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Guérin, Sylvain

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Guérin

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Sylvain

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Université Laval. Département d'ophtalmologie et d'oto-rhino-laryngologie - chirurgie cervico-faciale

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ncf10225154

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Voici les éléments 1 - 10 sur 11
  • PublicationAccès libre
    Autologous transplantation of rabbit limbal epithelia cultured on fibrin gels for ocular surface reconstruction
    (Éditeur non identifié, 2006-02-01) Guérin, Sylvain; Germain, Lucie; Giasson, Claude J.; Giroux-Talbot, Mariève; Auger, François A.; Bazin, Richard; Carrier, Patrick; Deschambeault, Alexandre
    Purpose: Regeneration of the corneal epithelium could be severely impaired in patients suffering from limbal stem cell deficiency. The purpose of this study was to evaluate the restoration of the corneal epithelium by grafting onto denuded corneas autologous limbal cells cultured on fibrin gels. The rabbit model was chosen to allow the microscopic evaluation over time after grafting. Methods: Rabbit limbal epithelial cells (RLECs) were isolated and cultured from small limbal biopsies (3 mm2). The epithelium was separated from stroma after dispase digestion and put in culture on lethally irradiated fibroblasts used as a feeder layer. At the first passage, RLECs were cultured on a fibrin gel matrix. At confluence, the cultured epithelia were grafted in vivo on denuded autologous rabbit corneas. At different postoperative times, grafted and control (without graft or grafted with fibrin gels only) rabbit corneas were compared in vivo with a slit lamp microscope, and in situ by histological and immunohistological microscopy of harvested biopsies. Results: A small limbal biopsy was sufficient to generate enough RLECs to prepare several grafts and to perform cell analysis. Only two weeks were required to produce a cultured epithelium suitable for autologous transplantation. One month after grafting, a normal corneal phenotype was observed on the ocular surface of grafted rabbits in contrast to the control rabbits (ungrafted or grafted with fibrin gel only) where histological signs of conjunctivalization were found. The absence of goblet cells and negative staining for keratin 4 confirmed that the cultured cells persisted and that the epithelium regenerated after grafting was not from conjunctival origin. Conclusions: Our results demonstrate that an autologous epithelium cultured on a physiologically biodegradable matrix can be prepared from a small biopsy and grafted on denuded cornea. The autologous graft allows epithelial regeneration from cultured cells and promotes corneal healing of unilateral total stem cell deficiency.
  • PublicationRestreint
    Tissue Engineering of Cornea
    (Marcel Dekker, 2004-06-23) Giasson, Claude-J.; Guérin, Sylvain; Salesse, Christian; Germain, Lucie; Auger, François A.; Carrier, Patrick
    The 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.
  • PublicationRestreint
    Tissue engineering of skin and cornea : Development of new models for in vitro studies
    (Academy of Sciences, 2010-06-02) Guérin, Sylvain; Germain, Lucie; Larouche, Danielle; Bisson, Francis; Paquet, Claudie; Robitaille, Hubert; Auger, François A.; Gaudreault, Manon.; Martel, Israël; Duranceau, Louise; Proulx, Stéphanie; Carrier, Patrick; Simard-Bisson, Carolyne; Fradette, Julie
    Human beings are greatly preoccupied with the unavoidable nature of aging. While the biological processes of senescence and aging are the subjects of intense investigations, the molecular mechanisms linking aging with disease and death are yet to be elucidated. Tissue engineering offers new models to study the various processes associated with aging. Using keratin 19 as a stem cell marker, our studies have revealed that stem cells are preserved in human skin reconstructed by tissue engineering and that the number of epithelial stem cells varies according to the donor's age. As with skin, human corneas can also be engineered in vitro. Among the epithelial cells used for reconstructing skin and corneas, significant age-dependent variations in the expression of the transcription factor Sp1 were observed. Culturing skin epithelial cells with a feeder layer extended their life span in culture, likely by preventing Sp1 degradation in epithelial cells, therefore demonstrating the pivotal role played by this transcription factor in cell proliferation. Finally, using the human tissue-engineered skin as a model, we linked Hsp27 activation with skin differentiation.
  • PublicationRestreint
    Can we produce a human corneal equivalent by tissue engineering?
    (Elsevier, 2000-07-31) Guérin, Sylvain; Salesse, Christian; Germain, Lucie; Auger, François A.; Carrier, Patrick
    Tissue 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.
  • PublicationRestreint
    Reconstructed 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, Rina
    The 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 libre
