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Paquet, Claudie

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Paquet
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Claudie
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Laboratoire de pathologie (HSS), CHU de Québec (HDQ-CHUL-HSFA-HEJ-HSS) (Université Laval)
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Voici les éléments 1 - 10 sur 10
  • Publication
    Restreint
    Considerations in the choice of a skin donor site for harvesting keratinocytes containing a high proportion of stem cells for culture in vitro
    (Butterworth-Heinemann, 2010-12-03) Germain, Lucie; Larouche, Danielle; Paquet, Claudie; Fugère, Claudia.; Genest, Hervé; Auger, François A.; Gauvin, Robert; Têtu, Félix-Andre; Bouchard, Maurice; Roy, Aphonse; Fradette, Julie; Lavoie, Amélie; Beauparlant, Annie.
    The treatment of severely burned patients has benefited from the grafting of skin substitutes obtained by expansion of epithelial cells in culture. The aim of this study was to evaluate whether the anatomic site chosen for harvesting skin had an impact on the quality of the derived cell cultures. Considering that hair follicles contain epithelial stem cells, we compared hairy skin sites featuring different densities and sizes of hair follicles for their capacity to generate high quality keratinocyte cultures. Three anatomic sites from adult subjects were compared: scalp, chest skin and p-auricular (comprising pre-auricular and post-auricular) skin. Keratin (K) 19 was used as a marker to evaluate the proportion of stem cells. Keratinocytes were isolated using the two-step thermolysin and trypsin cell extraction method, and cultured in vitro. The proportion of K19-positive cells harvested from p-auricular skin was about twice that of the scalp. This K19-positive cell content also remained higher during the first subcultures. In contrast to these in vitro results, the number of K19-positive cells estimated in situ on skin sections was about double in scalp as in p-auricular skin. Chest skin had the lowest number of K19-positive cells. These results indicate that in addition to the choice of an adult anatomic site featuring a high number of stem cells in situ, the quality of the cultures greatly depends on the ability to extract stem cells from the skin biopsy
  • Publication
    Restreint
    The feeder layer-mediated extended lifetime of cultured human skin keratinocytes is associated with altered levels of the transcription factors Sp1 and Sp3
    (Wiley, 2005-10-21) Masson-Gadais, Bénédicte; Guérin, Sylvain; Germain, Lucie; Paquet, Claudie; Fugère, Claudia.; Lefort, Natalie; Leclerc, Steeve
    Primary cultured epithelial cells that are used for basic research are often cultivated on plastic whereas those used for clinical purposes are usually cultured in the presence of a feeder layer. Here, we examined the influence of a feeder layer on the expression, affinity and DNA binding ability of the transcription factors, Sp1 and Sp3 in primary cultures of human skin keratinocytes. Co-culturing both newborn and adult skin keratinocytes with lethally irradiated 3T3 cells as a feeder layer contributed to maintain the cell's morphological and growth characteristics and delayed terminal differentiation in vitro. 3T3 also stabilized the DNA binding properties of Sp1 without altering its transcription. Stimulation of Sp1/Sp3 expression appears to be mediated through cell–cell interactions and by factors secreted by 3T3. Thus, we propose that the feeder layer delay terminal differentiation of primary cultured skin keratinocytes by preventing extinction of transcription factors, like Sp1 and Sp3, which play pivotal functions in the cell cycle.
