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Genna VG, Maurizi E, Rama P, Pellegrini G. Biology and medicine on ocular surface restoration: Advancements and limits of limbal stem cell deficiency treatments. Ocul Surf 2025; 35:57-67. [PMID: 39580144 DOI: 10.1016/j.jtos.2024.11.005] [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: 01/17/2024] [Revised: 11/08/2024] [Accepted: 11/13/2024] [Indexed: 11/25/2024]
Abstract
Ocular vision can be hampered by corneal damages, sensibly reducing patients' quality of life and having important social and economic consequences. Ocular surface diseases, which often lead to corneal opacities with visual impairment are the most severe forms of the Limbal Stem Cell Deficiency (LSCD). The present review provides an updated perspective on the available treatments for LSCD, focusing on clinical and biological features, as well as critical points to monitor during clinical translation. Recently developed surgical treatments for LSCD are described, along with their benefits and limitations, with the aim of addressing the issue of correct patient selection. Autologous surgical approaches have been attempted, such as conjunctival limbal autograft (CLAU), simple limbal epithelial transplantation (SLET), and others. Allogeneic limbal stem cell transplantation represents an alternative but carries risk of rejection and requires immunosuppression. Other potential treatments are based on induced pluripotent stem cells (iPSCs), but they require further investigation. The development of advanced therapy medicinal products (ATMPs) such as cultivated limbal epithelial transplantation (CLET), or the use of other epithelia as cultivated oral mucosal epithelial cell transplantation (COMET), has opened additional therapeutic possibilities. Some common critical issues in clinical translation are described, such as patient selection, biopsy procurement, or the use of human/animal derived components, which require rigorous validation to ensure safety and efficacy. Personalized medicine is a promising field for ocular surface restoration, where long-term follow-up studies and standardized criteria are crucial to evaluate the efficacy of these treatments and their cost-effectiveness in providing high-value healthcare.
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Affiliation(s)
| | - Eleonora Maurizi
- Centre for Regenerative Medicine ''S. Ferrari'', University of Modena and Reggio Emilia, Modena, Italy
| | - Paolo Rama
- Department of Ophthalmology, IRCCS Policlinico San Matteo, Pavia, Italy
| | - Graziella Pellegrini
- Centre for Regenerative Medicine ''S. Ferrari'', University of Modena and Reggio Emilia, Modena, Italy.
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2
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Suanno G, Genna VG, Maurizi E, Dieh AA, Griffith M, Ferrari G. Cell therapy in the cornea: The emerging role of microenvironment. Prog Retin Eye Res 2024; 102:101275. [PMID: 38797320 DOI: 10.1016/j.preteyeres.2024.101275] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2023] [Revised: 05/22/2024] [Accepted: 05/23/2024] [Indexed: 05/29/2024]
Abstract
The cornea is an ideal testing field for cell therapies. Its highly ordered structure, where specific cell populations are sequestered in different layers, together with its accessibility, has allowed the development of the first stem cell-based therapy approved by the European Medicine Agency. Today, different techniques have been proposed for autologous and allogeneic limbal and non-limbal cell transplantation. Cell replacement has also been attempted in cases of endothelial cell decompensation as it occurs in Fuchs dystrophy: injection of cultivated allogeneic endothelial cells is now in advanced phases of clinical development. Recently, stromal substitutes have been developed with excellent integration capability and transparency. Finally, cell-derived products, such as exosomes obtained from different sources, have been investigated for the treatment of severe corneal diseases with encouraging results. Optimization of the success rate of cell therapies obviously requires high-quality cultured cells/products, but the role of the surrounding microenvironment is equally important to allow engraftment of transplanted cells, to preserve their functions and, ultimately, lead to restoration of tissue integrity and transparency of the cornea.
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Affiliation(s)
- Giuseppe Suanno
- Vita-Salute San Raffaele University, Milan, Italy; Eye Repair Unit, Division of Neuroscience, IRCCS San Raffaele Scientific Institute, Milan, Italy
| | | | - Eleonora Maurizi
- Centre for Regenerative Medicine ''S. Ferrari'', University of Modena and Reggio Emilia, Modena, Italy
| | - Anas Abu Dieh
- Maisonneuve-Rosemont Hospital Research Centre, Montreal, Quebec, Canada
| | - May Griffith
- Maisonneuve-Rosemont Hospital Research Centre, Montreal, Quebec, Canada.
| | - Giulio Ferrari
- Vita-Salute San Raffaele University, Milan, Italy; Eye Repair Unit, Division of Neuroscience, IRCCS San Raffaele Scientific Institute, Milan, Italy; Ophthalmology Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy.
