studying the expression of transcription factors such as Oct4, Pax6 and ΔNp63α, selected differentiation markers, and signaling or epigenetic markers such as Notch receptors and Prdm3. Namely, proliferative non-differentiated cells, committed cells, and cells that constitute a stratified epithelium with a limbal epithelial-like structure. RNAseq based transcriptome analysis showed that 4891 genes were differentially expressed among these stages displaying distinctive gene signatures: proliferative cells had 1278 genes as gene signature, and seem to be early epithelial progenitors with an Oct4+, KLF4+, Myc+, ΔNp63α+, ABCG2+, Vimentin+, Zeb1+, VANGL1+, Krt3-, Krt12- phenotype. Committed cells had a gene signature with 417 genes and displayed markers indicative of the beginning of corneal differentiation, and genes characteristic of proliferative cells; we found the possible participation of Six3 and Six4 transcription factors along this stage. The third stage matches with a stratified corneal epithelium (gene signature comprising 979 genes) and is typified by an increase in the expression of WNT10A and NOTCH 2 and 3 signaling and Cux1 transcription factor, besides Pax6, KLF4 or Sox9. The differentiated cells express about 50% of the genes that belong to the Epidermal Differentiation Complex (EDC). Analysis of the differences between corneal epithelium and epidermis could be crucial to understand the regulatory mechanisms that lead to the expression of the differentiated phenotype.
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Affiliation(s)
- María Teresa Ortiz-Melo
- Department of Cell Biology, Centro de Investigación y de Estudios Avanzados del IPN, Apdo. Postal 14-740. México City, 07000, Mexico; Unidad de Biotecnología y Prototipos, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Ap. Postal 314, 54000, Tlalnepantla, Edo. de México, Mexico; Posgrado en Ciencias Biológicas, Universidad Nacional Autónoma de México. Unidad de Posgrado, Edificio A, 1° Piso, Circuito de Posgrados, Ciudad Universitaria, Coyoacán, C.P. 04510, Ciudad de México, Mexico
| | - Maria Jimena Garcia-Murillo
- Department of Cell Biology, Centro de Investigación y de Estudios Avanzados del IPN, Apdo. Postal 14-740. México City, 07000, Mexico
| | - Víctor Manuel Salazar-Rojas
- Unidad de Biotecnología y Prototipos, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Ap. Postal 314, 54000, Tlalnepantla, Edo. de México, Mexico
| | - Jorge E Campos
- Unidad de Biotecnología y Prototipos, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Ap. Postal 314, 54000, Tlalnepantla, Edo. de México, Mexico
| | - Federico Castro-Muñozledo
- Department of Cell Biology, Centro de Investigación y de Estudios Avanzados del IPN, Apdo. Postal 14-740. México City, 07000, Mexico.
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15
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Corneal Stem Cells as a Source of Regenerative Cell-Based Therapy. Stem Cells Int 2020; 2020:8813447. [PMID: 32765614 PMCID: PMC7388005 DOI: 10.1155/2020/8813447] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2020] [Revised: 07/03/2020] [Accepted: 07/10/2020] [Indexed: 12/15/2022] Open
Abstract
In the past few years, intensive research has focused on corneal stem cells as an unlimited source for cell-based therapy in regenerative ophthalmology. Today, it is known that the cornea has at least two types of stem cells: limbal epithelial stem cells (LESCs) and corneal stromal stem cells (CSSCs). LESCs are used for regeneration of corneal surface, while CSSCs are used for regeneration of corneal stroma. Until now, various approaches and methods for isolation of LESCs and CSSCs and their successful transplantation have been described and tested in several preclinical studies and clinical trials. This review describes in detail phenotypic characteristics of LESCs and CSSCs and discusses their therapeutic potential in corneal regeneration. Since efficient and safe corneal stem cell-based therapy is still a challenging issue that requires continuous cooperation between researchers, clinicians, and patients, this review addresses the important limitations and suggests possible strategies for improvement of corneal stem cell-based therapy.
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16
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Bonzano C, Canciani B, Olivari S, Papadia M, Bagnis A, Cutolo CA, Bonzano E, Pagani P, Cancedda R, Traverso CE. CFSE: A New Method for Identifying Human Limbal Stem Cells and Following Their Migration in Human Cornea. In Vivo 2020; 33:1851-1855. [PMID: 31662512 DOI: 10.21873/invivo.11678] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2019] [Revised: 08/31/2019] [Accepted: 09/03/2019] [Indexed: 11/10/2022]
Abstract
AIM To develop a method capable of identifying human corneal limbal stem cells (LSCs) and follow their proliferation and migration in the epithelium. MATERIALS AND METHODS Ten fresh matched pairs of cadaveric normal human corneas were obtained from donors. Carboxyfluorescein diacetate succinimidyl ester (CFSE) was used to target LSCs. The distribution of CFSE-positive cell clusters was analyzed by fluorescence microscopy by counterstaining with 4',6-diamidino-2-phenylindole (DAPI). Fluorescence was digitally recorded for seven days, and the rate of cell movement was determined. RESULTS CFSE-labeled cells were tracked in corneas. Analysis of time sequences revealed that they moved centripetally. Daily average CFSE-labeled LSC movement was 0.073±0.01 cm (±SD). CONCLUSION CFSE allowed us to identify LSCs and to track their centripetal migration from the limbal basal layer to the anterior ocular surface. This experimental system appears to be a valuable tool for further studies on corneal epithelial cell migration and proliferation.
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Affiliation(s)
- Chiara Bonzano
- Eye Clinic, DiNOGMI, University of Genoa and IRCCS San Martino Polyclinic Hospital, Genoa, Italy
| | - Barbara Canciani
- Laboratory of Regenerative Medicine, Department of Oncology, Biology and Genetics, IRCCS San Martino Polyclinic Hospital, Genoa, Italy
| | - Sara Olivari
- Eye Clinic, DiNOGMI, University of Genoa and IRCCS San Martino Polyclinic Hospital, Genoa, Italy
| | | | - Alessandro Bagnis
- Eye Clinic, DiNOGMI, University of Genoa and IRCCS San Martino Polyclinic Hospital, Genoa, Italy
| | - Carlo Alberto Cutolo
- Eye Clinic, DiNOGMI, University of Genoa and IRCCS San Martino Polyclinic Hospital, Genoa, Italy
| | - Elisabetta Bonzano
- School of Experimental Medicine, University of Pavia and Department of Radiation Oncology, IRCCS San Matteo Polyclinic Foundation, Pavia, Italy
| | - Paola Pagani
- Eye Clinic, DiNOGMI, University of Genoa and IRCCS San Martino Polyclinic Hospital, Genoa, Italy
| | - Ranieri Cancedda
- Laboratory of Regenerative Medicine, Department of Oncology, Biology and Genetics, IRCCS San Martino Polyclinic Hospital, Genoa, Italy
| | - Carlo Enrico Traverso
- Eye Clinic, DiNOGMI, University of Genoa and IRCCS San Martino Polyclinic Hospital, Genoa, Italy
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17
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Banayan N, Georgeon C, Grieve K, Ghoubay D, Baudouin F, Borderie V. [In vivo confocal microscopy and optical coherence tomography as innovative tools for the diagnosis of limbal stem cell deficiency (French translation of the article)]. J Fr Ophtalmol 2018; 41:968-980. [PMID: 30473234 DOI: 10.1016/j.jfo.2018.07.004] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2018] [Revised: 07/10/2018] [Accepted: 07/13/2018] [Indexed: 01/22/2023]
Abstract
The limbus is the anatomical and functional barrier between corneal and conjunctival epithelia. It is characterized by presence of the limbal stem cell niche which allows corneal homeostasis to be maintained. Limbal stem cell deficiency is characterized by a dual process: insufficient regeneration of corneal epithelium, which cannot therefore assure its function of physiological support, associated with corneal invasion by conjunctival proliferation. Diagnosis is currently made via routine clinical examination, corneal impression cytology and in vivo confocal microscopy (IVCM). Slit lamp examination shows abnormal limbal anatomy, thin and irregular epithelium with late fluorescein staining, and superficial vascularization. With its high resolution, IVCM allows identification of limbal and corneal epithelial changes at a cellular level in en face views, parallel to the corneal surface, but with a restricted viewing field of the corneal surface. It shows a poor transition between the corneal and conjunctival epithelia, associated with a loss of the normal corneal epithelial stratification, low basal cell and sub-basal nerve plexus densities, even with sub-epithelial fibrosis. Optical coherence tomography in central cornea and at the limbus, with scans in different orientations, allows a quick, global and non-invasive analysis of normal eyes and those with limbal stem cell deficiency. It shows a thin limbal epithelium, lacking normal thickening, featuring absence of stromal undulations and limbal crypts in cross-sections and sections parallel to the limbus, lack of visible limbal crypts in en face sections, loss of clear transition between the hyporeflective corneal epithelium and the hyperreflective conjunctival epithelium, and hyperreflective sub-epithelial fibrosis.
