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Growth of limbal epithelial cells on corneal stroma. A. The de-epithelial donor corneal stroma contained Bowman's layer with entire basement membrane shown by HE and Laminin 5 staining; B-E. Limbal epithelial cells grew from one to multiple layers from a fresh limbal explant. doi:10.1371/journal.pone.0049571.g002
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Tissue engineering holds great promise for corneal transplantation to treat blinding diseases. This study was to explore the use of natural corneal stroma as an optimal substrate to construct a native like corneal equivalent. Human corneal epithelium was cultivated from donor limbal explants on corneal stromal discs prepared by FDA approved Horizon...
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Context 1
... epithelium with 5-7 multi-layers was observed in all limbal explant cultures on the corneal stromal discs, which resembles the native-like human corneal epithelial mor- phology and structure in vivo, as examined by cross-sections of the cultures. As shown in Fig 2A by Hematoxylin and Eosin (HE) Staining and laminin 5 immunostaining, the prepared corneal stromal discs contain an intact basement membrane that overlies Bowman's layer after the donors' corneal epithelium has been completely removed. HLECs grew well on the stromal discs from one layer in early days (Fig 2B) to form multi-layer epithelium in 2-3 weeks (Fig 2 C-E). ...
Context 2
... shown in Fig 2A by Hematoxylin and Eosin (HE) Staining and laminin 5 immunostaining, the prepared corneal stromal discs contain an intact basement membrane that overlies Bowman's layer after the donors' corneal epithelium has been completely removed. HLECs grew well on the stromal discs from one layer in early days (Fig 2B) to form multi-layer epithelium in 2-3 weeks (Fig 2 C-E). The cells in the basal layer were polygonal and well-organised, and cells became more flattened in the suprabasal and superficial layers. ...
Context 3
... shown in Fig 2A by Hematoxylin and Eosin (HE) Staining and laminin 5 immunostaining, the prepared corneal stromal discs contain an intact basement membrane that overlies Bowman's layer after the donors' corneal epithelium has been completely removed. HLECs grew well on the stromal discs from one layer in early days (Fig 2B) to form multi-layer epithelium in 2-3 weeks (Fig 2 C-E). The cells in the basal layer were polygonal and well-organised, and cells became more flattened in the suprabasal and superficial layers. ...
Context 4
... 5, a ligand of integrins a3b1 and a6b4, is essential constituent components of the lamina lucida of the corneal basement membrane. The positive staining of Laminin 5 in Fig 2 showed the integrated basement membrane of our native-like corneal equivalent. Like other basement mem- branes in the body, the corneal basement membrane anchors epithelial cells and provides positional information for healing, repair and tissue regeneration [16,17]. ...
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Purpose
Although the existence of the limbal stem cell ( LSC ) niche is accepted, precise knowledge of its 3D architecture remains incomplete.
Methods
The LSC niche was explored on freshly excised corneoscleral rims from human donors (n = 47), pigs (n = 15) and mice (n = 27) with full‐field optical coherence microscopy ( FFOCM ).
Results
Limbal c...
Citations
... Marquage par immunofluorescence indirecte de l'involucrine sur des coupes de l'épithélium cornéen humain (3,4).D'après(Lin et al. 2012) ...