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3
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Tedesco L, Rossi F, Ruocco C, Ragni M, Carruba MO, Valerio A, Nisoli E. An original amino acid formula favours in vitro corneal epithelial wound healing by promoting Fn1, ITGB1, and PGC-1α expression. Exp Eye Res 2022; 219:109060. [PMID: 35390334 DOI: 10.1016/j.exer.2022.109060] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2022] [Revised: 03/17/2022] [Accepted: 03/30/2022] [Indexed: 11/04/2022]
Abstract
Corneal disorders are frequent, involving most diabetic patients; among its manifestations, they include delayed wound healing. Since maintenance of mitochondrial homeostasis is fundamental for the cell, stimulation of mitochondrial biogenesis represents a unique therapeutic tool for preventing and treating disorders with a deficit in energy metabolism. We have recently demonstrated that a branched-chain amino acid (BCAA)-enriched mixture (BCAAem) supported mitochondrial biogenesis in cardiac and skeletal muscle, reduced liver damage caused by alcohol, and prevented the doxorubicin-dependent mitochondrial damage in cardiomyocytes. The present study aimed to investigate a new amino acid mixture, named six amino acids (6AA), to promote corneal epithelial wound healing by regulating mitochondrial biogenesis. A murine epithelium cell line (TKE2) exposed to this mixture showed increased mitochondrial biogenesis markers, fibronectin 1 (Fn1) and integrin beta 1 (ITGB1) involved in extracellular matrix synthesis and cell migration. Most importantly, the 6AA mixture completely restored the wound in scratch assays, confirming the potential of this new formula in eye disorders like keratopathy. Moreover, our results demonstrate for the first time that peroxisome proliferator-receptor γ coactivator 1 α (PGC-1α) is expressed in TKE2 cells, which controls mitochondrial function and corneal repair process. These results could be relevant for the treatment mainly focused on corneal re-epithelialisation.
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Affiliation(s)
- Laura Tedesco
- Center for Study and Research on Obesity, Department of Medical Biotechnology and Translational Medicine, University of Milan, via Vanvitelli, 32 - 20129, Milan, Italy.
| | - Fabio Rossi
- Center for Study and Research on Obesity, Department of Medical Biotechnology and Translational Medicine, University of Milan, via Vanvitelli, 32 - 20129, Milan, Italy
| | - Chiara Ruocco
- Center for Study and Research on Obesity, Department of Medical Biotechnology and Translational Medicine, University of Milan, via Vanvitelli, 32 - 20129, Milan, Italy
| | - Maurizio Ragni
- Center for Study and Research on Obesity, Department of Medical Biotechnology and Translational Medicine, University of Milan, via Vanvitelli, 32 - 20129, Milan, Italy
| | - Michele O Carruba
- Center for Study and Research on Obesity, Department of Medical Biotechnology and Translational Medicine, University of Milan, via Vanvitelli, 32 - 20129, Milan, Italy
| | - Alessandra Valerio
- Department of Molecular and Translational Medicine, Brescia University, Brescia, Italy
| | - Enzo Nisoli
- Center for Study and Research on Obesity, Department of Medical Biotechnology and Translational Medicine, University of Milan, via Vanvitelli, 32 - 20129, Milan, Italy
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4
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Corneal Epithelial Stem Cells-Physiology, Pathophysiology and Therapeutic Options. Cells 2021; 10:cells10092302. [PMID: 34571952 PMCID: PMC8465583 DOI: 10.3390/cells10092302] [Citation(s) in RCA: 32] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2021] [Revised: 08/27/2021] [Accepted: 08/28/2021] [Indexed: 12/12/2022] Open
Abstract
In the human cornea, regeneration of the epithelium is regulated by the stem cell reservoir of the limbus, which is the marginal region of the cornea representing the anatomical and functional border between the corneal and conjunctival epithelium. In support of this concept, extensive limbal damage, e.g., by chemical or thermal injury, inflammation, or surgery, may induce limbal stem cell deficiency (LSCD) leading to vascularization and opacification of the cornea and eventually vision loss. These acquired forms of limbal stem cell deficiency may occur uni- or bilaterally, which is important for the choice of treatment. Moreover, a variety of inherited diseases, such as congenital aniridia or dyskeratosis congenita, are characterized by LSCD typically occurring bilaterally. Several techniques of autologous and allogenic stem cell transplantation have been established. The limbus can be restored by transplantation of whole limbal grafts, small limbal biopsies or by ex vivo-expanded limbal cells. In this review, the physiology of the corneal epithelium, the pathophysiology of LSCD, and the therapeutic options will be presented.