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Affiliation(s)
- N Banayan
- Service d'ophtalmologie 5, centre hospitalier national d'ophtalmologie des Quinze-Vingts, 28, rue de Charenton, 75012 Paris, France
| | - C Georgeon
- Service d'ophtalmologie 5, centre hospitalier national d'ophtalmologie des Quinze-Vingts, 28, rue de Charenton, 75012 Paris, France
| | - K Grieve
- Inserm UMR S 968, institut de la vision, Sorbonne université, CHNO des 15-20, 75012 Paris, France
| | - D Ghoubay
- Service d'ophtalmologie 5, centre hospitalier national d'ophtalmologie des Quinze-Vingts, 28, rue de Charenton, 75012 Paris, France; Inserm UMR S 968, institut de la vision, Sorbonne université, CHNO des 15-20, 75012 Paris, France
| | - F Baudouin
- Inserm UMR S 968, institut de la vision, Sorbonne université, CHNO des 15-20, 75012 Paris, France
| | - V Borderie
- Service d'ophtalmologie 5, centre hospitalier national d'ophtalmologie des Quinze-Vingts, 28, rue de Charenton, 75012 Paris, France; Inserm UMR S 968, institut de la vision, Sorbonne université, CHNO des 15-20, 75012 Paris, France.
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18
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Banayan N, Georgeon C, Grieve K, Ghoubay D, Baudouin F, Borderie V. In vivo confocal microscopy and optical coherence tomography as innovative tools for the diagnosis of limbal stem cell deficiency. J Fr Ophtalmol 2018; 41:e395-e406. [DOI: 10.1016/j.jfo.2018.09.003] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2018] [Revised: 09/10/2018] [Accepted: 09/12/2018] [Indexed: 12/13/2022]
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19
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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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20
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Wahlig S, Yam GHF, Chong W, Seah XY, Kocaba V, Ang M, Htoon HM, Tun TA, Ong HS, Mehta JS. Quantification of the Posterior Cornea Using Swept Source Optical Coherence Tomography. Transl Vis Sci Technol 2018; 7:2. [PMID: 30197834 PMCID: PMC6126962 DOI: 10.1167/tvst.7.5.2] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2018] [Accepted: 07/15/2018] [Indexed: 12/13/2022] Open
Abstract
Purpose We define optical coherence tomography (OCT) measurement parameters of the corneal endothelium/Descemet's membrane (DM) complex and peripheral transition zone (TZ) and describe these measurements in an ethnically Chinese population. Methods OCT images of the anterior segment and iridocorneal angle were obtained from 129 healthy Chinese subjects (129 eyes), aged 40 to 81 years. The scleral spur (SS) and Schwalbe's line (SL) were identified in each image. Endothelium/DM diameter, referred to as endothelial arc length (EAL), is the SL-to-SL distance. The SS-to-SL distance encompasses the TZ and trabecular meshwork (TM). Since the TZ cannot be visualized by OCT, a ratio of TZ-to-TZ+TM width was calculated from scanning electron microscopy (SEM) images obtained from 5 cadaveric corneas. The SS-to-SL distance was multiplied by this ratio to approximate in vivo TZ width. Results From SEM measurements, the relationship TZ = 0.20*(TZ+TM) was determined. From OCT measurements, mean EAL was 12.15 ± 0.58 mm and mean TZ width was 156 ± 20 μm. For eyes with horizontal and vertical images, vertical EAL was significantly greater than horizontal EAL (P = 0.03). Conclusions Corneal endothelium/DM diameter and TZ width can be obtained from OCT images. Although only combined TZ+TM is visualized on OCT, TZ width can be reasonably approximated. Translational Relevance Emerging procedures, like endothelial cell injection and DM transplantation (DMT), require accurate measurements of endothelium/DM size for preoperative planning. Size of the TZ, which may contain progenitor cells, also could contribute to endothelial regeneration in these procedures.
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Affiliation(s)
- Stephen Wahlig
- Singapore Eye Research Institute (SERI), Singapore.,Duke University School of Medicine, Durham, NC, USA
| | - Gary Hin-Fai Yam
- Singapore Eye Research Institute (SERI), Singapore.,Singapore National Eye Center (SNEC), Singapore
| | | | - Xin-Yi Seah
- Singapore Eye Research Institute (SERI), Singapore
| | - Viridiana Kocaba
- Singapore Eye Research Institute (SERI), Singapore.,Department of Ophthalmology, Edouard Herriot Hospital, Hospices Civils de Lyon, Lyon, France.,Université de Lyon, F-69000 Lyon, France; Université Lyon 1, F-69100 Villeurbanne, France
| | - Marcus Ang
- Singapore Eye Research Institute (SERI), Singapore.,Singapore National Eye Center (SNEC), Singapore.,Eye-ACP, Duke-NUS Graduate Medical School, Singapore.,Moorfields Eye Hospital, London, UK
| | - Hla Myint Htoon
- Singapore Eye Research Institute (SERI), Singapore.,Eye-ACP, Duke-NUS Graduate Medical School, Singapore
| | - Tin A Tun
- Singapore Eye Research Institute (SERI), Singapore.,Singapore National Eye Center (SNEC), Singapore
| | - Hon Shing Ong
- Singapore Eye Research Institute (SERI), Singapore.,Singapore National Eye Center (SNEC), Singapore.,Moorfields Eye Hospital, London, UK
| | - Jodhbir S Mehta
- Singapore Eye Research Institute (SERI), Singapore.,Singapore National Eye Center (SNEC), Singapore.,Eye-ACP, Duke-NUS Graduate Medical School, Singapore
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21
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Guo ZH, Zhang W, Jia YYS, Liu QX, Li ZF, Lin JS. An Insight into the Difficulties in the Discovery of Specific Biomarkers of Limbal Stem Cells. Int J Mol Sci 2018; 19:ijms19071982. [PMID: 29986467 PMCID: PMC6073450 DOI: 10.3390/ijms19071982] [Citation(s) in RCA: 30] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2018] [Revised: 06/25/2018] [Accepted: 06/29/2018] [Indexed: 12/13/2022] Open
Abstract
Keeping the integrity and transparency of the cornea is the most important issue to ensure normal vision. There are more than 10 million patients going blind due to the cornea diseases worldwide. One of the effective ways to cure corneal diseases is corneal transplantation. Currently, donations are the main source of corneas for transplantation, but immune rejection and a shortage of donor corneas are still serious problems. Graft rejection could cause transplanted cornea opacity to fail. Therefore, bioengineer-based corneas become a new source for corneal transplantation. Limbal stem cells (LSCs) are located at the basal layer in the epithelial palisades of Vogt, which serve a homeostatic function for the cornea epithelium and repair the damaged cornea. LSC-based transplantation is one of the hot topics currently. Clinical data showed that the ratio of LSCs to total candidate cells for a transplantation has a significant impact on the effectiveness of the transplantation. It indicates that it is very important to accurately identify the LSCs. To date, several putative biomarkers of LSCs have been widely reported, whereas their specificity is controversial. As reported, the identification of LSCs is based on the characteristics of stem cells, such as a nuclear-to-cytoplasm ratio (N/C) ≥ 0.7, label-retaining, and side population (SP) phenotype. Here, we review recently published data to provide an insight into the circumstances in the study of LSC biomarkers. The particularities of limbus anatomy and histochemistry, the limits of the current technology level for LSC isolation, the heterogeneity of LSCs and the influence of enzyme digestion are discussed. Practical approaches are proposed in order to overcome the difficulties in basic and applied research for LSC-specific biomarkers.
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Affiliation(s)
- Zhi Hou Guo
- School of Medicine, Huaqiao University, Quanzhou 362021, China.
| | - Wei Zhang
- School of Medicine, Huaqiao University, Quanzhou 362021, China.
| | | | - Qing Xiu Liu
- School of Medicine, Huaqiao University, Quanzhou 362021, China.
| | - Zhao Fa Li
- School of Medicine, Huaqiao University, Quanzhou 362021, China.
| | - Jun Sheng Lin
- School of Medicine, Huaqiao University, Quanzhou 362021, China.
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22
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Emerging Therapeutic Strategies for Limbal Stem Cell Deficiency. J Ophthalmol 2018; 2018:7894647. [PMID: 30050691 PMCID: PMC6040301 DOI: 10.1155/2018/7894647] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2017] [Accepted: 04/19/2018] [Indexed: 12/12/2022] Open
Abstract
Identification and characterization of the limbal epithelial stem cells (LESCs) has proven to be a major accomplishment in anterior ocular surface biology. These cells have been shown to be a subpopulation of limbal epithelial basal cells, which serve as the progenitor population of the corneal epithelium. LESCs have been demonstrated to play an important role in maintaining corneal epithelium homeostasis. Many ocular surface diseases, including intrinsic (e.g., Sjogren's syndrome) or extrinsic (e.g., alkali or thermal burns) insults, which impair LESCs, can lead to limbal stem cell deficiency (LSCD). LSCD is characterized by an overgrowth of conjunctival-derived epithelial cells, corneal neovascularization, and chronic inflammation, eventually leading to blindness. Treatment of LSCD has been challenging, especially in bilateral total LSCD. Recently, advances in LESC research have led to novel therapeutic approaches for treating LSCD, such as transplantation of the cultured limbal epithelium. These novel therapeutic approaches have demonstrated efficacy for ocular surface reconstruction and restoration of vision in patients with LSCD. However, they all have their own limitations. Here, we describe the current status of LSCD treatment and discuss the advantages and disadvantages of the available therapeutic modalities.
