Personne : Guérin, Sylvain
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Université Laval. Département d'ophtalmologie et d'oto-rhino-laryngologie - chirurgie cervico-faciale
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- PublicationAccès libreReconstruction of a human cornea by the self-assembly approach of tissue engineering using the three native cell types(Éditeur non identifié, 2010-10-29) Giasson, Claude-J.; Guérin, Sylvain; Germain, Lucie; Audet, Caroline; Auger, François A.; Uwamaliya, Jeanne d'Arc; Proulx, Stéphanie; Carrier, Patrick; Deschambeault, AlexandrePurpose: The purpose of this study was to produce and characterize human tissue-engineered corneas reconstructed using all three corneal cell types (epithelial, stromal, and endothelial cells) by the self-assembly approach. Methods: Fibroblasts cultured in medium containing serum and ascorbic acid secreted their own extracellular matrix and formed sheets that were superposed to reconstruct a stromal tissue. Endothelial and epithelial cells were seeded on each side of the reconstructed stroma. After culturing at the air-liquid interface, the engineered corneas were fixed for histology and transmission electron microscopy (TEM). Immunofluorescence labeling of epithelial keratins, basement membrane components, Na+/K+-ATPase α1, and collagen type I was also performed. Results: Epithelial and endothelial cells adhered to the reconstructed stroma. After 10 days at the air-liquid interface, the corneal epithelial cells stratified (4 to 5 cell layers) and differentiated into well defined basal and wing cells that also expressed Na+/K+-ATPase α1 protein, keratin 3/12, and basic keratins. Basal epithelial cells from the reconstructed epithelium formed many hemidesmosomes and secreted a well defined basement membrane rich in laminin V and collagen VII. Endothelial cells formed a monolayer of tightly-packed cells and also expressed the function related protein Na+/K+-ATPase α1. Conclusions: This study demonstrates the feasibility of producing a complete tissue-engineered human cornea, similar to native corneas, using untransformed fibroblasts, epithelial and endothelial cells, without the need for exogenous biomaterial.
- PublicationAccès libreRole of the extracellular matrix proteins in the resistance of SP6.5 uveal melanoma cells toward cisplatin(Spandidos Publications, 2005-03-01) Paquet-Bouchard, Carine; Guérin, Sylvain; Germain, Lucie; Bérubé, Mélanie; Giroux-Talbot, Mariève; Petitclerc, Éric; Collin, CharlesUveal melanoma is the most frequent primary intraocular tumor in the adult population. This malignancy has a high mortality rate and responds poorly to existing chemotherapy. Recently, the tumor environment has been found to exert a profound influence on drug response through cell interaction with components from the extracellular matrix (ECM). In the present study, we investigated whether individual components from the ECM may affect cell survival and/or cell death induced by the cytotoxic agent cisplatin on the SP6.5 uveal melanoma cell line. Tumor cells were shown by immunofluorescence analyses to be surrounded by the ECM proteins fibronectin (FN), type IV collagen (CIV) and laminin (LM), both at the primary and metastatic sites. Binding of SP6.5 cells to FN, LM and CIV is primarily dictated by the expression of membrane bound integrins from the beta1 family as revealed by cell adhesion assays conducted on ECM-coated culture plates. Analysis of cell death by flow cytometry demonstrated that culturing SP6.5 cells in the presence of FN, CIV and LM, substantially reduced the percentage of cells undergoing apoptosis after cisplatin treatment when compared with those seeded on a non-permissive matrix. These results suggest that adhesion of the SP6.5 uveal melanoma cells to the ECM proteins FN, CIV and LM might therefore confer resistance to the chemotherapeutic agent cisplatin. The cellular resistance induced by the ECM proteins toward cisplatin could explain in part the local recurrence of metastasis derived from uveal melanoma often observed clinically after chemotherapy.