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5
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Okada Y, Zhang Y, Zhang L, Yeh LK, Wang YC, Saika S, Liu CY. Shp2-mediated MAPK pathway regulates ΔNp63 in epithelium to promote corneal innervation and homeostasis. J Transl Med 2020; 100:630-642. [PMID: 31653968 PMCID: PMC7102931 DOI: 10.1038/s41374-019-0338-2] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2019] [Revised: 08/19/2019] [Accepted: 08/30/2019] [Indexed: 12/16/2022] Open
Abstract
Corneal nerve fibers serving sensory, reflex, and neurotrophic functions sustain corneal homeostasis and transparency to promote normal visual function. It is not known whether corneal epithelium is also important for the corneal innervation. Herein, we generated a compound transgenic mouse strain, K14rtTA;tetO-Cre (TC);Shp2flox/flox, in which Shp2 was conditionally knocked out from K14-positive cells including corneal epithelium (Shp2K14ce-cko) upon doxycycline (dox) administration. Our data reveal that Shp2K14ce-cko caused corneal denervation. More specifically, corneal epithelium thickness and corneal sensitivity reduced dramatically in Shp2K14ce-cko mice. In addition, corneal epithelial wound healing after debridement was delayed substantially in the mutant mice. These defects manifested in Shp2K14ce-cko mice resemble the symptoms of human neurotrophic keratopathy. Our in vitro study shows that neurite outgrowth of the mouse primary trigeminal ganglion cells (TGCs) was inhibited when as cocultured with mouse corneal epithelial cells (TKE2) transfected by Shp2-, Mek1/2-, or ∆Np63-targeted siRNA but not by Akt1/2-targeted siRNA. Furthermore, ∆Np63 RNA interference downregulated Ngf expression in TKE2 cells. Cotransfection experiments reveal that Shp2 tightly monitored ΔNp63 protein levels in HEK293 and TKE2 cells. Taken together, our data suggest that the Shp2-mediated MAPK pathway regulated ΔNp63, which in turn positively regulated Ngf in epithelium to promote corneal innervation and epithelial homeostasis.
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Affiliation(s)
- Yuka Okada
- Indiana University School of Optometry, Bloomington, IN, USA.
- Department of Ophthalmology, Wakayama Medical University, School of Medicine, Wakayama, Japan.
| | - Yujin Zhang
- Indiana University School of Optometry, Bloomington, IN, USA
| | - Lingling Zhang
- Indiana University School of Optometry, Bloomington, IN, USA
| | - Lung-Kun Yeh
- Department of Ophthalmology, Chang-Gung Memorial Hospital, Linko, Taiwan
| | - Yen-Chiao Wang
- Indiana University School of Optometry, Bloomington, IN, USA
| | - Shizuya Saika
- Department of Ophthalmology, Wakayama Medical University, School of Medicine, Wakayama, Japan
| | - Chia-Yang Liu
- Indiana University School of Optometry, Bloomington, IN, USA.
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Khot MI, Downey CL, Armstrong G, Svavarsdottir HS, Jarral F, Andrew H, Jayne DG. The role of ABCG2 in modulating responses to anti-cancer photodynamic therapy. Photodiagnosis Photodyn Ther 2019; 29:101579. [PMID: 31639455 DOI: 10.1016/j.pdpdt.2019.10.014] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2019] [Revised: 10/03/2019] [Accepted: 10/11/2019] [Indexed: 01/10/2023]
Abstract
The ATP-binding cassette (ABC) superfamily G member 2 (ABCG2) transmembrane protein transporter is known for conferring resistance to treatment in cancers. Photodynamic therapy (PDT) is a promising anti-cancer method involving the use of light-activated photosensitisers to precisely induce oxidative stress and cell death in cancers. ABCG2 can efflux photosensitisers from out of cells, reducing the capacity of PDT and limiting the efficacy of treatment. Many studies have attempted to elucidate the relationship between the expression of ABCG2 in cancers, its effect on the cellular retention of photosensitisers and its impact on PDT. This review looks at the studies which investigate the effect of ABCG2 on a range of different photosensitisers in different pre-clinical models of cancer. This work also evaluates the approaches that are being investigated to address the role of ABCG2 in PDT with an outlook on potential clinical validation.
