|
1
|
Moreno IY, Parsaie A, Gesteira TF, Coulson-Thomas VJ. Characterization of the Limbal Epithelial Stem Cell Niche. Invest Ophthalmol Vis Sci 2023; 64:48. [PMID: 37906057 PMCID: PMC10619699 DOI: 10.1167/iovs.64.13.48] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2023] [Accepted: 10/09/2023] [Indexed: 11/02/2023] Open
Abstract
Purpose Limbal epithelial stem cells (LESCs) reside within a LSC niche (LSCN). We recently identified that hyaluronan (HA) is a major constituent of the LSCN, and that HA is necessary for maintaining LESCs in the "stem cell" state, both in vitro and in vivo. Herein, we characterized the LSCN to identify key components of the HA-specific LSCN. Methods The cornea and limbal rim were dissected from mouse corneas, subjected to mRNA extraction, and sequenced using a NextSeq 500 (Illumina) and data processed using CLC Genomics Workbench 20 (Qiagen) and the STRING database to identify key components of the LSCN. Their expression was confirmed by real-time PCR, Western blotting, and immunohistochemistry. Furthermore, the differential expression of key compounds in different corneal cell types were determined with single-cell RNA sequencing. Results We identified that the hyaladherins inter-alpha-inhibitor (IαI), TSG-6 and versican are highly expressed in the limbus. Specifically, HA/HC complexes are present in the LSCN, in the stroma underlying the limbal epithelium, and surrounding the limbal vasculature. For IαI, heavy chains 5 and 2 (HC5 and HC2) were found to be the most highly expressed HCs in the mouse and human limbus and were associate with HA-forming HA/HC-specific matrices. Conclusions The LSCN contains HA/HC complexes, which have been previously correlated with stem cell niches. The identification of HA/HC complexes in the LSCN could serve as a new therapeutic avenue for treating corneal pathology. Additionally, HA/HC complexes could be used as a substrate for culturing LESCs before LESC transplantation.
Collapse
Affiliation(s)
- Isabel Y. Moreno
- College of Optometry, University of Houston, Houston, Texas, United States
| | - Arian Parsaie
- College of Optometry, University of Houston, Houston, Texas, United States
- College of Natural Science and Mathematics, University of Houston, Houston, Texas, United States
| | - Tarsis F. Gesteira
- College of Optometry, University of Houston, Houston, Texas, United States
| | | |
Collapse
|
|
2
|
Young BM, Shankar K, Tho CK, Pellegrino AR, Heise RL. Laminin-driven Epac/Rap1 regulation of epithelial barriers on decellularized matrix. Acta Biomater 2019; 100:223-234. [PMID: 31593773 DOI: 10.1016/j.actbio.2019.10.009] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2019] [Revised: 10/03/2019] [Accepted: 10/03/2019] [Indexed: 12/28/2022]
Abstract
Decellularized tissues offer a unique tool for developing regenerative biomaterials or in vitro platforms for the study of cell-extracellular matrix (ECM) interactions. One main challenge associated with decellularized lung tissue is that ECM components can be stripped away or altered by the detergents used to remove cellular debris. Without characterizing the composition of lung decellularized ECM (dECM) and the cellular response caused by the altered composition, it is difficult to utilize dECM for regeneration and specifically, engineering the complexities of the alveolar-capillary barrier. This study takes steps towards uncovering if dECM must be enhanced with lost ECM proteins to achieve proper epithelial barrier formation. To achieve this, the epithelial barrier function was assessed on dECM coatings with and without the systematic addition of several key basement membrane proteins. After comparing barrier function on collagen I, fibronectin, laminin, and dECM in varying combinations as an in vitro coating, the alveolar epithelium exhibited superior barrier function when dECM was supplemented with laminin as evidenced by trans-epithelial electrical resistance (TEER) and permeability assays. Increased barrier resistance with laminin addition was associated with upregulation of Claudin-18, E-cadherin, and junction adhesion molecule (JAM)-A, and stabilization of zonula occludens (ZO)-1 at junction complexes. The Epac/Rap1 pathway was observed to play a role in the ECM-mediated barrier function determined by protein expression and Epac inhibition. These findings revealed potential ECM coatings and molecular therapeutic targets for improved regeneration with decellularized scaffolds. STATEMENT OF SIGNIFICANCE: Efforts to produce a transplantable organ-scale biomaterial for lung regeneration has not been entirely successful to date, due to incomplete cell-cell junction formation, ultimately leading to severe edema in vivo. To fully understand the process of alveolar junction formation on ECM-derived biomaterials, this research has characterized and tailored decellularized ECM (dECM) to mitigate reductions in barrier strength or cell attachment caused by abnormal ECM compositions or detergent damage to dECM. These results indicate that laminin-driven Epac signaling plays a vital role in the stabilization of the alveolar barrier. Addition of laminin or Epac agonists during alveolar regeneration can reduce epithelial permeability within bioengineered lungs.
