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Fradette, Julie

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Fradette

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Julie

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Université Laval. Département de chirurgie

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ncf11860418

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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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High definition confocal imaging modalities for the characterization of tissue-engineered substitutes

2018-04-01, Fradette, Julie, Mayrand, Dominique

Optimal imaging methods are necessary in order to perform a detailed characterization of thick tissue samples from either native or engineered tissues. Tissue-engineered substitutes are featuring increasing complexity including multiple cell types and capillary-like networks. Therefore, technical approaches allowing the visualization of the inner structural organization and cellular composition of tissues are needed. This chapter describes an optical clearing technique which facilitates the detailed characterization of whole-mount samples from skin and adipose tissues (ex vivo tissues and in vitro tissue-engineered substitutes) when combined with spectral confocal microscopy and quantitative analysis on image renderings.

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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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A nanoparticle ink allowing the high precision visualization of tissue engineered scaffolds by MRI

2023-03-25, Leon-Chaviano, Samila, Kiseleva, Mariia, Legros, Philippe, Collin, Simon, Théophraste, Lescot, Henoumont, Céline, Gossuin, Yves, Laurent, Sophie, Fradette, Julie, Bégin-Drolet, André, Ruel, Jean, Fortin, Marc-André

Hydrogels are widely used as cell scaffolds in several biomedical applications. Once implanted in vivo, cell scaffolds must often be visualized, and monitored overtime. However, cell scaffolds appear poorly contrasted in most biomedical imaging modalities such as magnetic resonance imaging (MRI). MRI is the imaging technique of choice for high-resolution visualization of low-density, water-rich tissues. Attempts to enhance hydrogel contrast in MRI are performed with “negative” contrast agents that produce several image artifacts impeding the delineation of the implant’s contours. In this study, a magnetic ink based on ultra-small iron oxide nanoparticles (USPIONs; <5 nm diameter cores) is developed and integrated into biocompatible alginate hydrogel used in cell scaffolding applications. Relaxometric properties of the magnetic hydrogel are measured, as well as biocompatibility and MR-visibility (T1-weighted mode; in vitro and in vivo). A 2-week MR follow-up study is performed in the mouse model, demonstrating no image artifacts, and the retention of “positive” contrast overtime, which allows very precise delineation of tissue grafts with MRI. Finally, a 3D-contouring procedure developed to facilitate graft delineation and geometrical conformity assessment is applied on an inverted template alginate pore network. This proof-of-concept establishes the possibility to reveal precisely engineered hydrogel structures using this USPIONs ink high-visibility approach.

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Localization of merkel cells at hairless and hairy human skin sites using keratin 18

1995-09-01, Michel, Martine, Germain, Lucie, Godbout, Marie-Josée, Fradette, Julie

Les cellules de Merkel sont des cellules cutanées particulières, possédant non seulement des granules de sécrétion contenant des neuropeptides, mais également des desmosomes et des tonofilaments formés des kératines 8, 18, 19 et 20. Les cellules de Merkel sont plutôt rares dans la peau d'adulte. Toutefois, elles ont été observées en plus grande quantité dans les régions sans poils, telles la paume des mains et la plante des pieds. Leur présence a aussi été rapportée dans les follicules pileux. Les cellules de Merkel sont souvent innervées par des fibres nerveuses sensorielles et on leur reconnaît un rôle de mécanorécepteurs dans la peau. Toutefois, leur origine et fonction précises ne sont pas encore clairement établies. Le but de cette étude était de localiser les cellules de Merkel dans des régions anatomiques pourvues ou non de follicules pileux, par immunohistochimie avec les anticorps Ks18.174 et Ks19.1 respectivement dirigés contre les kératines 18 et 19. Dans la paume des mains et la plante des pieds, les cellules de Merkel ont été identifiées dans la couche basale de l'épiderme, à la base des papilles. Pour étudier la localisation des cellules de Merkel dans une région possédant des poils, nous avons choisi la peau provenant de réduction mammaire car on y trouve de petits follicules pileux représentatifs des poils couvrant une grande partie de notre corps. Des cellules de Merkel ont été observées dans l'épiderme interfolliculaire et dans le follicule pileux, où elles ont été localisées dans la région de l'isthme.Mots clés : peau, humain, cellule de Merkel, kératines, follicule pileux.

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The type I keratin 19 possesses distinct and context-dependent assembly properties

1998-12-25, Germain, Lucie, Fradette, Julie, Seshaiah, Partha, Coulombe, Pierre A.