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Affiliation(s)
- M Ibrahim Khot
- School of Medicine, St James's University Hospital, University of Leeds, Leeds, UK.
| | - Candice L Downey
- School of Medicine, St James's University Hospital, University of Leeds, Leeds, UK
| | - Gemma Armstrong
- School of Medicine, St James's University Hospital, University of Leeds, Leeds, UK
| | | | - Fazain Jarral
- School of Medicine, St James's University Hospital, University of Leeds, Leeds, UK
| | - Helen Andrew
- School of Medicine, St James's University Hospital, University of Leeds, Leeds, UK
| | - David G Jayne
- School of Medicine, St James's University Hospital, University of Leeds, Leeds, UK
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7
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ROS-mediated Different Homeostasis of Murine Corneal Epithelial Progenitor Cell Line under Oxidative Stress. Sci Rep 2016; 6:36481. [PMID: 27805062 PMCID: PMC5090348 DOI: 10.1038/srep36481] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2016] [Accepted: 10/12/2016] [Indexed: 01/31/2023] Open
Abstract
The role of ROS in stem cell biology has not been fully illustrated and understood. Here we compared the different responses and investigated the mechanism underlying oxidative stress induced by hydrogen peroxide (H2O2) between murine corneal epithelial progenitor cell line (TKE2) and mature murine corneal epithelial cells (MCE). TKE2 showed a different homeostasis and strong resistance to H2O2. TKE2 reduced the production of ROS, inhibited ROS generation enzyme NADPH oxidase 4 (NOX4), and increased dual specificity phosphatase 6 (DUSP6). Furthermore, TKE2 activated nuclear factor (erythroid-derived 2)-like 2 (NRF2) signaling pathway, regulated miR-125B1 and miR-29B1, and elevated levels of antioxidants glutathione S-transferase P (GSTP) and superoxide dismutases (SOD). The association with ROS of the cells was also verified by RNA interference approach and pharmacological antagonization. In addition, TKE2 enhanced the autophagy after exposure to H2O2. The novel evidence suggests that TKE2 cells have different homeostasis and strong antioxidant properties against oxidative stress via the regulation of ROS formation and pathway.
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Prasanphanich AF, White DE, Gran MA, Kemp ML. Kinetic Modeling of ABCG2 Transporter Heterogeneity: A Quantitative, Single-Cell Analysis of the Side Population Assay. PLoS Comput Biol 2016; 12:e1005188. [PMID: 27851764 PMCID: PMC5113006 DOI: 10.1371/journal.pcbi.1005188] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2016] [Accepted: 10/10/2016] [Indexed: 12/13/2022] Open
Abstract
The side population (SP) assay, a technique used in cancer and stem cell research, assesses the activity of ABC transporters on Hoechst staining in the presence and absence of transporter inhibition, identifying SP and non-SP cell (NSP) subpopulations by differential staining intensity. The interpretation of the assay is complicated because the transporter-mediated mechanisms fail to account for cell-to-cell variability within a population or adequately control the direct role of transporter activity on staining intensity. We hypothesized that differences in dye kinetics at the single-cell level, such as ABCG2 transporter-mediated efflux and DNA binding, are responsible for the differential cell staining that demarcates SP/NSP identity. We report changes in A549 phenotype during time in culture and with TGFβ treatment that correlate with SP size. Clonal expansion of individually sorted cells re-established both SP and NSPs, indicating that SP membership is dynamic. To assess the validity of a purely kinetics-based interpretation of SP/NSP identity, we developed a computational approach that simulated cell staining within a heterogeneous cell population; this exercise allowed for the direct inference of the role of transporter activity and inhibition on cell staining. Our simulated SP assay yielded appropriate SP responses for kinetic scenarios in which high transporter activity existed in a portion of the cells and little differential staining occurred in the majority of the population. With our approach for single-cell analysis, we observed SP and NSP cells at both ends of a transporter activity continuum, demonstrating that features of transporter activity as well as DNA content are determinants of SP/NSP identity.