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23
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Ghaemi SR, Delalat B, Harding FJ, Irani YD, Williams KA, Voelcker NH. Identification and In Vitro Expansion of Buccal Epithelial Cells. Cell Transplant 2018; 27:957-966. [PMID: 29860901 PMCID: PMC6050911 DOI: 10.1177/0963689718773330] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022] Open
Abstract
Ex vivo-expanded buccal mucosal epithelial (BME) cell transplantation has
been used to reconstruct the ocular surface. Methods for enrichment and maintenance of BME
progenitor cells in ex vivo cultures may improve the outcome of BME cell
transplantation. However, the parameter of cell seeding density in this context has
largely been neglected. This study investigates how varying cell seeding density
influences BME cell proliferation and differentiation on tissue culture polystyrene
(TCPS). The highest cell proliferation activity was seen when cells were seeded at
5×104 cells/cm2. Both below and above this density, the cell
proliferation rate decreased sharply. Differential immunofluorescence analysis of surface
markers associated with the BME progenitor cell population (p63, CK19, and ABCG2), the
differentiated cell marker CK10 and connexin 50 (Cx50) revealed that the initial cell
seeding density also significantly affected the progenitor cell marker expression profile.
Hence, this study demonstrates that seeding density has a profound effect on the
proliferation and differentiation of BME stem cells in vitro, and this is
relevant to downstream cell therapy applications.
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Affiliation(s)
- Soraya Rasi Ghaemi
- 1 Future Industries Institute, University of South Australia, Mawson Lakes, SA, Australia
| | - Bahman Delalat
- 1 Future Industries Institute, University of South Australia, Mawson Lakes, SA, Australia.,2 Manufacturing, Commonwealth Scientific and Industrial Research Organisation (CSIRO), Clayton, VIC, Australia.,3 Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia
| | - Frances J Harding
- 1 Future Industries Institute, University of South Australia, Mawson Lakes, SA, Australia
| | - Yazad D Irani
- 4 Department of Ophthalmology, Flinders University, Bedford Park, SA, Australia
| | - Keryn A Williams
- 4 Department of Ophthalmology, Flinders University, Bedford Park, SA, Australia
| | - Nicolas H Voelcker
- 1 Future Industries Institute, University of South Australia, Mawson Lakes, SA, Australia.,2 Manufacturing, Commonwealth Scientific and Industrial Research Organisation (CSIRO), Clayton, VIC, Australia.,3 Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia
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24
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Patruno M, Perazzi A, Martinello T, Gomiero C, Maccatrozzo L, Iacopetti I. Investigations of the corneal epithelium in Veterinary Medicine: State of the art on corneal stem cells found in different mammalian species and their putative application. Res Vet Sci 2018; 118:502-507. [PMID: 29758534 DOI: 10.1016/j.rvsc.2018.05.006] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2018] [Revised: 03/29/2018] [Accepted: 05/07/2018] [Indexed: 02/08/2023]
Abstract
The existence of progenitor cells that can readily differentiate into a specific cell type is a common cellular strategy for physiological tissue growth and repair mechanisms. In the mammalian cornea, many aspects regarding the nature and location of these cells are still unclear. In the human limbus (peripheral area of the cornea) progenitor cells have been found and characterized but in non-human mammals, the picture is not so clear. In this review, we examine current knowledge about the morphology of limbus and the localization of corneal epithelial stem cells in all species studied so far, comparing data with humans. We have also explored different research directions in the veterinary field in order to discuss the: i) currently used protocols and ii) best range of treatments for ocular pathologies in which corneal stem cells are involved.
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Affiliation(s)
- M Patruno
- Department of Comparative Biomedicine and Food Science, University of Padova, Viale dell'Università 16, 35020, Legnaro - Agripolis, Padova, Italy.
| | - A Perazzi
- Department of Animal Medicine, Production and Health, University of Padova, Viale dell'Università 16, 35020, Legnaro - Agripolis, Padova, Italy
| | - T Martinello
- Department of Comparative Biomedicine and Food Science, University of Padova, Viale dell'Università 16, 35020, Legnaro - Agripolis, Padova, Italy
| | - C Gomiero
- Department of Comparative Biomedicine and Food Science, University of Padova, Viale dell'Università 16, 35020, Legnaro - Agripolis, Padova, Italy
| | - L Maccatrozzo
- Department of Comparative Biomedicine and Food Science, University of Padova, Viale dell'Università 16, 35020, Legnaro - Agripolis, Padova, Italy
| | - I Iacopetti
- Department of Animal Medicine, Production and Health, University of Padova, Viale dell'Università 16, 35020, Legnaro - Agripolis, Padova, Italy
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Guo Y, Ma X, Wu W, Shi M, Ma J, Zhang Y, Zhao E, Yang X. Coordinated microRNA/mRNA expression profiles reveal a putative mechanism of corneal epithelial cell transdifferentiation from skin epidermal stem cells. Int J Mol Med 2017; 41:877-887. [PMID: 29207049 PMCID: PMC5752239 DOI: 10.3892/ijmm.2017.3304] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2016] [Accepted: 11/15/2017] [Indexed: 01/09/2023] Open
Abstract
Skin epidermal stem cells (SESCs), which share a single origin with corneal epithelial cells (CECs), are considered to be one of the most ideal seed cells for the construction of tissue engineered corneas. However, the mechanism underlying the transdifferentiation of SESCs to CECs has not been fully elucidated. In the present study, to identify critical microRNAs (miRNAs/miRs) and genes that regulate the transdifferentiation of SESCs to CECs, SESCs and CECs were collected from sheep and used for small RNA sequencing and mRNA microarray analyses. Among the differentially expressed miRNAs and genes, 36 miRNAs were downregulated and 123 genes were upregulated in the CECs compared with those in the SESCs. miR-10b exhibited the largest change in expression between the cell types. Target genes of the 36 downregulated miRNAs were predicted and a computational approach demonstrated that these target genes may be involved in several signaling pathways, including the 'PI3K signaling pathway', the 'Wnt signaling pathway' and the 'MAPK signaling pathway', as well as in 'focal adhesion'. Comparison of these target genes to the 123 upregulated genes identified 43 intersection genes. A regulatory network of these 43 intersection genes and its correlative miRNAs were constructed, and three genes (dedicator of cytokinesis 9, neuronal differentiation 1 and activated leukocyte cell adhesion molecule) were found to have high interaction frequencies. The expression levels of 7 randomly selected miRNAs and the 3 intersection genes were further validated by reverse transcription-quantitative polymerase chain reaction. It was found that miR-10b, the Wnt signaling pathway and the 3 intersection genes may act together and serve a critical role in the transdifferentiation process. This study identified miRNAs and genes that were expressed in SESCs and CECs that may assist in uncovering its underlying molecular mechanism, as well as promote corneal tissue engineering using epidermal stem cells for clinical applications.
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Affiliation(s)
- Yanjie Guo
- Life Science College, Luoyang Normal University, Luoyang, Henan 471934, P.R. China
| | - Xiya Ma
- Life Science College, Luoyang Normal University, Luoyang, Henan 471934, P.R. China
| | - Weini Wu
- Life Science College, Luoyang Normal University, Luoyang, Henan 471934, P.R. China
| | - Mingyan Shi
- Life Science College, Luoyang Normal University, Luoyang, Henan 471934, P.R. China
| | - Junlong Ma
- Life Science College, Luoyang Normal University, Luoyang, Henan 471934, P.R. China
| | - Yaping Zhang
- Life Science College, Luoyang Normal University, Luoyang, Henan 471934, P.R. China
| | - Erkang Zhao
- Life Science College, Luoyang Normal University, Luoyang, Henan 471934, P.R. China
| | - Xueyi Yang
- Life Science College, Luoyang Normal University, Luoyang, Henan 471934, P.R. China
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Characterization of Ex Vivo Expanded Oral Mucosal Epithelium Cells on Acellular Porcine Corneal Stroma for Ocular Surface Reconstruction. J Ophthalmol 2017; 2017:6761714. [PMID: 28567305 PMCID: PMC5439182 DOI: 10.1155/2017/6761714] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2016] [Revised: 02/27/2017] [Accepted: 03/12/2017] [Indexed: 01/28/2023] Open
Abstract
Purpose To ex vivo expand oral mucosal epithelium cells (OMECs) on acellular porcine corneal stroma (APCS) without using feeder cells and serum and to compare the morphologic and phenotypic characteristics of cultured oral cells on APCS to those of cells on deluded human amniotic membrane (HAM). Methods SD rat oral mucosal biopsies were cultured on APCS and HAM. Reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry were used to analyze the characterization of stem cells and epithelial differentiation of the outgrowth products. Results Stratified and optimal transplantable OMECs were obtained after being cultured three to four weeks. Both RT-PCR and immunohistochemistry showed that cultured OMECs expressed markers of epithelial differentiation cytokeratin K3 and epithelial stem cell markers of p63 and ABCG2. Conclusions OMECs can be successfully cultured on APCS without using xenobiotic feeder cells and serum. Characterization showed that these sheets retain the morphologic and phenotypic characteristics of OMECs within differentiated cells and stem cells. The optimal transplantable sheets can prove to be particularly beneficial to both bilateral limbal stem cell deficiency and deep corneal lesions.
