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Personne :
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 36
  • 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
    Dissociation, quantification and culture of normal human merkel cells among epidermal cell populations derived from glabrous and hairy skin sites
    (Springer, 2003-06-23) Germain, Lucie; Larouche, Danielle; Couture, Véronique; Fugère, Claudia.; Guignard, Rina; Fradette, Julie; Caouette-Laberge, Louise; Beauparlant, Annie.; Roy, Alphonse
    Merkel cells constitute a unique population that remains difficult to characterize in human skin because of their scarcity. Our aim was to develop tools for the study of Merkel cells in vitro. As a first step, we evaluated the possibility of harvesting human Merkel cells with the two-step extraction method that is widely used to extract and culture keratinocytes. Merkel cells were identified in the epithelial portion of hairy or glabrous skin biopsies by keratin (K)18 and K20 labeling. The totality of cutaneous epithelial cells were isolated from either hairy or glabrous skin biopsies following enzymatic dissociation of both the epidermis and the hair follicles. Flow cytometry was performed to quantify the small Merkel cell population. The analysis revealed that K18-labeled cells represented between 4.0 and 7.6% of freshly dissociated basal epidermal cells. No significant differences were seen between samples derived from glabrous palmar and hairy anatomic sites from children and adults, respectively. We also reported on the presence of Merkel cells in primary and first subcultures of human epidermal cells. The next step will be to enrich the isolated human Merkel cells and improve their culture conditions. An amplification of the number of Merkel cells will allow further studies to unravel long-standing questions regarding their origin, proliferative capacity, and functions in cutaneous biology
  • PublicationRestreint
    Vibrissa hair bulge houses two populations of skin epithelial stem cells distinct by their keratin profile
    (Federation of American Societies for Experimental Biology, 2007-12-27) Germain, Lucie; Tong, Xuemei; Larouche, Danielle; Fradette, Julie; Coulombe, Pierre A.
    Defining the properties of postnatal stem cells is of interest given their relevance for tissue homeostasis and therapeutic applications, such as skin tissue engineering for burn patients. In hair follicles, the bulge region of the outer root sheath houses stem cells. We show that explants from the prominent bulge area, but not the bulb, in rodent vibrissa follicles can produce epidermis in a skin model of tissue engineering. Using morphological criteria and keratin expression, we typified epithelial stem cells of vibrissa bulge. Two types of slow-cycling cells (Bb, Bs1) featuring a high colony-forming capacity occur in the bulge. Bb cells are located in the outermost basal layer, express K5, K15, K17, and K19, and feature a loosely organized keratin network. Bs1 cells localize to the suprabasal layers proximal to Bb cells and express K5/K17, corre lating with a network of densely bundled filaments. These prominent bundles are missing in K17-null mice, which lack vibrissa. Atypically, both the Bb and Bs1 keratinocytes lack K14 expression. These findings show heterogeneity within the hair follicle stem cell reposi tory, establish that a subset of slow-cycling cells are suprabasal in location, and point to a special role for K5/K17 filaments in a newly defined subset of stem cells. Our results are discussed in the context of long-term survival of engineered tissues after grafting that requires the presence of stem cells.