Collapse
Affiliation(s)
- Bethany M Young
- Department of Biomedical Engineering, Virginia Commonwealth University, 800 E. Leigh St, Room 1071, Richmond, VA 23219, United States
| | - Keerthana Shankar
- Department of Biomedical Engineering, Virginia Commonwealth University, 800 E. Leigh St, Room 1071, Richmond, VA 23219, United States
| | - Cindy K Tho
- Department of Biomedical Engineering, Virginia Commonwealth University, 800 E. Leigh St, Room 1071, Richmond, VA 23219, United States
| | - Amanda R Pellegrino
- Department of Biomedical Engineering and Nursing, Duquesne University, 600 Forbes Ave, Pittsburg, Pennsylvania 15282, United States
| | - Rebecca L Heise
- Department of Biomedical Engineering, Virginia Commonwealth University, 800 E. Leigh St, Room 1071, Richmond, VA 23219, United States; Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, 1101 East Marshall St, Richmond, Virginia 23298, United States.
| |
Collapse
|
|
3
|
Couture C, Desjardins P, Zaniolo K, Germain L, Guérin SL. Enhanced wound healing of tissue-engineered human corneas through altered phosphorylation of the CREB and AKT signal transduction pathways. Acta Biomater 2018; 73:312-325. [PMID: 29656072 DOI: 10.1016/j.actbio.2018.04.021] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2017] [Revised: 04/05/2018] [Accepted: 04/10/2018] [Indexed: 12/22/2022]
Abstract
The cornea is a transparent organ, highly specialized and unique that is continually subjected to abrasive forces and occasional mechanical or chemical trauma because of its anatomical localization. Upon injury, the extracellular matrix (ECM) rapidly changes to promote wound healing through integrin-dependent activation of specific signal transduction mediators whose contribution is to favor faster closure of the wound by altering the adhesive and migratory properties of the cells surrounding the damaged area. In this study, we exploited the human tissue-engineered cornea (hTECs) as a model to study the signal transduction pathways that participate to corneal wound healing. By exploiting both gene profiling and activated kinases arrays, we could demonstrate the occurrence of important alterations in the level of expression and activation of a few mediators from the PI3K/Akt and CREB pathways in response to the ECM remodeling taking place during wound healing of damaged hTECs. Pharmacological inhibition of CREB with C646 considerably accelerated wound closure compared to controls. This process was considerably accelerated further when both C646 and SC79, an Akt agonist, were added together to wounded hTECs. Therefore, our study demonstrate that proper corneal wound healing requires the activation of Akt together with the inhibition of CREB and that wound healing in vitro can be altered by the use of pharmacological inhibitors (such as C646) or agonists (such as SC79) of these mediators. STATEMENT OF SIGNIFICANCE Corneal wounds account for a large proportion of all visual disabilities in North America. To our knowledge, this is the first time that a tissue-engineered human cornea (hTEC) entirely produced using normal untransformed human cells is used as a biomaterial to study the signal transduction pathways that are critical to corneal wound healing. Through the use of this biomaterial, we demonstrated that human corneal epithelial cells engaged in wound healing reduce phosphorylation of the signal transduction mediator CREB while, in the mean time, they increase that of AKT. By increasing the activation of AKT together with a decrease in CREB activation, we could considerably reduce wound closure time in our punch-damaged hTECs. Considering the increasing interest given to the reconstruction of different types of tissues, we believe these results will have a strong impact on the field of tissue-engineering and biomaterials. Altering the activation status of the Akt and CREB proteins might prove to be a therapeutically interesting avenue and may also find applications in wound healing of other tissues beside the cornea, such as the skin.
