Sainte-Foy-lès-Lyon, France
Sainte-Foy-lès-Lyon, France

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Metral E.,Gattefosse | Metral E.,CEA Grenoble | Metral E.,Laboratoire des Substituts Cutanes | Metral E.,University Grenoble Alpes | And 5 more authors.
International Journal of Molecular Sciences | Year: 2017

The epidermis basal layer is composed of two keratinocyte populations: Keratinocyte Stem cells (KSC) and Transitory Amplifying (TA) cells that arise from KSC division. Unfortunately, no specific marker exists to differ between KSC and TA cells. Here, we aimed at comparing two different methods that pretended to isolate these two populations: (i) the rapid adhesion method on coated substrate and (ii) the flow cytometry method, which is based on the difference in cell surface expressions of the α6 integrin and transferrin receptor (CD71). Then, we compared different parameters that are known to discriminate KSC and TA populations. Interestingly, we showed that both methods allow enrichment in stem cells. However, cell sorting by flow cytometry (α6high/CD71low) phenotype leads to a better enrichment of KSC since the colony forming efficiency is five times increased versus total cell suspension, whereas it is only 1.4 times for the adhesion method. Moreover, α6high/CD71low cells give rise to a thicker pluristratified epithelium with lower seeding density and display a low Ki67 positive cells number, showing that they have reached the balance between proliferation and differentiation. We clearly demonstrated that cells isolated by a rapid adherent method are not the same population as KSC isolated by flow cytometry following α6high/CD71low phenotype. © 2017 by the authors; licensee MDPI, Basel, Switzerland.


Dos Santos M.,Laboratoire des Substituts Cutanes | Dos Santos M.,University of Lyon | Metral E.,Laboratoire des Substituts Cutanes | Boher A.,LabSkin Creations | And 4 more authors.
Matrix Biology | Year: 2015

Skin aging is a complex phenomenon in which several mechanisms operate simultaneously. Among them, intrinsic aging is a time-dependent process, which leads to gradual skin changes affecting its structure and function such as thinning down of both epidermal and dermal compartments and a flattening and fragility of the dermo-epidermal junction. Today, several approaches have been proposed for the generation of aged skin in vitro, including skin explants from aged donors and three-dimensional skin equivalent treated by aging-inducing chemical compounds or engineered with human cells isolated from aged donors. The aim of this study was to develop and validate a new in vitro model of aging based on skin equivalent demonstrating the same phenotypic changes that were observed in chronological aging. By using prolonged culture as a proxy for cellular aging, we extended to 120 days the culture time of a skin equivalent model based on collagen-glycosaminoglycan-chitosan porous polymer and engineered with human skin cells from photo-protected sites of young donors. Morphological, immunohistological and ultrastructural analysis at different time points of the culture allowed characterizing the phenotypic changes observed in our model in comparison to samples of non photo-exposed normal human skin from different ages. We firstly confirmed that long-term cultured skin equivalents are still morphologically consistent and functionally active even after 120. days of culture. However, similar to in vivo chronological skin aging a significant decrease of the epidermis thickness as well as the number of keratinocyte expressing proliferation marker Ki67 are observed in extended culture time skin equivalent. Epidermal differentiation markers loricrin, filaggrin, involucrin and transglutaminase, also strongly decreased. Ultrastructural analysis of basement membrane showed typical features of aged skin such as duplication of lamina densa and alterations of hemidesmosomes. Moreover, the expression of hyaluronan and its surface receptor CD44 drastically decreased as observed during chronological skin aging. Finally, we found that the level of p16INK4A expression significantly increased supporting cellular senescence process associated to our model. To conclude, the major morphological and ultrastructural epidermal modifications observed in both our extended culture skin equivalent model and skin biopsies from old donors validate the relevance of our model for studying chronological aging, understanding and elucidating age-related modifications of basic skin biological processes. In addition, our model provides a unique tool for identifying new targeted molecules intended at improving the appearance of aging skin. © 2015.