  • PublicationRestreint
    Minimal contraction for tissue-engineered skin substitutes when matured at the air–liquid interface
    (John Wiley & Sons, 2013-06-03) Germain, Lucie; Larouche, Danielle; Auger, François A.; Marcoux, Hugo-Bastien; Gauvin, Robert; Guignard, Rina
    The structural stability of skin substitutes is critical to avoid aesthetic and functional problems after grafting, such as contractures and hypertrophic scars. The present study was designed to assess the production steps having an influence on the contractile behaviour of the tissue-engineered skin made by the self-assembly approach, where keratinocytes are cultured on tissue-engineered dermis comprised of fibroblasts and the endogenous extracellular matrix they organized. Thus, different aspects were investigated, such as the assembly method of the engineered dermis (various sizes and anchoring designs) and the impact of epithelial cell differentiation (culture submerged in the medium or at the air–liquid interface). To evaluate the structural stability at the end of the production, the substitutes were detached from their anchorages and deposited on a soft substrate, and contraction was monitored over 1 week. Collected data were analysed using a mathematical model to characterize contraction. We observed that the presence of a differentiated epidermis significantly reduced the amount of contraction experienced by the engineered tissues, independently of the assembly method used for their production. When the epidermis was terminally differentiated, the average contraction was only 24 4% and most of the contraction occurred within the first 12 h following deposition on the substrate. This is 2.2-fold less compared to when the epidermis was cultured under the submerged condition, or when tissue-engineered dermis was not overlaid with epithelial cells. This study highlights that the maturation at the air–liquid interface is a critical step in the reconstruction of a tissue engineered skin that possesses high structural stability
  • 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
    Reconstructed human skin produced in vitro and grafted on athymic mice
    (Ovid, 2002-06-15) Li, Hui; Germain, Lucie; Xu, Wen; Larouche, Danielle; Juhász, Julianna; Auger, François A.; Pouliot, Roxane
    Background. The best alternative to a split-thickness graft for the wound coverage of patients with extensive burns should be in vitro reconstructed autologous skin made of both dermis and epidermis and devoid of exogenous extracellular matrix proteins and synthetic material. We have designed such a reconstructed human skin (rHS) and present here its first in vivo grafting on athymic mice. Methods. The rHS was made by culturing newborn or adult keratinocytes on superimposed fibrous sheets obtained after culturing human fibroblasts with ascorbic acid. Ten days after keratinocyte seeding, reconstructed skins were either cultured at the air-liquid interface or grafted on athymic mice. We present the macroscopic, histologic, and phenotypic properties of such tissues in vitro and in vivo after grafting on nude mice. Results. After maturation in vitro, the reconstructed skin exhibited a well-developed human epidermis that expressed differentiated markers and basement membrane proteins. Four days after grafting, a complete take of all grafts was obtained. Histological analysis revealed that the newly generated epidermis of newborn rHS was thicker than that of adult rHS after 4 days but similar 21 days after grafting. The basement membrane components (bullous pemphigoid antigens, laminin, and type IV and VII collagens) were detected at the dermo-epidermal junction, showing a continuous line 4 days after grafting. Ultrastructural studies revealed that the basement membrane was continuous and well organized 21 days after transplantation. The macroscopic aspect of the reconstructed skin revealed a resistant, supple, and elastic tissue. Elastin staining and elastic fibers were detected as a complex network in the rHS that contributes to the good elasticity of this new reconstructed tissue. Conclusions. This new rHS model gives supple and easy to handle skins while demonstrating an adequate wound healing on mice. These results are promising for the development of this skin substitute for permanent coverage of burn wounds.
  • PublicationRestreint
    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.
  • PublicationRestreint
    Tissue reorganization in response to mechanical load increases functionality
    (2005-02-28) Bergeron, François; Langelier, Ève.; Grenier, Guillaume.; Germain, Lucie; Larouche, Danielle; Dupuis, Daniel; Rancourt, Denis; Auger, François A.; Gauvin, Robert; Baker, Kathleen; Rémy-Zolghadri, Murielle
    In the rapidly growing field of tissue engineering, the functional properties of tissue substitutes are recognized as being of the utmost importance. The present study was designed to evaluate the effects of static mechanical forces on the functionality of the produced tissue constructs. Living tissue sheets reconstructed by the self-assembly approach from human cells, without the addition of synthetic material or extracellular matrix (ECM), were subjected to mechanical load to induce cell and ECM alignment. In addition, the effects of alignment on the function of substitutes reconstructed from these living tissue sheets were evaluated. Our results show that tissue constructs made from living tissue sheets, in which fibroblasts and ECM were aligned, presented higher mechanical resistance. This was assessed by the modulus of elasticity and ultimate strength as compared with tissue constructs in which components were randomly oriented. Moreover, tissue-engineered vascular media made from a prealigned living tissue sheet, produced with smooth muscle cells, possessed greater contractile capacity compared with those produced from living tissue sheets that were not prealigned. These results show that the mechanical force generated by cells during tissue organization is an asset for tissue component alignment. Therefore, this work demonstrates a means to improve the functionality (mechanical and vasocontractile properties) of tissues reconstructed by tissue engineering by taking advantage of the biomechanical forces generated by cells under static strain.
  • 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.