|
1
|
Braunsperger MV, Martin G, Herzig T, Kußberger I, Gießl A, Steimle S, Schlötzer-Schrehardt U, Schlunck G, Reinhard T, Polisetti N. Proteomic Insights into Human Limbal Epithelial Progenitor-Derived Small Extracellular Vesicles. Stem Cell Rev Rep 2025:10.1007/s12015-025-10877-w. [PMID: 40238075 DOI: 10.1007/s12015-025-10877-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 04/04/2025] [Indexed: 04/18/2025]
Abstract
Limbal epithelial stem/progenitor cells (LEPC), supported by limbal mesenchymal stromal cells (LMSC) and limbal melanocytes (LM) within a specialized niche, are responsible for maintaining the corneal epithelium. Small extracellular vesicles (sEV) emerged as critical mediators of intercellular communication in various stem cell niches, yet their role in maintaining human limbal niche homeostasis remains poorly understood. In this study, tangential flow filtration and size exclusion chromatography were used to isolate sEV from LEPC-, LMSC- and LM-conditioned media. The isolated sEV from LEPC exhibited properties characteristic for sEV as confirmed by nanoparticle tracking analysis for size and concentration, by electron microscopy for morphology, and by western blot analysis of canonical EV markers including the cell-specific protein (cytokeratin 17/19). Quantitative and comparative proteomic profiling revealed distinct molecular signatures of LEPC-derived sEV, enriched in factors associated with keratinocyte development, extracellular matrix organization, and niche regulation. These findings suggest that LEPC-derived sEV may serve as important signaling mediators within the limbal niche microenvironment, though additional studies are needed to determine their specific functional roles in maintaining niche homeostasis.
Collapse
Affiliation(s)
- Moritz Vincent Braunsperger
- Eye Center, Medical Center - Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106, Freiburg, Germany
| | - Gottfried Martin
- Eye Center, Medical Center - Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106, Freiburg, Germany
| | - Tabea Herzig
- Eye Center, Medical Center - Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106, Freiburg, Germany
| | - Isabell Kußberger
- Eye Center, Medical Center - Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106, Freiburg, Germany
| | - Andreas Gießl
- Department of Ophthalmology, University Hospital Erlangen, Friedrich-Alexander-University of Erlan-gen-Nürnberg, Schwabachanlage 6, D-91054, Erlangen, Germany
| | - Stefan Steimle
- Cryo-EM Facility (CEF), University of Freiburg, Albertstrasse 21, 79104, Freiburg, Germany
- Institute of Physical Chemistry, University of Freiburg, Albertstrasse 21, 79104, Freiburg, Germany
| | - Ursula Schlötzer-Schrehardt
- Department of Ophthalmology, University Hospital Erlangen, Friedrich-Alexander-University of Erlan-gen-Nürnberg, Schwabachanlage 6, D-91054, Erlangen, Germany
| | - Günther Schlunck
- Eye Center, Medical Center - Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106, Freiburg, Germany
| | - Thomas Reinhard
- Eye Center, Medical Center - Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106, Freiburg, Germany
| | - Naresh Polisetti
- Eye Center, Medical Center - Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106, Freiburg, Germany.
| |
Collapse
|
|
2
|
Wang Y, Ge H, Chen P, Wang Y. Wnt/β-catenin signaling in corneal epithelium development, homeostasis, and pathobiology. Exp Eye Res 2024; 246:110022. [PMID: 39117134 DOI: 10.1016/j.exer.2024.110022] [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: 04/18/2024] [Revised: 07/07/2024] [Accepted: 08/05/2024] [Indexed: 08/10/2024]
Abstract
The corneal epithelium is located on the most anterior surface of the eyeball and protects against external stimuli. The development of the corneal epithelium and the maintenance of corneal homeostasis are essential for the maintenance of visual acuity. It has been discovered recently via the in-depth investigation of ocular surface illnesses that the Wnt/β-catenin signaling pathway is necessary for the growth and stratification of corneal epithelial cells as well as the control of endothelial cell stability. In addition, the Wnt/β-catenin signaling pathway is directly linked to the development of common corneal illnesses such as keratoconus, fungal keratitis, and corneal neovascularization. This review mainly summarizes the role of the Wnt/β-catenin signaling pathway in the development, homeostasis, and pathobiology of cornea, hoping to provide new insights into the study of corneal epithelium and the treatment of related diseases.
Collapse
Affiliation(s)
- Yihui Wang
- School of Basic Medicine, Qingdao University, Qingdao 266071, Shandong Province, China
| | - Huanhuan Ge
- School of Basic Medicine, Qingdao University, Qingdao 266071, Shandong Province, China
| | - Peng Chen
- School of Basic Medicine, Qingdao University, Qingdao 266071, Shandong Province, China; Institute of Stem Cell Regeneration Medicine, School of Basic Medicine, Qingdao University, Qingdao, 266071, China.
| | - Ye Wang
- Qingdao Central Hospital, University of Health and Rehabilitation Sciences (Qingdao Central Medical Group), Qingdao, Shandong 266042, China.
| |
Collapse
|
|
3
|
Long Q, Huang C, Zhang L, Jiang H, Zhao S, Zhang L, Zheng X, Ou S, Gu H. A novel tissue-engineered corneal epithelium based on ultra-thin amniotic membrane and mesenchymal stem cells. Sci Rep 2024; 14:17407. [PMID: 39075142 PMCID: PMC11286932 DOI: 10.1038/s41598-024-68219-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2024] [Accepted: 07/22/2024] [Indexed: 07/31/2024] Open
Abstract
Currently, in vitro cultured corneal epithelial transplantation is effective in treating limbal stem cell dysfunction (LSCD). Selecting carriers is crucial for constructing the corneal epithelium through tissue engineering. In this study, the traditional amniotic membrane (AM) was modified, and mesenchymal stem cells (MSCs) were inoculated into the ultra-thin amniotic membrane (UAM) stroma to construct a novel UAM-MSC tissue-engineered corneal epithelial carrier, that could effectively simulate the limbal stem cells (LSCs) microenvironment. The structure of different carriers cultured tissue-engineered corneal epithelium and the managed rabbit LSCD model corneas were observed through hematoxylin-eosin staining. Cell phenotypes were evaluated through fluorescence staining, Western blotting, and RT-qPCR. Additionally, cell junction genes and expression markers related to anti-neovascularization were evaluated using RT-qPCR. Corneal epithelium cell junctions were observed via an electron microscope. The tissue-engineered corneal epithelium culture medium was analyzed through mass spectrometry. Tissue-engineered corneal epithelial cells expanded by LSCs on UAM-MSCs had good transparency. Simultaneously, progenitor cell (K14, PNCA, p63) and corneal epithelial (PAX6) gene expression in tissue-engineered corneal epithelium constructed using UAM-MSCs was higher than that in corneal epithelial cells amplified by UAM and de-epithelialized amniotic membrane. Electron microscopy revealed that corneal epithelial cells grafted with UAM-MSCs were closely connected. In conclusion, the UAM-MSCs vector we constructed could better simulate the limbal microenvironment; the cultured tissue-engineered corneal epithelium had better transparency, anti-neovascularization properties, closer intercellular connections, and closer resemblance to the natural corneal epithelial tissue phenotype.
Collapse
Affiliation(s)
- Qiurong Long
- Guizhou Medical University, Guiyang, Guizhou, China
- The Affiliated Hospital of Guizhou Medical University, No.9 Beijing Road, Yunyan District, Guiyang, Guizhou, China
| | - Chao Huang
- Guizhou Medical University, Guiyang, Guizhou, China
| | - Liying Zhang
- Guizhou Medical University, Guiyang, Guizhou, China
- The Affiliated Hospital of Guizhou Medical University, No.9 Beijing Road, Yunyan District, Guiyang, Guizhou, China
| | - Hao Jiang
- Guizhou Medical University, Guiyang, Guizhou, China
- The Affiliated Hospital of Guizhou Medical University, No.9 Beijing Road, Yunyan District, Guiyang, Guizhou, China
| | - Su Zhao
- Guizhou Medical University, Guiyang, Guizhou, China
- The Affiliated Hospital of Guizhou Medical University, No.9 Beijing Road, Yunyan District, Guiyang, Guizhou, China
| | - Lingli Zhang
- Guizhou Medical University, Guiyang, Guizhou, China
| | - Xueer Zheng
- Guizhou Medical University, Guiyang, Guizhou, China
| | - Shangkun Ou
- Guizhou Medical University, Guiyang, Guizhou, China.
- The Affiliated Hospital of Guizhou Medical University, No.9 Beijing Road, Yunyan District, Guiyang, Guizhou, China.
| | - Hao Gu
- Guizhou Medical University, Guiyang, Guizhou, China.
- The Affiliated Hospital of Guizhou Medical University, No.9 Beijing Road, Yunyan District, Guiyang, Guizhou, China.
| |
Collapse
|
|
4
|
Swarup A, Phansalkar R, Morri M, Agarwal A, Subramaniam V, Li B, Wu AY. Single-cell transcriptomic analysis of corneal organoids during development. Stem Cell Reports 2023; 18:2482-2497. [PMID: 38039970 PMCID: PMC10724212 DOI: 10.1016/j.stemcr.2023.10.022] [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: 04/21/2023] [Revised: 10/27/2023] [Accepted: 10/30/2023] [Indexed: 12/03/2023] Open
Abstract
Corneal organoids are useful tools for disease modeling and tissue transplantation; however, they have not yet been well studied during maturation. We characterized human iPSC-derived corneal organoids at 1, 2, 3, and 4 months of development using single-cell RNA sequencing to determine the cellular heterogeneity at each stage. We found pluripotent cell clusters committed to epithelial cell lineage at 1 month; early corneal epithelial, endothelial, and stromal cell markers at 2 months; keratocytes as the largest cell population at 3 months; and a large epithelial cell population at 4 months. We compared organoid to fetal corneal development at different stages and found that 4-month organoids closely resemble the corneal cellular complexity of the fetal (16 post conception week) and adult cornea. Using RNA velocity trajectory analysis, we found that less differentiated cells appear to give rise to corneal epithelial cells during development.
Collapse
Affiliation(s)
- Aditi Swarup
- Department of Ophthalmology, Stanford University School of Medicine, Stanford, CA, USA
| | - Ragini Phansalkar
- Department of Ophthalmology, Stanford University School of Medicine, Stanford, CA, USA
| | - Maurizio Morri
- Chan Zuckerberg Biohub, Stanford University, San Francisco, CA, USA
| | - Aditi Agarwal
- Chan Zuckerberg Biohub, Stanford University, San Francisco, CA, USA
| | - Varun Subramaniam
- Department of Ophthalmology, Stanford University School of Medicine, Stanford, CA, USA
| | - BaoXiang Li
- Department of Ophthalmology, Stanford University School of Medicine, Stanford, CA, USA
| | - Albert Y Wu
- Department of Ophthalmology, Stanford University School of Medicine, Stanford, CA, USA; Institute of Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA.
| |
Collapse
|
|
5
|
Clahsen T, Hadrian K, Notara M, Schlereth SL, Howaldt A, Prokosch V, Volatier T, Hos D, Schroedl F, Kaser-Eichberger A, Heindl LM, Steven P, Bosch JJ, Steinkasserer A, Rokohl AC, Liu H, Mestanoglu M, Kashkar H, Schumacher B, Kiefer F, Schulte-Merker S, Matthaei M, Hou Y, Fassbender S, Jantsch J, Zhang W, Enders P, Bachmann B, Bock F, Cursiefen C. The novel role of lymphatic vessels in the pathogenesis of ocular diseases. Prog Retin Eye Res 2023; 96:101157. [PMID: 36759312 DOI: 10.1016/j.preteyeres.2022.101157] [Citation(s) in RCA: 20] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2022] [Revised: 12/13/2022] [Accepted: 12/17/2022] [Indexed: 02/10/2023]
Abstract
Historically, the eye has been considered as an organ free of lymphatic vessels. In recent years, however, it became evident, that lymphatic vessels or lymphatic-like vessels contribute to several ocular pathologies at various peri- and intraocular locations. The aim of this review is to outline the pathogenetic role of ocular lymphatics, the respective molecular mechanisms and to discuss current and future therapeutic options based thereon. We will give an overview on the vascular anatomy of the healthy ocular surface and the molecular mechanisms contributing to corneal (lymph)angiogenic privilege. In addition, we present (i) current insights into the cellular and molecular mechanisms occurring during pathological neovascularization of the cornea triggered e.g. by inflammation or trauma, (ii) the role of lymphatic vessels in different ocular surface pathologies such as dry eye disease, corneal graft rejection, ocular graft versus host disease, allergy, and pterygium, (iii) the involvement of lymphatic vessels in ocular tumors and metastasis, and (iv) the novel role of the lymphatic-like structure of Schlemm's canal in glaucoma. Identification of the underlying molecular mechanisms and of novel modulators of lymphangiogenesis will contribute to the development of new therapeutic targets for the treatment of ocular diseases associated with pathological lymphangiogenesis in the future. The preclinical data presented here outline novel therapeutic concepts for promoting transplant survival, inhibiting metastasis of ocular tumors, reducing inflammation of the ocular surface, and treating glaucoma. Initial data from clinical trials suggest first success of novel treatment strategies to promote transplant survival based on pretransplant corneal lymphangioregression.