L'épithélium cornéen, tout comme l'épiderme, forme une barrière complexe qui protège l'organisme contre les agressions de l'environnement. Dans l'épiderme, cette fonction est assurée en grande partie par la couche cornée qui correspond à l'étape finale, de mieux en mieux caractérisée, de la différenciation du kératinocyte. Par contre, le programme de différenciation de l'épithélium de la cornée est peu étudié. Pourtant, les premières manifestations de diverses pathologies cornéennes apparaissent au niveau de l'épithélium, et leur dépistage précoce pourrait modifier notablement leur prise en charge. Cela pourrait être le cas du kératocône, une ectasie cornéenne plurifactorielle et rare, associée à de nombreux remaniements de surface engendrant une dégradation progressive et irréversible de la vision. L'objectif de mon travail de thèse a été de caractériser le programme de différenciation terminale de l'épithélium cornéen en situation normale et dans le cas du kératocône en prenant comme référence les gènes de différenciation tardive de l' épiderme. Tout d'abord, j'ai analysé l'expression de 3 groupes de gènes représentatifs des étapes de la différenciation épidermique (gènes des kératines, des protéines des complexes jonctionnels et des composants de l'enveloppe cornée, une structure péricellulaire résistante qui remplace la membrane plasmique des kératinocytes épidermiques lors de l'étape ultime de leur différenciation) dans l'épithélium cornéen contrôle en utilisant des techniques de RT-qPCR, Western Blot et immunohistologie. J'ai montré que l'épithélium de la cornée exprime un grand nombre des gènes de l'épiderme, mais pas ceux de la cornéodesmosine, la filaggrine, l'hornerine et des kératines 1/10. En parallèle, j'ai recherché les mêmes gènes dans l'épithélium de la cornée de deux cohortes de patients atteints de kératocône. J'ai montré que la kératine 16 est exprimée dans les cornées contrôles et disparaît dans le kératocône. Curieusement, de nombreux gènes codant pour les composants de l'enveloppe cornée sont exprimés dans l'épithélium cornéen comme ceux de l'involucrine, des Small Proline- Rich Proteins (SPRRs) et des protéines de la plaque desmosomale. De plus leur expression diminue dans l'épithélium cornéen kératoconique, avec une localisation subcellulaire différente pour l'involucrine. Pourtant, l'enveloppe cornée n'est pas produite par les cellules épithéliales différenciées de la cornée. Les protéines SPRRs étant aussi impliquées dans la détoxification des espèces réactives de l'oxygène in vitro, nous avons supposé qu'elles jouent ce rôle dans l'épithélium cornéen. Enfin, j'ai complété mes analyses avec l'étude des principaux éléments de la voie NRF2 de protection au stress oxydatif : heme oxygenases 1 et 2, nuclear factor erythroid 2-related factor 2, lysyl oxydase, KEAP1, etc. Plusieurs de ces gènes sont exprimés plus faiblement dans les cornées du kératocône par rapport aux cornées contrôles. Ces résultats ont permis, d'une part, de caractériser le programme de différenciation des cellules épithéliales de la cornée et, d'autre part, de mieux comprendre la physiopathologie du kératocône en proposant l'implication d'un déficit de la réponse au stress oxydatif.
... 6 As there is no current consensus about distinct speciesspecific markers to detect SCs and their subsequent differentiation stages (eg, transient amplifying cells, TACs) in the cornea, only a combination of differentiation, and putative SC-associated markers can provide a suitable approach for identifications of SCs. 11,18 Cytokeratin (CK) proteins belong to the intermediate filament family of cytoskeletal proteins and contribute crucially to the maintenance of cell structure and intercellular adhesion. 19 Cytokeratins are valuable diagnostic makers, as their changes are associated with epithelial cell differentiation, response to epithelial injury and epithelial regeneration. ...
... 20 In humans, p63, nerve growth factor (NGF), ABCG2 and higher expression levels of epithelial growth factor receptor (EGFR) have been reported to be positive markers for the detection of corneal SCs. 18,21,22 So far, only p63 has been studied in the equine cornea and positive cells have been detected in the limbus and the crypt-like structure. 3,7 The aim of this study was to analyze the structure of the equine corneal epithelium including the corneo-scleral junction using light-and electron microscopy. ...
... 10,27 Interestingly, this is in contrast to the human cornea, where the E-cadherin/β-catenin complex has been described in suprabasal epithelial cell layers and in less differentiated limbal cells. 18,21,28 Therefore, species-specific differences seem to exist for the localization of the E-cadherin/β-catenin complex in the corneal and limbal epithelium. ...
Objective
The morphology of the corneal epithelium in two age groups of horses is described. Distribution patterns of proliferation‐, differentiation‐, stem cell‐associated markers and cell junction proteins were assessed.
Methods
Corneal samples from 12 horses (six foals and six adult horses) were analyzed after H&E staining and immunohistochemistry using the following antibodies: E‐cadherin, β‐catenin, Connexin 43 (Cx43), tight junction protein 1 (TJP1), cytokeratin (CK) 14, CK 19, CK 3, CK 10, vimentin, Ki67, p63, nerve growth factor (NGF), ABCG2, and epithelial growth factor receptor. Semiquantitative analysis of crypt, limbal, peripheral, and central zone was performed. Semithin and ultrathin sections were used for ultrastructural evaluation of the epithelium.