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Shaharuddin B, Ahmad S, Md Latar N, Ali S, Meeson A. A Human Corneal Epithelial Cell Line Model for Limbal Stem Cell Biology and Limbal Immunobiology. Stem Cells Transl Med 2016; 6:761-766. [PMID: 28297580 PMCID: PMC5442771 DOI: 10.5966/sctm.2016-0175] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2016] [Accepted: 09/01/2016] [Indexed: 11/16/2022] Open
Abstract
Limbal stem cell (LSC) deficiency is a visually debilitating condition caused by abnormal maintenance of LSCs. It is treated by transplantation of donor-derived limbal epithelial cells (LECs), the success of which depends on the presence and quality of LSCs within the transplant. Understanding the immunobiological responses of these cells within the transplants could improve cell engraftment and survival. However, human corneal rings used as a source of LSCs are not always readily available for research purposes. As an alternative, we hypothesized that a human telomerase-immortalized corneal epithelial cell (HTCEC) line could be used as a model for studying LSC immunobiology. HTCEC constitutively expressed human leukocyte antigen (HLA) class I but not class II molecules. However, when stimulated by interferon-γ, HTCECs then expressed HLA class II antigens. Some HTCECs were also migratory in response to CXCL12 and expressed stem cell markers, Nanog, Oct4, and Sox2. In addition because both HTCECs and LECs contain side population (SP) cells, which are an enriched LSC population, we used these SP cells to show that some HTCEC SP cells coexpressed ABCG2 and ABCB5. HTCEC SP and non-side population (NSP) cells also expressed CXCR4, but the SP cells expressed higher levels. Both were capable of colony formation, but the NSP colonies were smaller and contained fewer cells. In addition, HTCECs expressed ΔNp63α. These results suggest the HTCEC line is a useful model for further understanding LSC biology by using an in vitro approach without reliance on a supply of human tissue. Stem Cells Translational Medicine 2017;6:761-766.
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Affiliation(s)
- Bakiah Shaharuddin
- Institute of Genetic Medicine, Newcastle University, Newcastle Upon‐Tyne, United Kingdom
- Advanced Medical and Dental Institute, Universiti Sains Malaysia, Pulau Pinang, Malaysia
| | - Sajjad Ahmad
- St. Paul's Eye Unit, Royal Liverpool University Hospital, Liverpool, United Kingdom
- Department of Eye and Vision Sciences, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, United Kingdom
| | - Nani Md Latar
- Institute of Genetic Medicine, Newcastle University, Newcastle Upon‐Tyne, United Kingdom
- Department of Surgery, Faculty of Medicine, Universiti Kebangsaan, Malaysia Medical Centre, Kuala Lumpur, Malaysia
| | - Simi Ali
- Institute of Cellular Medicine, Newcastle University, Newcastle Upon‐Tyne, United Kingdom
| | - Annette Meeson
- Institute of Genetic Medicine, Newcastle University, Newcastle Upon‐Tyne, United Kingdom
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Limbal Stem Cell Deficiency: Current Treatment Options and Emerging Therapies. Stem Cells Int 2015; 2016:9798374. [PMID: 26788074 PMCID: PMC4691643 DOI: 10.1155/2016/9798374] [Citation(s) in RCA: 108] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2015] [Accepted: 08/18/2015] [Indexed: 12/15/2022] Open
Abstract
Severe ocular surface disease can result in limbal stem cell deficiency (LSCD), a condition leading to decreased visual acuity, photophobia, and ocular pain. To restore the ocular surface in advanced stem cell deficient corneas, an autologous or allogenic limbal stem cell transplantation is performed. In recent years, the risk of secondary LSCD due to removal of large limbal grafts has been significantly reduced by the optimization of cultivated limbal epithelial transplantation (CLET). Despite the great successes of CLET, there still is room for improvement as overall success rate is 70% and visual acuity often remains suboptimal after successful transplantation. Simple limbal epithelial transplantation reports higher success rates but has not been performed in as many patients yet. This review focuses on limbal epithelial stem cells and the pathophysiology of LSCD. State-of-the-art therapeutic management of LSCD is described, and new and evolving techniques in ocular surface regeneration are being discussed, in particular, advantages and disadvantages of alternative cell scaffolds and cell sources for cell based ocular surface reconstruction.
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29
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Kolli S, Ahmad S, Mudhar HS, Meeny A, Lako M, Figueiredo FC. Successful application of ex vivo expanded human autologous oral mucosal epithelium for the treatment of total bilateral limbal stem cell deficiency. Stem Cells 2015; 32:2135-46. [PMID: 24590515 DOI: 10.1002/stem.1694] [Citation(s) in RCA: 86] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2013] [Accepted: 02/09/2014] [Indexed: 11/07/2022]
Abstract
Ocular surface reconstruction with ex vivo expanded limbal stem cells (LSCs) is a widely used clinical treatment for patients with limbal stem cell deficiency (LSCD). This is not applicable to patients with bilateral LSCD where there are no remaining LSCs. Cultivated oral mucosa epithelium (OME) has been used as an alternative source of autologous epithelial stem cells for ocular reconstruction in few clinical trials. However, successful generation of stratified OME epithelium has only been achieved in the presence of animal feeder cells and/or animal-derived products in the culture media, likely to contribute to increased risk of pathogen transmission and graft rejection. In this study, we report generation of multilayered OME epithelium that shares many of the characteristics of corneal epithelium using a fully compliant good manufacturing practice, feeder- and animal product-free method. Proof of concept was achieved by transplantation of autologous ex vivo expanded OME in two patients with histologically confirmed bilateral total LSCD that resulted in successful reversal of LSCD in the treated eye up to 24 months.
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Affiliation(s)
- Sai Kolli
- Institute of Genetic Medicine, Newcastle University, Newcastle, United Kingdom; Department of Ophthalmology, Royal Victoria Infirmary, Newcastle University, Newcastle, United Kingdom
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Sanchez RF, Daniels JT. Mini-Review: Limbal Stem Cells Deficiency in Companion Animals: Time to Give Something Back? Curr Eye Res 2015; 41:425-32. [PMID: 26287764 DOI: 10.3109/02713683.2015.1056801] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Experimental animals have been used extensively in the goal of developing sight-saving therapies for humans. One example is the development of transplantation of cultured limbal epithelial stem cells (LESC) to restore vision following ocular surface injury or disease. With clinical trials of cultured LESC therapy underway in humans and a potential companion animal population suffering from similar diseases, it is perhaps time to give something back. Comparatively to humans, what is known about the healthy limbus and corneal surface physiology of companion animals is still very little. Blinding corneal diseases in animals such as symblepharon in cats with Feline Herpes Virus-1 infections require a basic understanding of the functional companion animal limbus and corneal stem cells. Our understanding of many other vision threatening conditions such as scarring of the cornea post-inflammation with lymphocytic-plasmacytic infiltrate in dogs (aka chronic superficial keratitis) or pigment proliferation with Pigmentary Keratitis of Pugs would benefit from a better understanding of the animal cornea in health and disease. This is also vital when new therapeutic approaches are considered. This review will explore the current challenges and future research directions that will be required to increase our understanding of corneal diseases in animals and consider the potential development and delivery of cultured stem cell therapy to veterinary ocular surface patients.