Collapse
|
|
4
|
Loh IP, Fan Gaskin JC, Sherwin T, McGhee CNJ. Extreme Descemet's membrane rupture with hydrops in keratoconus: Clinical and histological manifestations. Am J Ophthalmol Case Rep 2018; 10:271-275. [PMID: 29780950 PMCID: PMC5956727 DOI: 10.1016/j.ajoc.2018.04.003] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2017] [Revised: 03/28/2018] [Accepted: 04/03/2018] [Indexed: 11/24/2022] Open
Abstract
Purpose To study the clinical and histological manifestations of an extreme Descemet's membrane rupture as a result of keratoconus. Observations Using Periodic acid-Schiff assay to study a keratoconic cornea with an extreme rupture showed that the ruptured Descemet's membrane had retracted and folded into scrolls and ridges. The dimensions of the rupture were estimated to be 3.7mm2, and the central cornea was extremely thinned with a thickness of only 260μm. Stromal scarring and loosely packed lamellae were present anterior to the scrolls and ridges. Antibodies targetting the major components of Descemet's membrane, Laminin and type IV collagen, displayed intense labelling adjacent to the scrolls where the stroma was denuded and differential expression patterns lined the ridges. Environmental scanning electron microscopy showed possible collagen deposition at the site of rupture. Conclusions and importance The specific staining patterns of laminin and type IV collagen suggest these components have an important role in re-endothelisation of the cornea. This is the first known report of spatial resolution of the topography of the Descemet's membrane rupture established by environmental scanning electron microscopic image montage.
Collapse
Affiliation(s)
- I-Ping Loh
- Department of Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and Health Sciences, University of Auckland, Park Road, Auckland, New Zealand
| | - Jennifer C Fan Gaskin
- Department of Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and Health Sciences, University of Auckland, Park Road, Auckland, New Zealand
| | - Trevor Sherwin
- Department of Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and Health Sciences, University of Auckland, Park Road, Auckland, New Zealand
| | - Charles N J McGhee
- Department of Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and Health Sciences, University of Auckland, Park Road, Auckland, New Zealand
| |
Collapse
|
|
5
|
Wan PX, Wang BW, Wang ZC. Importance of the stem cell microenvironment for ophthalmological cell-based therapy. World J Stem Cells 2015; 7:448-460. [PMID: 25815128 PMCID: PMC4369500 DOI: 10.4252/wjsc.v7.i2.448] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/27/2014] [Revised: 09/17/2014] [Accepted: 10/29/2014] [Indexed: 02/06/2023] Open
Abstract
Cell therapy is a promising treatment for diseases that are caused by cell degeneration or death. The cells for clinical transplantation are usually obtained by culturing healthy allogeneic or exogenous tissue in vitro. However, for diseases of the eye, obtaining the adequate number of cells for clinical transplantation is difficult due to the small size of tissue donors and the frequent needs of long-term amplification of cells in vitro, which results in low cell viability after transplantation. In addition, the transplanted cells often develop fibrosis or degrade and have very low survival. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPS) are also promising candidates for cell therapy. Unfortunately, the differentiation of ESCs can bring immune rejection, tumorigenicity and undesired differentiated cells, limiting its clinical application. Although iPS cells can avoid the risk of immune rejection caused by ES cell differentiation post-transplantation, the low conversion rate, the risk of tumor formation and the potentially unpredictable biological changes that could occur through genetic manipulation hinder its clinical application. Thus, the desired clinical effect of cell therapy is impaired by these factors. Recent research findings recognize that the reason for low survival of the implanted cells not only depends on the seeded cells, but also on the cell microenvironment, which determines the cell survival, proliferation and even reverse differentiation. When used for cell therapy, the transplanted cells need a specific three-dimensional structure to anchor and specific extra cellular matrix components in addition to relevant cytokine signaling to transfer the required information to support their growth. These structures