Results
The height of the epithelium varied between age groups and crypts were consistently present. In the peripheral and central epithelium, three types of basal cells resembling a pseudostratified epithelium were characterized. Potential stem cell markers (CK 14, p63, NGF, and ABCG2) were present in all zones with decreasing frequency toward the center. Cornea‐specific differentiation marker CK 3 was not expressed in the most basal cell layer of the limbal epithelium. E‐cadherin, β‐catenin, and Cx43 revealed a similar apico‐lateral signal pattern throughout the entire epithelium; only TJP1 was additionally seen at the basal surface.
Conclusions
This study presents a systematic semiquantitative evaluation of the equine corneal epithelium, showing the presence of crypts as potential stem cell niche with CK 14, p63, NGF, and ABCG2 as relevant markers for cells with regenerative capacity. The pseudostratified arrangement of the basal layer was a unique finding.
... The first successful clinical outcome of cultivated corneal epithelial transplantation in two patients was published by Pellegrini and colleagues in 1997. After this initial report, various researchers have reporting transplantation of in vitro cultivated corneal epithelial cells on scaffolds, such as amniotic membrane, fibrin, and collagen hydrogel, with a great impact for clinic corneal transplantation [39,40]. Tsai et al. 2000 in their review concluded that transplantation of autologous limbal epithelial cells cultured on amniotic membrane is a simple and effective method of reconstructing the corneal [41]. ...
Blinding corneal scarring is usually treated with allogeneic graft tissue. Nevertheless, the global shortage of donors leaves millions of patients in need of therapy. Traditional tissue engineering strategies involves the combination of cells, growth factors, and scaffolds that can supply cellular biological components allowing to restore the tissue function. The mesenchymal stem cells found in the limbal stroma (L-MSCs) have a self-renewal potential for multilineage differentiation. Thus, in this work we compared the potential of human amniotic membrane (hAM) and porcine small intestine submucosa (SIS) as scaffolds for L-MSCs, aiming at potential applications in corneal regeneration. For that, L-MSCs were seeded on hAM and SIS and we analyzed their viability, actin cytoskeleton, nuclei morphology, cell density, adhesion and surface markers. Our results showed that cells adhered and integrated into both membranes with a high cell density, an important characteristic for cell therapy. However, due to its transparency, the hAM allowed a better observation of L-MSCs. In addition, the analysis of surface markers expression on L-MSCs after two weeks showed a slight increase in the percentages of negative markers for MSCs grown on SIS membrane. Thus, considering a long-term culture, the hAM was considered better in maintaining the MSCs phenotype. Regarding the function as scaffolds, SIS was as efficient as the amniotic membrane, considering that these two types of biological matrices maintained the cell viability, actin cytoskeleton, nuclei morphology and mesenchymal phenotype, without causing cell death. Therefore, our data in vitro provides evidence for future pre-clinical studies were these membranes can be used as a support to transport mesenchymal stem cells to the injured area, creating a kind of temporary curative, allowing the release of bioactive molecules, such as cytokines and growth factors and then promoting the tissue regeneration, both in human and veterinary medicine.
... Corneal epithelium layers have also been established in vitro with great success using cell-based [114,115], membrane-based [116,117], or scaffold-supported approaches [116,118] (Fig. 5). Furthermore, engineered epithelial tissue sheets, established by the use of polymeric substrates, have been clinically evaluated using autologous oral mucosal epithelium [114]. ...