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Affiliation(s)
- Rick F Sanchez
- a Royal Veterinary College, Queen Mother Hospital for Animals , Hatfield , UK and
| | - Julie T Daniels
- b Department of Ocular Biology and Therapeutics , UCL Institute of Ophthalmology , London , UK
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Villarreal Villarreal R, Vela Barrera ID, Villarreal Guerra P, Moreno Treviño MG, Rivera Silva G. Células madre limbocorneales: actualidades y aplicaciones terapéuticas. REVISTA MEXICANA DE OFTALMOLOGÍA 2015. [DOI: 10.1016/j.mexoft.2014.10.004] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/23/2022] Open
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32
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Bron A, Argüeso P, Irkec M, Bright F. Clinical staining of the ocular surface: Mechanisms and interpretations. Prog Retin Eye Res 2015; 44:36-61. [DOI: 10.1016/j.preteyeres.2014.10.001] [Citation(s) in RCA: 90] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2014] [Revised: 10/08/2014] [Accepted: 10/08/2014] [Indexed: 10/24/2022]
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33
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Li YH, Cheng CY, Wang NK, Tan HY, Tsai YJ, Hsiao CH, Ma DHK, Yeh LK. Characterization of the modified chitosan membrane cross-linked with genipin for the cultured corneal epithelial cells. Colloids Surf B Biointerfaces 2014; 126:237-44. [PMID: 25576808 DOI: 10.1016/j.colsurfb.2014.12.029] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2014] [Revised: 12/13/2014] [Accepted: 12/16/2014] [Indexed: 12/22/2022]
Abstract
OBJECTIVES To modify a chitosan membrane (CM) by cross-linking the chitosan with genipin, a naturally occurring cross-linker extracted from Gardenia jasminoides fructus, with the aim of developing a new cell culture support and to observe the phenotypes of cultured human corneal epithelial cells (HCECs) on genipin-cross-linked chitosan membrane (GCM). METHODS We tested the cross-linking characteristics and mechanical strength of the GCM. CMs modified by cross-linking with different concentrations of genipin were prepared to investigate the rate of membrane degradation. The biocompatibility of the GCMs was investigated by determining the viability of HCECs cultured on them in vitro. The morphology of the HCECs cultured on CM or GCM was analyzed by confocal microscopy and scanning electron microscopy (SEM). Immunocytochemical staining was conducted to determine the phenotypes of the cultured cells. RESULTS The fixation index of the GCM was 31 ± 3% after treatment of CM with 0.5mM genipin. A stress-strain test showed that the GCM could tolerate three times the mechanical force of noncross-linked CM. The biodegradation rate of GCM was much slower than for CM. A 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay showed that cell viability was not affected by cross-linking with 5.0mM genipin. SEM showed that the cultured HCECs adhered to and grew well on the surface of the GCM. Immunocytochemical staining showed keratin 3 (K3) and connexin 43 (Cx-43) immunoreactive HCECs on the GCM and their proliferative ability was not significantly affected by strong immunoreactivity of Ki-67 and p63 markers. CONCLUSIONS GCM has potential as a scaffold for corneal epithelium in ocular surface surgery and greater mechanical strength and slower degradation than unmodified CM.
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Affiliation(s)
- Ya-Han Li
- Department of Ophthalmology, Chang-Gung Memorial Hospital, Linkou, Taiwan
| | - Ching-Yi Cheng
- Department of Cosmetic Science, Graduate Institute of Health Industry Technology, Research Center for Industry of Human Ecology, Chang Gung University of Science and Technology, Kwei-Shan, Tao-Yuan, Taiwan
| | - Nan-Kai Wang
- Department of Ophthalmology, Chang-Gung Memorial Hospital, Linkou, Taiwan; Chang-Gung University College of Medicine, Taiwan
| | - Hsin-Yuan Tan
- Department of Ophthalmology, Chang-Gung Memorial Hospital, Linkou, Taiwan; Chang-Gung University College of Medicine, Taiwan
| | - Yueh-Ju Tsai
- Department of Ophthalmology, Chang-Gung Memorial Hospital, Linkou, Taiwan; Chang-Gung University College of Medicine, Taiwan
| | - Ching-Hsi Hsiao
- Department of Ophthalmology, Chang-Gung Memorial Hospital, Linkou, Taiwan; Chang-Gung University College of Medicine, Taiwan
| | - David Hui-Kang Ma
- Department of Ophthalmology, Chang-Gung Memorial Hospital, Linkou, Taiwan; Chang-Gung University College of Medicine, Taiwan
| | - Lung-Kun Yeh
- Department of Ophthalmology, Chang-Gung Memorial Hospital, Linkou, Taiwan; Chang-Gung University College of Medicine, Taiwan.
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Gichuhi S, Ohnuma SI, Sagoo MS, Burton MJ. Pathophysiology of ocular surface squamous neoplasia. Exp Eye Res 2014; 129:172-82. [PMID: 25447808 PMCID: PMC4726664 DOI: 10.1016/j.exer.2014.10.015] [Citation(s) in RCA: 91] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2014] [Accepted: 10/17/2014] [Indexed: 12/22/2022]
Abstract
The incidence of ocular surface squamous neoplasia (OSSN) is strongly associated with solar ultraviolet (UV) radiation, HIV and human papilloma virus (HPV). Africa has the highest incidence rates in the world. Most lesions occur at the limbus within the interpalpebral fissure particularly the nasal sector. The nasal limbus receives the highest intensity of sunlight. Limbal epithelial crypts are concentrated nasally and contain niches of limbal epithelial stem cells in the basal layer. It is possible that these are the progenitor cells in OSSN. OSSN arises in the basal epithelial cells spreading towards the surface which resembles the movement of corneo-limbal stem cell progeny before it later invades through the basement membrane below. UV radiation damages DNA producing pyrimidine dimers in the DNA chain. Specific CC → TT base pair dimer transformations of the p53 tumour-suppressor gene occur in OSSN allowing cells with damaged DNA past the G1-S cell cycle checkpoint. UV radiation also causes local and systemic photoimmunosuppression and reactivates latent viruses such as HPV. The E7 proteins of HPV promote proliferation of infected epithelial cells via the retinoblastoma gene while E6 proteins prevent the p53 tumour suppressor gene from effecting cell-cycle arrest of DNA-damaged and infected cells. Immunosuppression from UV radiation, HIV and vitamin A deficiency impairs tumour immune surveillance allowing survival of aberrant cells. Tumour growth and metastases are enhanced by; telomerase reactivation which increases the number of cell divisions a cell can undergo; vascular endothelial growth factor for angiogenesis and matrix metalloproteinases (MMPs) that destroy the intercellular matrix between cells. Despite these potential triggers, the disease is usually unilateral. It is unclear how HPV reaches the conjunctiva.
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Affiliation(s)
- Stephen Gichuhi
- London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK; Department of Ophthalmology, University of Nairobi, P.O Box 19676-00202, Nairobi, Kenya.
| | - Shin-ichi Ohnuma
- UCL Institute of Ophthalmology, 11-43 Bath Street, London EC1V 9EL, UK.
| | - Mandeep S Sagoo
- UCL Institute of Ophthalmology, 11-43 Bath Street, London EC1V 9EL, UK; Moorfields Eye Hospital, 162 City Road, London EC1V 2PD, UK; St. Bartholomew's Hospital, W Smithfield, London EC1A 7BE, UK.
| | - Matthew J Burton
- London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK; Moorfields Eye Hospital, 162 City Road, London EC1V 2PD, UK.
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Yoon JJ, Ismail S, Sherwin T. Limbal stem cells: Central concepts of corneal epithelial homeostasis. World J Stem Cells 2014; 6:391-403. [PMID: 25258661 PMCID: PMC4172668 DOI: 10.4252/wjsc.v6.i4.391] [Citation(s) in RCA: 81] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/25/2014] [Revised: 08/20/2014] [Accepted: 09/01/2014] [Indexed: 02/06/2023] Open
Abstract
A strong cohort of evidence exists that supports the localisation of corneal stem cells at the limbus. The distinguishing characteristics of limbal cells as stem cells include slow cycling properties, high proliferative potential when required, clonogenicity, absence of differentiation marker expression coupled with positive expression of progenitor markers, multipotency, centripetal migration, requirement for a distinct niche environment and the ability of transplanted limbal cells to regenerate the entire corneal epithelium. The existence of limbal stem cells supports the prevailing theory of corneal homeostasis, known as the XYZ hypothesis where X represents proliferation and stratification of limbal basal cells, Y centripetal migration of basal cells and Z desquamation of superficial cells. To maintain the mass of cornea, the sum of X and Y must equal Z and very elegant cell tracking experiments provide strong evidence in support of this theory. However, several recent studies have suggested the existence of oligopotent stem cells capable of corneal maintenance outside of the limbus. This review presents a summary of data which led to the current concepts of corneal epithelial homeostasis and discusses areas of controversy surrounding the existence of a secondary stem cell reservoir on the corneal surface
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36
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Sareen D, Saghizadeh M, Ornelas L, Winkler MA, Narwani K, Sahabian A, Funari VA, Tang J, Spurka L, Punj V, Maguen E, Rabinowitz YS, Svendsen CN, Ljubimov AV. Differentiation of human limbal-derived induced pluripotent stem cells into limbal-like epithelium. Stem Cells Transl Med 2014; 3:1002-12. [PMID: 25069777 DOI: 10.5966/sctm.2014-0076] [Citation(s) in RCA: 62] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Limbal epithelial stem cell (LESC) deficiency (LSCD) leads to corneal abnormalities resulting in compromised vision and blindness. LSCD can be potentially treated by transplantation of appropriate cells, which should be easily expandable and bankable. Induced pluripotent stem cells (iPSCs) are a promising source of transplantable LESCs. The purpose of this study was to generate human iPSCs and direct them to limbal differentiation by maintaining them on natural substrata mimicking the native LESC niche, including feederless denuded human amniotic membrane (HAM) and de-epithelialized corneas. These iPSCs were generated with nonintegrating vectors from human primary limbal epithelial cells. This choice of parent cells was supposed to enhance limbal cell differentiation from iPSCs by partial retention of parental epigenetic signatures in iPSCs. When the gene methylation patterns were compared in iPSCs to parental LESCs using Illumina global methylation arrays, limbal-derived iPSCs had fewer unique methylation changes than fibroblast-derived iPSCs, suggesting retention of epigenetic memory during reprogramming. Limbal iPSCs cultured for 2 weeks on HAM developed markedly higher expression of putative LESC markers ABCG2, ΔNp63α, keratins 14, 15, and 17, N-cadherin, and TrkA than did fibroblast iPSCs. On HAM culture, the methylation profiles of select limbal iPSC genes (including NTRK1, coding for TrkA protein) became closer to the parental cells, but fibroblast iPSCs remained closer to parental fibroblasts. On denuded air-lifted corneas, limbal iPSCs even upregulated differentiated corneal keratins 3 and 12. These data emphasize the importance of the natural niche and limbal tissue of origin in generating iPSCs as a LESC source with translational potential for LSCD treatment.