    Irradiated human dermal fibroblasts are as efficient as mouse fibroblasts as a feeder layer to improve human epidermal cell culture lifespan
    (Molecular Diversity Preservation International, 2013-02-26) Guérin, Sylvain; Germain, Lucie; Larouche, Danielle; Bisson, Francis; Rochefort, Éloise; Zaniolo, Karine; Damour, Odile; Auger, François A.; Simard-Bisson, Carolyne; Lavoie, Amélie
    A fibroblast feeder layer is currently the best option for large scale expansion of autologous skin keratinocytes that are to be used for the treatment of severely burned patients. In a clinical context, using a human rather than a mouse feeder layer is desirable to reduce the risk of introducing animal antigens and unknown viruses. This study was designed to evaluate if irradiated human fibroblasts can be used in keratinocyte cultures without affecting their morphological and physiological properties. Keratinocytes were grown either with or without a feeder layer in serum-containing medium. Our results showed that keratinocytes grown either on an irradiated human feeder layer or irradiated 3T3 cells (i3T3) can be cultured for a comparable number of passages. The average epithelial cell size and morphology were also similar. On the other hand, keratinocytes grown without a feeder layer showed heavily bloated cells at early passages and stop proliferating after only a few passages. On the molecular aspect, the expression level of the transcription factor Sp1, a useful marker of keratinocytes lifespan, was maintained and stabilized for a high number of passages in keratinocytes grown with feeder layers whereas Sp1 expression dropped quickly without a feeder layer. Furthermore, gene profiling on microarrays identified potential target genes whose expression is differentially regulated in the absence or presence of an i3T3 feeder layer and which may contribute at preserving the growth characteristics of these cells. Irradiated human dermal fibroblasts therefore provide a good human feeder layer for an effective expansion of keratinocytes in vitro that are to be used for clinical purposes.
  • PublicationRestreint
    Tissue engineering of human cornea
    (CRC Press, 2014-03-27) Guillemette, Maxime.; Giasson, Claude-J.; Guérin, Sylvain; Germain, Lucie; Auger, François A.; Gaudreault, Manon.; Proulx, Stéphanie; Carrier, Patrick; Chirila, Traian
    The cornea is a well-organized tissue composed of three cell types (epithelial, stromal and endothelial cells), each having an important role for its functionality. This chapter will address different tissue engineering approaches to the reconstruction of either partial or full-thickness living corneal substitutes that can be used either as in vitro models for woundhealing studies, or in vivo, eventually replacing the donor cornea for transplantation in humans. Isolation of the proper cells, followed by appropriate culture conditions, and assembly into a three-dimensional tissue construct, are the first steps required for producing a functional corneal substitute.
  • PublicationRestreint
    Contribution of Sp1 to telomerase expression and activity in skin keratinocytes cultured with a feeder layer
    (Wistar Institute of Anatomy and Biology, 2014-06-24) Guérin, Sylvain; Germain, Lucie; Rochette, Patrick J.; Bisson, Francis; Zaniolo, Karine; Paquet, Claudie; Damour, Odile; Bourget, Jean-Michel; Boudreau, François; Landreville, Solange; Auger, François A.
    The growth of primary keratinocytes is improved by culturing them with a feeder layer. The aim of this study was to assess whether the feeder layer increases the lifespan of cultured epithelial cells by maintaining or improving telomerase activity and expression. The addition of an irradiated fibroblast feeder layer of either human or mouse origin (i3T3) helped maintain telomerase activity as well as expression of the transcription factor Sp1 in cultured keratinocytes. In contrast, senescence occurred earlier, together with a reduction of Sp1 expression and telomerase activity, in keratinocytes cultured without a feeder layer. Telomerase activity was consistently higher in keratinocytes grown on the three different feeder layers tested relative to cells grown without them. Suppression of Sp1 expression by RNA inhibition (RNAi) reduced both telomerase expression and activity in keratinocytes and also abolished their long-term growth capacity suggesting that Sp1 is a key regulator of both telomerase gene expression and cell cycle progression of primary cultured human skin keratinocytes. The results of the present study therefore suggest that the beneficial influence of the feeder layer relies on its ability to preserve telomerase activity in cultured human keratinocytes through the maintenance of stable levels of Sp1 expression.
  • PublicationAccès libre
    Impact 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, Alexandre
    Purpose: 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.
  • PublicationAccès libre
    Characterization 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, Alexandre
    Purpose. 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.