Experimental cell research studying three-dimensional (3D) tissues in space and on Earth using new techniques to simulate microgravity is currently a hot topic in Gravitational Biology and Biomedicine. This review will focus on the current knowledge of the use of stem cells and specialized cells for tissue engineering under simulated microgravity conditions. We will report on recent advancements in the ability to construct 3D aggregates from various cell types using devices originally created to prepare for spaceflights such as the random positioning machine (RPM), the clinostat, or the NASA-developed rotating wall vessel (RWV) bioreactor, to engineer various tissues such as preliminary vessels, eye tissue, bone, cartilage, multicellular cancer spheroids, and others from different cells. In addition, stem cells had been investigated under microgravity for the purpose to engineer adipose tissue, cartilage, or bone. Recent publications have discussed different changes of stem cells when exposed to microgravity and the relevant pathways involved in these biological processes. Tissue engineering in microgravity is a new technique to produce organoids, spheroids, or tissues with and without scaffolds. These 3D aggregates can be used for drug testing studies or for coculture models. Multicellular tumor spheroids may be interesting for radiation experiments in the future and to reduce the need for in vivo experiments. Current achievements using cells from patients engineered on the RWV or on the RPM represent an important step in the advancement of techniques that may be applied in translational Regenerative Medicine.
... As an alternative to donor cornea, synthetic keratoprosthesis are an option; however, complications can be severe (Akpek, Alkharashi, Hwang, Ng, & Lindsley, 2014;Ilhan-Sarac & Akpek, 2005). With no other biological options for surgery, corneal tissue engineering has become an increasingly important research focus (Ghezzi, Rnjak-Kovacina, & Kaplan, 2015;Lin et al., 2012;Shah, Brugnano, Sun, Vase, & Orwin, 2008). ...
With insufficient options to meet the clinical demand for cornea transplants, one emerging area of emphasis is on cornea tissue engineering. In the present study, the goal was to combine the corneal stroma and epithelium into one co-culture system, in order to monitor both human corneal stromal stem cell (hCSSC) and human corneal epithelial cell (hCE) growth and differentiation into keratocytes and differentiated epithelium in these 3D tissue systems in vitro. Co-culture conditions were first optimized, including the medium, air/liquid interface culture, and surface topography and chemistry of biomaterial scaffold films based on silk protein. The silk was used as scaffolding for both stromal and epithelial tissue layers because it is cell compatible, can be surface patterned, and is optically clear. Next, the effects of proliferating and differentiating hCEs and hCSSCs in this in vitro system were studied, including the effects on cell proliferation, matrix formation by immunochemistry, and gene expression by RT-qPCR. The incorporation of both cell types into the co-culture system demonstrated more complete differentiation and growth for both cell types compared to the corneal stromal cells and corneal epithelial cells alone. Silk films for corneal epithelial culture were optimized to combine a 4.0 micron-scale surface pattern with bulk-loaded collagen type IV. Differentiation of each cell type was in evidence based on increased expression of corneal stroma and epithelial proteins and transcript levels after 6 weeks in co-culture on the optimized silk scaffolds. This article is protected by copyright. All rights reserved.
... Among the different strategies utilized by different groups, the transplantation of limbal epithelia whether surgically obtained from the contralateral limbus [205,214], from donor tissue [215,216], or bioengineered epithelia [217][218][219][220] are, by now, the most valuable tools for corneal surface reconstruction and the relief and cure of limbal stem cell deficiency (LSCD). Independently from variability in patient selection, the type of culture techniques, source of donor tissue, biocompatibility of materials and surgical technique, reconstruction of the corneal surface has shown a clinical success of about 60-80 % [214,[221][222][223][224]. In most cases, the outcome seems to be related to the presence of limbal stem cells in the tissue used as source for the manufacturing of the grafts, and supports the benefit of the use of limbal stem cells in ophthalmic therapy. ...
... Based on the formation of multilayered epithelia expressing corneal differentiation markers [218,220,225] and its successful clinical application [231,232], the use of stem cells different from limbal stem cells shows a high potential for corneal reconstruction. However, because stem cell markers persist in the transient amplifying cell population and in the early differentiating cells [105,106], surgeons are not certain about the cell types transplanted onto the patients. ...