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Affiliation(s)
- Dhruv Sareen
- Regenerative Medicine Institute, Eye Program, and Departments of Biomedical Sciences, Neurosurgery, Genomics Core, and Surgery, Cedars-Sinai Medical Center, Los Angeles, California, USA; Norris Comprehensive Cancer Center Bioinformatics Core and Division of Hematology, University of Southern California, Los Angeles, California, USA; David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA
| | - Mehrnoosh Saghizadeh
- Regenerative Medicine Institute, Eye Program, and Departments of Biomedical Sciences, Neurosurgery, Genomics Core, and Surgery, Cedars-Sinai Medical Center, Los Angeles, California, USA; Norris Comprehensive Cancer Center Bioinformatics Core and Division of Hematology, University of Southern California, Los Angeles, California, USA; David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA
| | - Loren Ornelas
- Regenerative Medicine Institute, Eye Program, and Departments of Biomedical Sciences, Neurosurgery, Genomics Core, and Surgery, Cedars-Sinai Medical Center, Los Angeles, California, USA; Norris Comprehensive Cancer Center Bioinformatics Core and Division of Hematology, University of Southern California, Los Angeles, California, USA; David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA
| | - Michael A Winkler
- Regenerative Medicine Institute, Eye Program, and Departments of Biomedical Sciences, Neurosurgery, Genomics Core, and Surgery, Cedars-Sinai Medical Center, Los Angeles, California, USA; Norris Comprehensive Cancer Center Bioinformatics Core and Division of Hematology, University of Southern California, Los Angeles, California, USA; David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA
| | - Kavita Narwani
- Regenerative Medicine Institute, Eye Program, and Departments of Biomedical Sciences, Neurosurgery, Genomics Core, and Surgery, Cedars-Sinai Medical Center, Los Angeles, California, USA; Norris Comprehensive Cancer Center Bioinformatics Core and Division of Hematology, University of Southern California, Los Angeles, California, USA; David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA
| | - Anais Sahabian
- Regenerative Medicine Institute, Eye Program, and Departments of Biomedical Sciences, Neurosurgery, Genomics Core, and Surgery, Cedars-Sinai Medical Center, Los Angeles, California, USA; Norris Comprehensive Cancer Center Bioinformatics Core and Division of Hematology, University of Southern California, Los Angeles, California, USA; David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA
| | - Vincent A Funari
- Regenerative Medicine Institute, Eye Program, and Departments of Biomedical Sciences, Neurosurgery, Genomics Core, and Surgery, Cedars-Sinai Medical Center, Los Angeles, California, USA; Norris Comprehensive Cancer Center Bioinformatics Core and Division of Hematology, University of Southern California, Los Angeles, California, USA; David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA
| | - Jie Tang
- Regenerative Medicine Institute, Eye Program, and Departments of Biomedical Sciences, Neurosurgery, Genomics Core, and Surgery, Cedars-Sinai Medical Center, Los Angeles, California, USA; Norris Comprehensive Cancer Center Bioinformatics Core and Division of Hematology, University of Southern California, Los Angeles, California, USA; David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA
| | - Lindsay Spurka
- Regenerative Medicine Institute, Eye Program, and Departments of Biomedical Sciences, Neurosurgery, Genomics Core, and Surgery, Cedars-Sinai Medical Center, Los Angeles, California, USA; Norris Comprehensive Cancer Center Bioinformatics Core and Division of Hematology, University of Southern California, Los Angeles, California, USA; David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA
| | - Vasu Punj
- Regenerative Medicine Institute, Eye Program, and Departments of Biomedical Sciences, Neurosurgery, Genomics Core, and Surgery, Cedars-Sinai Medical Center, Los Angeles, California, USA; Norris Comprehensive Cancer Center Bioinformatics Core and Division of Hematology, University of Southern California, Los Angeles, California, USA; David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA
| | - Ezra Maguen
- Regenerative Medicine Institute, Eye Program, and Departments of Biomedical Sciences, Neurosurgery, Genomics Core, and Surgery, Cedars-Sinai Medical Center, Los Angeles, California, USA; Norris Comprehensive Cancer Center Bioinformatics Core and Division of Hematology, University of Southern California, Los Angeles, California, USA; David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA
| | - Yaron S Rabinowitz
- Regenerative Medicine Institute, Eye Program, and Departments of Biomedical Sciences, Neurosurgery, Genomics Core, and Surgery, Cedars-Sinai Medical Center, Los Angeles, California, USA; Norris Comprehensive Cancer Center Bioinformatics Core and Division of Hematology, University of Southern California, Los Angeles, California, USA; David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA
| | - Clive N Svendsen
- Regenerative Medicine Institute, Eye Program, and Departments of Biomedical Sciences, Neurosurgery, Genomics Core, and Surgery, Cedars-Sinai Medical Center, Los Angeles, California, USA; Norris Comprehensive Cancer Center Bioinformatics Core and Division of Hematology, University of Southern California, Los Angeles, California, USA; David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA
| | - Alexander V Ljubimov
- Regenerative Medicine Institute, Eye Program, and Departments of Biomedical Sciences, Neurosurgery, Genomics Core, and Surgery, Cedars-Sinai Medical Center, Los Angeles, California, USA; Norris Comprehensive Cancer Center Bioinformatics Core and Division of Hematology, University of Southern California, Los Angeles, California, USA; David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA
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Petsch C, Schlötzer-Schrehardt U, Meyer-Blazejewska E, Frey M, Kruse FE, Bachmann BO. Novel collagen membranes for the reconstruction of the corneal surface. Tissue Eng Part A 2014; 20:2378-89. [PMID: 24621144 DOI: 10.1089/ten.tea.2013.0552] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
No standardized biomaterial exists for the surgical treatment of persistent corneal erosions and ulcerations. We analyzed the suitability and biocompatibility of defined noncross-linked and UV/riboflavin cross-linked equine type I collagen membranes for the reconstruction of the corneal surface. Isolated human oral mucosa epithelial cells, a cell type in clinical use for the treatment of ocular surface diseases, were subcultivated on both types of membranes and examined concerning cell adhesion, proliferation, and differentiation. Biocompatibility was evaluated following superficial and intrastromal corneal transplantation in New Zealand white rabbits. In cell cultures all collagen membranes supported adhesion of oral mucosa epithelial cells leading to the formation of multilayered epithelial cell sheets. After intrastromal corneal implantation clinical signs of degradation were seen in all variants of collagen membranes, which was fastest in noncross-linked variants. The histological and ultrastructural level invasion of keratocytes and production of new collagen fibers inside the collagen membranes could be detected in noncross-linked variants. After superficial corneal implantation covering of the membranes by corneal epithelium over time was visible. Ultrastructural analysis showed a slower rate of degradation and less invading keratocytes in cross-linked variants compared with noncross-linked collagen membranes. Cross-linked and noncross-linked variants of the collagen membrane proofed to be suitable to serve as a carrier for epithelial stem cells in vitro and showed a high biocompatibility in vivo. These results indicate that the tested collagen membranes might be suitable for the reconstruction of the corneal surface in patients with nonhealing ulcerations. Whether membranes with faster or slower degradation properties are preferable for the treatment of persistent corneal ulcerations might depend on the underlying corneal pathology and the degree of concomitant inflammation.