present in the matrix in which the stem cells reside are known as the stem cell microenvironment. The microenvironment interaction with the stem cells provides the necessary homeostasis for cell maintenance and growth. A large number of studies suggest that to explore how to reconstruct the stem cell microenvironment and strengthen its combination with the transplanted cells are key steps to successful cell therapy. In this review, we will describe the interactions of the stem cell microenvironment with the stem cells, discuss the importance of the stem cell microenvironment for cell-based therapy in ocular diseases, and introduce the progress of stem cell-based therapy for ocular diseases.
Collapse
|
|
6
|
Yoon JJ, Ismail S, Sherwin T. Limbal stem cells: Central concepts of corneal epithelial homeostasis. World J Stem Cells 2014; 6:391-403. [PMID: 25258661 PMCID: PMC4172668 DOI: 10.4252/wjsc.v6.i4.391] [Citation(s) in RCA: 81] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/25/2014] [Revised: 08/20/2014] [Accepted: 09/01/2014] [Indexed: 02/06/2023] Open
Abstract
A strong cohort of evidence exists that supports the localisation of corneal stem cells at the limbus. The distinguishing characteristics of limbal cells as stem cells include slow cycling properties, high proliferative potential when required, clonogenicity, absence of differentiation marker expression coupled with positive expression of progenitor markers, multipotency, centripetal migration, requirement for a distinct niche environment and the ability of transplanted limbal cells to regenerate the entire corneal epithelium. The existence of limbal stem cells supports the prevailing theory of corneal homeostasis, known as the XYZ hypothesis where X represents proliferation and stratification of limbal basal cells, Y centripetal migration of basal cells and Z desquamation of superficial cells. To maintain the mass of cornea, the sum of X and Y must equal Z and very elegant cell tracking experiments provide strong evidence in support of this theory. However, several recent studies have suggested the existence of oligopotent stem cells capable of corneal maintenance outside of the limbus. This review presents a summary of data which led to the current concepts of corneal epithelial homeostasis and discusses areas of controversy surrounding the existence of a secondary stem cell reservoir on the corneal surface
Collapse
|
|
7
|
Torricelli AAM, Singh V, Santhiago MR, Wilson SE. The corneal epithelial basement membrane: structure, function, and disease. Invest Ophthalmol Vis Sci 2013; 54:6390-400. [PMID: 24078382 DOI: 10.1167/iovs.13-12547] [Citation(s) in RCA: 173] [Impact Index Per Article: 14.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
The corneal epithelial basement membrane (BM) is positioned between basal epithelial cells and the stroma. This highly specialized extracellular matrix functions not only to anchor epithelial cells to the stroma and provide scaffolding during embryonic development but also during migration, differentiation, and maintenance of the differentiated epithelial phenotype. Basement membranes are composed of a diverse assemblage of extracellular molecules, some of which are likely specific to the tissue where they function; but in general they are composed of four primary components--collagens, laminins, heparan sulfate proteoglycans, and nidogens--in addition to other components such as thrombospondin-1, matrilin-2, and matrilin-4 and even fibronectin in some BM. Many studies have focused on characterizing BM due to their potential roles in normal tissue function and disease, and these structures have been well characterized in many tissues. Comparatively few studies, however, have focused on the function of the epithelial BM in corneal physiology. Since the normal corneal stroma is avascular and has relatively low keratocyte density, it is expected that the corneal BM would be different from the BM in other tissues. One function that appears critical in homeostasis and wound healing is the barrier function to penetration of cytokines from the epithelium to stroma (such as transforming growth factor β-1), and possibly from stroma to epithelium (such as keratinocyte growth factor). The corneal epithelial BM is also involved in many inherited and acquired corneal diseases. This review examines this structure in detail and discusses the importance of corneal epithelial BM in homeostasis, wound healing, and disease.