  • Publication
    Restreint
    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
    Restreint
    Human epithelial stem cells persist within tissue-engineered skin produced by the self-assembly approach
    (Mary Ann Liebert, 2013-02-15) Germain, Lucie; Goyer, Benjamin; Larouche, Danielle; Kim, Dong Hyun; Paquet, Claudie; Fugère, Claudia.; Dunnwald, Martine; Robitaille, Hubert; Sauvé, Sarah; Desgagné, Maxime; Fradette, Julie; Lavoie, Amélie; Beauparlant, Annie.; Pouliot, Roxane
    To adequately and permanently restore organ function after grafting, human tissue-engineered skin substitutes (TESs) must ultimately contain and preserve functional epithelial stem cells (SCs). It is therefore essential that a maximum of SCs be preserved during each in vitro step leading to the production of TESs such as the culture process and the elaboration of a skin cell bank by cryopreservation. To investigate the presence and functionality of epithelial SCs within the human TESs made by the self-assembly approach, slow-cycling cells were identified using 5′-bromo-2′-deoxyuridine (BrdU) in the three-dimensional construct. A subset of basal epithelial cells retained the BrdU label and was positive for the SC-associated marker keratin 19 within TESs after a chase of 21 days in culture post-BrdU labeling. Moreover, keratinocytes harvested from TESs gave rise to SC-like colonies in secondary monolayer subcultures, indicating that SCs were preserved within TESs. To evaluate the effect of cryopreservation with dimethyl sulfoxide and storage in liquid nitrogen on SCs, human epithelial cells were extracted from skin samples, amplified in culture, and used to produce TESs, before cryopreservation as well as after thawing. We found that the proportion and the growth potential of epithelial SCs in monolayer culture and in TESs remained constant before and after cryopreservation. Further, the functionality of these substitutes was demonstrated by successfully grafting human TESs on athymic mice for 6 months. We conclude that human epithelial skin SCs are adequately preserved upon human tissue reconstruction. Thus, these TESs produced by the self-assembly approach are suitable for clinical applications.
  • Publication
    Restreint
    La médecine régénératrice : les cellules souches, les interactions cellulaires et matricielles dans la reconstruction cutanée et cornéenne par génie tissulaire
    (Elsevier Masson, 2008-06-02) Germain, Lucie; Larouche, Danielle; Paquet, Claudie; Auger, François A.; Proulx, Stéphanie; Carrier, Patrick; Lavoie, Amélie; Beauparlant, Annie.
    Le génie tissulaire vise à produire des tissus ou organes in vitro pour le remplacement permanent des tissus endommagés. À cette fin, la production de tissus autologues possède l’avantage d’éviter tout risque de rejet suite à leur transplantation sur un patient. La maîtrise des conditions de culture des cellules souches humaines postnatales est essentielle à la réalisation de tels tissus. Il est aussi souhaitable que leur organisation histologique et leur fonctionnalité se rapprochent de celles des tissus natifs. De plus, les cellules souches jouent un rôle essentiel au niveau du remplacement des cellules épithéliales différenciées dans les tissus qui doivent constamment se renouveler, tels que la peau et la cornée. Nous avons décrit une méthode qui permet de produire des organes vivants in vitro à partir de cellules humaines postnatales sans ajouter de biomatériaux. Cette méthode d’auto-assemblage repose sur la capacité qu’ont les cellules mésenchymateuses de s’organiser en tissu en présence des conditions de culture adéquates. Grâce à différentes techniques, ces tissus peuvent ensuite être assemblés en organes plus complexes tels que les vaisseaux sanguins, les valves cardiaques, la peau ou encore la cornée. Ces divers tissus pourront éventuellement être utilisés pour le remplacement d’organes malades ou endommagés et fourniront de nouvelles alternatives pour la médecine régénératrice de demain. Cet article de revue sera concentré sur la peau et la cornée. L’importance d’utiliser des conditions d’isolement et de culture qui permettent de conserver les cellules souches et de contrôler l’organisation des tissus afin d’assurer la qualité et la fonctionnalité des organes reconstitués par génie tissulaire sera discutée.
  • Publication
    Restreint
    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.
  • Publication
    Accès libre
    Des­ Canadiens­ «­ précurseurs­ de ­la ­recherche­ sur­ les ­cellules­ souches­ hématopoïétiques­ »­ lauréats­ du ­prix­ Lasker
    (EDK, 2006-02-15) Germain, Lucie; Larouche, Danielle; Paquet, Claudie
    À la fin de l'année 2005, le prestigieux prix Lasker pour la recherche médicale fondamentale a été décerné à deux scientifiques canadiens, les Docteurs Ernest A. McCulloch et James E. Till, qui se sont illustrés par leurs travaux portant notamment sur la capacité des cellules souches à s’auto-renouveler et à se différencier en cellules pouvant présenter de multiples phénotypes.