- PublicationRestreintA role for DLK in microtubule reorganization to the cell periphery and in the maintenance of desmosomal and tight junction integrity(Elsevier Science, 2016-10-07) Bidoggia, Julie; Guérin, Sylvain; Germain, Lucie; Larouche, Danielle; Blouin, Richard; Hirai, Syu-Ichi; Simard-Bisson, CarolyneDual leucine zipper-bearing kinase (DLK) is an inducer of keratinocyte differentiation, a complex process also involving microtubule reorganization to the cell periphery. However, signaling mechanisms involved in this process remain to be elucidated. Here, we demonstrate that DLK enhances and is required for microtubule reorganization to the cell periphery in human cell culture models and in Dlk knockout mouse embryos. In tissue-engineered skins with reduced DLK expression, cortical distribution of two microtubule regulators, LIS1 and HSP27, is impaired as well as desmosomal and tight junction integrity. Altered cortical distribution of desmosomal and tight junction proteins was also confirmed in Dlk knockout mouse embryos. Finally, desmosomal and tight junction defects were also observed after microtubule disruption in nocodazole-treated tissue-engineered skins, thus confirming a role for microtubules in the maintenance of these types of cell junctions. Globally, this study demonstrates that DLK is a key regulator of microtubule reorganization to the cell periphery during keratinocyte differentiation and that this process is required for the maintenance of desmosomal and tight junction integrity.
- PublicationRestreintCan we produce a human corneal equivalent by tissue engineering?(Elsevier, 2000-07-31) Guérin, Sylvain; Salesse, Christian; Germain, Lucie; Auger, François A.; Carrier, PatrickTissue engineering is progressing rapidly. Bioengineered substitutes are already available for experimental applications and some clinical purposes such as skin replacement. This review focuses on the development of reconstructed human cornea in vitro by tissue engineering. Key elements to consider in the corneal reconstruction, such as the source for epithelial cells and keratocytes, are discussed and the various steps of production are presented. Since one application of this human model is to obtain a better understanding of corneal wound healing, the mechanisms of this phenomenon as well as the function played both by membrane-bound integrins and components from the extracellular matrix have also been addressed. The analysis of integrins by immunohistofluorescence labelling of our reconstructed human cornea revealed that β1, α3, α5, and α6 integrin subunits were expressed but α4 was not. Laminin, type VII collagen and fibronectin were also detected. Finally, the future challenges of corneal reconstruction by tissue engineering are discussed and the tremendous applications of such tissue produced in vitro for experimental as well as clinical purposes are considered.
- PublicationRestreintReconstructed human cornea produced in vitro by tissue engineering(Karger, 1999-05-01) Grandbois, Éric; Guérin, Sylvain; Germain, Lucie; Giasson, Marcelle; Boisjoly, Hélène; Auger, François A.; Guignard, RinaThe aim of the present study was to produce a reconstructed human cornea in vitro by tissue engineering and to characterize the expression of integrins and basement membrane proteins in this reconstructed cornea. Epithelial cells and fibroblasts were isolated from human corneas (limbus or centre) and cultured on plastic substrates in vitro. Reconstructed human corneas were obtained by culturing epithelial cells on collagen gels containing fibroblasts. Histological (Masson’s trichrome staining) and immunohistological (laminin, type VII collagen, fibronectin as well as β1, α3, α4, α5, and α6 integrin subunits) studies were performed. Human corneal epithelial cells from the limbus yielded colonies of small fast-growing cells when cultured on plastic substrates. They could be subcultured for several passages in contrast to central corneal cells. In reconstructed cornea, the epithelium had 4–5 cell layers by the third day of culture; basal cells were cuboidal. The basement membrane components were already detected after 3 days of culture. Integrin stainings, except for the α4 integrin, were also positive after 3 days. They were mostly detected at the epithelium-stroma junction. Such in vitro tissue-engineered human cornea, which shows appropriate histology and expression of basement membrane components and integrins, provides tools for further physiological, toxicological and pharmacological studies as well as being an attractive model for gene expression studies.
