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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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Considerations in the choice of a skin donor site for harvesting keratinocytes containing a high proportion of stem cells for culture in vitro

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

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Tissue engineering of skin and cornea : Development of new models for in vitro studies

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.

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Regeneration of skin and cornea by tissue engineering

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

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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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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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Normal human merkel cells are present in epidermal cell populations isolated and cultured from glabrous and hairy skin sites

2015-12-08, Caouette, Louise, Germain, Lucie, Larouche, Danielle, Couture, Véronique, Fugère, Claudia., Guignard, Rina, Fradette, Julie, Roy, Alphonse, Beauparlant, Annie.

The Merkel cell is a highly specialized cell that primarily acts as a slowly adapting mechanoreceptor. Merkel cells are scarce in normal skin but can be identified by the expression of distinct keratin filaments. Merkel cells constitute a very unique population and many questions still remain as to their origin, number, proliferative capacity, and functions in cutaneous biology. The dissociation of epidermal cells from skin is a widely used technique to extract and culture keratinocytes. We took advantage of a two-step extraction method to quantify keratin-20-expressing Merkel cells among total cutaneous cells obtained from either hairy or glabrous skin biopsies. Flow cytometry analysis revealed that keratin-20-labeled Merkel cells represent between 3.6% and 5.7% of freshly dissociated basal epidermal cells. No significant differences were seen between samples derived from glabrous palmar and hairy anatomic sites, from children and adult, respectively. We also report on the presence of Merkel cells in primary and first subcultures of epidermal cells indicating their capacity to remain viable after extraction from skin of various anatomic sites. To our knowledge, this is the first demonstration of nontumorigenic human Merkel cells in culture in vitro. The persistence of a small number of Merkel cells in culture suggests that, with the development of appropriate culture conditions, these cells could be amplified and further studied to unravel long-standing questions relative to their paracrine function or epithelial origin.

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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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Human epithelial stem cells persist within tissue-engineered skin produced by the self-assembly approach

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.

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Stem cells of the skin and cornea : their clinical applications in regenerative medicine

2011-02-01, Germain, Lucie, Larouche, Danielle, Gauvin, Robert, Proulx, Stéphanie, Fradette, Julie

Purpose of review: The use of stem cells is of great interest for the treatment of various pathologies and ultimately for the restoration of organ function. Progress pointing towards future treatments of skin and corneal epithelial stem cell defects are reviewed, including the transplantation of living tissue-engineered substitutes. Recent findings: This article focuses on substitutes optimized for permanent replacement of skin and cornea. New skin substitutes for burn care are currently under development. More complex tissue-engineered skin substitutes in which stroma, adipose tissue, capillaries, and neurons are combined with the epithelium are being developed. Some dermal/epidermal substitutes have been applied to the treatment of patients. Cultured corneal epithelial cells have been characterized and more complete corneal substitutes are being designed. Long-term clinical results on the transplantation of cultured corneal stem cells for the treatment of limbal stem cell deficiency have been reported. Summary: Advances in tissue engineering for the development of substitutes that will benefit patients suffering from skin or corneal stem cell deficiencies are reviewed. These products are often a combination of cells, scaffolds and other factors. Key considerations in the development of corneal and skin substitutes for clinical applications are discussed.