Collapse
Affiliation(s)
- Thomas Clahsen
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany; Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany
| | - Karina Hadrian
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany; Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany
| | - Maria Notara
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany; Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany
| | - Simona L Schlereth
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany; Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany
| | - Antonia Howaldt
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
| | - Verena Prokosch
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
| | - Thomas Volatier
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
| | - Deniz Hos
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany; Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany
| | - Falk Schroedl
- Center for Anatomy and Cell Biology, Institute of Anatomy and Cell Biology - Salzburg, Paracelsus Medical University, Salzburg, Austria
| | - Alexandra Kaser-Eichberger
- Center for Anatomy and Cell Biology, Institute of Anatomy and Cell Biology - Salzburg, Paracelsus Medical University, Salzburg, Austria
| | - Ludwig M Heindl
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
| | - Philipp Steven
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany; Cluster of Excellence: Cellular Stress Responses in Ageing-Associated Diseases, CECAD, University of Cologne, Cologne, Germany
| | - Jacobus J Bosch
- Centre for Human Drug Research and Leiden University Medical Center, Leiden, the Netherlands
| | | | - Alexander C Rokohl
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
| | - Hanhan Liu
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
| | - Mert Mestanoglu
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
| | - Hamid Kashkar
- Institute for Molecular Immunology, Center for Molecular Medicine Cologne (CMMC), CECAD Research Center, Faculty of Medicine and University Hospital of Cologne, University of Cologne, Cologne, Germany
| | - Björn Schumacher
- Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany; Cluster of Excellence: Cellular Stress Responses in Ageing-Associated Diseases, CECAD, University of Cologne, Cologne, Germany
| | - Friedemann Kiefer
- European Institute for Molecular Imaging (EIMI), University of Münster, 48149, Münster, Germany
| | - Stefan Schulte-Merker
- Institute for Cardiovascular Organogenesis and Regeneration, Faculty of Medicine, WWU Münster, Münster, Germany
| | - Mario Matthaei
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
| | - Yanhong Hou
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany; Eye Institute and Department of Ophthalmology, Eye & ENT Hospital, Fudan University, 83 Fenyang Road, Xuhui District, Shanghai, China
| | - Sonja Fassbender
- IUF‒Leibniz Research Institute for Environmental Medicine, Duesseldorf, Germany; Immunology and Environment, Life & Medical Sciences (LIMES) Institute, University of Bonn, Bonn, Germany
| | - Jonathan Jantsch
- Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
| | - Wei Zhang
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
| | - Philip Enders
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
| | - Björn Bachmann
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
| | - Felix Bock
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany; Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany
| | - Claus Cursiefen
- Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany; Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany; Cluster of Excellence: Cellular Stress Responses in Ageing-Associated Diseases, CECAD, University of Cologne, Cologne, Germany.
| |
Collapse
|
|
6
|
Swamynathan SK, Swamynathan S. Corneal epithelial development and homeostasis. Differentiation 2023; 132:4-14. [PMID: 36870804 PMCID: PMC10363238 DOI: 10.1016/j.diff.2023.02.002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2022] [Revised: 01/27/2023] [Accepted: 02/20/2023] [Indexed: 03/06/2023]
Abstract
The corneal epithelium (CE), the most anterior cellular structure of the eye, is a self-renewing stratified squamous tissue that protects the rest of the eye from external elements. Each cell in this exquisite three-dimensional structure needs to have proper polarity and positional awareness for the CE to serve as a transparent, refractive, and protective tissue. Recent studies have begun to elucidate the molecular and cellular events involved in the embryonic development, post-natal maturation, and homeostasis of the CE, and how they are regulated by a well-coordinated network of transcription factors. This review summarizes the status of related knowledge and aims to provide insight into the pathophysiology of disorders caused by disruption of CE development, and/or homeostasis.
Collapse
Affiliation(s)
| | - Sudha Swamynathan
- Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15213, USA
| |
Collapse
|
|
7
|
Bisevac J, Katta K, Petrovski G, Moe MC, Noer A. Wnt/β-Catenin Signaling Activation Induces Differentiation in Human Limbal Epithelial Stem Cells Cultured Ex Vivo. Biomedicines 2023; 11:1829. [PMID: 37509479 PMCID: PMC10377110 DOI: 10.3390/biomedicines11071829] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2023] [Revised: 06/05/2023] [Accepted: 06/13/2023] [Indexed: 07/30/2023] Open
Abstract
Human limbal epithelial stem cells (hLESCs) continuously replenish lost or damaged human corneal epithelial cells. The percentage of stem/progenitor cells in autologous ex vivo expanded tissue is essential for the long-term success of transplantation in patients with limbal epithelial stem cell deficiency. However, the molecular processes governing the stemness and differentiation state of hLESCs remain uncertain. Therefore, we sought to explore the impact of canonical Wnt/β-catenin signaling activation on hLESCs by treating ex vivo expanded hLESC cultures with GSK-3 inhibitor LY2090314. Real-time qRT-PCR and microarray data reveal the downregulation of stemness (TP63), progenitor (SOX9), quiescence (CEBPD), and proliferation (MKI67, PCNA) genes and the upregulation of genes for differentiation (CX43, KRT3) in treated- compared to non-treated samples. The pathway activation was shown by AXIN2 upregulation and enhanced levels of accumulated β-catenin. Immunocytochemistry and Western blot confirmed the findings for most of the above-mentioned markers. The Wnt/β-catenin signaling profile demonstrated an upregulation of WNT1, WNT3, WNT5A, WNT6, and WNT11 gene expression and a downregulation for WNT7A and DKK1 in the treated samples. No significant differences were found for WNT2, WNT16B, WIF1, and DKK2 gene expression. Overall, our results demonstrate that activation of Wnt/β-catenin signaling in ex vivo expanded hLESCs governs the cells towards differentiation and reduces proliferation and stem cell maintenance capability.
Collapse
Affiliation(s)
- Jovana Bisevac
- Center for Eye Research and Innovative Diagnostics, Department of Ophthalmology, Oslo University Hospital, P.O. Box 4956 Nydalen, 0424 Oslo, Norway
- Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, 0318 Oslo, Norway
| | - Kirankumar Katta
- Department of Immunology, Oslo University Hospital, Hf Rikshospitalet, 0424 Oslo, Norway
| | - Goran Petrovski
- Center for Eye Research and Innovative Diagnostics, Department of Ophthalmology, Oslo University Hospital, P.O. Box 4956 Nydalen, 0424 Oslo, Norway
- Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, 0318 Oslo, Norway
| | - Morten Carstens Moe
- Center for Eye Research and Innovative Diagnostics, Department of Ophthalmology, Oslo University Hospital, P.O. Box 4956 Nydalen, 0424 Oslo, Norway
- Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, 0318 Oslo, Norway
| | - Agate Noer
- Center for Eye Research and Innovative Diagnostics, Department of Ophthalmology, Oslo University Hospital, P.O. Box 4956 Nydalen, 0424 Oslo, Norway
| |
Collapse
|
|
8
|
Polisetti N, Martin G, Cristina Schmitz HR, Schlötzer-Schrehardt U, Schlunck G, Reinhard T. Characterization of Porcine Ocular Surface Epithelial Microenvironment. Int J Mol Sci 2023; 24:ijms24087543. [PMID: 37108705 PMCID: PMC10145510 DOI: 10.3390/ijms24087543] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2023] [Revised: 04/13/2023] [Accepted: 04/14/2023] [Indexed: 04/29/2023] Open
Abstract
The porcine ocular surface is used as a model of the human ocular surface; however, a detailed characterization of the porcine ocular surface has not been documented. This is due, in part, to the scarcity of antibodies produced specifically against the porcine ocular surface cell types or structures. We performed a histological and immunohistochemical investigation on frozen and formalin-fixed, paraffin-embedded ocular surface tissue from domestic pigs using a panel of 41 different antibodies related to epithelial progenitor/differentiation phenotypes, extracellular matrix and associated molecules, and various niche cell types. Our observations suggested that the Bowman's layer is not evident in the cornea; the deep invaginations of the limbal epithelium in the limbal zone are analogous to the limbal interpalisade crypts of human limbal tissue; and the presence of goblet cells in the bulbar conjunctiva. Immunohistochemistry analysis revealed that the epithelial progenitor markers cytokeratin (CK)15, CK14, p63α, and P-cadherin were expressed in both the limbal and conjunctival basal epithelium, whereas the basal cells of the limbal and conjunctival epithelium did not stain for CK3, CK12, E-cadherin, and CK13. Antibodies detecting marker proteins related to the extracellular matrix (collagen IV, Tenascin-C), cell-matrix adhesion (β-dystroglycan, integrin α3 and α6), mesenchymal cells (vimentin, CD90, CD44), neurons (neurofilament), immune cells (HLA-ABC; HLA-DR, CD1, CD4, CD14), vasculature (von Willebrand factor), and melanocytes (SRY-homeobox-10, human melanoma black-45, Tyrosinase) on the normal human ocular surface demonstrated similar immunoreactivity on the normal porcine ocular surface. Only a few antibodies (directed against N-cadherin, fibronectin, agrin, laminin α3 and α5, melan-A) appeared unreactive on porcine tissues. Our findings characterize the main immunohistochemical properties of the porcine ocular surface and provide a morphological and immunohistochemical basis useful to research using porcine models. Furthermore, the analyzed porcine ocular structures are similar to those of humans, confirming the potential usefulness of pig eyes to study ocular surface physiology and pathophysiology.
Collapse
Affiliation(s)
- Naresh Polisetti
- Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106 Freiburg, Germany
| | - Gottfried Martin
- Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106 Freiburg, Germany
| | - Heidi R Cristina Schmitz
- CEMT-Freiburg, Experimental Surgery, Hospital-Medical Center, Faculty of Medicine, University of Freiburg, Breisacher Str. 66, 79106 Freiburg, Germany
| | - Ursula Schlötzer-Schrehardt
- Department of Ophthalmology, University Hospital Erlangen, Friedrich-Alexander-University of Erlangen-Nürnberg, Schwabachanlage 6, 91054 Erlangen, Germany
| | - Günther Schlunck
- Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106 Freiburg, Germany
| | - Thomas Reinhard
- Eye Center, Medical Center, Faculty of Medicine, University of Freiburg, Killianstrasse 5, 79106 Freiburg, Germany
| |
Collapse
|
|
9
|
Tavakkoli F, Eleiwa TK, Elhusseiny AM, Damala M, Rai AK, Cheraqpour K, Ansari MH, Doroudian M, H Keshel S, Soleimani M, Djalilian AR, Sangwan VS, Singh V. Corneal stem cells niche and homeostasis impacts in regenerative medicine; concise review. Eur J Ophthalmol 2023:11206721221150065. [PMID: 36604831 DOI: 10.1177/11206721221150065] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
The limbal stem cells niche (LSCN) is an optimal microenvironment that provides the limbal epithelial stem cells (LESCs) and strictly regulates their proliferation and differentiation. Disturbing the LSCN homeostasis can lead to limbal stem cell dysfunction (LSCD) and subsequent ocular surface aberrations, such as corneal stromal inflammation, persistent epithelial defects, corneal neovascularisation, lymphangiogenesis, corneal opacification, and conjunctivalization. As ocular surface disorders are considered the second main cause of blindness, it becomes crucial to explore different therapeutic strategies for restoring the functions of the LSCN. A major limitation of corneal transplantation is the current shortage of donor tissue to meet the requirements worldwide. In this context, it becomes mandatory to find an alternative regenerative medicine, such as using cultured limbal epithelial/stromal stem cells, inducing the production of corneal like cells by using other sources of stem cells, and using tissue engineering methods aiming to produce the three-dimensional (3D) printed cornea. Limbal epithelial stem cells have been considered the magic potion for eye treatment. Epithelial and stromal stem cells in the limbal niche hold the responsibility of replenishing the corneal epithelium. These stem cells are being used for transplantation to maintain corneal epithelial integrity and ultimately sustain optimal vision. In this review, we summarised the characteristics of the LSCN and their current and future roles in restoring corneal homeostasis in eyes with LSCD.