PubMed | LabSkin Creations, Laboratoire des Substituts Cutanes and University of Lyon
Type: | Journal: Matrix biology : journal of the International Society for Matrix Biology | Year: 2015

Skin aging is a complex phenomenon in which several mechanisms operate simultaneously. Among them, intrinsic aging is a time-dependent process, which leads to gradual skin changes affecting its structure and function such as thinning down of both epidermal and dermal compartments and a flattening and fragility of the dermo-epidermal junction. Today, several approaches have been proposed for the generation of aged skin in vitro, including skin explants from aged donors and three-dimensional skin equivalent treated by aging-inducing chemical compounds or engineered with human cells isolated from aged donors. The aim of this study was to develop and validate a new in vitro model of aging based on skin equivalent demonstrating the same phenotypic changes that were observed in chronological aging. By using prolonged culture as a proxy for cellular aging, we extended to 120 days the culture time of a skin equivalent model based on collagen-glycosaminoglycan-chitosan porous polymer and engineered with human skin cells from photo-protected sites of young donors. Morphological, immunohistological and ultrastructural analysis at different time points of the culture allowed characterizing the phenotypic changes observed in our model in comparison to samples of non photo-exposed normal human skin from different ages. We firstly confirmed that long-term cultured skin equivalents are still morphologically consistent and functionally active even after 120 days of culture. However, similar to in vivo chronological skin aging a significant decrease of the epidermis thickness as well as the number of keratinocyte expressing proliferation marker Ki67 are observed in extended culture time skin equivalent. Epidermal differentiation markers loricrin, filaggrin, involucrin and transglutaminase, also strongly decreased. Ultrastructural analysis of basement membrane showed typical features of aged skin such as duplication of lamina densa and alterations of hemidesmosomes. Moreover, the expression of hyaluronan and its surface receptor CD44 drastically decreased as observed during chronological skin aging. Finally, we found that the level of p16INK4A expression significantly increased supporting cellular senescence process associated to our model. To conclude, the major morphological and ultrastructural epidermal modifications observed in both our extended culture skin equivalent model and skin biopsies from old donors validate the relevance of our model for studying chronological aging, understanding and elucidating age-related modifications of basic skin biological processes. In addition, our model provides a unique tool for identifying new targeted molecules intended at improving the appearance of aging skin.


Rodriguez J.,Laboratoire des Substituts Cutanes | Rodriguez J.,French Institute of Health and Medical Research | Rodriguez J.,Edouard Herriot Hospital | Boucher F.,University of Lyon | And 13 more authors.
Stem Cell Research and Therapy | Year: 2015

Background: The use of stem cells from adipose tissue or adipose-derived stem cells (ASCs) in regenerative medicine could be an interesting alternative to bone marrow stem cells because they are easily accessible and available in large quantities. The aim of this study was to evaluate the potential effect of ASCs on the healing of 12 mm diameter-excisional wounds (around 110 mm2) in nude mice. Methods: Thirty nude mice underwent surgery to create one 12-mm excisional wound per mouse (spontaneous healing, n = 6; Cytocare® 532, n = 12; ASCs, n = 12). The Galiano wound model was chosen to avoid shrinkage and thus slow the spontaneous healing (SH) of mouse skin, making it closer to the physiology of human skin healing. Transparent dressings were used to enable daily healing time measurements to be taken. Immunohistochemistry, histological and blood perfusion analysis were carried out on the healed skin. Results: The in vivo results showed the effectiveness of using ASCs on reducing the time needed for complete healing to 21.2 days for SH, 17.4 days for vehicle alone (Cytocare® 532) and 14.6 days with the addition of ASCs (p < 0.001). Moreover, cutaneous perfusion of the healed wound was significantly improved in ASC-treated mice compared to SH group, as shown by laser Doppler flowmetry and the quantitation of blood vessels using immunohistochemistry of αsmooth muscle actin. Conclusions: The tolerance and efficacy of cryopreserved ASCs to accelerate the complete closure of the wound by increasing the maturation of the skin and its blood perfusion, shows their therapeutic benefit in the wound healing context. © 2015 Rodriguez et al.


Houcine A.,University Paul Sabatier | Delalleau A.,Pixience | Heraud S.,Laboratoire des substituts cutanes | Guiraud B.,Pierre Fabre | And 6 more authors.
Skin Research and Technology | Year: 2016