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Affiliation(s)
- Adam F. Prasanphanich
- The Wallace H. Coulter Department of Biomedical Engineering Georgia Institute of Technology and Emory University, Atlanta, Georgia, United States of America
| | - Douglas E. White
- The Wallace H. Coulter Department of Biomedical Engineering Georgia Institute of Technology and Emory University, Atlanta, Georgia, United States of America
| | - Margaret A. Gran
- The Wallace H. Coulter Department of Biomedical Engineering Georgia Institute of Technology and Emory University, Atlanta, Georgia, United States of America
| | - Melissa L. Kemp
- The Wallace H. Coulter Department of Biomedical Engineering Georgia Institute of Technology and Emory University, Atlanta, Georgia, United States of America
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9
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Kubota M, Shui YB, Liu M, Bai F, Huang AJ, Ma N, Beebe DC, Siegfried CJ. Mitochondrial oxygen metabolism in primary human lens epithelial cells: Association with age, diabetes and glaucoma. Free Radic Biol Med 2016; 97:513-519. [PMID: 27445101 PMCID: PMC4996752 DOI: 10.1016/j.freeradbiomed.2016.07.016] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/24/2015] [Revised: 06/14/2016] [Accepted: 07/18/2016] [Indexed: 12/13/2022]
Abstract
PURPOSE The hypoxic environment around the lens is important for maintaining lens transparency. Lens epithelial cells (LECs) play a key role in lens metabolism. We measured oxygen consumption to assess the role of human LECs in maintaining hypoxia around the lens, as well as the impact of systemic and ocular diagnosis on these cells. METHODS Baseline cellular respiration was measured in rabbit LECs (NN1003A), canine kidney epithelial cells (MDCK), trabecular meshwork cells (TM-5), and bovine corneal endothelial cells (CCEE) using a XF96 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA), which measures oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in vitro. Following informed written consent, lens capsule epithelial cells were obtained from patients during cataract surgery and were divided into small explants in 96-well plates. Capsules were removed when LECs became confluent. OCR was normalized to the number of cells per well using rabbit LECs as a standard. The effect of patient age, sex, race, and presence of diabetes or glaucoma on oxygen consumption was assessed by using the Mann-Whitney U test and multivariate regression analysis. RESULTS Primary LECs were obtained from 69 patients. The OCR from donors aged 70 and over was lower than that of those under 70 years (2.21±1.037 vs. 2.86±1.383 fmol/min/cell; p<0.05). Diabetic patients had lower OCR than non-diabetic patients (2.02±0.911 vs. 2.79±1.332fmol/min/cell; p<0.05), and glaucoma patients had lower OCR than non-glaucoma patients (2.27±1.19 vs. 2.83±1.286 fmol/min/cell; p<0.05). Multivariate regression analysis confirmed that donors aged 70 and over (p<0.05), diabetic patients (p<0.01), and glaucoma patients (p<0.05) had significantly lower OCR, independent of other variables. Gender and race had no significant effect on OCR. CONCLUSIONS The lower oxygen consumption rate of human LECs in older donors and patients with diabetes or glaucoma could contribute to cataract development. Diabetes and glaucoma are particularly important factors associated with decreased OCR, independent of age. Ongoing studies are examining pO2 at the anterior surface of the lens in vivo and oxygen consumption in the patient's LECs.