Collapse
|
|
8
|
Nieto-Miguel T, Galindo S, Reinoso R, Corell A, Martino M, Pérez-Simón JA, Calonge M. In vitro simulation of corneal epithelium microenvironment induces a corneal epithelial-like cell phenotype from human adipose tissue mesenchymal stem cells. Curr Eye Res 2013; 38:933-44. [PMID: 23767776 DOI: 10.3109/02713683.2013.802809] [Citation(s) in RCA: 63] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
PURPOSE Transplantation of autologous corneal stem cells in not possible in cases of bilateral limbal stem cell deficiency (LSCD). To restore the ocular surface in these patients, an autologous extraocular source of stem cells is desirable to avoid dependence on deceased donor tissue and host immunosuppression of allogenic transplants. While bone marrow-derived mesenchymal stem cells (MSCs) can acquire certain characteristics of corneal epithelial cells, subcutaneous adipose tissue (AT) is more readily available and accessible. The aim of this study was to determine if extraocular human AT-derived MSCs (hAT-MSCs) can acquire in vitro some features of corneal epithelial-like cells. METHODS hAT-MSCs were isolated from human lipoaspirates and expanded up to 3-4 passages. We studied the immunophenotype of MSCs and demonstrated its multipotent capacity to differentiate toward osteoblasts, adipocytes and chondrocytes. To test the capacity of differentiation of hAT-MSCs toward corneal epithelial-like cells, hAT-MSCs were cultured on substrata of plastic or collagen IV. We used basal culture medium (BM), BM conditioned with human corneal epithelial cells (HCEcBM) and BM conditioned with limbal fibroblasts (LFcBM). RESULTS The hAT-MSCs incubated for 15 days with HCEcBM acquired more polygonal and complex morphology as evaluated by phase-contrast microscopy and flow cytometry. Additionally, the expression of transforming growth factor-β receptor CD105 and corneal epithelial marker CK12 got increased as evaluated by flow cytometry, real-time reverse-transcription polymerase chain reaction, western blot and immunostaining. These changes were absent in hAT-MSCs incubated with unconditioned BM or with LFcBM. CONCLUSIONS Corneal epithelial-like cells can be induced from extraocular hAT-MSCs by subjecting them to an in vitro microenvironment containing conditioning signals derived from differentiated human corneal epithelial cells. Our results suggest that hAT-MSCs could provide a novel source of stem cells that hold the potential to restore sight lost in patients suffering from bilateral ocular surface failure due to LSCD.
Collapse
Affiliation(s)
- Teresa Nieto-Miguel
- Ocular Surface Group, IOBA-Institute for Applied Ophthalmobiology, University of Valladolid, Valladolid, Spain.
| | | | | | | | | | | | | |
Collapse
|
|
9
|
Ordonez P, Di Girolamo N. Limbal epithelial stem cells: role of the niche microenvironment. Stem Cells 2012; 30:100-7. [PMID: 22131201 DOI: 10.1002/stem.794] [Citation(s) in RCA: 90] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
The cornea contains a reservoir of self-regenerating epithelial cells that are essential for maintaining its transparency and good vision. The study of stem cells in this functionally important organ has grown over the past four decades, partly due to the ease with which this tissue is visualized, its accessibility with minimally invasive instruments, and the fact that its stem cells are segregated within a transitional zone between two functionally diverse epithelia. While human, animal, and ex vivo models have been instrumental in progressing the corneal stem cell field, there is still much to be discovered about this exquisitely sensitive window for sight. This review will provide an overview of the human cornea, where its stem cells reside and how components of the microenvironment including extracellular matrix proteins and their integrin receptors are thought to govern corneal stem cell homeostasis.