- PublicationAccès libreInfluence of Sp1/Sp3 expression on corneal epithelial cells proliferation and differentiation properties in reconstructed tissues(Association for Research in Vision and Ophthalmology, 2003-04-01) Guérin, Sylvain; Germain, Lucie; Giasson, Marcelle; Gaudreault, Manon.; Leclerc, Steeve; Carrier, Patrick; Larouche, KathyPURPOSE : Primary cultured epithelial cells are widely used for the production of tissue-engineered substitutes and are gaining popularity as a model for gene expression studies. However, as such cells are passaged in culture, they often lose their ability to proliferate by progressing toward terminal cell differentiation, a process likely to be determined by altered expression of transcription factors that have functions critical for cell adhesion and differentiation. This study was designed to determine whether the variable life span of primary cultured human corneal epithelial cells (HCECs) might be the consequence of varying expression levels of the well-known transcription factors Sp1 and Sp3 (Sp1/Sp3). METHODS : HCECs were obtained from donor eyes and cultured on irradiated Swiss-3T3. Sp1/Sp3 expression was monitored by Western blot and electrophoretic mobility shift assay (EMSA). The Sp1/Sp3 regulatory influence was evaluated by transfection of HCECs with a recombinant plasmid bearing the Sp1/Sp3-dependent poly(ADP-ribose) polymerase (rPARP) promoter fused to the CAT reporter gene. HCECs that expressed various levels of Sp1/Sp3 were also used for the production of corneal substitutes. RESULTS : Expression of Sp1/Sp3 was dramatically inconsistent between HCECs isolated from the eyes of different donors. Both factors were highly expressed during one passage and then totally disappeared as cells terminally differentiated. Proper stratification of HCECs on reconstructed tissue substitutes could be obtained only with cells that also had a delayed peak of Sp1/Sp3 expression when cultured in vitro. CONCLUSIONS : Expression of Sp1/Sp3 may represent a good predictor for selecting HCECs that are most likely to proliferate, stratify, and differentiate properly when used for the production of reconstructed corneal substitutes.
- PublicationRestreintThe tissue-engineered human cornea as a model to study expression of matrix metalloproteinases during corneal wound healing(Elsevier BV, 2015-11-22) Guérin, Sylvain; Germain, Lucie; Couture, Camille; Zaniolo, Karine; Lake, Jennifer; Patenaude, Julien; Carrier, PatrickCorneal injuries remain a major cause of consultation in the ophthalmology clinics worldwide. Repair of corneal wounds is a complex mechanism that involves cell death, migration, proliferation, differentiation, and extracellular matrix (ECM) remodeling. In the present study, we used a tissue-engineered, two-layers (epithelium and stroma) human cornea as a biomaterial to study both the cellular and molecular mechanisms of wound healing. Gene profiling on microarrays revealed important alterations in the pattern of genes expressed by tissue-engineered corneas in response to wound healing. Expression of many MMPs-encoding genes was shown by microarray and qPCR analyses to increase in the migrating epithelium of wounded corneas. Many of these enzymes were converted into their enzymatically active form as wound closure proceeded. In addition, expression of MMPs by human corneal epithelial cells (HCECs) was affected both by the stromal fibroblasts and the collagen-enriched ECM they produce. Most of all, results from mass spectrometry analyses provided evidence that a fully stratified epithelium is required for proper synthesis and organization of the ECM on which the epithelial cells adhere. In conclusion, and because of the many characteristics it shares with the native cornea, this human two layers corneal substitute may prove particularly useful to decipher the mechanistic details of corneal wound healing.