Background: The reliability of the biophysical properties of skin equivalents (SEs) remains a challenge for medical applications and for product efficacy tests following the European Directive 2003/15/EC2 on the prohibition of animal experiments for cosmetic products. Methods: We propose to adapt the biophysical in vivo testing techniques to compare full thickness model growth vs. time. The interest in using such techniques lies in possible comparisons between in vivo and in vitro skin as well as monitoring samples over the culture time. Results: High frequency ultrasound technique, optical coherence tomography (OCT), and laser scanning microscopy were used to analyze SEs morphology at days D42 and D60 whereas their microstructure was assessed through transmission electron microscopy and classical histology. A correlation between these observations and mechanical measurements has been proposed so as to underline the consequence of both the development of the dermis elastic fibers and the epidermis differentiation. Conclusion: Ultrasounds measurements show a highly homogeneous dermis whereas the OCT technique clearly distinguishes the stratum corneum and the living epidermis. The increase in the thicknesses of these layers as well as the growth in elastin and collagen fibers results in strong modifications of the samples mechanical properties. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd


PubMed | University Paul Sabatier, Laboratoire des substituts cutanes, Pixience and Pierre Fabre
Type: Journal Article | Journal: Skin research and technology : official journal of International Society for Bioengineering and the Skin (ISBS) [and] International Society for Digital Imaging of Skin (ISDIS) [and] International Society for Skin Imaging (ISSI) | Year: 2016

The reliability of the biophysical properties of skin equivalents (SEs) remains a challenge for medical applications and for product efficacy tests following the European Directive 2003/15/EC2 on the prohibition of animal experiments for cosmetic products.We propose to adapt the biophysical in vivo testing techniques to compare full thickness model growth vs. time. The interest in using such techniques lies in possible comparisons between in vivo and in vitro skin as well as monitoring samples over the culture time.High frequency ultrasound technique, optical coherence tomography (OCT), and laser scanning microscopy were used to analyze SEs morphology at days D42 and D60 whereas their microstructure was assessed through transmission electron microscopy and classical histology. A correlation between these observations and mechanical measurements has been proposed so as to underline the consequence of both the development of the dermis elastic fibers and the epidermis differentiation.Ultrasounds measurements show a highly homogeneous dermis whereas the OCT technique clearly distinguishes the stratum corneum and the living epidermis. The increase in the thicknesses of these layers as well as the growth in elastin and collagen fibers results in strong modifications of the samples mechanical properties.


PubMed | French Institute of Health and Medical Research, Laboratoire Central dAnatomie Pathologique, Laboratoire des substituts cutanes, French National Center for Scientific Research and University of Lyon
Type: | Journal: Stem cell research & therapy | Year: 2015

The use of stem cells from adipose tissue or adipose-derived stem cells (ASCs) in regenerative medicine could be an interesting alternative to bone marrow stem cells because they are easily accessible and available in large quantities. The aim of this study was to evaluate the potential effect of ASCs on the healing of 12 mm diameter-excisional wounds (around 110 mm(2)) in nude mice.Thirty nude mice underwent surgery to create one 12-mm excisional wound per mouse (spontaneous healing, n=6; Cytocare 532, n=12; ASCs, n=12). The Galiano wound model was chosen to avoid shrinkage and thus slow the spontaneous healing (SH) of mouse skin, making it closer to the physiology of human skin healing. Transparent dressings were used to enable daily healing time measurements to be taken. Immunohistochemistry, histological and blood perfusion analysis were carried out on the healed skin.The in vivo results showed the effectiveness of using ASCs on reducing the time needed for complete healing to 21.2 days for SH, 17.4 days for vehicle alone (Cytocare 532) and 14.6 days with the addition of ASCs (p<0.001). Moreover, cutaneous perfusion of the healed wound was significantly improved in ASC-treated mice compared to SH group, as shown by laser Doppler flowmetry and the quantitation of blood vessels using immunohistochemistry of smooth muscle actin.The tolerance and efficacy of cryopreserved ASCs to accelerate the complete closure of the wound by increasing the maturation of the skin and its blood perfusion, shows their therapeutic benefit in the wound healing context.


PubMed | Service de Chirurgie Plastique; Hopital Edouard Herriot ; Lyon, Laboratoire des Substituts Cutanes, French National Center for Scientific Research and Lyon University
Type: Journal Article | Journal: Adipocyte | Year: 2015