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Affiliation(s)
- Corinna Petsch
- 1 Department of Ophthalmology, University of Erlangen-Nürnberg , Erlangen, Germany
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Kang YG, Shin JW, Park SH, Oh MJ, Park HS, Shin JW, Kim SH. Effects of flow-induced shear stress on limbal epithelial stem cell growth and enrichment. PLoS One 2014; 9:e93023. [PMID: 24658122 PMCID: PMC3962472 DOI: 10.1371/journal.pone.0093023] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2014] [Accepted: 02/27/2014] [Indexed: 12/13/2022] Open
Abstract
The roles of limbal epithelial stem cells (LESCs) are widely recognized, but for these cells to be utilized in basic research and potential clinical applications, researchers must be able to efficiently isolate them and subsequently maintain their stemness in vitro. We aimed to develop a biomimetic environment for LESCs involving cells from their in vivo niche and the principle of flow-induced shear stress, and to subsequently demonstrate the potential of this novel paradigm. LESCs, together with neighboring cells, were isolated from the minced limbal tissues of rabbits. At days 8 and 9 of culture, the cells were exposed to a steady flow or intermittent flow for 2 h per day in a custom-designed bioreactor. The responses of LESCs and epithelial cells were assessed at days 12 and 14. LESCs and epithelial cells responded to both types of flow. Proliferation of LESCs, as assessed using a BrdU assay, was increased to a greater extent under steady flow conditions. Holoclones were found under intermittent flow, indicating that differentiation into transient amplifying cells had occurred. Immunofluorescent staining of Bmi-1 suggested that steady flow has a positive effect on the maintenance of stemness. This finding was confirmed by real-time PCR. Notch-1 and p63 were more sensitive to intermittent flow, but this effect was transient. K3 and K12 expression, indicative of differentiation of LESCs into epithelial cells, was induced by flow and lasted longer under intermittent flow conditions. In summary, culture of LESCs in a bioreactor under a steady flow paradigm, rather than one of intermittent flow, is beneficial for both increasing proliferation and maintaining stemness. Conversely, intermittent flow appears to induce differentiation of LESCs. This novel experimental method introduces micro-mechanical stimuli to traditional culture techniques, and has potential for regulating the proliferation and differentiation of LESCs in vitro, thereby facilitating research in this field.
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Affiliation(s)
- Yun Gyeong Kang
- Department of Biomedical Engineering, Inje University, Gyeongnam, Korea
| | - Ji Won Shin
- Cardiovascular and Metabolic Disease Center, Inje University, Busan, Korea
| | - So Hee Park
- Department of Biomedical Engineering, Inje University, Gyeongnam, Korea
| | - Min-Jae Oh
- Department of Biomedical Engineering, Inje University, Gyeongnam, Korea
| | | | - Jung-Woog Shin
- Department of Biomedical Engineering, Inje University, Gyeongnam, Korea
- Cardiovascular and Metabolic Disease Center, Inje University, Busan, Korea
- Department of Health Science and Technology/Institute of Aged Life Redesign/UHRC, Inje University, Gyeongnam, Korea
- * E-mail: (J-WS); (S-HK)
| | - Su-Hyang Kim
- Department of Biomedical Engineering, Inje University, Gyeongnam, Korea
- * E-mail: (J-WS); (S-HK)
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Pellegrini G, Rama P, Matuska S, Lambiase A, Bonini S, Pocobelli A, Colabelli RG, Spadea L, Fasciani R, Balestrazzi E, Vinciguerra P, Rosetta P, Tortori A, Nardi M, Gabbriellini G, Traverso CE, Macaluso C, Losi L, Percesepe A, Venturi B, Corradini F, Panaras A, Di Rocco A, Guatelli P, De Luca M. Biological parameters determining the clinical outcome of autologous cultures of limbal stem cells. Regen Med 2013; 8:553-67. [PMID: 23725042 DOI: 10.2217/rme.13.43] [Citation(s) in RCA: 100] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022] Open
Abstract
AIM Limbal cultures restore the corneal epithelium in patients with ocular burns. We investigated the biological parameters instrumental for their clinical success. METHODS We report a long-term multicenter prospective study on 152 patients carrying corneal destruction due to severe ocular burns, treated with autologous limbal cells cultured on fibrin and clinical-grade 3T3-J2 feeder cells. Clinical results were statistically evaluated both by parametric and nonparametric methods. RESULTS Clinical outcomes were scored as full success, partial success and failure in 66.05, 19.14 and 14.81% of eyes, respectively. The total number of clonogenic cells, colony size, growth rate and presence of conjunctival cells could not predict clinical results. Instead, the clinical data provided conclusive evidence that graft quality and likelihood of a successful outcome rely on an accurate evaluation of the number of stem cells detected before transplantation as holoclones expressing high levels of the p63 transcription factor. No adverse effects related to the feeder layer have been observed and the regenerated epithelium was completely devoid of any 3T3-J2 contamination. CONCLUSION Cultures of limbal stem cells can be safely used to successfully treat massive destruction of the human cornea. We emphasize the importance of a discipline for defining the suitability and the quality of cultured epithelial grafts, which are relevant to the future clinical use of any cultured cell type.
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Affiliation(s)
- Graziella Pellegrini
- Centre for Regenerative Medicine 'Stefano Ferrari', University of Modena & Reggio Emilia, Modena, Italy.
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Forni MF, Loureiro RR, Cristovam PC, Bonatti JA, Sogayar MC, Gomes JÁP. Comparison Between Different Biomaterial Scaffolds for Limbal-Derived Stem Cells Growth and Enrichment. Curr Eye Res 2012; 38:27-34. [DOI: 10.3109/02713683.2012.733053] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
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Eslani M, Baradaran-Rafii A, Ahmad S. Cultivated Limbal and Oral Mucosal Epithelial Transplantation. Semin Ophthalmol 2012; 27:80-93. [DOI: 10.3109/08820538.2012.680641] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
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Crawford AZ, McGhee CNJ. Management of limbal stem cell deficiency in severe ocular chemical burns. Clin Exp Ophthalmol 2012; 40:227-9. [PMID: 22490111 DOI: 10.1111/j.1442-9071.2012.02775.x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
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Martínez-Conesa EM, Espel E, Reina M, Casaroli-Marano RP. Characterization of ocular surface epithelial and progenitor cell markers in human adipose stromal cells derived from lipoaspirates. Invest Ophthalmol Vis Sci 2012; 53:513-20. [PMID: 22199247 DOI: 10.1167/iovs.11-7550] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
PURPOSE The goal of this study was to characterize and compare mesenchymal stem cells from adult human adipose tissue (ADS cells) with progenitor cell lines from the human corneoscleral limbus and to analyze their potential for the expression of epithelial markers. METHODS Stem cell markers (CD34, CD90, p63, and ABCG2) and epithelial cell markers (CK3/76, CK12, CK76, CK19, and CK1/5/10/14) were analyzed by immunostaining, flow cytometry, Western blot analysis, and PCR methods. The authors assayed adhesion and proliferation on different extracellular matrix proteins. RESULTS ADS cells expressed a set of progenitor cell markers, including p63 and ABCG2. CK12 expression in ADS cell cultures increased spontaneously and progressively by differential adhesion, which demonstrates the cells' potential and capability to acquire epithelial-like cell characteristics. The authors observed an increase in the adhesion and proliferation of ADS cells seeded onto different basement membrane extracellular matrix proteins. Laminin substrates reduced the proliferative state of ADS cells. CONCLUSIONS The expression of putative stem cell markers (CD90, ABCG2, and p63) and cytokeratins (CK12 and CK76) supports the hypothesis that ADS cells have self-renewal capacity and intrinsic plasticity that enables them to acquire some epithelial-like characteristics. Therefore, adult ADS cells could be a potential source for cell therapy in ocular surface regeneration.
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Comparison of stem cell properties in cell populations isolated from human central and limbal corneal epithelium. Cornea 2011; 30:1155-62. [PMID: 21849892 DOI: 10.1097/ico.0b013e318213796b] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
PURPOSE The limbus of the cornea is said to be the niche for limbal stem cells (LSCs) and the primary source of corneal epithelial maintenance. Previously, we aimed to have shown that central human epithelial cells are capable of corneal regeneration after wounding. In this study, we aimed to investigate whether central epithelial cells in human corneas have LSC properties. METHODS Human corneal epithelial cells were separated from the central cornea and the limbus. Isolated cells were collected for sphere-forming assay, and spheres formed subsequently were analyzed using immunohistochemistry. Fluorescence-activated cell sorting (FACS) was also used to analyze epithelial cells from central cornea, limbal rim, older donors, younger donors, and dissociated spheres. These analyses were based on cell size and Hoechst 33342 dye efflux ability, and side populations and non-side populations were isolated for colony growth measurement and sphere-forming assay. RESULTS Human central and limbal epithelial cells were capable of forming spheres, in a 1:2 ratio, that were positive for p63 immunolabeling. In FACS, central and limbal epithelial cells showed no significant difference in cell size and dye efflux ability. There were almost 10 times more large cells with good dye efflux ability from younger donors than from older donors, and the gated side population showed more than 4 times faster rate of colony growth than the non-side population. Dissociated sphere cells, however, did not follow a similar pattern to tissue-derived cells using FACS analysis. In these, there were more than twice as many large cells than small cells with good dye efflux ability. CONCLUSIONS Both limbal and central epithelial cells are capable of forming spheres in cultures that have stem cell properties. Central and limbal epithelial cells cannot be differentiated using FACS, but younger donor tissues give rise to greater numbers of large cells with high dye efflux. Therefore, results indicate that human central corneal epithelium contains cells with stem/progenitor properties, and these stem properties decline with age.