- PublicationAccès libreCharacterization of wound reepithelialization using a new human tissue–engineered corneal wound healing model(Association for Research in Vision and Ophthalmology, 2008-04-01) Giasson, Claude-J.; Guérin, Sylvain; Germain, Lucie; Giroux-Talbot, Mariève; Auger, François A.; Carrier, Patrick; Deschambeault, AlexandrePurpose. The reepithelialization of the corneal surface is an important process for restoring the imaging properties of this tissue. The purpose of the present study was to characterize and validate a new human in vitro three-dimensional corneal wound healing model by studying the expression of basement membrane components and integrin subunits that play important roles during epithelial cell migration and to verify whether the presence of exogenous factors could accelerate the reepithelialization. Methods. Tissue-engineered human cornea was wounded with a 6-mm biopsy punch, and the reepithelialization from the surrounding margins was studied. Biopsy samples of the reepithelialized surface were harvested 3 days after wounding and were processed for histologic, electron microscopic, and immunofluorescence analyses. The effects of fibrin and epithelial growth factor (EGF) on wound reepithelialization were also studied. Results. Results demonstrated that this in vitro model allowed the migration of human corneal epithelial cells on a natural extracellular matrix. During reepithelialization, epithelial cell migration followed a consistent wavelike pattern similar to that reported for human corneal wound healing in vivo. This model showed a histologic appearance similar to that of native tissue as well as expression and modulation of basement membrane components and the integrin subunits known to be main actors during the wound healing process. It also allowed quantification of the reepithelialization rate, which was significantly accelerated in the presence of fibrin or EGF. The results indicated that αvβ6 integrin expression was increased in the migrating epithelial cells compared with the surrounding corneal tissue. Conclusions. The similarity observed with the in vivo wound healing process supports the use of this tissue-engineered model for investigating the basic mechanisms involved in corneal reepithelialization. Moreover, this model may also be used as a tool to screen agents that affect reepithelialization or to evaluate the effect of growth factors before animal testing.
- PublicationAccès libreImpact of cell source on human cornea reconstructed by tissue engineering(IOVS, 2009-06-01) Giasson, Claude-J.; Guérin, Sylvain; Germain, Lucie; Audet, Caroline; Giroux-Talbot, Mariève; Auger, François A.; Gauvin, Robert; Carrier, Patrick; Deschambeault, AlexandrePurpose: To investigate the effect of the tissue origin of stromal fibroblasts and epithelial cells on reconstructed corneas in vitro. Methods: Four types of constructs were produced by the self-assembly approach using the following combinations of human cells: corneal fibroblasts/corneal epithelial cells, corneal fibroblasts/skin epithelial cells, skin fibroblasts/corneal epithelial cells, skin fibroblasts/skin epithelial cells. Fibroblasts were cultured with ascorbic acid to produce stromal sheets on which epithelial cells were cultured. After 2 weeks at the air-liquid interface, the reconstructed tissues were photographed, absorption spectra were measured, and tissues were fixed for histologic analysis. Cytokine expression in corneal- or skin-fibroblast-conditioned media was determined with the use of protein array membranes. The effect of culturing reconstructed tissues with conditioned media, or media supplemented with a cytokine secreted mainly by corneal fibroblasts, was determined. Results: The tissue source from which epithelial and mesenchymal cells were isolated had a great impact on the macroscopic and histologic features (epithelium thickness and differentiation) and the functional properties (transparency) of the reconstructed tissues. The reconstructed cornea had ultraviolet-absorption characteristics resembling those of native human cornea. The regulation of epithelial differentiation and thickness was mesenchyme-dependent and mediated by diffusible factors. IL-6, which is secreted in greater amounts by corneal fibroblasts than skin fibroblasts, decreased the expression of the differentiation marker DLK in the reconstructed epidermis. Conclusions: The tissue origin of fibroblasts and epithelial cells plays a significant role in the properties of the reconstructed tissues. These human models are promising tools for gaining a thorough understanding of epithelial-stromal interactions and regulation of epithelia homeostasis.
- PublicationRestreintTissue Engineering of Cornea(Marcel Dekker, 2004-06-23) Giasson, Claude-J.; Guérin, Sylvain; Salesse, Christian; Germain, Lucie; Auger, François A.; Carrier, PatrickThe cornea is the transparent barrier between the eye and the environment. Tissue-engineered corneas are currently developed to replace wounded or diseased corneas. Various experimental applications are also foreseen for these tissues reconstructed in vitro by tissue engineering. This article covers the ﬁrst human corneas reconstructed by tissue engineering from normal human cells and the different models used for the production of human and animal corneas in vitro. Corneal injury and the activation of the complex wound«hea]ing mechanisms are also addressed. Finally, we will attempt to provide the reader with a brief look toward the future of corneal tissue engineering, including the challenges that lie ahead as well as the potential experimental and clinical applications of this ﬁeld.