Obesity is linked to adipose tissue hypertrophy (increased adipocyte cell size) and hyperplasia (increased cell number). Comparative analyses of gene datasets allowed us to identify 1426 genes which may represent common adipose phenotype in humans and mice. Among them we identified several adipocyte-specific genes dysregulated in obese adipose tissue, involved in either fatty acid storage (acyl CoA synthase ACSL1, hormone-sensitive lipase LIPE, aquaporin 7 AQP7, perilipin PLIN) or cell adhesion (fibronectin FN1, collagens COL1A1, COL1A3, metalloprotein MMP9, or both (scavenger receptor FAT/CD36). Using real-time analysis of cell surface occupancy on xCELLigence system we developed a new method to study lipid uptake and differentiation of mouse 3T3L1 fibroblasts and human adipose stem cells. Both processes are regulated by insulin and fatty acids such as oleic acid. We showed that fatty acid addition to culture media increased the differentiation rate and was required for full differentiation into unilocular adipocytes. Significant activation of lipogenesis, i.e. lipid accumulation, by either insulin or oleic acid was monitored in times ranging from 1 to 24h, depending on differentiation state, whereas significant effects on adipogenesis, i.e., surperimposed lipid accumulation and gene transcriptional regulations were measured after 3 to 4 d. Combination of selected times for analysis of lipid contents, cell counts, size fractionations, and gene transcriptional regulations showed that FAT/CD36 specific inhibitor AP5258 significantly increased cell survival of oleic acid-treated mouse and human adipocytes, and partially restored the transcriptional response to oleic acid in the presence of insulin through JNK pathway. Taken together, these data open new perspectives to study the molecular mechanisms commonly dysregulated in mouse and human obesity at the level of lipogenesis linked to hypertrophy and adipogenesis linked to hyperplasia.


Claus S.,University of Lyon | Claus S.,Laboratoire des Substituts Cutanes | Aubert-Foucher E.,University of Lyon | Demoor M.,University of Caen Lower Normandy | And 8 more authors.
Journal of Cellular Biochemistry | Year: 2010

Articular cartilage is a specialized connective tissue containing chondrocytes embedded in a network of extracellular macromolecules such as type II collagen and presents poor capacity to self-repair. Autologous chondrocyte transplantation (ACT) is worldwide used for treatment of focal damage to articular cartilage. However, dedifferentiation of chondrocytes occurs during the long term culture necessary for mass cell production. The aim of this study was to investigate if addition of bone morphogenetic protein (BMP)-2, a strong inducer of chondrogenic expression, to human chondrocytes immediately after their isolation from cartilage, could help to maintain their chondrogenic phenotype in long-term culture conditions. Human articular chondrocytes were cultured according to the procedure used for ACT. Real-time PCR and Western blotting were performed to evaluate the cellular phenotype. Exogenous BMP-2 dramatically improves the chondrogenic character of knee articular chondrocytes amplified over two passages, as assessed by the BMP-2 stimulation on type II procollagen expression and synthesis. This study reveals that BMP-2 could potentially serve as a therapeutic agent for supporting the chondrogenic phenotype of human articular chondrocytes expanded in the conditions generally used for ACT. © 2010 Wiley-Liss, Inc.


Claus S.,University of Lyon | Mayer N.,University of Lyon | Aubert-Foucher E.,University of Lyon | Chajra H.,Symatese Biomateriaux | And 6 more authors.
Tissue Engineering - Part C: Methods | Year: 2012

Objective: Articular cartilage has a poor capacity for spontaneous repair. Tissue engineering approaches using biomaterials and chondrocytes offer hope for treatments. Our goal was to test whether collagen sponges could be used as scaffolds for reconstruction of cartilage with human articular chondrocytes. We investigated the effects on the nature and abundance of cartilage matrix produced of sequential addition of chosen soluble factors during cell amplification on plastic and cultivation in collagen scaffolds. Design: Isolated human articular chondrocytes were amplified for two passages with or without a cocktail of fibroblast growth factor (FGF)-2 and insulin (FI). The cells were then cultured in collagen sponges with or without a cocktail of bone morphogenetic protein (BMP)-2, insulin, and triiodothyronine (BIT). The constructs were cultivated for 36 days in vitro or for another 6-week period in a nude mouse-based contained-defect organ culture model. Gene expression was analyzed using polymerase chain reaction, and protein production was analyzed using Western-blotting and immunohistochemistry. Results: Dedifferentiation of chondrocytes occured during cell expansion on plastic, and FI stimulated this dedifferentiation. We found that addition of BIT could trigger chondrocyte redifferentiation and cartilage-characteristic matrix production in the collagen sponges. The presence of FI during cell expansion increased the chondrocyte responsiveness to BIT. © 2012 Mary Ann Liebert, Inc.

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