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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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Keratin 19 as a stem cell marker in vivo and in vitro

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.

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Reconstructed human skin produced in vitro and grafted on athymic mice

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.

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Human post-natal stem cells in organs produced by tissue engineering for clinical applications

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.

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Characterization of a 150 kDa accessory receptor for TGF-beta 1 on keratinocytes : direct evidence for a GPI anchor and ligand binding of the released form.

2001-09-05, Tam, Betty Y. Y., Germain, Lucie, Larouche, Danielle, Hooper, N.M, Philip, Anie

Fibroblasts play a critical role in wound repair and in the development of fibrotic diseases, and transforming growth factor-β (TGF-β) has been shown to profoundly modulate fibroblast function. However, there is limited information on the TGF-β receptor types, isoform specificity, and complex formation in skin fibroblasts. Here, we report that normal adult human skin fibroblasts display two isoform-specific, cell surface glycosyl phosphatidylinositol (GPI)-anchored, TGF-β binding proteins in addition to the type I, II, and III TGF-β receptors. The identities of these proteins are confirmed on the basis of their affinity for TGF-β isoforms, immunoprecipitation with specific antireceptor antibodies, and other biochemical analyses. Immunoprecipitation results also indicated oligomeric complex formation between type I and II and between type II and III TGF-β receptors. Furthermore, by using affinity labeling and two-dimensional electrophoresis, we demonstrate the occurrence of type I and II heterodimers and type I homodimers of TGF-β receptors on these cells. Because the type I receptor does not bind TGF-β in the absence of type II receptor, these results indicate that one molecule of TGF-β induces the formation of a heterooligomeric complex containing more than one molecule each of type I and II TGF-β receptors on these cells. These cells respond to TGF-β by markedly down-regulating all five binding proteins and by potently augmenting DNA synthesis. These results allow the expansion of the proposed heteromeric TGF-β receptor signaling paradigm using mutantcells that are unresponsive to TGF-β and cell lines that have been transfected to overexpress these receptors, to include normal TGF-β-responsive cells. In addition, the definition of TGF-β receptor profiles in human skin fibroblasts provides important information for studying their alterations in these cells in various skin diseases.

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Dissociation, quantification and culture of normal human merkel cells among epidermal cell populations derived from glabrous and hairy skin sites

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

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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.

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A novel single-step self-assembly approach for the fabrication of tissue-engineered vascular constructs

2009-12-28, Germain, Lucie, Ahsan, Taby, Larouche, Danielle, Auger, François A., Gauvin, Robert, Dubé, Jean, Tanguay, Robert M., Lévesque, Philippe

There is a clinical need for a functional tissue-engineered blood vessel because small-caliber arterial graft (<5 mm) applications are limited by the availability of suitable autologous vessels and suboptimal performances of synthetic grafts. This study presents an analysis of the mechanical properties of tissue-engineered vascular constructs produced using a novel single-step self-assembly approach. Briefly, the tissue-engineered vascular media were produced by culturing smooth muscle cell in the presence of sodium l-ascorbate until the formation of a cohesive tissue sheet. This sheet was then rolled around a tubular support to create a media construct. Alternatively, the tissue-engineered vascular adventitia was produced by rolling a tissue sheet obtained from dermal fibroblasts or saphenous vein fibroblasts. The standard self-assembly approach to obtain the two-layer tissue-engineered vascular constructs comprising both media and adventitia constructs consists of two steps in which tissue-engineered vascular media were first rolled on a tubular support and a tissue-engineered vascular adventitia was then rolled on top of the first layer. This study reports an original alternative method for assembling tissue-engineered vascular constructs comprising both media and an adventitia in a single step by rolling a continuous tissue sheet containing both cell types contiguously. This tissue sheet was produced by growing smooth muscle cells alongside fibroblasts (saphenous vein fibroblasts or dermal fibroblasts) in the same culture dish separated by a spacer, which is removed later in the culture period. The mechanical strength assessed by uniaxial tensile testing, burst pressure measurements, and viscoelastic behavior evaluated by stepwise stress relaxation tests reveals that the new single-step fabrication method significantly improves the mechanical properties of tissue-engineered vascular construct for both ultimate tensile strength and all the viscoelastic moduli.

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Vibrissa hair bulge houses two populations of skin epithelial stem cells distinct by their keratin profile

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.

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La médecine régénératrice : les cellules souches, les interactions cellulaires et matricielles dans la reconstruction cutanée et cornéenne par génie tissulaire

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.

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Caractérisation des cellules souches présentes dans les follicules pileux et analyse de leur potentiel de différenciation in vivo et in vitro à l'aide de peaux reconstruites par génie tissulaire

2005, Larouche, Danielle, Germain, Lucie

Les cellules souches sont à la base de la régénération des tissus adultes et du maintien de leur homéostasie. Afin d'étudier le processus de différenciation des cellules souches cutanées et d'approfondir nos connaissances sur les conditions qui contrôlent leur différenciation en épiderme et/ou en follicules pileux in vitro différents substituts cutanés ont été mis au point par génie tissulaire. D'une part, la portion dermique des peaux reconstruites a été produite par la superposion de feuillets de matrice extracellulaire produits par des fibroblastes humains ou murins cultivés en présence d'acide ascorbique. D'autre part, des kératinocytes (humains ou murins) ou des follicules pileux immatures (murins) ont été implantés sur les dermes reconstruits. Les propriétés histologiques et immunohistochimiques des tissus ont été évaluées après leur maturation in vitro ou sur l'animal. Nos résultats ont révélé que les interactions mésenchyme-épithéliales créées dans la peau reconstruite par génie tissulaire influencent significativement la voie de différenciation qu'empruntent les kératinocytes engendrés par les cellules souches et qu' il est possible, en recréant les conditions adéquates, de reconstruire des substituts cutanées aux propriétés histologiques similaires à la peau native ainsi que des peaux reconstruites capables de soutenir la formation de follicules pileux après la greffe. Des études in situ ont également été menées sur les cellules souches de la vibrisse de la souris. Nous avons observé que les kératinocytes présentant un cycle cellulaire long forment deux populations distinctes dans la région du renflement: une est en contact avec la membrane basilaire et l'autre ne l'est pas (appelée Bs1). Chacune de ces populations possède un aspect histologique distinct et présente une organisation, du réseau de kératine qui lui est spécifique et qui est associée à l'expression de kératines particulières. L'étude des vibrisses des souris dont le gène de la kératine 17 (K 17) a été invalidé a révélé que la K 1 7 est essentielle à la formation des vibrisses et à l'intégrité ultrastructurale des cellules Bs 1. En conclusion, mes travaux apportent de nouvelles données sur les conditions qui régissent la différenciation des cellules souches cutanées et sur les caractéristiques intrinsèques des cellules souches de la vibrisse.