Stem cell niche may be described as an anatomically defined and protected location that provides housing, positioning information and signaling inputs necessary to support normal stem cell activity. Based on the distribution of the differentiation-linked keratins, proliferative potential and wound healing abilities in ocular surface, it was proposed that an anatomical structure, the limbus, was the presumptive site of residence of corneal stem cells. Further analysis determined that the limbus contains a specific anatomical structure that probably provides the microenvironmental characteristics that correspond to the stem cell niche. This structure was termed as the Limbal Epithelial Crypt (LEC). Accumulated evidences show that the LEC is the site where stem cells interact directly and/or indirectly with at least six different cell types: epithelial, stromal, Langerhans cells, melanocytes, and telocytes. In addition, a rich and distinctive vasculature as well as an extensive neural network exist at limbal niches. These cell types, together with growth factors, cytokines and specific components of the Extracellular Matrix establish the conditions for the regulated growth, migration and delayed differentiation of the corneal stem cells. In spite of the wide variety of molecular markers described for limbal epithelial cells, it has been extremely difficult to isolate stem cells. This is explained by the persistence of stem cell markers in the transient amplifying cell population and in the early differentiating cells. Consequently, the use of stem cells in ophthalmic therapy shows variable outcomes and research must be increased before a formal clinical use. Nevertheless, the analysis of the different cells and factors involved in stem cell regulation, may help the development of new therapies based on the interference or stimulation of the signaling pathways and microenvironmental components that control limbal stem cells.
... The epithelium was detached from the limbal explants with dispase and seeded on amniotic membrane with a final yield of 2-4 layers of epithelium. This suggests that limbal cells may have been differentiated as what happens when stem cells become multilayered [49]. In two cases at the time of surgery, a 6-mm central disk was punched out of the amniotic membrane upon which the cells were cultured, which would have eliminated a significant number of transplanted cells. ...
Objective. To evaluate outcomes of cultivated limbal epithelial transplantation (CLET) for management of ocular surface failure due to limbal stem cell deficiency (LSCD). Design. Prospective, noncomparative, interventional case series and extensive comparison with recent similar studies. Participants. Twenty eyes with LSCD underwent CLET (11 autologous; 9 allogeneic) and were followed up for 3 years. Etiologies were divided into 3 prognostic categories: Group 1, chemical injuries (7 eyes); Group 2, immune-based inflammation (4 eyes); and Group 3, noninflammatory diseases (9 eyes). Intervention. Autologous and allogeneic limbal epithelial cells were cultivated on amniotic membranes and transplanted. Evaluations were based on clinical parameters, survival analysis, and in vivo confocal microscopy (IVCM). European Union Tissues/Cells Directive and good manufacturing procedures were followed.
Main Outcome Measures. Improved clinical parameters, absence of epithelial defects, and improved central corneal epithelial phenotype. Results. Success rate was 80% at 1-2 years and 75% at 3 years. Autografts and allografts had similar survival. Success rate was significantly lower in prognostic Group 1 (42.9%) than in Groups 2-3 (100% each). All clinical parameters improved substantially. By IVCM, 80% of cases improved in epithelial status. Conclusions. CLET improved corneal epithelium quality, with subsequent improvement in symptoms, quality of life, and vision. These results confirm that CLET is a valid therapy for ocular surface failure.
... [37][38][39] Alternatively, human donor corneal stromal tissues have been proposed as substrates for human corneal epithelium growth, displaying, in vitro, features similar to the native limbal epithelium. 40 The lack of corneal tissue donor availability significantly affects its clinical potential for corneal reconstruction. Among natural polymers, reconstituted type I collagen hydrogels can successfully encapsulate human corneal limbal epithelial cells, resulting in functional stratified epithelial layers. ...
A lack of healthy transplantable tissue to treat corneal blindness has led researchers to investigate the development and application of different scaffold materials for corneal tissue engineering and regeneration. In this study, hydrogels fabricated from decellularized corneal extracellular matrix were developed as a new approach to corneal stroma tissue regeneration. Porcine corneas were decellularized using a technique that combined freeze-thaw cycles with a nuclease treatment. The corneas were then freeze-dried, milled and digested in an acidic pepsin solution that was used to form a hydrogel after adjusting the pH and gelation temperature. The resultant corneal matrix hydrogels (CMHs) were seeded with human corneal stromal cells and cultured for several days. When compared to collagens hydrogels, CMHs had superior optical transparency, similar mechanical properties and were better able to retain the stromal cells native keratocyte phenotype. The CMHs also supported cell viability and proliferation and contained sulfated glycosaminoglycan's (sGAG), a vital constituent for maintaining corneal transparency. These results suggest the CMHs could provide an exceptional biomaterial for corneal stroma regeneration.