Collapse
Affiliation(s)
- Fatemeh Tavakkoli
- Department of Community Health, College of Health Technology, Cihan University, Erbil, Iraq.,SSR Stem Cell Biology Laboratory, Brien Holden Eye Research Centre, Centre for Ocular Regeneration, Hyderabad Eye Research Foundation, L.V. Prasad Eye Institute, Hyderabad, India.,Centre for Genetic Disorders, Banaras Hindu University, Varanasi, India
| | - Taher K Eleiwa
- Department of Ophthalmology, Benha University, Benha, Egypt
| | - Abdelrahman M Elhusseiny
- Department of Ophthalmology, Harvey and Bernice Jones Eye Institute, University of Arkansas for Medical Sciences, Little Rock, AR, USA
| | - Mukesh Damala
- SSR Stem Cell Biology Laboratory, Brien Holden Eye Research Centre, Centre for Ocular Regeneration, Hyderabad Eye Research Foundation, L.V. Prasad Eye Institute, Hyderabad, India.,School of Life Sciences, University of Hyderabad, Hyderabad, India
| | - Amit K Rai
- Centre for Genetic Disorders, Banaras Hindu University, Varanasi, India
| | - Kasra Cheraqpour
- Translational Eye Research Center, Farabi Eye Hospital, 48439Tehran University of Medical Sciences, Tehran, Iran
| | - Mohammad H Ansari
- Ophthalmic Research Center, Department of Ophthalmology, Labbafinejad Medical Center, 556492Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Mohammad Doroudian
- Department of Cell and Molecular Biology, Faculty of Biological Sciences, 145440Kharazmi University, Tehran, Iran
| | - Saeed H Keshel
- Department of Tissue Engineering and Applied Cell Sciences, 556492Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Mohammad Soleimani
- Department of Ophthalmology, 159636Illinois Eye and Ear Infirmary, University of Illinois at Chicago, Chicago, IL, USA
| | - Ali R Djalilian
- Department of Ophthalmology, 159636Illinois Eye and Ear Infirmary, University of Illinois at Chicago, Chicago, IL, USA
| | | | - Vivek Singh
- SSR Stem Cell Biology Laboratory, Brien Holden Eye Research Centre, Centre for Ocular Regeneration, Hyderabad Eye Research Foundation, L.V. Prasad Eye Institute, Hyderabad, India
| |
Collapse
|
|
10
|
Transcriptomic Landscape and Functional Characterization of Human Induced Pluripotent Stem Cell-Derived Limbal Epithelial Progenitor Cells. Cells 2022; 11:cells11233752. [PMID: 36497012 PMCID: PMC9737332 DOI: 10.3390/cells11233752] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2022] [Revised: 11/04/2022] [Accepted: 11/18/2022] [Indexed: 11/25/2022] Open
Abstract
Limbal stem cell deficiency (LSCD) is a complex, multifactorial disease affecting limbal epithelial progenitor cells (LEPC), which are essential for maintaining corneal stability and transparency. Human induced pluripotent stem cell-derived (hiPSC-) LEPC are a promising cell source for the treatment of LSCD. However, their similarity to native tissue-derived (T-) LEPC and their functional characterization has not been studied in detail. Here, we show that hiPSC-LEPC and T-LEPC have rather similar gene expression patterns, colony-forming ability, wound-healing capacity, and melanosome uptake. In addition, hiPSC-LEPC exhibited lower immunogenicity and reduced the proliferation of peripheral blood mononuclear cells compared with T-LEPC. Similarly, the hiPSC-LEPC secretome reduced the proliferation of vascular endothelial cells more than the T-LEPC secretome. Moreover, hiPSC-LEPC successfully repopulated decellularized human corneolimbal (DHC/L) scaffolds with multilayered epithelium, while basal deposition of fibrillary material was observed. These findings suggest that hiPSC-LEPC exhibited functional properties close to native LEPC and that hiPSC-LEPC-DHC/L scaffolds might be feasible for transplantation in patients suffering from LSCD in the future. Although hiPSC-LEPC-based stem cell therapy is promising, the current study also revealed new challenges, such as abnormal extracellular matrix deposition, that need to be overcome before hiPSC-LEPC-based stem cell therapies are viable.
Collapse
|
|
11
|
Ramos T, Parekh M, Meleady P, O’Sullivan F, Stewart RMK, Kaye SB, Hamill K, Ahmad S. Specific decellularized extracellular matrix promotes the plasticity of human ocular surface epithelial cells. Front Med (Lausanne) 2022; 9:974212. [PMID: 36457571 PMCID: PMC9705355 DOI: 10.3389/fmed.2022.974212] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2022] [Accepted: 10/21/2022] [Indexed: 11/25/2023] Open
Abstract
The ocular surface is composed of two phenotypically and functionally different epithelial cell types: corneal and the conjunctival epithelium. Upon injury or disease, ocular surface homeostasis is impaired resulting in migration of conjunctival epithelium on to the corneal surface. This can lead to incomplete transdifferentiation toward corneal epithelial-like cells in response to corneal basement membrane cues. We show that corneal extracellular matrix (ECM) proteins induce conjunctival epithelial cells to express corneal associated markers losing their conjunctival associated phenotype at both, mRNA and protein level. Corneal epithelial cells behave the same in the presence of conjunctival ECM proteins, expressing markers associated with conjunctival epithelium. This process of differentiation is accompanied by an intermediate step of cell de-differentiation as an up-regulation in the expression of epithelial stem cell markers is observed. In addition, analysis of ECM proteins by laminin screening assays showed that epithelial cell response is laminin-type dependent, and cells cultured on laminin-511 showed lower levels of lineage commitment. The phosphorylation and proteolysis levels of proteins mainly involved in cell growth and differentiation showed lower modifications in cells with lower lineage commitment. These observations showed that the ECM proteins may serve as tools to induce cell differentiation, which may have potential applications for the treatment of ocular surface injuries.
Collapse
Affiliation(s)
- Tiago Ramos
- Department of Eye and Vision Science, University of Liverpool, Liverpool, United Kingdom
- Faculty of Brain Sciences, Institute of Ophthalmology, University College London, London, United Kingdom
| | - Mohit Parekh
- Faculty of Brain Sciences, Institute of Ophthalmology, University College London, London, United Kingdom
| | - Paula Meleady
- Primary Department, National Institute for Cellular Biotechnology, Dublin City University, Dublin, Ireland
| | - Finbarr O’Sullivan
- Primary Department, National Institute for Cellular Biotechnology, Dublin City University, Dublin, Ireland
| | - Rosalind M. K. Stewart
- Department of Eye and Vision Science, University of Liverpool, Liverpool, United Kingdom
- St Paul’s Eye Unit, Royal Liverpool University Hospital, Liverpool, United Kingdom
- Department of Ophthalmology, Aberdeen Royal Infirmary, Aberdeen, United Kingdom
| | - Stephen B. Kaye
- Department of Eye and Vision Science, University of Liverpool, Liverpool, United Kingdom
- St Paul’s Eye Unit, Royal Liverpool University Hospital, Liverpool, United Kingdom
| | - Kevin Hamill
- Department of Eye and Vision Science, University of Liverpool, Liverpool, United Kingdom
| | - Sajjad Ahmad
- Department of Eye and Vision Science, University of Liverpool, Liverpool, United Kingdom
- Faculty of Brain Sciences, Institute of Ophthalmology, University College London, London, United Kingdom
- St Paul’s Eye Unit, Royal Liverpool University Hospital, Liverpool, United Kingdom
- External Eye Disease Service, Moorfields Eye Hospital, London, United Kingdom
| |
Collapse
|
|
12
|
Di Girolamo N, Park M. Cell identity changes in ocular surface Epithelia. Prog Retin Eye Res 2022:101148. [DOI: 10.1016/j.preteyeres.2022.101148] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2022] [Revised: 10/13/2022] [Accepted: 11/09/2022] [Indexed: 11/21/2022]
|
|
13
|
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).
Collapse
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
| |
Collapse
|
|
14
|
Robertson SYT, Roberts JS, Deng SX. Regulation of Limbal Epithelial Stem Cells: Importance of the Niche. Int J Mol Sci 2021; 22:11975. [PMID: 34769405 PMCID: PMC8584795 DOI: 10.3390/ijms222111975] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2021] [Revised: 10/29/2021] [Accepted: 11/03/2021] [Indexed: 12/11/2022] Open
Abstract
Limbal epithelial stem/progenitor cells (LSCs) reside in a niche that contains finely tuned balances of various signaling pathways including Wnt, Notch, BMP, Shh, YAP, and TGFβ. The activation or inhibition of these pathways is frequently dependent on the interactions of LSCs with various niche cell types and extracellular substrates. In addition to receiving molecular signals from growth factors, cytokines, and other soluble molecules, LSCs also respond to their surrounding physical structure via mechanotransduction, interaction with the ECM, and interactions with other cell types. Damage to LSCs or their niche leads to limbal stem cell deficiency (LSCD). The field of LSCD treatment would greatly benefit from an understanding of the molecular regulation of LSCs in vitro and in vivo. This review synthesizes current literature around the niche factors and signaling pathways that influence LSC function. Future development of LSCD therapies should consider all these niche factors to achieve improved long-term restoration of the LSC population.
Collapse
Affiliation(s)
| | | | - Sophie X. Deng
- Jules Stein Eye Institute, University of California, Los Angeles, CA 94143, USA; (S.Y.T.R.); (J.S.R.)
| |
Collapse
|
|
15
|
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.
Collapse
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.
| |
Collapse
|
|
16
|
Amin S, Jalilian E, Katz E, Frank C, Yazdanpanah G, Guaiquil VH, Rosenblatt MI, Djalilian AR. The Limbal Niche and Regenerative Strategies. Vision (Basel) 2021; 5:vision5040043. [PMID: 34698278 PMCID: PMC8544688 DOI: 10.3390/vision5040043] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2021] [Revised: 08/27/2021] [Accepted: 09/16/2021] [Indexed: 12/17/2022] Open
Abstract
The protective function and transparency provided by the corneal epithelium are dependent on and maintained by the regenerative capacity of limbal epithelial stem cells (LESCs). These LESCs are supported by the limbal niche, a specialized microenvironment consisting of cellular and non-cellular components. Disruption of the limbal niche, primarily from injuries or inflammatory processes, can negatively impact the regenerative ability of LESCs. Limbal stem cell deficiency (LSCD) directly hampers the regenerative ability of the corneal epithelium and allows the conjunctival epithelium to invade the cornea, which results in severe visual impairment. Treatment involves restoring the LESC population and functionality; however, few clinically practiced therapies currently exist. This review outlines the current understanding of the limbal niche, its pathology and the emerging approaches targeted at restoring the limbal niche. Most emerging approaches are in developmental phases but show promise for treating LSCD and accelerating corneal regeneration. Specifically, we examine cell-based therapies, bio-active extracellular matrices and soluble factor therapies in considerable depth.
Collapse
Affiliation(s)
- Sohil Amin
- Department of Ophthalmology and Visual Sciences, University of Illinois Chicago, Chicago, IL 60612, USA; (S.A.); (E.J.); (E.K.); (C.F.); (G.Y.); (V.H.G.); (M.I.R.)
| | - Elmira Jalilian
- Department of Ophthalmology and Visual Sciences, University of Illinois Chicago, Chicago, IL 60612, USA; (S.A.); (E.J.); (E.K.); (C.F.); (G.Y.); (V.H.G.); (M.I.R.)
| | - Eitan Katz
- Department of Ophthalmology and Visual Sciences, University of Illinois Chicago, Chicago, IL 60612, USA; (S.A.); (E.J.); (E.K.); (C.F.); (G.Y.); (V.H.G.); (M.I.R.)
| | - Charlie Frank
- Department of Ophthalmology and Visual Sciences, University of Illinois Chicago, Chicago, IL 60612, USA; (S.A.); (E.J.); (E.K.); (C.F.); (G.Y.); (V.H.G.); (M.I.R.)
| | - Ghasem Yazdanpanah
- Department of Ophthalmology and Visual Sciences, University of Illinois Chicago, Chicago, IL 60612, USA; (S.A.); (E.J.); (E.K.); (C.F.); (G.Y.); (V.H.G.); (M.I.R.)
- Richard and Loan Hill Department of Bioengineering, University of Illinois Chicago, Chicago, IL 60612, USA
| | - Victor H. Guaiquil
- Department of Ophthalmology and Visual Sciences, University of Illinois Chicago, Chicago, IL 60612, USA; (S.A.); (E.J.); (E.K.); (C.F.); (G.Y.); (V.H.G.); (M.I.R.)
| | - Mark I. Rosenblatt
- Department of Ophthalmology and Visual Sciences, University of Illinois Chicago, Chicago, IL 60612, USA; (S.A.); (E.J.); (E.K.); (C.F.); (G.Y.); (V.H.G.); (M.I.R.)
| | - Ali R. Djalilian
- Department of Ophthalmology and Visual Sciences, University of Illinois Chicago, Chicago, IL 60612, USA; (S.A.); (E.J.); (E.K.); (C.F.); (G.Y.); (V.H.G.); (M.I.R.)