  • Publication
    Restreint
    Tissue-engineered skin preserving the potential of epithelial cells to differentiate into hair after grafting.
    (liebertonline.com, 2018-01-09) Fortier, Kristine; Germain, Lucie; Larouche, Danielle; Paquet, Claudie
    The aim of this study was to evaluate whether tissue-engineered skin produced in vitro was able to sustain growth of hair follicles in vitro and after grafting. Different tissues were designed. Dissociated newborn mouse keratinocytes or newborn mouse hair buds (HBs) were added onto dermal constructs consisting of a tissue-engineered cell-derived matrix elaborated from either newborn mouse or adult human fibroblasts cultured with ascorbic acid. After 7–21 days of maturation at the air–liquid interface, no hair was noticed in vitro. Epidermal differentiation was observed in all tissue-engineered skin. However, human fibroblast-derived tissue-engineered dermis (hD) promoted a thicker epidermis than mouse fibroblast-derived tissue-engineered dermis (mD). In association with mD, HBs developed epithelial cyst-like inclusions presenting outer root sheath-like attributes. In contrast, epidermoid cyst-like inclusions lined by a stratified squamous epithelium were present in tissues composed of HBs and hD. After grafting, pilo-sebaceous units formed and hair grew in skin elaborated from HBs cultured 10–26 days submerged in culture medium in association with mD. However, the number of normal hair follicles decreased with longer culture time. This hair-forming capacity after grafting was not observed in tissues composed of hD overlaid with HBs. These results demonstrate that epithelial stem cells can be kept in vitro in a permissive tissue-engineered dermal environment without losing their potential to induce hair growth after grafting.
  • Publication
    Restreint
    Regeneration of skin and cornea by tissue engineering
    (Springer, 2018-02-01T16:37:44Z) Germain, Lucie; Larouche, Danielle; Paquet, Claudie; Auger, François A.; Carrier, Patrick; Fradette, Julie; Audet, Julie; Stanford, William L.
    Progress in tissue engineering has led to the development of technologies allowing the reconstruction of autologous tissues from the patient’s own cells. Thus, tissue-engineered epithelial substitutes produced from cultured skin epithelial cells undergo long-term regeneration after grafting, indicating that functional stem cells were preserved during culture and following grafting. However, these cultured epithelial sheets reconstruct only the upper layer of the skin and lack the mechanical properties associated to the connective tissue of the dermis. We have designed a reconstructed skin entirely made from human cutaneous cells comprising both the dermis and the epidermis, as well as a well-organized basement membrane by a method named the self-assembly approach. In this chapter, protocols to generate reconstructed skin and corneal epithelium suitable for grafting are described in details. The methods include extraction and culture of human skin keratinocytes, human skin fibroblasts as well as rabbit and human corneal epithelial cells, and a complete description of the skin reconstructed by the self-assembly approach and of corneal epithelium reconstructed over a fibrin gel
  • Publication
    Restreint
    Identification of epithelial stem cells in vivo and in vitro using keratin 19 and BrdU
    (Humana Press, 2010-01-01) Germain, Lucie; Larouche, Danielle; Paquet, Claudie; Simard-Bisson, Carolyne; Lavoie, Amélie
    Progress in the identification of skin stem cells and the improvement of culture methods open the possibility to use stem cells in regenerative medicine. Based on their quiescent nature, the development of label retention assays allowed the localization of skin stem cells in the bulge region of the pilosebaceous units and in the bottom of rete ridges in glabrous skin. The development of markers such as keratin 19 also permits their study in human tissues. In this chapter, protocols to identify skin stem cells based on their slow-cycling property and their expression of keratin 19 will be described in detail. The methods include the labeling of skin stem cells within mouse or rat tissues in vivo, the labeling of proliferative human cells in vitro using 5-bromo-2-deoxyuridine (BrdU), and the detection of keratin 19 and BrdU by immunofluorescence or immunoperoxidase staining.