Personne : Larouche, Danielle
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Département d'épidémiologie, Faculté de Médecine, Université Laval
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- PublicationRestreintIdentification 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, AmélieProgress 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.
- PublicationAccès libreHuman 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, JulieThis 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 libreMarkers 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, EricThe 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.
- PublicationRestreintDissociation, 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, AlphonseMerkel 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
- PublicationRestreintKeratin 19 as a stem cell marker in vivo and in vitro(Humana Press, 2005-01-01) Fortier, Kristine; Germain, Lucie; Larouche, Danielle; Hayward, Cindy JeanThe 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.
- PublicationRestreintRegeneration 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