- Correspondence:
| |
Collapse
|
|
17
|
Ligocki AJ, Fury W, Gutierrez C, Adler C, Yang T, Ni M, Bai Y, Wei Y, Lehmann GL, Romano C. Molecular characteristics and spatial distribution of adult human corneal cell subtypes. Sci Rep 2021; 11:16323. [PMID: 34381080 PMCID: PMC8357950 DOI: 10.1038/s41598-021-94933-8] [Citation(s) in RCA: 31] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2020] [Accepted: 07/19/2021] [Indexed: 12/13/2022] Open
Abstract
Bulk RNA sequencing of a tissue captures the gene expression profile from all cell types combined. Single-cell RNA sequencing identifies discrete cell-signatures based on transcriptomic identities. Six adult human corneas were processed for single-cell RNAseq and 16 cell clusters were bioinformatically identified. Based on their transcriptomic signatures and RNAscope results using representative cluster marker genes on human cornea cross-sections, these clusters were confirmed to be stromal keratocytes, endothelium, several subtypes of corneal epithelium, conjunctival epithelium, and supportive cells in the limbal stem cell niche. The complexity of the epithelial cell layer was captured by eight distinct corneal clusters and three conjunctival clusters. These were further characterized by enriched biological pathways and molecular characteristics which revealed novel groupings related to development, function, and location within the epithelial layer. Moreover, epithelial subtypes were found to reflect their initial generation in the limbal region, differentiation, and migration through to mature epithelial cells. The single-cell map of the human cornea deepens the knowledge of the cellular subsets of the cornea on a whole genome transcriptional level. This information can be applied to better understand normal corneal biology, serve as a reference to understand corneal disease pathology, and provide potential insights into therapeutic approaches.
Collapse
Affiliation(s)
- Ann J Ligocki
- Regeneron Pharmaceuticals, Inc., Tarrytown, NY, 10591, USA
| | - Wen Fury
- Regeneron Pharmaceuticals, Inc., Tarrytown, NY, 10591, USA
| | | | | | - Tao Yang
- Regeneron Pharmaceuticals, Inc., Tarrytown, NY, 10591, USA
| | - Min Ni
- Regeneron Pharmaceuticals, Inc., Tarrytown, NY, 10591, USA
| | - Yu Bai
- Regeneron Pharmaceuticals, Inc., Tarrytown, NY, 10591, USA
| | - Yi Wei
- Regeneron Pharmaceuticals, Inc., Tarrytown, NY, 10591, USA
| | | | - Carmelo Romano
- Regeneron Pharmaceuticals, Inc., Tarrytown, NY, 10591, USA.
| |
Collapse
|
|
18
|
Abdul-Al M, Kyeremeh GK, Saeinasab M, Heidari Keshel S, Sefat F. Stem Cell Niche Microenvironment: Review. Bioengineering (Basel) 2021; 8:bioengineering8080108. [PMID: 34436111 PMCID: PMC8389324 DOI: 10.3390/bioengineering8080108] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2021] [Revised: 07/14/2021] [Accepted: 07/16/2021] [Indexed: 12/13/2022] Open
Abstract
The cornea comprises a pool of self-regenerating epithelial cells that are crucial to preserving clarity and visibility. Limbal epithelial stem cells (LESCs), which live in a specialized stem cell niche (SCN), are crucial for the survival of the human corneal epithelium. They live at the bottom of the limbal crypts, in a physically enclosed microenvironment with a number of neighboring niche cells. Scientists also simplified features of these diverse microenvironments for more analysis in situ by designing and recreating features of different SCNs. Recent methods for regenerating the corneal epithelium after serious trauma, including burns and allergic assaults, focus mainly on regenerating the LESCs. Mesenchymal stem cells, which can transform into self-renewing and skeletal tissues, hold immense interest for tissue engineering and innovative medicinal exploration. This review summarizes all types of LESCs, identity and location of the human epithelial stem cells (HESCs), reconstruction of LSCN and artificial stem cells for self-renewal.
Collapse
Affiliation(s)
- Mohamed Abdul-Al
- Department of Biomedical and Electronics Engineering, School of Engineering, University of Bradford, Bradford BD71DP, UK; (M.A.-A.); (G.K.K.)
| | - George Kumi Kyeremeh
- Department of Biomedical and Electronics Engineering, School of Engineering, University of Bradford, Bradford BD71DP, UK; (M.A.-A.); (G.K.K.)
| | - Morvarid Saeinasab
- Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad 91779 48974, Iran;
| | - Saeed Heidari Keshel
- Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran 19839 69411, Iran;
| | - Farshid Sefat
- Department of Biomedical and Electronics Engineering, School of Engineering, University of Bradford, Bradford BD71DP, UK; (M.A.-A.); (G.K.K.)
- Interdisciplinary Research Centre in Polymer Science & Technology (Polymer IRC), University of Bradford, Bradford BD71DP, UK
- Correspondence:
| |
Collapse
|
|
19
|
Chen SY, Zhu Y, Zhang Y, Hsu D, Tseng SCG. HC-HA/PTX3 from amniotic membrane reverts senescent limbal niche cells to Pax6+ neural crest progenitors to support limbal epithelial progenitors. Stem Cells 2021; 39:280-295. [PMID: 33373496 PMCID: PMC7986837 DOI: 10.1002/stem.3323] [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: 04/08/2020] [Accepted: 12/07/2020] [Indexed: 12/11/2022]
Abstract
Quiescence and self‐renewal of human corneal epithelial progenitor/stem cells (LEPC) are regulated by the limbal niche, presumably through close interaction with limbal (stromal) niche cells (LNC). Paired box homeotic gene 6 (Pax6), a conserved transcription factor essential for eye development, is essential for proper differentiation of limbal and corneal epithelial stem cells. Pax6 haploinsufficiency causes limbal stem cell deficiency, which leads to subsequent corneal blindness. We previously reported that serial passage of nuclear Pax6+ LNC resulted in the gradual loss of nuclear Pax6+ and neural crest progenitor status, the latter of which was reverted upon recovery of Pax6. These findings suggest Pax6 plays a pivotal role in supporting the self‐renewal of LEPC in limbal niche. Herein, we show that HC‐HA/PTX3, a unique matrix purified from amniotic membrane (AM) and consists of heavy chain 1of inter‐α‐trypsin inhibitor covalently linked to hyaluronic acid and complexed with pentraxin 3, is capable of reverting senescent LNC to nuclear Pax6+ neural crest progenitors that support self‐renewal of LEPC. Such reversion is causally linked to early cell aggregation mediated by activation of C‐X‐C chemokine receptor type 4 (CXCR4)‐mediated signaling followed by activation of bone morphogenetic protein (BMP) signaling. Furthermore, CXCR4‐mediated signaling, but not BMP signaling, controls recovery of the nuclear Pax6+ neural crest progenitors. These findings not only explain why AM helps in vivo and ex vivo expansion of human LEPC, but they also illuminate the potential role of HC‐HA/PTX3 as a surrogate matrix niche that complements stem cell‐based therapies in regenerative medicine.
Collapse
Affiliation(s)
- Szu-Yu Chen
- R&D Department, Tissue Tech, Inc, Miami, Florida, USA
| | - Yingting Zhu
- R&D Department, Tissue Tech, Inc, Miami, Florida, USA
| | - Yuan Zhang
- R&D Department, Tissue Tech, Inc, Miami, Florida, USA
| | - David Hsu
- R&D Department, Tissue Tech, Inc, Miami, Florida, USA
| | | |
Collapse
|
|
20
|
González S, Halabi M, Ju D, Tsai M, Deng SX. Role of Jagged1-mediated Notch Signaling Activation in the Differentiation and Stratification of the Human Limbal Epithelium. Cells 2020; 9:cells9091945. [PMID: 32842657 PMCID: PMC7564045 DOI: 10.3390/cells9091945] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2020] [Revised: 08/12/2020] [Accepted: 08/21/2020] [Indexed: 12/22/2022] Open
Abstract
The Notch signaling pathway plays a key role in proliferation and differentiation. We investigated the effect of Jagged 1 (Jag1)-mediated Notch signaling activation in the human limbal stem/progenitor cell (LSC) population and the stratification of the limbal epithelium in vitro. After Notch signaling activation, there was a reduction in the amount of the stem/progenitor cell population, epithelial stratification, and expression of proliferation markers. There was also an increase of the corneal epithelial differentiation. In the presence of Jag1, asymmetric divisions were decreased, and the expression pattern of the polarity protein Par3, normally present at the apical-lateral membrane of basal cells, was dispersed in the cells. We propose a mechanism in which Notch activation by Jag1 decreases p63 expression at the basal layer, which in turn reduces stratification by decreasing the number of asymmetric divisions and increases differentiation.
Collapse
|
|
21
|
Tseng SCG, Chen SY, Mead OG, Tighe S. Niche regulation of limbal epithelial stem cells: HC-HA/PTX3 as surrogate matrix niche. Exp Eye Res 2020; 199:108181. [PMID: 32795525 DOI: 10.1016/j.exer.2020.108181] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2020] [Revised: 06/15/2020] [Accepted: 07/31/2020] [Indexed: 12/13/2022]
Abstract
Homeostasis of the corneal epithelium is ultimately maintained by stem cells that reside in a specialized microenvironment within the corneal limbus termed palisades of Vogt. This limbal niche nourishes, protects, and regulates quiescence, self-renewal, and fate decision of limbal epithelial stem/progenitor cells (LEPCs) toward corneal epithelial differentiation. This review focuses on our current understanding of the mechanism by which limbal (stromal) niche cells (LNCs) regulate the aforementioned functions of LEPCs. Based on our discovery and characterization of a unique extracellular matrix termed HC-HA/PTX3 (Heavy chain (HC1)-hyaluronan (HA)/pentraxin 3 (PTX3) complex, "-" denotes covalent linkage; "/" denotes non-covalent binding) in the birth tissue, i.e., amniotic membrane and umbilical cord, we put forth a new paradigm that HC-HA/PTX3 serves as a surrogate matrix niche by maintaining the in vivo nuclear Pax6+ neural crest progenitor phenotype to support quiescence and self-renewal but prevent corneal fate decision of LEPCs. This new paradigm helps explain how limbal stem cell deficiency (LSCD) develops in aniridia due to Pax6-haplotype deficiency and further explains why transplantation of HC-HA/PTX3-containing amniotic membrane prevents LSCD in acute chemical burns and Stevens Johnson syndrome, augments the success of autologous LEPCs transplantation in patients suffering from partial or total LSCD, and assists ex vivo expansion (engineering) of a graft containing LEPCs. We thus envisage that this new paradigm based on regenerative matrix HC-HA/PTX3 as a surrogate niche can set a new standard for regenerative medicine in and beyond ophthalmology.
Collapse
Affiliation(s)
- Scheffer C G Tseng
- Research & Development Department, TissueTech, Inc., Miami, FL, 33126, USA; Ocular Surface Center and Ocular Surface Research & Education Foundation, Miami, FL, 33126, USA.
| | - Szu-Yu Chen
- Research & Development Department, TissueTech, Inc., Miami, FL, 33126, USA
| | - Olivia G Mead
- Research & Development Department, TissueTech, Inc., Miami, FL, 33126, USA
| | - Sean Tighe
- Research & Development Department, TissueTech, Inc., Miami, FL, 33126, USA; Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, FL, 33136, USA; Department of Ophthalmology, Herbert Wertheim College of Medicine, Florida International University, Miami, FL, 33199, USA
| |
Collapse
|
|
22
|
Seyed-Safi AG, Daniels JT. The limbus: Structure and function. Exp Eye Res 2020; 197:108074. [PMID: 32502532 DOI: 10.1016/j.exer.2020.108074] [Citation(s) in RCA: 25] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2020] [Revised: 05/14/2020] [Accepted: 05/17/2020] [Indexed: 12/12/2022]
Abstract
Limbal function is a key determinant of corneal epithelial integrity. Lineage tracing studies in mice have highlighted that the centripetal movement of epithelial progenitors from the limbus drives both the steady-state maintenance of the corneal epithelium and its regeneration following injury. It is well established that this is facilitated by a population of limbal epithelial stem cells within the limbus. It is becoming increasingly apparent that the behaviour of these stem cells and their ability to respond to the needs of the tissue are closely linked to their immediate microenvironment - the stem cell niche. Increasing understanding of the structural features of this niche and the signalling networks that they coordinate is required to enhance the therapeutic application of these cells in the treatment of limbal stem cell deficiency. Importantly, an improved characterisation of the hierarchy of limbal epithelial progenitors using both new and old putative markers will enable a greater appreciation for the effects of many of these limbal niche factors on stem cell fate.