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Affiliation(s)
- M Kubota
- Departments of Ophthalmology & Visual Sciences, Washington University School of Medicine, St. Louis, MO, United States; Departments of Ophthalmology, Keio University School of Medicine, Tokyo, Japan.
| | - Y B Shui
- Departments of Ophthalmology & Visual Sciences, Washington University School of Medicine, St. Louis, MO, United States.
| | - M Liu
- Departments of Ophthalmology & Visual Sciences, Washington University School of Medicine, St. Louis, MO, United States.
| | - F Bai
- Departments of Ophthalmology & Visual Sciences, Washington University School of Medicine, St. Louis, MO, United States.
| | - A J Huang
- Departments of Ophthalmology & Visual Sciences, Washington University School of Medicine, St. Louis, MO, United States.
| | - N Ma
- Departments of Ophthalmology & Visual Sciences, Washington University School of Medicine, St. Louis, MO, United States; Departments of Ophthalmology, Tangdu Hospital, Fourth Military Medical University, Xi'an, China.
| | - D C Beebe
- Departments of Ophthalmology & Visual Sciences, Washington University School of Medicine, St. Louis, MO, United States; Departments of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO, United States
| | - C J Siegfried
- Departments of Ophthalmology & Visual Sciences, Washington University School of Medicine, St. Louis, MO, United States.
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Poleshko AG, Volotovski ID. The role of ABCG2 in maintaining the viability and proliferative activity of bone marrow mesenchymal stem cells in hypoxia. Biophysics (Nagoya-shi) 2016. [DOI: 10.1134/s0006350916020159] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
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11
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Pellegrini G, Lambiase A, Macaluso C, Pocobelli A, Deng S, Cavallini GM, Esteki R, Rama P. From discovery to approval of an advanced therapy medicinal product-containing stem cells, in the EU. Regen Med 2016; 11:407-20. [PMID: 27091398 PMCID: PMC5561870 DOI: 10.2217/rme-2015-0051] [Citation(s) in RCA: 38] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
In 1997, the human corneal epithelium was reconstructed in vitro and transplanted on patients. Later, it became a routine treatment, before regulations considered advanced therapy medicinal products and drugs on the same lines. Manufacturing, before and after good manufacturing practice setting, was established in different facilities and the clinical application in several hospitals. Advanced therapy medicinal products, including stem cells, are unique products with different challenges than other drugs: some uncertainties, in addition to benefit, cannot be avoided. This review will focus on all recent developments in the stem cell-based corneal therapy.
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Affiliation(s)
- Graziella Pellegrini
- Centre for Regenerative Medicine "Stefano Ferrari", University of Modena and Reggio Emilia, via G.Gottardi 100, Modena, 41125, Italy; Holostem Terapie Avanzate, Modena, Italy
| | - Alessandro Lambiase
- Department of Sense Organs, University of Rome "Sapienza", viale Regina Elena, Rome, Italy
| | - Claudio Macaluso
- Unit of Ophthalmology, University of Parma, Via Gramsci 14, 43126 Parma, Italy; IMEM - CNR (Italian National Reserach Council), Parco Area delle Scienze 37/A - 43124 Parma, Italy
| | - Augusto Pocobelli
- Ophthalmology Unit-Eye Bank, S. Giovanni Addolorata Hospital, via S. Stefano Rotondo 9, Rome, Italy
| | - Sophie Deng
- Cornea Division Stein Eye Institute, UCLA 100 Stein Plaza Los Angeles, CA 90095, USA
| | - Gian Maria Cavallini
- Ophthalmology Unit, Policlinico University Hospital, University of Modena & Reggio Emilia, via Del Pozzo 71, Modena, 41125, Italy
| | - Roza Esteki
- Centre for Regenerative Medicine "Stefano Ferrari", University of Modena and Reggio Emilia, via G.Gottardi 100, Modena, 41125, Italy; Holostem Terapie Avanzate, Modena, Italy
| | - Paolo Rama
- Cornea & Ocular Surface Unit San Raffaele Scientific Institute Via Olgettina, 60-20132 Milano, Italy
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12
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Masuda H, Maruyama T, Gargett CE, Miyazaki K, Matsuzaki Y, Okano H, Tanaka M. Endometrial side population cells: potential adult stem/progenitor cells in endometrium. Biol Reprod 2015; 93:84. [PMID: 26316062 DOI: 10.1095/biolreprod.115.131490] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2015] [Accepted: 08/19/2015] [Indexed: 12/18/2022] Open
Abstract
Uterine endometrium is one of the most important organs for species preservation. However, the physiology of human endometrium remains poorly understood, because the human endometrium undergoes rapid and large changes during each menstrual cycle and it is very difficult to investigate human endometrium as one organ. This remarkable regenerative capacity of human endometrium strongly suggests the existence of adult stem cells, and physiology of endometrium cannot be explained without adult stem cells. Therefore, investigating endometrial stem/progenitor cells should lead to a breakthrough in understanding the normal endometrial physiology and the pathophysiology of endometrial neoplastic disorders, such as endometriosis and endometrial cancer. Several cell populations have been discovered as putative endometrial stem/progenitor cells. Emerging evidence reveals that the endometrial side population (SP) is one of the potential endometrial stem/progenitor populations. Of all the endometrial stem/progenitor cell candidates, the endometrial SP (ESP) is best investigated in vitro and in vivo, and has the largest number of references. In this review, we provide an overview of the accumulating evidence for the ESP cells, both directly from human endometria and from cultured endometrial cells. Furthermore, SP cells are compared to other potential stem/progenitor cells, and we discuss their stem cell properties. We also discuss the difficulties and unsolved issues in endometrial stem cell biology.