Keratins (K), the cytoplasmic intermediate filament (IF) proteins of epithelial cells, are encoded by a multigene family and expressed in a tissue- and differentiation-specific manner. In human skin, keratinocytes of the basal layer of epidermis and the outer root sheath of hair follicles express K5 and K14 as their main keratins. A small subpopulation of basal cells exhibiting stem-cell like characteristics express, in addition, K19. At 40 kDa, this keratin is the smallest IF protein due to an exceptionally short carboxyl-terminal domain. We examined the assembly properties of K19 and contrasted them to K14 in vitro and in vivo. Relative to K5-K14, we find that K5-K19 form less stable tetramers that polymerize into shorter and narrower IFs in vitro. When transiently co-expressed in cultured baby hamster kidney cells, the K5 and K19 combination fails to form a filamentous array, whereas the K5-K14 and K8-K19 ones readily do so. Transient expression of K19 in the epithelial cell lines T51B-Ni and A431 results in its integration into the endogenous keratin network with minimal if any perturbation. Collectively, these results indicate that K19 possesses assembly properties that are distinct from those of K14 and suggest that it may impart unique properties to the basal cells expressing it in skin epithelia.

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IFATS Collection : using human adipose-derived stem/stromal cells for the production of new skin substitutes

2008-07-10, Germain, Lucie, Vincent, Caroline, Trottier, Valérie, Marceau-Fortier, Guillaume, Fradette, Julie

The ability to harvest and culture stem cell populations from various human postnatal tissues is central to regenerative medicine applications, including tissue engineering. The discovery of multipotent mesenchymal stem cells within the stromal fraction of adipose tissue prompted their use for the healing and reconstruction of many tissues. Here, we examined the influence of adipose-derived stem/stromal cells (ASCs) on skin's regenerative processes, from a tissue engineering perspective. Using a self-assembly approach, human skin substitutes were produced. They featured a stromal compartment containing human extracellular matrix endogenously produced from either dermal fibroblasts or adipose-derived stem/stromal cells differentiated or not toward the adipogenic lineage. Human keratinocytes were seeded on each stroma and cultured at the air-liquid interface to reconstruct a bilayered skin substitute. These new skin substitutes, containing an epidermis and a distinctive stroma devoid of synthetic biomaterial, displayed characteristics similar to human skin. The influence of the type of stromal compartment on epidermal morphogenesis was assessed by the evaluation of tissue histology, the expression of key protein markers of the epidermal differentiation program (keratin [K] 14, K10, transglutaminase), the expression of dermo-epidermal junction components (laminins, collagen VII), and the presence of basement membrane and hemidesmosomes. Our findings suggest that adipose-derived stem/stromal cells could usefully substitute dermal fibroblasts for skin reconstruction using the self-assembly method. Finally, by exploiting the adipogenic potential of ASCs, we also produced a more complete trilayered skin substitute consisting of the epidermis, the dermis, and the adipocyte-containing hypodermis, the skin's deepest layer. Disclosure of potential conflicts of interest is found at the end of this article.

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Characterization of in vitro engineered human adipose tissues : relevant adipokine secretion and impact of TNF-α

2015-09-14, Roy, Alphonse, Proulx, Maryse, Côté, Jean-François, Safoine, Meryem, Aubin, Kim, Audet-Casgrain, Marie-Alice, Fradette, Julie, Têtu, Félix-André

Representative modelling of human adipose tissue functions is central to metabolic research. Tridimensional models able to recreate human adipogenesis in a physiological tissue-like context in vitro are still scarce. We describe the engineering of white adipose tissues reconstructed from their cultured adipose-derived stromal precursor cells. We hypothesize that these reconstructed tissues can recapitulate key functions of AT under basal and pro-inflammatory conditions. These tissues, featuring human adipocytes surrounded by stroma, were stable and metabolically active in long-term cultures (at least 11 weeks). Secretion of major adipokines and growth factors by the reconstructed tissues was determined and compared to media conditioned by human native fat explants. Interestingly, the secretory profiles of the reconstructed adipose tissues indicated an abundant production of leptin, PAI-1 and angiopoietin-1 proteins, while higher HGF levels were detected for the human fat explants. We next demonstrated the responsiveness of the tissues to the pro-inflammatory stimulus TNF-α, as reflected by modulation of MCP-1, NGF and HGF secretion, while VEGF and leptin protein expression did not vary. TNF-α exposure induced changes in gene expression for adipocyte metabolism-associated mRNAs such as SLC2A4, FASN and LIPE, as well as for genes implicated in NF-κB activation. Finally, this model was customized to feature adipocytes representative of progressive stages of differentiation, thereby allowing investigations using newly differentiated or more mature adipocytes. In conclusion, we produced tridimensional tissues engineered in vitro that are able to recapitulate key characteristics of subcutaneous white adipose tissue. These tissues are produced from human cells and their neo-synthesized matrix elements without exogenous or synthetic biomaterials. Therefore, they represent unique tools to investigate the effects of pharmacologically active products on human stromal cells, extracellular matrix and differentiated adipocytes, in addition to compounds modulating adipogenesis from precursor cells.

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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 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.