Collapse
|
|
23
|
Zhang C, Mei H, Robertson SYT, Lee HJ, Deng SX, Zheng JJ. A Small-Molecule Wnt Mimic Improves Human Limbal Stem Cell Ex Vivo Expansion. iScience 2020; 23:101075. [PMID: 32361505 PMCID: PMC7200314 DOI: 10.1016/j.isci.2020.101075] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2019] [Revised: 02/11/2020] [Accepted: 04/14/2020] [Indexed: 12/20/2022] Open
Abstract
Ex vivo cultured limbal stem/progenitor cells is an effective alternative to other surgical treatments for limbal stem cell deficiency, but a standard xenobiotic-free method for culturing the LSCs in vitro needs to be optimized. Because Wnt ligands are required for LSC expansion and preservation in vitro, to create a small-molecule Wnt mimic, we created a consolidated compound by linking a Wnt inhibitor that binds to the Wnt co-receptor Frizzled to a peptide derived from the N-terminal Dickkopf-1 that binds to Lrp (low-density lipoprotein receptor-related protein) 5/6, another Wnt co-receptor. This Wnt mimic not only enhances cellular Wnt signaling activation, but also improves the progenitor cell phenotype of in vitro cultured limbal epithelial cells. As the maintenance of stem cell characteristics in the process of culture expansion is essential for the success of ocular surface reconstruction, the small molecules generated in this study may be helpful in the development of pharmaceutical reagents for treating corneal wounds.
Collapse
Affiliation(s)
- Chi Zhang
- Stein Eye Institute, Department of Ophthalmology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA
| | - Hua Mei
- Department of Ophthalmology, University of North Carolina School of Medicine, Chapel Hill, NC 27517, USA
| | - Sarah Y T Robertson
- Stein Eye Institute, Department of Ophthalmology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA
| | - Ho-Jin Lee
- Department of Natural Sciences, Southwest Tennessee Community College, Memphis, TN 38134, USA
| | - Sophie X Deng
- Stein Eye Institute, Department of Ophthalmology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA.
| | - Jie J Zheng
- Stein Eye Institute, Department of Ophthalmology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA.
| |
Collapse
|
|
24
|
Keratin 12 mRNA expression could serve as an early corneal marker for limbal explant cultures. Cytotechnology 2020; 72:239-245. [PMID: 32016711 PMCID: PMC7192984 DOI: 10.1007/s10616-020-00373-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2019] [Accepted: 01/23/2020] [Indexed: 10/31/2022] Open
Abstract
This investigation aimed to identify early corneal marker and conjunctival epithelial differentiation through transcriptional analysis of limbal explant cultures and study early differentiation patterns of known corneal and conjunctival differentiation markers. 2 mm punch biopsies of limbal region were obtained from 6 donors of the Lions Cornea Bank Saar-Lorloux/Trier-Westpfalz. Limbal explants were dissected into corneal and conjunctival biopsy sections. Biopsies were placed with epithelial side down into 12 Wells. As soon as the outgrowing cells had reached confluence, they were harvested. mRNA expression of corneal differentiation markers KRT12, KRT3, DSG1, PAX6, ADH7 and ALDH1A1, conjunctival markers KRT19, KRT13 and stem cell marker ABCG2 were measured via qPCR. KRT12 and PAX6 protein expressions were evaluated using Western Blot. Results suggested that KRT12 mRNA expression was significantly higher in outgrowing cells from the corneal side of the biopsies as in those from the conjunctival side (p = 0.0043). There was no significant difference in mRNA expression of other analyzed markers comparing with marker expression of outgrown cells from both limbal biopsies (p > 0.13). KRT12 and PAX6 Western Blot analysis showed no difference in cells harvested from both sides. In conclusion, KRT12 mRNA might be a marker to measure corneal origin of cells from limbal biopsies with unknown composition of corneal and conjunctival progenitor cells. KRT3, DSG1, PAX6, ADH7, ALDH1A1, KRT19, KRT13 and ABCG2 mRNA as well as KRT12 and PAX6 protein expression could not contribute to differentiate corneal from conjunctival cell identity from limbal biopsies.
Collapse
|
|
25
|
Reza HM, Saleh R, Jain P, Rahman GMS, Bepari AK. C-MAF Expression in Adult Human Ocular Surface and its Implication in Pterygium Pathogenesis. Rep Biochem Mol Biol 2020; 8:419-423. [PMID: 32582801 PMCID: PMC7275840] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2019] [Accepted: 12/22/2019] [Indexed: 06/11/2023]
Abstract
BACKGROUND c-MAF, a transcription factor that belongs to the b-Zip Maf transcription factor family, was found to be critical for lens development in vertebrates. It is a well-known fact that the adult human ocular surface expresses c-MAF, however, its role in the limbus, cornea and conjunctiva remains unknown. Thus, the present study aimed to investigate c-MAF expression within the human ocular surface, and its potential role in pterygium pathogenesis. METHODS We performed immunohistochemical staining to detect c-MAF expression in frozen adult human tissue sections, including the limbus, cornea and conjunctiva, and cultured cells from eye cadavers. We then compared c-MAF expression to the expression of a known protein, P63. Lastly, we performed RT-PCR, and immunohistochemistry for c-MAF expression in healthy adult human conjunctiva and pterygium. RESULTS We found differential c-MAF expression between adult human limbus, cornea and conjunctiva tissues. Further, we observed that c-MAF is downregulated in the pterygium compared to healthy conjunctiva. CONCLUSION Overall, our results suggest that c-MAF may play a context-specific role in maintaining limbal, corneal and conjunctival homeostasis, and may be critical for preventing pterygium development in humans.
Collapse
Affiliation(s)
- Hasan Mahmud Reza
- Department of Pharmaceutical Sciences, North South University, Dhaka 1229, Bangladesh.
| | - Razwa Saleh
- Department of Pharmaceutical Sciences, North South University, Dhaka 1229, Bangladesh.
| | - Preeti Jain
- Department of Pharmaceutical Sciences, North South University, Dhaka 1229, Bangladesh.
| | | | - Asim Kumar Bepari
- Department of Pharmaceutical Sciences, North South University, Dhaka 1229, Bangladesh.
| |
Collapse
|
|
26
|
Selecting Appropriate Reference Genes for Quantitative Real-Time Polymerase Chain Reaction Studies in Isolated and Cultured Ocular Surface Epithelia. Sci Rep 2019; 9:19631. [PMID: 31873107 PMCID: PMC6927975 DOI: 10.1038/s41598-019-56054-1] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2019] [Accepted: 12/05/2019] [Indexed: 12/20/2022] Open
Abstract
The introduction of tissue engineering has allowed scientists to push the boundaries and treat seriously damaged ocular surface epithelia. They have managed to do this through the development of biological substitutes that restore, maintain or improve tissue function. To ensure the generation of a therapeutically safe and effective graft, knowledge on the transcriptional profile of native and cultured ocular surface epithelia is of undeniable value. Gene expression studies are, however, only as reliable as their proper selection of internal reaction controls or reference genes. In this study, we determined the expression stability of a number of reference genes: 18s rRNA, ACTB, ATP5B, CyC1, EIF4A2, GAPDH, RPL13A, SDHA, TOP1, UBC, and YWHAZ in primary isolates as well as in ex vivo cultured ocular surface epithelia explants (day 0 and/or day 14). Expression stability of the reference genes was assessed with both the geNorm and NormFinder software that use a pairwise comparison and a model-based approach, respectively. Our results extend the general recommendation of using multiple reference genes for normalization purposes to our model systems and provide an overview of several references genes that are likely to be stable in similar culture protocols.
Collapse
|
|
27
|
Decellularized Human Stromal Lenticules Combine with Corneal Epithelial-Like Cells: A New Resource for Corneal Tissue Engineering. Stem Cells Int 2019; 2019:4252514. [PMID: 31885607 PMCID: PMC6925757 DOI: 10.1155/2019/4252514] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2019] [Accepted: 09/10/2019] [Indexed: 12/17/2022] Open
Abstract
The lack of donor corneal tissue or the immunological rejection remains a challenge for individuals with limbal stem cell deficiency (LSCD) who are treated with keratoplasty. Numerous lenticules which were extracted by small incision lenticule extraction (SMILE) appear to be useful materials for keratoplasty. In order to reduce the incidence of allograft rejection, lenticules would be decellularized. Lenticules which were treated with liquid nitrogen and nucleases had no cellular and nuclear materials remained. Human induced pluripotent stem cells (iPSCs) can be generated from the patient who requires keratoplasty, offering an autologous alternative and eliminating the risk of graft rejection. We found that BMP-4, RA, N-2 supplement, hEGF, B27, decellularized human stromal lenticules, conditioned medium, or induction medium promoted the differentiation of human iPSCs with high purity. The results showed that human iPSCs cultured for 4 days in differentiation medium A, 14 days in condition medium, and 1 week in induction medium on decellularized human stromal lenticules developed markedly higher expression of the markers P63, CK3, and CK12 than did those in the other methods. The level of gene expression of the epithelial and pluripotency markers and analysis by scanning electron microscopy and immunohistochemistry also showed successful differentiation. After inducing differentiation in vitro, corneal epithelial-like cells were induced. In the study, we investigated the possibility of a new resource for corneal tissue engineering.
Collapse
|
|
28
|
Notch Inhibition Prevents Differentiation of Human Limbal Stem/Progenitor Cells in vitro. Sci Rep 2019; 9:10373. [PMID: 31316119 PMCID: PMC6637172 DOI: 10.1038/s41598-019-46793-6] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2019] [Accepted: 06/06/2019] [Indexed: 12/26/2022] Open
Abstract
Notch signaling has been shown to regulate the homeostasis and wound healing of the corneal epithelium. We investigated the effect of Notch inhibition in the human limbal stem/progenitor cells (LSCs) in vitro by using small molecules. Treatment of the LSCs with DAPT and SAHM1 reduced the proliferation rate and maintained the undifferentiated state of the LSCs in a concentration dependent manner. Stratification and differentiation of the corneal epithelium were not reduced after Notch inhibition, indicating that the function of the corneal basal cells is retained. Our findings suggest that Notch signaling plays a role in the proliferation and maintenance of LSCs.
Collapse
|
|
29
|
González S, Oh D, Baclagon ER, Zheng JJ, Deng SX. Wnt Signaling Is Required for the Maintenance of Human Limbal Stem/Progenitor Cells In Vitro. Invest Ophthalmol Vis Sci 2019; 60:107-112. [PMID: 30640975 PMCID: PMC6333110 DOI: 10.1167/iovs.18-25740] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022] Open
Abstract
Purpose A chemical approach to examine the role of Wnt signaling in maintaining the stemness and/or proliferation of limbal stem/progenitor cells (LSCs). Methods LSCs were isolated from human donor eyes and cultured as single cells for 12 to 14 days with the following small molecules: IIIC3, an antagonist of the Wnt signaling inhibitor Dickkopf (DKK), and IC15, a Wnt signaling inhibitor. Proliferation of LSCs in the presence of IIIC3 and IC15 was determined by the number of cells and colonies established. Maintenance of stemness was determined by p63α, cytokeratin (K)12, and K14 expression. Results Activation of Wnt, through IIIC3-mediated DKK inhibition, resulted in similar colony forming efficiency (CFE) as in the untreated LSCs, but significantly increased the number of cultivated cells 7.21% with 5 μM. Inhibition of Wnt with IC15 significantly reduced the CFE (P ≤ 0.01) and the number of cultivated cells by 16% to 29%. Percentage of cells expressing high levels of p63α (p63αbright) and quantity of small cells (≤12 μm), which contain the LSCs, increased 4.71% and 11.26% (both P < 0.05), respectively, with 5 μM IIIC3. All concentrations of IIIC3 and IC15 retained the K14 undifferentiated marker (97%), while differentiation, as detected by expression of K12, was found in up to 2% of cells in 1 μM IIIC3, 1 μM IC15, or 5 μM IIIC3. Conclusions Wnt signaling is required in LSC proliferation and maintenance of an undifferentiated state. The current study is a proof of concept that the Wnt pathway could be modulated in LSCs to enhance or decrease the efficiency of human LSC expansion.
Collapse
Affiliation(s)
- Sheyla González
- Stein Eye Institute, University of California, Los Angeles, California, United States
| | - Denise Oh
- Stein Eye Institute, University of California, Los Angeles, California, United States
| | - Elfren R Baclagon
- Stein Eye Institute, University of California, Los Angeles, California, United States
| | - Jie J Zheng
- Stein Eye Institute, University of California, Los Angeles, California, United States
| | - Sophie X Deng
- Stein Eye Institute, University of California, Los Angeles, California, United States
| |
Collapse
|
|
30
|
Strategies for reconstructing the limbal stem cell niche. Ocul Surf 2019; 17:230-240. [PMID: 30633966 DOI: 10.1016/j.jtos.2019.01.002] [Citation(s) in RCA: 45] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2018] [Revised: 10/21/2018] [Accepted: 01/07/2019] [Indexed: 12/19/2022]
Abstract
The epithelial cell layer that covers the surface of the cornea provides a protective barrier while maintaining corneal transparency. The rapid and effective turnover of these epithelial cells depends, in part, on the limbal epithelial stem cells (LESCs) located in a specialized microenvironment known as the limbal niche. Many disorders affecting the regeneration of the corneal epithelium are related to deficiency and/or dysfunction of LESCs and the limbal niche. Current approaches for regenerating the corneal epithelium following significant injuries such as burns and inflammatory attacks are primarily aimed at repopulating the LESCs. This review summarizes and assesses the clinical feasibility and efficacy of current and emerging approaches for reconstruction of the limbal niche. In particular, the application of mesenchymal stem cells along with appropriate biological scaffolds appear to be promising strategies for long-term revitalization of the limbal niche.
