Personne :
Gaudreault, Manon.

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Département d'oto-rhino-laryngologie et d'ophtalmologie, Faculté de médecine, Université Laval
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  • Publication
    Accès libre
    Influence 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, Kathy
    PURPOSE : 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.
  • Publication
    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.
  • Publication
    Rescue of the transcription factors Sp1 and NFI in human skin keratinocytes through a feeder-layer-dependent suppression of the proteasome activity
    (Academic Press, 2012-03-12) Masson-Gadais, Bénédicte; Guérin, Sylvain; Germain, Lucie; Rochette, Patrick J.; Duval, Céline; Gaudreault, Manon.; Vigneault, François; Touzel-Deschênes, Lydia
    Co-culturing human skin keratinocytes along with a feeder layer has proven to considerably improve their proliferative properties by delaying massive induction of terminal differentiation. Through a yet unclear mechanism, we recently reported that irradiated 3T3 (i3T3) fibroblasts used as a feeder layer increase the nuclear content of Sp1, a positive transcription factor (TF) that plays a critical role in many cellular functions including cell proliferation, into both adult skin keratinocytes and newborn skin keratinocytes. In this study, we examined the influence of i3T3 on the expression and DNA binding of NFI, another TF important for cell proliferation and cell cycle progression, and attempted to decipher the mechanism by which the feeder layer contributes at maintaining higher levels of these TFs in skin keratinocytes. Our results indicate that co-culturing both adult skin keratinocytes and newborn skin keratinocytes along with a feeder layer dramatically increases glycosylation of NFI and may prevent it from being degraded by the proteasome.
  • Publication
    Accès libre
  • Publication
    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.
  • Publication
    Accès libre
    Transcriptional regulation of the human α6 integrin gene by the transcription factor NFI during corneal wound healing
    (Association for Research in Vision and Ophthalmology, 2008-09-01) Guérin, Sylvain; Germain, Lucie; Gingras, Marie-Ève; Gaudreault, Manon.; Leclerc, Steeve; Vigneault, François; Carrier, Patrick
    Purpose. Wound healing of the corneal epithelium is highly influenced by regulation of integrin gene expression. A recent study demonstrated that laminin (LM), a major constituent of the extracellular matrix (ECM), reduces expression of the human α6 integrin subunit gene by altering the properties of the transcription factor (TF) Sp1. In this work, a target site was identified for the TF nuclear factor I (NFI) on the human α6 gene, and its regulatory influence was characterized in corneal epithelial cells. Methods. Plasmids bearing the α6 promoter fused to the CAT gene were transfected into human (HCECs) and rabbit (RCECs) corneal epithelial cells grown on LM. The DNA-binding site for NFI in the α6 promoter was identified by DNase I footprinting. Expression and DNA binding of NFI was monitored by Western blot, RT-PCR, and electrophoretic mobility shift assays (EMSAs), and its function was investigated through RNAi and NFI overexpression assays. Results. All NFI isoforms were found to be expressed in HCECs and RCECs. Transfection analyses revealed that NFI is a repressor of α6 expression in both types of cells. LM increases expression of NFI, whereas inhibition of each NFI isoform increases promoter activity suggesting that NFI is a key repressor of α6 transcription. In addition, the negative influence of NFI appears to be potentiated by the degradation of Sp1 when cells are grown on LM. Conclusions. Repression of α6 expression therefore contributes to the final steps of corneal wound healing by both reducing proliferation and allowing attachment of the epithelium to the basal membrane.
  • Publication
    Expression of the alpha 5 integrin gene in corneal epithelial cells cultured on tissue-engineered human extracellular matrices
    (IPC Science and Technology Press, 2013-05-30) Guérin, Sylvain; Salesse, Christian; Germain, Lucie; Deschambault, Alexandre; Zaniolo, Karine; Lake, Jennifer; Gaudreault, Manon.; Bazin, Richard; Carrier, Patrick
    The integrin α5β1 plays a major role in corneal wound healing by promoting epithelial cell adhesion and migration over the fibronectin matrix secreted as a cellular response to corneal damage. Expression of α5 is induced when rabbit corneal epithelial cells (RCECs) are grown in the presence of fibronectin. Here, we examined whether α5 expression is similarly altered when RCECs or human corneal epithelial cells (HCECs) are grown on a reconstructed stromal matrix used as an underlying biomaterial. Mass spectrometry and immunofluorescence analyses revealed that the biomaterial matrix produced by culturing human corneal fibroblasts with ascorbic acid (ECM/35d) contains several types of collagens, fibronectin, tenascin and proteoglycans. Results from transfection of CAT/α5-promoter plasmids, Western blot and EMSA analyses indicated that ECM/35d significantly increase expression of α5 in HCECs as a result of alteration in the expression and DNA binding of the transcription factors NFI, Sp1, AP-1 and PAX6. The biological significance of this biomaterial substitute on the expression of the α5 gene may therefore contribute to better understand the function played by the α5β1 integrin during corneal wound healing.