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Kim SW, Seo KY, Rhim T, Kim EK. Effect of Retinoic Acid on Epithelial Differentiation and Mucin Expression in Primary Human Corneal Limbal Epithelial Cells. Curr Eye Res 2011; 37:33-42. [DOI: 10.3109/02713683.2011.620728] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
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Abstract
The cornea, the most anterior segment of the eye, provides us with exquisite vision. Unlike other vital tissues, it is poorly protected from the environment and is thus reliant on a self-renewal program to preserve integrity. This function is reserved for corneal epithelial stem cells located in the basal layer of the limbus, a narrow transition zone that segregates the peripheral cornea from the adjacent conjunctiva. Under physiological conditions, these cells replenish the corneal epithelium when mature or traumatized cells are lost. However, when the limbus is extensively damaged, stem cell activity is compromised, resulting in a condition known as limbal stem cell deficiency (LSCD). This disease is characterized by corneal neovascularization and persistent epithelial defects which impair vision. Over the past 20 years a myriad of treatment options have been developed for LSCD, most of which incorporate stem cell transplantation. Due to the disadvantages associated with the use of allogeneic and xenogeneic material, researchers are currently focusing on refining techniques involving autologous limbal tissue transplantation and are delving into the possibility that stem cells found in other organs can provide an alternative source of corneal epithelium. Determining where donor stem cells reside on the recipient's ocular surface and how long they remain viable will provide further insights into improving current therapeutic options for patients with LSCD.
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Su L, Cui H, Xu C, Xie X, Chen Q, Gao X. Putative rabbit conjunctival epithelial stem/progenitor cells preferentially reside in palpebral conjunctiva. Curr Eye Res 2011; 36:797-803. [PMID: 21812662 DOI: 10.3109/02713683.2011.593727] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
PURPOSE Conjunctival epithelia play an important role in forming a physical protective barrier of the ocular surface. To protect the integrity of the ocular surface, the conjunctival epithelial cells (CECs) must be self-renewing from local stem cells. In this study, the distribution of conjunctival epithelial stem/progenitor cells was determined. METHODS In a long-term bromodeoxyuridine (BrdU) label-retaining study, adult New Zealand white rabbits were injected intraperitoneally with BrdU 50 mg/kg/d for 7 days and follow-up for 6 weeks. In the short-term BrdU label-retaining study, rabbits were injected intraperitoneally with BrdU 50 mg/kg once and BrdU-labeled CECs were detected after 1, 3, 5, and 7 days, respectively. In vitro, growth kinetics, proliferating cell nuclear antigen (PCNA) immunocytochemical staining and colony-forming abilities of rabbit palpebral, fornical and bulbar CECs cultured in an identical environment were investigated and compared between different parts. RESULTS In the long-term BrdU label-retaining study, BrdU-labeled cells concentrated at the mucocutaneous junction. In the short-term BrdU label-retaining study, the peak concentration of BrdU label-retaining conjunctival epithelial cells progressively migrated from the mucocutaneous junction to the fornix in rabbits 1, 3, 5 and 7 days postinjection respectively, but a focus of BrdU-labeled cells remained in the mucocutaneous junction at all postinjection intervals. Rabbit palpebral CECs grew faster and assigned more PCNA-positive cells and higher colony-forming abilities than the other two parts in vitro. CONCLUSIONS Long-term retention of BrdU-labeled cells at the mucocutaneous junction indicated that slow-cycling stem cells may locate at this position. The epithelial stem/progenitor cells at the mucocutaneous junction may serve as an origination of transient amplifying cells that migrate towards the fornix. Rabbit palpebral CECs (including mucocutaneous junction epithelial cells) possess higher self-renewing ability in vitro. Collectively, conjunctival epithelial stem/progenitor cells in the rabbit may preferentially reside in palpebral conjunctiva, especially the mucocutaneous junction.
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Affiliation(s)
- Li Su
- Department of Ophthalmology, Shanghai Tenth People's Hospital, Affiliate of Tongji University School of Medicine, Shanghai, PR China
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Varghese VM, Prasad T, Kumary TV. Optimization of culture conditions for an efficient xeno-feeder free limbal cell culture system towards ocular surface regeneration. Microsc Res Tech 2011; 73:1045-52. [PMID: 20196106 DOI: 10.1002/jemt.20831] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Ex vivo expansion of limbal stem cells from a small biopsy and its subsequent transplantation is the golden choice of treatment for limbal stem cell deficiency. Use of murine 3T3 feeder layer is a prerequisite for this ex vivo expansion. There is an ever-increasing demand for feeder free cultures to avoid xenotoxicity and transmission of xeno-diseases to human system. This study was aimed to establish an efficient xeno-feeder free limbal culture system towards ocular surface regeneration. To study the effect of initial dispase treatment and culture system used, migratory distance of cells from explants was analyzed from phase contrast images using "interactive measurements" of Qwin software (Leica). Expression of p63 in different culture systems was studied by immunofluorescent staining, followed by quantitative confocal microscopy (Carl Zeiss). Results showed dispase treatment was not necessary for establishing limbal explant culture. A combination of Iscove's modified Dulbecco's medium and Panserin 801 resulted in formation of autofeeder layer with maintenance of progenitor characteristics, thus mimicking natural tissue architecture. Further analysis of this culture system showed that cells could be cultured till confluency. Immunofluorescent staining of ABCG2 revealed presence of stem cell marker in the confluent cell layer. Scanning Electron Micrographs demonstrated homogenous population of tightly packed cells in this culture system. Replacement of bovine serum with autologous serum did not affect morphology or growth of cells in this culture system. This study will be a major step in the development of xeno-feeder free epithelial equivalents towards ocular surface reconstruction.
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Affiliation(s)
- Viji Mary Varghese
- Tissue Culture Laboratory, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, Kerala, India
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Induction of corneal epithelium-like cells from cynomolgus monkey embryonic stem cells and their experimental transplantation to damaged cornea. Cornea 2011; 29:432-8. [PMID: 20164754 DOI: 10.1097/ico.0b013e3181b9ffcc] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
PURPOSE We previously reported the successful transplantation of corneal epithelium-like cells derived from mouse embryonic stem (ES) cells onto injured mouse cornea. Here, we tested whether nonhuman primate ES cells have ability to differentiate into corneal epithelial cells and whether monkey ES cell-derived corneal epithelium-like cells were applicable for the experimental transplantation to damaged cornea. METHODS Monkey ES cells were cultivated on type IV collagen-coated dishes for various days to induce differentiation into corneal epithelium-like cells. The differentiation was evaluated by reverse transcription-polymerase chain reaction and immunostaining. The corneal epithelium-like cells were transplanted to the injured mouse cornea. Reconstitution of the corneal epithelium was evaluated by immunostaining. RESULTS The cells cultured on type IV collagen showed cobblestone-like appearance resembling epithelial cells. They expressed messenger RNA of pax6, p63, E-cadherin, CD44, proliferating cell nuclear antigen, keratin 3, and keratin 12. Protein expressions of pax6, keratin 3/12, p63, proliferating cell nuclear antigen, E-cadherin, and CD44 were confirmed by immunostaining. When the corneal epithelium-like cells were transplanted, they adhered to the corneal stroma, leading to formation of multiple cell layers. The grafted cells were stained with anti-human nuclear protein antibody, which cross-reacted with nuclei of monkey cells but not with those of mouse cells. They retained the expressions of keratin 3/12, E-cadherin, and CD44. CONCLUSIONS We induced corneal epithelium-like cells from monkey ES cells with moderate efficiency. The cells were successfully transplanted onto the injured mouse cornea. This is the first demonstration that nonhuman primate ES cells were induced to differentiate into corneal epithelium-like cells, which were applicable for transplantation to an animal model of corneal injury.
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Abstract
INTRODUCTION Blindness affects 50 million individuals worldwide; a significant proportion of them require a cell or tissue-based repair or replacement strategy to mend their damaged or diseased cornea. This review will focus on the epithelial stem cell (ESC) population of the cornea, where they reside, how they are identified and what alternative cells can be used as functional substitutes. SOURCE OF DATA Data for this review were collated after performing literature searches by inserting key words (cornea, limbal, stem cells (SCs), epithelium, stroma, and endothelium) into the search engine PubMed. AREAS OF AGREEMENT The prevailing notion is that corneal ESCs reside in an exclusive niche and their activation is dictated by niche-specific signals. AREAS OF CONTROVERSY Recent studies refute this hypothesis, as the central cornea of many animal species also possesses similar proliferative and clonogenic activity. The other area of controversy is in relation to the use of autologous and/or allogeneic cell therapies which are mostly contaminated with xenogeneic factors, potentially exposing the recipient to potentially harmful foreign infection. GROWING POINTS Due to the shortage of donor corneal biomaterial, alternative cellular sources are being sought, discovered and trialed. AREAS TIMELY FOR DEVELOPING RESEARCH With the exception of conjunctival and oral mucosal epithelium, no other cell type has been successfully used to treat patients with severe corneal epithelial defects. Embryonic and foetal SCs may have the greatest potential of all; however there are moral, legal, religious and scientific hurdles to overcome before they become routinely used in the clinic.
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Affiliation(s)
- Nick Di Girolamo
- Inflammation and Infection Research Centre, School of Medical Sciences, University of New South Wales, Sydney, NSW 2052, Australia.
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