Collapse
|
|
31
|
Liu L, Nielsen FM, Emmersen J, Bath C, Østergaard Hjortdal J, Riis S, Fink T, Pennisi CP, Zachar V. Pigmentation Is Associated with Stemness Hierarchy of Progenitor Cells Within Cultured Limbal Epithelial Cells. Stem Cells 2018; 36:1411-1420. [PMID: 29781179 DOI: 10.1002/stem.2857] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2018] [Revised: 04/26/2018] [Accepted: 05/07/2018] [Indexed: 12/15/2022]
Abstract
Ex vivo cultured human limbal epithelial stem/progenitor cells (hLESCs) are the main source for regenerative therapy of limbal stem cell deficiency (LSCD), which is worldwide one of the major causes of corneal blindness. Despite many stemness-associated markers have been identified within the limbal niche, the phenotype of the earliest hLESCs has not been hitherto identified. We sought to confirm or refute the use of tumor protein p63 (p63) and ATP binding cassette subfamily B member 5 (ABCB5) as surrogate markers for hLESCs early within the limbal differentiation hierarchy. Based on a robust fluorescence-activated cell sorting and subsequent RNA isolation protocol, a comprehensive transcriptomic profile was obtained from four subpopulations of cultured hLESCs. The subpopulations were defined by co-expression of two putative stem/progenitor markers, the p63 and ABCB5, and the corneal differentiation marker cytokeratin 3. A comparative transcriptomic analysis yielded novel data that indicated association between pigmentation and differentiation, with the p63 positive populations being the most pigmented and immature of the progenitors. In contrast, ABCB5, either alone or in co-expression patterns, identified more committed progenitor cells with less pigmentation. In conclusion, p63 is superior to ABCB5 as a marker for stemness. Stem Cells 2018;36:1411-1420.
Collapse
Affiliation(s)
- Lei Liu
- Laboratory for Stem Cell Research, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark.,Department of Pediatric Surgery, First Hospital of Jilin University, Changchun, Jilin, People's Republic of China
| | - Frederik Mølgaard Nielsen
- Laboratory for Stem Cell Research, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark
| | - Jeppe Emmersen
- Laboratory for Stem Cell Research, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark
| | - Chris Bath
- Department of Ophthalmology, Aalborg University Hospital, Aalborg, Denmark
| | | | - Simone Riis
- Laboratory for Stem Cell Research, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark
| | - Trine Fink
- Laboratory for Stem Cell Research, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark
| | - Cristian Pablo Pennisi
- Laboratory for Stem Cell Research, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark
| | - Vladimir Zachar
- Laboratory for Stem Cell Research, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark
| |
Collapse
|
|
32
|
Menzel-Severing J, Zenkel M, Polisetti N, Sock E, Wegner M, Kruse FE, Schlötzer-Schrehardt U. Transcription factor profiling identifies Sox9 as regulator of proliferation and differentiation in corneal epithelial stem/progenitor cells. Sci Rep 2018; 8:10268. [PMID: 29980721 PMCID: PMC6035181 DOI: 10.1038/s41598-018-28596-3] [Citation(s) in RCA: 36] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2018] [Accepted: 06/26/2018] [Indexed: 02/08/2023] Open
Abstract
Understanding transcription factor (TF) regulation of limbal epithelial stem/progenitor cells (LEPCs) may aid in using non-ocular cells to regenerate the corneal surface. This study aimed to identify and characterize TF genes expressed specifically in LEPCs isolated from human donor eyes by laser capture microdissection. Using a profiling approach, preferential limbal expression was found for SoxE and SoxF genes, particularly for Sox9, which showed predominantly cytoplasmic localization in basal LEPCs and nuclear localization in suprabasal and corneal epithelial cells, indicating nucleocytoplasmic translocation and activation during LEPC proliferation and differentiation. Increased nuclear localization of Sox9 was also observed in activated LEPCs following clonal expansion and corneal epithelial wound healing. Knockdown of SOX9 expression in cultured LEPCs by RNAi led to reduced expression of progenitor cell markers, e.g. keratin 15, and increased expression of differentiation markers, e.g. keratin 3. Furthermore, SOX9 silencing significantly suppressed the proliferative capacity of LEPCs and reduced levels of glycogen synthase kinase 3 beta (GSK-3ß), a negative regulator of Wnt/ß-catenin signaling. Sox9 expression, in turn, was significantly suppressed by treatment of LEPCs with exogenous GSK-3ß inhibitors and enhanced by small molecule inhibitors of Wnt signaling. Our results suggest that Sox9 and Wnt/ß-catenin signaling cooperate in mutually repressive interactions to achieve a balance between quiescence, proliferation and differentiation of LEPCs in the limbal niche. Future molecular dissection of Sox9-Wnt interaction and mechanisms of nucleocytoplasmic shuttling of Sox9 may aid in improving the regenerative potential of LEPCs and the reprogramming of non-ocular cells for corneal surface regeneration.
Collapse
Affiliation(s)
- Johannes Menzel-Severing
- Department of Ophthalmology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany
| | - Matthias Zenkel
- Department of Ophthalmology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany
| | - Naresh Polisetti
- Department of Ophthalmology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany
| | - Elisabeth Sock
- Institut für Biochemie, Emil-Fischer-Zentrum, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany
| | - Michael Wegner
- Institut für Biochemie, Emil-Fischer-Zentrum, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany
| | - Friedrich E Kruse
- Department of Ophthalmology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany
| | | |
Collapse
|
|
33
|
The Role of Limbal Epithelial Stem Cells in Regulating Corneal (Lymph)angiogenic Privilege and the Micromilieu of the Limbal Niche following UV Exposure. Stem Cells Int 2018; 2018:8620172. [PMID: 29853920 PMCID: PMC5964490 DOI: 10.1155/2018/8620172] [Citation(s) in RCA: 54] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2017] [Accepted: 04/18/2018] [Indexed: 12/02/2022] Open
Abstract
The cornea is a clear structure, void of blood, and lymphatic vessels, functioning as our window to the world. Limbal epithelial stem cells, occupying the area between avascular cornea and vascularized conjunctiva, have been implicated in tissue border maintenance, preventing conjunctivalisation and propagation of blood and lymphatic vessels into the cornea. Defects in limbal epithelial stem cells are linked to corneal neovascularisation, including lymphangiogenesis, chronic inflammation, conjunctivalisation, epithelial abnormalities including the presence of goblet cells, breaks in Bowman's membrane, persistent epithelial defects and ulceration, ocular surface squamous neoplasia, lipid keratopathy, pain, discomfort, and compromised vision. It has been postulated that pterygium is an example of focal limbal deficiency. Previous reports showing changes occurring in limbal epithelium during pterygium pathogenesis suggest that there is a link to stem cell damage. In this light, pterygium can serve as a model disease of UV-induced stem cell damage also characterised by corneal blood and lymphangiogenesis. This review focuses on the role of corneal and limbal epithelial cells and the stem cell niche in maintaining corneal avascularity and corneal immune privilege and how this may be deregulated following UV exposure. We present an overview of the PUBMED literature in the field as well as recent work from our laboratories.
Collapse
|
|
34
|
Abstract
PURPOSE OF REVIEW The aim of this review is to describe the underlying mechanisms of corneal epithelial homeostasis in addition to illustrating the vital role of the limbal epithelial stem cells (LESCs) and the limbal niche in epithelial regeneration and wound healing. RECENT FINDINGS The shedded corneal epithelial cells are constantly replenished by the LESCs which give rise to epithelial cells that proliferate, differentiate, and migrate centripetally. While some recent studies have proposed that epithelial stem cells may also be present in the central cornea, the predominant location for the stem cells is the limbus. The limbal niche is the specialized microenvironment consisting of cells, extracellular matrix, and signaling molecules that are essential for the function of LESCs. Disturbances to limbal niche can result in LESC dysfunction; therefore, limbal stem cell deficiency should also be considered a limbal niche deficiency. Current and in-development therapeutic strategies are aimed at restoring the limbal niche, by medical and/or surgical treatments, administration of trophic factors, and cell based therapies. SUMMARY The corneal epithelium is constantly replenished by LESCs that are housed within the limbal niche. The limbal niche is the primary determinant of the LESC function and novel therapeutic approaches should be focused on regeneration of this microenvironment.
Collapse
|
|
35
|
Limbal Stem Cells from Aged Donors Are a Suitable Source for Clinical Application. Stem Cells Int 2016; 2016:3032128. [PMID: 28042298 PMCID: PMC5155095 DOI: 10.1155/2016/3032128] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2016] [Revised: 10/02/2016] [Accepted: 10/09/2016] [Indexed: 12/13/2022] Open
Abstract
Limbal stem cells (LSC) are the progenitor cells that maintain the transparency of the cornea. Limbal stem cell deficiency (LSCD) leads to corneal opacity, inflammation, scarring, and blindness. A clinical approach to treat this condition consists in LSC transplantation (LSCT) after ex vivo expansion of LSC. In unilateral LSCD, an autologous transplant is possible, but cases of bilateral LSCD require allogenic LSCT. Cadaveric donors represent the most important source of LSC allografts for treatment of bilateral LSCD when living relative donors are not available. To evaluate the suitability of aged cadaveric donors for LSCT, we compared three pools of LSC from donors of different ages (<60 years, 60–75 years, and >75 years). We evaluated graft quality in terms of percent of p63-positive (p63+) cells by immunofluorescence, colony forming efficiency, and mRNA and protein expression of p63, PAX6, Wnt7a, E-cadherin, and cytokeratin (CK) 12, CK3, and CK19. The results showed that LSC cultures from aged donors can express ≥3% of p63+ cells—considered as the minimum value for predicting favorable clinical outcomes after LSCT—suggesting that these cells could be a suitable source of LSC for transplantation. Our results also indicate the need to evaluate LSC graft quality criteria for each donor.
Collapse
|
|
36
|
Kasinathan JR, Namperumalsamy VP, Veerappan M, Chidambaranathan GP. A novel method for a high enrichment of human corneal epithelial stem cells for genomic analysis. Microsc Res Tech 2016; 79:1165-1172. [PMID: 27862636 DOI: 10.1002/jemt.22771] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2016] [Revised: 08/11/2016] [Accepted: 08/18/2016] [Indexed: 12/13/2022]
Abstract
Understanding the molecular mechanisms that regulate the corneal epithelial stem cells (CESCs) in maintaining corneal homeostasis remains elusive largely due to the lack of a specific marker for their isolation. This study aims to enrich CESCs from human donor limbal epithelium and to evaluate the level of enrichment based on expression of ΔNp63α, a putative CESC marker. A two-stage enrichment of CESCs was carried out. (a) The limbal basal epithelial cells were isolated by differential enzymatic treatment and five-fold enrichment was achieved from 2% of CESCs present in the total limbal epithelium. The CESCs were quantified on the basis of two parameters-high expression of p63/ABCG2 and nucleus to cytoplasmic (N/C) ratio ≥0.7. (b) Cytospin smears of isolated basal cells were Giemsa stained and cells with N/C ratio ≥0.7 were separated by laser capture microdissection. This strategy resulted in an enrichment of CESCs to 78.57% based on two-parameter analysis using p63 and 76.66% using ABCG2. RT-PCR was carried out for ΔNp63 isoforms (α, β, and γ) and connexin-43, with GAPDH for normalization. The expression of ΔNp63α was restricted to the enriched population of CESCs in contrast to its absence in limbal basal cells with N/C ratio <0.7 and CCECs. The unique expression of ΔNp63α and 5.9-fold reduced connexin-43 expression in the enriched population of CESCs indicates its high purity. Further analysis of these cells will help in elucidating the molecular mechanisms associated with stemness and also in identifying a specific marker for CESCs.