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Affiliation(s)
- Hirotaka Masuda
- Department of Obstetrics and Gynecology, Keio University School of Medicine, Tokyo, Japan
| | - Tetsuo Maruyama
- Department of Obstetrics and Gynecology, Keio University School of Medicine, Tokyo, Japan
| | - Caroline E Gargett
- The Ritchie Centre, Hudson Monash Institute of Medical Research and Department of Obstetrics and Gynaecology, Monash University, Melbourne, Victoria, Australia
| | - Kaoru Miyazaki
- Department of Obstetrics and Gynecology, Keio University School of Medicine, Tokyo, Japan
| | - Yumi Matsuzaki
- Department of Life Science Laboratory of Tumor Biology, Shimane University Faculty of Medicine, Izumo, Shimane, Japan
| | - Hideyuki Okano
- Department of Physiology, Keio University School of Medicine, Tokyo, Japan
| | - Mamoru Tanaka
- Department of Obstetrics and Gynecology, Keio University School of Medicine, Tokyo, Japan
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13
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Characterisation of human limbal side population cells isolated using an optimised protocol from an immortalised epithelial cell line and primary limbal cultures. Stem Cell Rev Rep 2014; 10:240-50. [PMID: 24174130 DOI: 10.1007/s12015-013-9481-0] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
The challenges in limbal stem cell biology largely remain in the process of identification, isolation and expansion of these adult corneal epithelial stem cells of the eye. Due to the absence of specific limbal stem cell markers, identification and isolation of putative limbal stem cells is a complicated task. The side population assay is an isolation method that utilises the ability of stem cells to efflux the DNA-binding dye Hoechst 33342 (or other vital dyes) combined with dual wavelength flow cytometry and is a valuable strategy to enrich for limbal stem cells. This assay has been used to successfully identify stem/ progenitor cell populations in a variety of tissues and cell lines. Here we optimise this assay to identify SP cell populations in both primary human limbal epithelial cultures and in an established human corneal epithelial cell line. The limbal SP fraction showed higher expression of ATP-binding cassette sub-family G member 2 (ABCG2), ΔNp63--a common limbal stem cell marker and the stem cell marker Sox2 compared to non-SP cells (NSP).
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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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Tan JJ, Azmi SM, Yong YK, Cheah HL, Lim V, Sandai D, Shaharuddin B. Tualang honey improves human corneal epithelial progenitor cell migration and cellular resistance to oxidative stress in vitro. PLoS One 2014; 9:e96800. [PMID: 24802273 PMCID: PMC4011849 DOI: 10.1371/journal.pone.0096800] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2013] [Accepted: 04/11/2014] [Indexed: 12/24/2022] Open
Abstract
Stem cells with enhanced resistance to oxidative stress after in vitro expansion have been shown to have improved engraftment and regenerative capacities. Such cells can be generated by preconditioning them with exposure to an antioxidant. In this study we evaluated the effects of Tualang honey (TH), an antioxidant-containing honey, on human corneal epithelial progenitor (HCEP) cells in culture. Cytotoxicity, gene expression, migration, and cellular resistance to oxidative stress were evaluated. Immunofluorescence staining revealed that HCEP cells were holoclonal and expressed epithelial stem cell marker p63 without corneal cytokeratin 3. Cell viability remained unchanged after cells were cultured with 0.004, 0.04, and 0.4% TH in the medium, but it was significantly reduced when the concentration was increased to 3.33%. Cell migration, tested using scratch migration assay, was significantly enhanced when cells were cultured with TH at 0.04% and 0.4%. We also found that TH has hydrogen peroxide (H2O2) scavenging ability, although a trace level of H2O2 was detected in the honey in its native form. Preconditioning HCEP cells with 0.4% TH for 48 h showed better survival following H2O2-induced oxidative stress at 50 µM than untreated group, with a significantly lower number of dead cells (15.3 ± 0.4%) were observed compared to the untreated population (20.5 ± 0.9%, p<0.01). Both TH and ascorbic acid improved HCEP viability following induction of 100 µM H2O2, but the benefit was greater with TH treatment than with ascorbic acid. However, no significant advantage was demonstrated using 5-hydroxymethyl-2-furancarboxaldehyde, a compound that was found abundant in TH using GC/MS analysis. This suggests that the cellular anti-oxidative capacity in HCEP cells was augmented by native TH and was attributed to its antioxidant properties. In conclusion, TH possesses antioxidant properties and can improve cell migration and cellular resistance to oxidative stress in HCEP cells in vitro.
