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Larouche, Danielle

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Larouche

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Danielle

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Université Laval. Département de chirurgie

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ncf10162624

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Voici les éléments 1 - 10 sur 13
  • PublicationRestreint
    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
  • PublicationRestreint
    Keratin 19 as a stem cell marker in vivo and in vitro
    (Humana Press, 2005-01-01) Fortier, Kristine; Germain, Lucie; Larouche, Danielle; Hayward, Cindy Jean
    The skin is a dynamic tissue in which terminally differentiated keratinocytes are replaced by the proliferation of new epithelial cells that will undergo differentiation. The rapid and continual turnover of skin throughout life depends on a cell population with unique characteristics: the stem cells. These cells are relatively undifferentiated, retain a high capacity for self-renewal throughout their lifetime, have a large proliferative potential, and are normally slow cycling. The long-term regeneration of grafted cultured epidermis indicates that epidermal stem cells are maintained in cultures. In animals they can be identified with 3H-thymidine or bromodeoxyuridine based on their property of slow cycling. The development of markers such as keratin 19 also permits their study in human tissues. In this chapter, protocols to study skin stem cells using their property of slow cycling and their expression of keratin 19 will be described in detail. The methods include the double labeling of tissues for keratin 19 and label-retaining cells (autoradiography of 3H-thymidine) in situ. The labeling of keratin 19 by immunofluorescence of by flow cytometry is described for cells in vitro.
  • 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
    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.
  • PublicationAccès libre
    Improved methods to produce tissue-engineered skin substitutes suitable for the permanent closure of full-thickness skin injuries
    (Mary Ann Liebert, 2016-11-01) Germain, Lucie; Larouche, Danielle; Auger, François A.; Martel, Israël; Cantin-Warren, Laurence; Ayoub, Akram; Gauvin, Robert; Guignard, Rina; Desgagné, Maxime; Moulin, Véronique; Lavoie, Amélie
    There is a clinical need for skin substitutes to replace full-thickness skin loss. Our group has developed a bilayered skin substitute produced from the patient’s own fibroblasts and keratinocytes referred to as Self-Assembled Skin Substitute (SASS). After cell isolation and expansion, the current time required to produce SASS is 45 days. We aimed to optimize the manufacturing process to standardize the production of SASS and to reduce production time. The new approach consisted in seeding keratinocytes on a fibroblast-derived tissue sheet before its detachment from the culture plate. Four days following keratinocyte seeding, the resulting tissue was stacked on two fibroblast-derived tissue sheets and cultured at the air–liquid interface for 10 days. The resulting total production time was 31 days. An alternative method adapted to more contractile fibroblasts was also developed. It consisted in adding a peripheral frame before seeding fibroblasts in the culture plate. SASSs produced by both new methods shared similar histology, contractile behavior in vitro and in vivo evolution after grafting onto mice when compared with SASSs produced by the 45-day standard method. In conclusion, the new approach for the production of high-quality human skin substitutes should allow an earlier autologous grafting for the treatment of severely burned patients.
  • PublicationAccès libre
    Human post-natal stem cells in organs produced by tissue engineering for clinical applications
    (Nova Biomedical Books, 2008-01-01) Germain, Lucie; Larouche, Danielle; Auger, François A.; Fradette, Julie
    This chapter will focus on the clinical applications of post-natal stem cells. Massive tissue loss frequently requires grafting for proper healing. Considering that there is a shortage of organ donors, the expansion of cells in vitro and the reconstruction of tissues or organs constitute a very valuable alternative solution. The first clinical application of such tissues has been the autologous culture of epidermal cells for the treatment of burn patients, and will be presented herein. Since the cutaneous epithelium forms squames that are lost, it is continuously renewed every 28 days and its long-term regeneration depends on stem cells. The importance of preserving stem cells during in vitro expansion and after grafting will thus be discussed. Clinical applications of cultured cells from other tissues, such as limbal stem cells for corneal epithelium (surface of the eye) replacement, will also be reviewed. Finally, the development of new promising technologies and methods taking advantage of other sources of stem cells that could be isolated after birth from tissues such as adipose depots will also be presented.
  • PublicationAccès libre
    Markers for an In vitro skin substitute
    (Artech House, 2018-02-01T16:41:46Z) Germain, Lucie; Jean, Jessica; Larouche, Danielle; Berthod, François; Pouliot, Roxane; Maguire, Tim; Novik, Eric
    The tissue engineering self-assembly approach allows the production of skin substitutes comprising both the dermis and epidermis, using methods promoting the secretion and organization of a dense extracellular matrix by skin cells. In a reconstructed epidermis, all cellular layers of the native tissue are present. An evaluation of the expression and localization of a number of specific protein markers revealed that the self-assembled, tissue-engineered skin substitute shares some common features with normal human skin, such as the expression of Ki-67, keratins 10 and 14, filaggrin, involucrin, transglutaminase, DLK, a3-integrin subunit, laminin-S, and collagens I, II, 1V, and VII. At the ultrastructural level, many differentiation markers can be observed, including desmosomes, as well as an organized basement membrane presenting hemidesmosomes, lamina densa, and lamina lucida. In this chapter, protocols to generate skin substitutes by the self-assembly approach will be presented and the methods including the labeling of the principal skin differentiation markers by immunofluorescence will be examined.
  • PublicationRestreint
    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
  • 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
    Hair follicles guide nerve migration in vitro and in vivo in tissue-engineered skin
    (Elsevier, 2011-03-03) Gingras, Marie; Germain, Lucie; Parenteau-Bareil, Rémi; Larouche, Danielle; Gagnon, Vicky; Berthod, François