Collapse
Affiliation(s)
- Jhansi Rani Kasinathan
- Department of Immunology and Stem Cell Biology, Aravind Medical Research Foundation, Dr. G. Venkataswamy Eye Research Institute, Madurai, Tamil Nadu, India
| | | | - Muthukkaruppan Veerappan
- Department of Immunology and Stem Cell Biology, Aravind Medical Research Foundation, Dr. G. Venkataswamy Eye Research Institute, Madurai, Tamil Nadu, India
| | - Gowri Priya Chidambaranathan
- Department of Immunology and Stem Cell Biology, Aravind Medical Research Foundation, Dr. G. Venkataswamy Eye Research Institute, Madurai, Tamil Nadu, India
| |
Collapse
|
|
37
|
Richardson R, Tracey-White D, Webster A, Moosajee M. The zebrafish eye-a paradigm for investigating human ocular genetics. Eye (Lond) 2016; 31:68-86. [PMID: 27612182 DOI: 10.1038/eye.2016.198] [Citation(s) in RCA: 117] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2016] [Accepted: 06/17/2016] [Indexed: 01/13/2023] Open
Abstract
Although human epidemiological and genetic studies are essential to elucidate the aetiology of normal and aberrant ocular development, animal models have provided us with an understanding of the pathogenesis of multiple developmental ocular malformations. Zebrafish eye development displays in depth molecular complexity and stringent spatiotemporal regulation that incorporates developmental contributions of the surface ectoderm, neuroectoderm and head mesenchyme, similar to that seen in humans. For this reason, and due to its genetic tractability, external fertilisation, and early optical clarity, the zebrafish has become an invaluable vertebrate system to investigate human ocular development and disease. Recently, zebrafish have been at the leading edge of preclinical therapy development, with their amenability to genetic manipulation facilitating the generation of robust ocular disease models required for large-scale genetic and drug screening programmes. This review presents an overview of human and zebrafish ocular development, genetic methodologies employed for zebrafish mutagenesis, relevant models of ocular disease, and finally therapeutic approaches, which may have translational leads in the future.
Collapse
Affiliation(s)
- R Richardson
- Department of Ocular Biology and Therapeutics, UCL Institute of Ophthalmology, London, UK
| | - D Tracey-White
- Department of Ocular Biology and Therapeutics, UCL Institute of Ophthalmology, London, UK
| | - A Webster
- Department of Ocular Biology and Therapeutics, UCL Institute of Ophthalmology, London, UK.,NIHR Biomedical Research Centre for Ophthalmology, Moorfields Eye Hospital NHS Foundation Trust, London, UK
| | - M Moosajee
- Department of Ocular Biology and Therapeutics, UCL Institute of Ophthalmology, London, UK.,NIHR Biomedical Research Centre for Ophthalmology, Moorfields Eye Hospital NHS Foundation Trust, London, UK
| |
Collapse
|
|
38
|
Vazirani J, Ali MH, Sharma N, Gupta N, Mittal V, Atallah M, Amescua G, Chowdhury T, Abdala-Figuerola A, Ramirez-Miranda A, Navas A, Graue-Hernández EO, Chodosh J. Autologous simple limbal epithelial transplantation for unilateral limbal stem cell deficiency: multicentre results. Br J Ophthalmol 2016; 100:1416-20. [DOI: 10.1136/bjophthalmol-2015-307348] [Citation(s) in RCA: 79] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2015] [Accepted: 01/03/2016] [Indexed: 11/03/2022]
|
|
39
|
Short-term uvb-irradiation leads to putative limbal stem cell damage and niche cell-mediated upregulation of macrophage recruiting cytokines. Stem Cell Res 2015; 15:643-654. [DOI: 10.1016/j.scr.2015.10.008] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/08/2015] [Accepted: 10/16/2015] [Indexed: 01/17/2023] Open
|
|
40
|
Mikhailova A, Jylhä A, Rieck J, Nättinen J, Ilmarinen T, Veréb Z, Aapola U, Beuerman R, Petrovski G, Uusitalo H, Skottman H. Comparative proteomics reveals human pluripotent stem cell-derived limbal epithelial stem cells are similar to native ocular surface epithelial cells. Sci Rep 2015; 5:14684. [PMID: 26423138 PMCID: PMC4589773 DOI: 10.1038/srep14684] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2015] [Accepted: 09/08/2015] [Indexed: 12/13/2022] Open
Abstract
Limbal epithelial stem cells (LESCs) are tissue-specific stem cells responsible for renewing the corneal epithelium. Acute trauma or chronic disease affecting LESCs may disrupt corneal epithelial renewal, causing vision threatening and painful ocular surface disorders, collectively referred to as LESC deficiency (LESCD). These disorders cannot be treated with traditional corneal transplantation and therefore alternative cell sources for successful cell-based therapy are needed. LESCs derived from human pluripotent stem cells (hPSCs) are a prospective source for ocular surface reconstruction, yet critical evaluation of these cells is crucial before considering clinical applications. In order to quantitatively evaluate hPSC-derived LESCs, we compared protein expression in native human corneal cells to that in hPSC-derived LESCs using isobaric tag for relative and absolute quantitation (iTRAQ) technology. We identified 860 unique proteins present in all samples, including proteins involved in cell cycling, proliferation, differentiation and apoptosis, various LESC niche components, and limbal and corneal epithelial markers. Protein expression profiles were nearly identical in LESCs derived from two different hPSC lines, indicating that the differentiation protocol is reproducible, yielding homogeneous cell populations. Their protein expression profile suggests that hPSC-derived LESCs are similar to the human ocular surface epithelial cells, and possess LESC-like characteristics.
Collapse
Affiliation(s)
| | - Antti Jylhä
- Department of Ophthalmology, School of Medicine, University of Tampere, Finland
| | | | - Janika Nättinen
- Department of Ophthalmology, School of Medicine, University of Tampere, Finland
| | | | - Zoltán Veréb
- Stem Cells and Eye Research Laboratory, Department of Ophthalmology, Faculty of Medicine, University of Szeged, Hungary
| | - Ulla Aapola
- Department of Ophthalmology, School of Medicine, University of Tampere, Finland
| | - Roger Beuerman
- Department of Ophthalmology, School of Medicine, University of Tampere, Finland.,Singapore Eye Research Institute and School of Medicine, Singapore
| | - Goran Petrovski
- Stem Cells and Eye Research Laboratory, Department of Ophthalmology, Faculty of Medicine, University of Szeged, Hungary
| | - Hannu Uusitalo
- Department of Ophthalmology, School of Medicine, University of Tampere, Finland.,Tampere University Hospital Eye Center, University of Tampere, Finland
| | | |
Collapse
|
|
41
|
Nakamura T, Inatomi T, Sotozono C, Koizumi N, Kinoshita S. Ocular surface reconstruction using stem cell and tissue engineering. Prog Retin Eye Res 2015; 51:187-207. [PMID: 26187034 DOI: 10.1016/j.preteyeres.2015.07.003] [Citation(s) in RCA: 50] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2015] [Revised: 07/08/2015] [Accepted: 07/08/2015] [Indexed: 12/22/2022]
Abstract
Most human sensory information is gained through eyesight, and integrity of the ocular surface, including cornea and conjunctiva, is known to be indispensable for good vision. It is believed that severe damage to corneal epithelial stem cells results in devastating ocular surface disease, and many researchers and scientists have tried to reconstruct the ocular surface using medical and surgical approaches. Ocular surface reconstruction via regenerative therapy is a newly developed medical field that promises to be the next generation of therapeutic modalities, based on the use of tissue-specific stem cells to generate biological substitutes and improve tissue functions. The accomplishment of these objectives depends on three key factors: stem cells, which have highly proliferative capacities and longevities; the substrates determining the environmental niche; and growth factors that support them appropriately. This manuscript describes the diligent development of ocular surface reconstruction using tissue engineering techniques, both past and present, and discusses and validates their future use for regenerative therapy in this field.
Collapse
Affiliation(s)
- Takahiro Nakamura
- Department of Frontier Medical Sciences and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
| | - Tsutomu Inatomi
- Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
| | - Chie Sotozono
- Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
| | - Noriko Koizumi
- Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
| | - Shigeru Kinoshita
- Department of Frontier Medical Sciences and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
| |
Collapse
|
|
42
|
Branch MJ, Yu WY, Sheridan C, Hopkinson A. Isolation of adult stem cell populations from the human cornea. Methods Mol Biol 2015; 1235:165-77. [PMID: 25388394 DOI: 10.1007/978-1-4939-1785-3_14] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/10/2023]
Abstract
Corneal blindness is a leading cause of vision loss globally. From a tissue engineering perspective, the cornea represents specific challenges in respect to isolating, stably expanding, banking, and effectively manipulating the various cell types required for effective corneal regeneration. The current research trend in this area focuses on a combined stem cell component with a biological or synthetic carrier or engineering scaffold. Corneal derived stem cells play an important role in such strategies as they represent an available supply of cells with specific abilities to further generate corneal cells in the long term. This chapter describes the isolation protocols of the epithelial stromal and endothelial stem cell populations.
Collapse
Affiliation(s)
- Matthew J Branch
- Ophthalmology DCN, University of Nottingham, Queen's Medical Center, Clifton Blvd., Nottingham, NG7 2UH, UK
| | | | | | | |
Collapse
|
|
43
|
Zhang ZH, Liu HY, Liu K, Xu X. Comparison of Explant and Enzyme Digestion Methods for Ex Vivo Isolation of Limbal Epithelial Progenitor Cells. Curr Eye Res 2015; 41:318-25. [PMID: 25860821 DOI: 10.3109/02713683.2015.1014566] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
PURPOSE To compare the effectiveness of two isolation systems for stemness, proliferation and mesenchymal contamination of ex vivo cultured limbal epithelial cells (LECs). METHODS Whole explant and dispase digestion methods were used to isolate LECs. For whole explant isolation, one limbal explant was cultivated up to four consecutive times (through LEC1 to LEC4). The performance of LECs isolated with both systems was evaluated according to the following parameters: immunofluorescent staining for adenosine 5'-triphosphate-binding cassette member 2 (ABCG2), p63, cytokeratin 3 (K3), Ki67, and vimentin, and flow cytometry analysis for ABCG2, Ki67 and vimentin. RESULTS Twelve LEC cultures were established using whole explant isolation, and nine LEC cultures were established using dispase digestion isolation. In immunofluorescent staining analysis, the ABCG2, p63 and Ki67 expressions were higher in LECs isolated with dispase compared to any LECs isolated with explant. Only the differences in ABCG2 and Ki67 were statistically significant. Further, LEC4 isolated with explant had the highest percentage of cells positive for vimentin, and LEC1 had the highest percentage of cells positive for K3. However, no significant differences were detected. In flow cytometry analysis, the expressions of ABCG2 and Ki67 were statistically higher for LECs isolated with dispase compared to any LECs isolated with explant. CONCLUSION Dispase digestion isolation technique was significantly superior to explant isolation techniques in terms of progenitor and proliferative cell contents.
Collapse
Affiliation(s)
- Zhi-Hua Zhang
- a Department of Ophthalmology , Shanghai First People's Hospital Affiliated to Shanghai Jiaotong University , Shanghai , China
| | - Hai-Yun Liu
- a Department of Ophthalmology , Shanghai First People's Hospital Affiliated to Shanghai Jiaotong University , Shanghai , China
| | - Kun Liu
- a Department of Ophthalmology , Shanghai First People's Hospital Affiliated to Shanghai Jiaotong University , Shanghai , China
| | - Xun Xu
- a Department of Ophthalmology , Shanghai First People's Hospital Affiliated to Shanghai Jiaotong University , Shanghai , China
| |
Collapse
|
|
44
|
West JD, Dorà NJ, Collinson JM. Evaluating alternative stem cell hypotheses for adult corneal epithelial maintenance. World J Stem Cells 2015; 7:281-99. [PMID: 25815115 PMCID: PMC4369487 DOI: 10.4252/wjsc.v7.i2.281] [Citation(s) in RCA: 46] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/29/2014] [Revised: 09/26/2014] [Accepted: 10/14/2014] [Indexed: 02/07/2023] Open
Abstract
In this review we evaluate evidence for three different hypotheses that explain how the corneal epithelium is maintained. The limbal epithelial stem cell (LESC) hypothesis is most widely accepted. This proposes that stem cells in the basal layer of the limbal epithelium, at the periphery of the cornea, maintain themselves and also produce transient (or transit) amplifying cells (TACs). TACs then move centripetally to the centre of the cornea in the basal layer of the corneal epithelium and also replenish cells in the overlying suprabasal layers. The LESCs maintain the corneal epithelium during normal homeostasis and become more active to repair significant wounds. Second, the corneal epithelial stem cell (CESC) hypothesis postulates that, during normal homeostasis, stem cells distributed throughout the basal corneal epithelium, maintain the tissue. According to this hypothesis, LESCs are present in the limbus but are only active during wound healing. We also consider a third possibility, that the corneal epithelium is maintained during normal homeostasis by proliferation of basal corneal epithelial cells without any input from stem cells. After reviewing the published evidence, we conclude that the LESC and CESC hypotheses are consistent with more of the evidence than the third hypothesis, so we do not consider this further. The LESC and CESC hypotheses each have difficulty accounting for one main type of evidence so we evaluate the two key lines of evidence that discriminate between them. Finally, we discuss how lineage-tracing experiments have begun to resolve the debate in favour of the LESC hypothesis. Nevertheless, it also seems likely that some basal corneal epithelial cells can act as long-term progenitors if limbal stem cell function is compromised. Thus, this aspect of the CESC hypothesis may have a lasting impact on our understanding of corneal epithelial maintenance, even if it is eventually shown that stem cells are restricted to the limbus as proposed by the LESC hypothesis.