Collapse
Affiliation(s)
- Paula Ordonez
- Inflammation and Infection Research Centre, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, Australia
| | | |
Collapse
|
|
10
|
Sakurai M, Hayashi R, Kageyama T, Yamato M, Nishida K. Induction of putative stratified epithelial progenitor cells in vitro from mouse-induced pluripotent stem cells. J Artif Organs 2011; 14:58-66. [PMID: 21298309 DOI: 10.1007/s10047-010-0547-3] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2010] [Accepted: 12/06/2010] [Indexed: 10/18/2022]
Abstract
Induced pluripotent stem (iPS) cells generally exhibit a normal karyotype, are transcriptionally and epigenetically similar to embryonic stem (ES) cells, and maintain the potential to differentiate into derivatives of all germ layers. Recently, the use of different types of cell or tissue derived from iPS cells for transplantation has become a possibility. However, the differentiation of epithelial lineages from iPS cells has not yet been demonstrated. We attempted to establish a culture technique for the induction of epithelial progenitors from mouse iPS cells. Mouse iPS cells were cultured on dishes coated with type IV collagen in keratinocyte culture medium (KCM) supplemented with or without bone morphogenic protein-4 (BMP-4) or combined with pretreatment of retinoic acid (RA) and BMP-4 in the undifferentiated state. Markers for undifferentiated cells (Oct3/4, Nanog) and for differentiation (p63, cytokeratin14) were analyzed by immunofluorescence staining and real-time RT-PCR. Putative epithelial progenitors were successfully induced in vitro from iPS cells. These progenitors expressed p63, a transcription factor necessary for maintenance of regenerative epithelia and cytokeratin 14 constitutively present in the basal layer of stratified epithelia. Enhancement of putative epithelial progenitor commitment was observed when cultured in KCM with BMP-4 following pretreatment of RA and BMP-4. The differentiation efficiency of putative epithelial progenitors from iPS cell cultures was similar to that of ES cell cultures. This report is the first to demonstrate in vitro differentiation of iPS cells into putative epithelial progenitors. These iPS-derived putative epithelial progenitors provide a powerful tool for understanding the mechanisms of epithelial lineage differentiation.
Collapse
Affiliation(s)
- Miharu Sakurai
- Department of Ophthalmology, Tohoku University School of Medicine, 1-1 Seiryomachi, Aoba-ku, Sendai, 980-8574, Japan
| | | | | | | | | |
Collapse
|
|
11
|
Induction of corneal epithelium-like cells from cynomolgus monkey embryonic stem cells and their experimental transplantation to damaged cornea. Cornea 2011; 29:432-8. [PMID: 20164754 DOI: 10.1097/ico.0b013e3181b9ffcc] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
PURPOSE We previously reported the successful transplantation of corneal epithelium-like cells derived from mouse embryonic stem (ES) cells onto injured mouse cornea. Here, we tested whether nonhuman primate ES cells have ability to differentiate into corneal epithelial cells and whether monkey ES cell-derived corneal epithelium-like cells were applicable for the experimental transplantation to damaged cornea. METHODS Monkey ES cells were cultivated on type IV collagen-coated dishes for various days to induce differentiation into corneal epithelium-like cells. The differentiation was evaluated by reverse transcription-polymerase chain reaction and immunostaining. The corneal epithelium-like cells were transplanted to the injured mouse cornea. Reconstitution of the corneal epithelium was evaluated by immunostaining. RESULTS The cells cultured on type IV collagen showed cobblestone-like appearance resembling epithelial cells. They expressed messenger RNA of pax6, p63, E-cadherin, CD44, proliferating cell nuclear antigen, keratin 3, and keratin 12. Protein expressions of pax6, keratin 3/12, p63, proliferating cell nuclear antigen, E-cadherin, and CD44 were confirmed by immunostaining. When the corneal epithelium-like cells were transplanted, they adhered to the corneal stroma, leading to formation of multiple cell layers. The grafted cells were stained with anti-human nuclear protein antibody, which cross-reacted with nuclei of monkey cells but not with those of mouse cells. They retained the expressions of keratin 3/12, E-cadherin, and CD44. CONCLUSIONS We induced corneal epithelium-like cells from monkey ES cells with moderate efficiency. The cells were successfully transplanted onto the injured mouse cornea. This is the first demonstration that nonhuman primate ES cells were induced to differentiate into corneal epithelium-like cells, which were applicable for transplantation to an animal model of corneal injury.