... [37][38][39] Alternatively, human donor corneal stromal tissues have been proposed as substrates for human corneal epithelium growth, displaying, in vitro, features similar to the native limbal epithelium. 40 The lack of corneal tissue donor availability significantly affects its clinical potential for corneal reconstruction. Among natural polymers, reconstituted type I collagen hydrogels can successfully encapsulate human corneal limbal epithelial cells, resulting in functional stratified epithelial layers. ...
Abstract To increase the epithelial proliferation of an auto-tissue-engineered lamellar cornea, 3.0×106 corneal epithelial cells were combined with 3×105 mouse embryonic stem cells pre-transfected with the HSV-tk gene (CECs+ESCs-TK group), and 3.3×106 corneal epithelial cells (CECs group) were seeded between the acellular porcine corneal stroma and the amniotic membrane using the centrifugal cell seeding method. After 4 days of perfusion culture (treatment with ganciclovir starting on day 2), a thicker corneal epithelium (4-5 layers) formed in the CECs+ESCs-TK group compared to that observed in the CECs group (2-3 layers). More stem/progenitor cell (K3-, p63+, ABCG2+ and integrin-β1+) and proliferation phenotypes (Ki67+) were measured in the CECs+ESCs-TK group compared to the CECs group using immunofluorescence staining, real-time qRT-PCR and flow cytometry. Consistent with these findings, the colony-forming efficiency and cellular doubling time were significantly different between the CECs+ESCs-TK group (16.18±3.98%, 28.45±2.03 h) and CECs group (11.96±2.60%, 36.3±1.15 h). In a rabbit lamellar transplantation model, the CECs+ESCs-TK group had better epithelium barrier functions and wound healing abilities compared with the CECs group. Furthermore, ESCs-TK could be completely and safely removed by ganciclovir. Thus, the ESCS-TK coculture system could serve as a potential strategy for corneal tissue engineering. Key words: corneal tissue engineering; proliferation; corneal epithelial cell; embryonic stem cells; transplantation.
... Using this process, 2-6 layers of stratified epithelium could be formed on different scaffolds [15][16][17][18][19][20][21][22], adhesive molecules and cell-cell junctions have been verified by immunofluorescence staining [17] and transmission electron microscopy [17][18][19]. The classical static culture method could promote epithelium growth and produce a favorable morphological outcome on amniotic membrane [23], compressed collagen [18], silk fibroin [24], corneal stromal lamella discs [25], and so on. ...
To construct an auto-tissue-engineered lamellar cornea (ATELC) for transplantation, based on acellular porcine corneal stroma and autologous corneal limbal explants, a dynamic culture process, which composed of a submersion culture, a perfusion culture and a dynamic air-liquid interface culture, was performed using appropriate parameters. The results showed that the ATELC-Dynamic possessed histological structure and DNA content that were similar to native lamellar cornea (NLC, p>0.05). Compared to NLC, the protein contents of zonula occludens-1, desmocollin-2 and integrin β4 in ATELC-Dynamic reached 93%, 89% and 73%, respectively. The basal cells of ATELC-Dynamic showed a better differentiation phenotype (K3-, P63+, ABCG2+) compared with that of ATELC in static air-lift culture (ATELC-Static, K3+, P63-, ABCG2-). Accordingly, the cell-cloning efficiency of ATELC-Dynamic (9.72±3.5%) was significantly higher than that of ATELC-Static (2.13±1.46%, p<0.05). The levels of trans-epithelial electrical resistance, light transmittance and areal modulus variation in ATELC-Dynamic all reached those of NLC (p>0.05). Rabbit lamellar keratoplasty showed that the barrier function of ATELC-Dynamic was intact, and there were no signs of epithelial shedding or neovascularization. Furthermore, the ATELC-Dynamic group had similar optical properties and wound healing processes compared with the NLC group. Thus, the sequential dynamic culture process that was designed according to corneal physiological characteristics could successfully reconstruct an auto-lamellar cornea with favorable morphological characteristics and satisfactory physiological function.