Collapse
Affiliation(s)
- John D West
- John D West, Natalie J Dorà, Genes and Development Group, Centre for Integrative Physiology, College of Medicine and Veterinary Medicine, University of Edinburgh, EH8 9XD Edinburgh, United Kingdom
| | - Natalie J Dorà
- John D West, Natalie J Dorà, Genes and Development Group, Centre for Integrative Physiology, College of Medicine and Veterinary Medicine, University of Edinburgh, EH8 9XD Edinburgh, United Kingdom
| | - J Martin Collinson
- John D West, Natalie J Dorà, Genes and Development Group, Centre for Integrative Physiology, College of Medicine and Veterinary Medicine, University of Edinburgh, EH8 9XD Edinburgh, United Kingdom
| |
Collapse
|
|
45
|
Mei H, Nakatsu MN, Baclagon ER, Deng SX. Frizzled 7 maintains the undifferentiated state of human limbal stem/progenitor cells. Stem Cells 2015; 32:938-45. [PMID: 24170316 DOI: 10.1002/stem.1582] [Citation(s) in RCA: 52] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2013] [Revised: 09/14/2013] [Accepted: 09/18/2013] [Indexed: 12/15/2022]
Abstract
Wnt signaling pathway plays an important role in the regulation of human limbal stem/progenitor cells (LSCs). To examine the possible function of Frizzled (Fz) receptors in LSCs, the expression of 10 Fz receptors was profiled in the limbus and cornea. Only Fz7 had preferential expression in the basal limbal epithelium which contains the LSCs. The expression of Fz7 was colocalized with the putative LSC markers including p63α, N-cadherin and keratin (K) 14, and was minimum in cells expressing the corneal maturation marker K12. The expression of Fz7 was higher in the enriched LSCs population and decreased in cultured LSCs when there was a loss of progenitor phenotype. When the Fz7 was knocked down (Fz(KD)) using shRNA in primary LSCs, the expression of putative LSC markers ABCG2, ΔNp63α, and K14 was decreased significantly. The colony forming efficiency of the Fz7(KD) LSCs was significantly decreased in the subsequent passage 1 and 2 compared to the control. Our finding suggests that Wnt signaling is one of the factors of LSC niche, and Fz7 helps to maintain the undifferentiated state of LSCs.
Collapse
Affiliation(s)
- Hua Mei
- Jules Stein Eye Institute, Department of Ophthalmology, David Geffen School of Medicine, University of California, Los Angeles, California, USA
| | | | | | | |
Collapse
|
|
46
|
Bath C, Fink T, Vorum H, Hjortdal J, Zachar V. Technical brief: Optimized pipeline for isolation of high-quality RNA from corneal cell subpopulations. Mol Vis 2014; 20:797-803. [PMID: 24940035 PMCID: PMC4057246] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2013] [Accepted: 06/10/2014] [Indexed: 11/16/2022] Open
Abstract
PURPOSE Attempts to determine the transcriptional profile of discrete subsets of limbal epithelial cells in situ using laser capture microdissection (LCM) face two major challenges. First, the transcriptional profile of cells within a tissue may rapidly change as the tissue is excised and exposed to cold ischemia. Second, there is a risk of degradation of the RNA as the cellular compartment is separated from the remaining tissue. An optimized protocol for LCM of corneal epithelium is presented to address these issues. METHODS Experiments using porcine eye globes were carried out to determine both optimal procedures and settings for tissue harvest, transport, storage, histology, LCM, and RNA isolation. The optimized protocol was validated using human corneal epithelium. RESULTS To facilitate preservation of the gene expression profile, we have developed a mechanical tool for dissection of cornea that, in combination with flash freezing, enables tissue to be stored within 5 min of enucleation of the eye. Furthermore, we describe how RNA from limbal crypt cells may be obtained using a procedure involving cryosectioning, histological staining, and LCM. CONCLUSION In this paper, we describe an optimized method for isolating high-quality RNA from cellular subpopulations confined to the limbal crypts of the cornea. The procedure yields RNA in amounts and quality suitable for downstream gene expression analyses, such as microarrays or next generation sequencing.
Collapse
Affiliation(s)
- Chris Bath
- Laboratory for Stem Cell Research, Department of Health Science and Technology, Aalborg University, Denmark,Department of Ophthalmology, Aalborg University Hospital, Denmark
| | - Trine Fink
- Laboratory for Stem Cell Research, Department of Health Science and Technology, Aalborg University, Denmark
| | - Henrik Vorum
- Department of Ophthalmology, Aalborg University Hospital, Denmark
| | - Jesper Hjortdal
- Department of Ophthalmology, Aarhus University Hospital, Denmark
| | - Vladimir Zachar
- Laboratory for Stem Cell Research, Department of Health Science and Technology, Aalborg University, Denmark
| |
Collapse
|
|
47
|
Honoré B, Vorum H. Proteomic analysis as a means to approach limbal stem cell biology in a search for stem cell markers. Proteomics Clin Appl 2014; 8:178-84. [PMID: 24497450 DOI: 10.1002/prca.201300049] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2013] [Revised: 10/06/2013] [Accepted: 12/02/2013] [Indexed: 12/13/2022]
Abstract
The cornea consists of three main layers: an outer surface epithelium, the stroma, and the endothelium. A clear cornea is necessary for optimal vision and is maintained and repaired from limbal epithelial stem cells located in the limbus between the cornea and the sclera. Diseases and injury may result in deficiency of the stem cells impairing their ability to renew the corneal epithelium. Patients with limbal stem cell deficiency experience chronic pain and ultimately blindness. Attempts to treat the disease are based on replacement of the stem cells by transplantation or by culturing the stem cells. We here review the proteomic techniques that so far have been used to approach characterization of limbal stem cells and markers to identify them. It is apparent that the field is in a rather inchoate state due to the scarcity and relative inaccessibility of the stem cells. However, the importance of revealing limbal stem cell biology and identifying stem cell biomarkers calls for greater use of emerging methodology. Strategies for future studies are discussed.
Collapse
Affiliation(s)
- Bent Honoré
- Department of Biomedicine, Aarhus University, Aarhus, Denmark
| | | |
Collapse
|
|
48
|
Veréb Z, Albert R, Póliska S, Olstad OK, Akhtar S, Moe MC, Petrovski G. Comparison of upstream regulators in human ex vivo cultured cornea limbal epithelial stem cells and differentiated corneal epithelial cells. BMC Genomics 2013; 14:900. [PMID: 24344983 PMCID: PMC3880589 DOI: 10.1186/1471-2164-14-900] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2013] [Accepted: 12/11/2013] [Indexed: 12/13/2022] Open
Abstract
Background The surface of the human eye is covered by corneal epithelial cells (CECs) which regenerate from a small population of limbal epithelial stem cells (LESCs). Cell therapy with LESCs is a non-penetrating treatment for preventing blindness due to LESC deficiency or dysfunction. Our aim was to identify new putative molecular markers and upstream regulators in the LESCs and associated molecular pathways. Results Genome-wide microarray transcriptional profiling was used to compare LESCs to differentiated human CECs. Ingenuity-based pathway analysis was applied to identify upstream regulators and pathways specific to LESCs. ELISA and flow cytometry were used to measure secreted and surface expressed proteins, respectively. More than 2 fold increase and decrease in expression could be found in 1830 genes between the two cell types. A number of molecules functioning in cellular movement (381), proliferation (567), development (552), death and survival (520), and cell-to-cell signaling (290) were detected having top biological functions in LESCs and several of these were confirmed by flow cytometric surface protein analysis. Custom-selected gene groups related to stemness, differentiation, cell adhesion, cytokines and growth factors as well as angiogenesis could be analyzed. The results show that LESCs play a key role not only in epithelial differentiation and tissue repair, but also in controlling angiogenesis and extracellular matrix integrity. Some pro-inflammatory cytokines were found to be important in stemness-, differentiation- and angiogenesis-related biological functions: IL-6 and IL-8 participated in most of these biological pathways as validated by their secretion from LESC cultures. Conclusions The gene and molecular pathways may provide a more specific understanding of the signaling molecules associated with LESCs, therefore, help better identify and use these cells in the treatment of ocular surface diseases.
Collapse
Affiliation(s)
| | | | | | | | | | | | - Goran Petrovski
- Stem Cells and Eye Research Laboratory, University of Debrecen, Debrecen, Hungary.
| |
Collapse
|
|
49
|
Kulkarni BB, Powe DG, Hopkinson A, Dua HS. Optimised laser microdissection of the human ocular surface epithelial regions for microarray studies. BMC Ophthalmol 2013; 13:62. [PMID: 24160452 PMCID: PMC4015997 DOI: 10.1186/1471-2415-13-62] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2013] [Accepted: 10/09/2013] [Indexed: 01/22/2023] Open
Abstract
Background The most important challenge of performing insitu transcriptional profiling of the human ocular surface epithelial regions is obtaining samples in sufficient amounts, without contamination from adjacent tissue, as the region of interest is microscopic and closely apposed to other tissues regions. We have effectively collected ocular surface (OS) epithelial tissue samples from the Limbal Epithelial Crypt (LEC), limbus, cornea and conjunctiva of post-mortem cadaver eyes with laser microdissection (LMD) technique for gene expression studies with spotted oligonucleotide microarrays and Gene 1.0 ST arrays. Methods Human donor eyes (4 pairs for spotted oligonucleotide microarrays, 3 pairs for Gene 1.0 ST arrays) consented for research were included in this study with due ethical approval of the Nottingham Research Ethics Committee. Eye retrieval was performed within 36 hours of post-mortem period. The dissected corneoscleral buttons were immersed in OCT media and frozen in liquid nitrogen and stored at −80°C till further use. Microscopic tissue sections of interest were taken on PALM slides and stained with Toluidine Blue for laser microdissection with PALM microbeam systems. Optimisation of the laser microdissection technique was crucial for efficient and cost effective sample collection. Results The starting concentration of RNA as stipulated by the protocol of microarray platforms was taken as the cut-off concentration of RNA samples in our studies. The area of LMD tissue processed for spotted oligonucleotide microarray study ranged from 86,253 μm2 in LEC to 392,887 μm2 in LEC stroma. The RNA concentration of the LMD samples ranged from 22 to 92 pg/μl. The recommended starting concentration of the RNA samples used for Gene 1.0 ST arrays was 6 ng/5 μl. To achieve the desired RNA concentration the area of ocular surface epithelial tissue sample processed for the Gene 1.0 ST array experiments was approximately 100,0000 μm2 to 130,0000 μm2. RNA concentration of these samples ranged from 10.88 ng/12 μl to 25.8 ng/12 μl, with the RNA integrity numbers (RIN) for these samples from 3.3 to 7.9. RNA samples with RIN values below 2, that had failed to amplify satisfactorily were discarded. Conclusions The optimised protocol for sample collection and laser microdissection improved the RNA yield of the insitu ocular surface epithelial regions for effective microarray studies on spotted oligonucleotide and affymetrix platforms.
Collapse
Affiliation(s)
| | | | | | - Harminder S Dua
- Division of Ophthalmology and Visual Sciences, B-Floor, Eye & ENT Building, Queen's Medical Centre, Derby Road, Nottingham, UK.
| |
Collapse
|
|
50
|
Molvaer RK, Andreasen A, Heegaard S, Thomsen JS, Hjortdal J, Urbak SF, Nielsen K. Interactive 3D computer model of the human corneolimbal region: crypts, projections and stem cells. Acta Ophthalmol 2013; 91:457-62. [PMID: 22682073 DOI: 10.1111/j.1755-3768.2012.02446.x] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
PURPOSE This study aims to clarify the existence of and to map the localization of different proposed stem cell niches in the corneal limbal region. MATERIALS AND METHODS One human eye was cut into 2200 consecutive sections. Every other section was stained with haematoxylin and eosin, digitized at low and high magnification, aligned, 3D reconstructed and visualized using interactive 3D visualization software. The visualization software has interactive tools that make free rotations in all directions possible and makes it possible to create virtual sections independent of the original cutting plan. In all, one low-magnification and 24 high-magnification interactive 3D models were created. Immunohistochemistry against stem cell markers p63 and ΔNp63α was performed as a supplement to the 3D models. RESULTS Using the interactive 3D models, we identified three types of stem cell niches in the limbal region: limbal epithelial crypts (LECs), limbal crypts (LCs) and focal stromal projections (FSPs). In all, eight LECs, 25 LCs and 105 FSPs were identified in the limbal region. The LECs, LCs and FSPs were predominantly located in the superior limbal region with seven LECs, 19 LCs and 93 FSPs in the superior limbal region and one LEC, six LCs and 12 FSPs in the inferior limbal region. Only few LECs, LCs and FSPs were localized nasally and temporally. CONCLUSION Interactive 3D models are a powerful tool that may help to shed more light on the existence and spatial localization of the different stem cell niches (LECs, LCs and FSPs) in the corneal limbal region.
Collapse
Affiliation(s)
- Rikke K Molvaer
- Department of Ophthalmology, Aarhus University Hospital, Aarhus, Denmark
| | | | | | | | | | | | | |
Collapse
|