Collapse
|
|
12
|
Kabosova A, Azar DT, Bannikov GA, Campbell KP, Durbeej M, Ghohestani RF, Jones JCR, Kenney MC, Koch M, Ninomiya Y, Patton BL, Paulsson M, Sado Y, Sage EH, Sasaki T, Sorokin LM, Steiner-Champliaud MF, Sun TT, Sundarraj N, Timpl R, Virtanen I, Ljubimov AV. Compositional differences between infant and adult human corneal basement membranes. Invest Ophthalmol Vis Sci 2007; 48:4989-99. [PMID: 17962449 DOI: 10.1167/iovs.07-0654] [Citation(s) in RCA: 142] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
PURPOSE Adult human corneal epithelial basement membrane (EBM) and Descemet's membrane (DM) components exhibit heterogeneous distribution. The purpose of the study was to identify changes of these components during postnatal corneal development. METHODS Thirty healthy adult corneas and 10 corneas from 12-day- to 3-year-old children were studied by immunofluorescence with antibodies against BM components. RESULTS Type IV collagen composition of infant corneal central EBM over Bowman's layer changed from alpha1-alpha2 to alpha3-alpha4 chains after 3 years of life; in the adult, alpha1-alpha2 chains were retained only in the limbal BM. Laminin alpha2 and beta2 chains were present in the adult limbal BM where epithelial stem cells are located. By 3 years of age, beta2 chain appeared in the limbal BM. In all corneas, limbal BM contained laminin gamma3 chain. In the infant DM, type IV collagen alpha1-alpha6 chains, perlecan, nidogen-1, nidogen-2, and netrin-4 were found on both faces, but they remained only on the endothelial face of the adult DM. The stromal face of the infant but not the adult DM was positive for tenascin-C, fibrillin-1, SPARC, and laminin-332. Type VIII collagen shifted from the endothelial face of infant DM to its stromal face in the adult. Matrilin-4 largely disappeared after the age of 3 years. CONCLUSIONS The distribution of laminin gamma3 chain, nidogen-2, netrin-4, matrilin-2, and matrilin-4 is described in the cornea for the first time. The observed differences between adult and infant corneal BMs may relate to changes in their mechanical strength, corneal cell adhesion and differentiation in the process of postnatal corneal maturation.
Collapse
Affiliation(s)
- Andrea Kabosova
- Ophthalmology Research Laboratories, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA
| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
13
|
Tuori A, Virtanen I, Uusitalo H. Lectin binding in the anterior segment of the bovine eye. ACTA ACUST UNITED AC 1994. [DOI: 10.1007/bf02388636] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
|
|
14
|
Affiliation(s)
- G E Marshall
- Tennent Institute of Ophthalmology, Western Infirmary, Glasgow
| | | | | |
Collapse
|