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Affiliation(s)
- Jun Jie Tan
- Advanced Medical and Dental Institute, Universiti Sains Malaysia, Kepala Batas, Penang, Malaysia
- * E-mail:
| | - Siti Maisura Azmi
- Advanced Medical and Dental Institute, Universiti Sains Malaysia, Kepala Batas, Penang, Malaysia
| | - Yoke Keong Yong
- Department of Human Anatomy, Faculty of Medicine and Health Sciences, Serdang, Selangor Darul Ehsan, Malaysia
| | - Hong Leong Cheah
- Advanced Medical and Dental Institute, Universiti Sains Malaysia, Kepala Batas, Penang, Malaysia
| | - Vuanghao Lim
- Advanced Medical and Dental Institute, Universiti Sains Malaysia, Kepala Batas, Penang, Malaysia
| | - Doblin Sandai
- Advanced Medical and Dental Institute, Universiti Sains Malaysia, Kepala Batas, Penang, Malaysia
| | - Bakiah Shaharuddin
- Advanced Medical and Dental Institute, Universiti Sains Malaysia, Kepala Batas, Penang, Malaysia
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Pellegrini G, Rama P, Di Rocco A, Panaras A, De Luca M. Concise Review: Hurdles in a Successful Example of Limbal Stem Cell-based Regenerative Medicine. Stem Cells 2014; 32:26-34. [DOI: 10.1002/stem.1517] [Citation(s) in RCA: 82] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023]
Affiliation(s)
- Graziella Pellegrini
- Centre for Regenerative Medicine “Stefano Ferrari”; University of Modena and Reggio Emilia; Modena Italy
| | - Paolo Rama
- San Raffaele Scientific Institute, Ophthalmology Department; Milano Italy
| | - Antonio Di Rocco
- Centre for Regenerative Medicine “Stefano Ferrari”; University of Modena and Reggio Emilia; Modena Italy
| | - Athanasios Panaras
- Centre for Regenerative Medicine “Stefano Ferrari”; University of Modena and Reggio Emilia; Modena Italy
| | - Michele De Luca
- Centre for Regenerative Medicine “Stefano Ferrari”; University of Modena and Reggio Emilia; Modena Italy
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Ordonez P, Di Girolamo N. Limbal epithelial stem cells: role of the niche microenvironment. Stem Cells 2012; 30:100-7. [PMID: 22131201 DOI: 10.1002/stem.794] [Citation(s) in RCA: 90] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
The cornea contains a reservoir of self-regenerating epithelial cells that are essential for maintaining its transparency and good vision. The study of stem cells in this functionally important organ has grown over the past four decades, partly due to the ease with which this tissue is visualized, its accessibility with minimally invasive instruments, and the fact that its stem cells are segregated within a transitional zone between two functionally diverse epithelia. While human, animal, and ex vivo models have been instrumental in progressing the corneal stem cell field, there is still much to be discovered about this exquisitely sensitive window for sight. This review will provide an overview of the human cornea, where its stem cells reside and how components of the microenvironment including extracellular matrix proteins and their integrin receptors are thought to govern corneal stem cell homeostasis.
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Affiliation(s)
- Paula Ordonez
- Inflammation and Infection Research Centre, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, Australia
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