14 research outputs found
A standardized procedure to obtain mesenchymal stem/stromal cells from minimally manipulated dental pulp and Whartonâs jelly samples
Transplantation of mesenchymal stem/stromal cells (MSCs) has emerged as an effective method to treat diseased or damaged organs and tissues, and hundreds of clinical trials using MSCs are currently under way to demonstrate the validity of such a therapeutic approach. However, most MSCs used for clinical trials are prepared in research laboratories with insufficient manufacturing quality control.In particular, laboratories lack standardized procedures for in vitro isolation of MSCs from tissue samples, resulting in heterogeneous populations of cells and variable experimental and clinical results. MSCs are now referred to as Human Cellular Tissue-based Products or Advanced Therapy Medicinal Products, and guidelines from the American Code of Federal Regulation of the Food and Drug Administration (21 CFR Part 1271) and from the European Medicines Agency (European Directive 1394/2007) define requirements for appropriate production of these cells. These guidelines, commonly called âGood Manufacturing Practicesâ (GMP), include recommendations about laboratory cell culture procedures to ensure optimal reproducibility, efficacy and safety of the final medicinal product. In particular, the Food and Drug Administration divides ex vivo cultured cells into âminimallyâ and âmore than minimallyâ manipulated samples, in function of the use or not of procedures âthat might alter the biological features of the cellsâ. Today, minimal manipulation conditions have not been defined for the collection and isolation of MSCs (Torre et al. 2015)(Ducret et al. 2015).Most if not all culture protocols that have been reported so far are unsatisfactory, because of the use of xeno- or allogeneic cell culture media, enzymatic treatment and long-term cell amplification that are known to alter the quality of MSCs. The aim of this study was to describe a standardized procedure for recovering MSCs with minimal handling from two promising sources, the dental pulp (DP) and the Whartonâs jelly (WJ) of the umbilical cord. The quality and homogeneity of the expanded cell populations were assessed by using flow cytometry with criteria that go beyond the International Society of Cellular Therapy (ISCT) guidelines for MSC characterization
The trans-well coculture of human synovial mesenchymal stem cells with chondrocytes leads to self-organization, chondrogenic differentiation, and secretion of TGFÎČ
Chondrogenic potential of bone marrow- and adipose tissue-derived adult human mesenchymal stem cells.
International audienceRegarding cartilage repair, tissue engineering is currently focusing on the use of adult mesenchymal stem cells (MSC) as an alternative to autologous chondrocytes. The potential of stem cells from various tissues to differentiate towards the chondrogenic phenotype has been investigated and it appears that the most common and studied sources are bone marrow (BM) and adipose tissue (AT) for historical and easy access reasons. In addition to three dimensional environment, the presence of member(s) of the transforming growth factor (TGF-ÎČ family and low oxygen tension have been reported to promote the in vitro differentiation of MSCs. Our work aimed at characterizing and comparing the degree of chondrogenic differentiation of MSCs isolated from BM and AT cultured in the same conditions. We also further aimed at and at determining whether hypoxia (2% oxygen) could affect the chondrogenic potential of AT-MSCs. Cells were first expanded in the presence of FGF-2, then harvested and centrifuged to allow formation of cell pellets, which were cultured in the presence of TGF-ÎČ3 and/or Bone Morphogenetic Protein-2 (BMP-2) and with 2 or 20% oxygen tension, for 24 days. Markers of the chondrocyte (COL2A1, AGC1, Sox9) and hypertrophic chondrocyte (COL10A1, MMP-13) were monitored by real-time PCR and/or by immunohistological staining. Our data show that BMP-2/TGF-ÎČ3 combination is the best culture condition to induce the chondrocyte phenotype in pellet cultures of BM and AT-MSCs. Particularly, a switch in the expression of the pre-chondrogenic type IIA form to the cartilage-specific type IIB form of COL2A1 was observed. A parallel increase in gene expression of COL10A1 and MMP-13 was also recorded. However when AT-MSCs were cultured in hypoxia, the expression of markers of hypertrophic chondrocytes decreased when BMP-2/TGF-ÎČ3 were present in the medium. Thus it seems that hypoxia participates to the control of AT-MSCs chondrogenesis. Altogether, these cellular model systems will help us to investigate further the potential of different adult stem cells for cartilage engineering
Reconstruction du cartilage nasal par ingĂ©nierie tissulaire Ă base de polyĂ©thylĂšne de haute densitĂ© et dâun hydrogel
International audienc
Réponse des chondrocytes humains à la bone morphogenetic protein-2 aprÚs leur dédifférenciation in vitro : utilisation potentielle de la bone morphogenetic protein-2 pour la thérapie cellulaire du cartilage
International audienc
DĂ©cryptage des signalisations molĂ©culaires contrĂŽlant la diffĂ©renciation des chondrocytes : retombĂ©es pour lâingĂ©nierie tissulaire du cartilage : le projet ANR-TecSan PROMOCART
International audienceProject PROMOCART (2007â2010) was adopted following the call 2006 ANR-TecSan. It was led by the laboratory of Biology and Engineering of Cartilage (Lyon), and carried out in partnership with the laboratory of Extracellular Matrix and Pathology (Caen), Lille Institute of Biology, SymatĂšse BiomatĂ©riaux company (Chaponost) and laboratory of Skin Substitutes (Lyon Hospital). Cartilage presents poor intrinsic healing capacity. Autologous chondrocyte implantation (ACI) is a worldwide used technique applied to focal defects of articular cartilage. However, it implies a step of cell amplification on plastic, which results in the loss of chondrocyte differentiation. The first objective of PROMOCART was to determine if bone morphogenetic protein (BMP)-2 could maintain or restore the differentiated phenotype of human chondrocytes. With the view of applying ACI to developing osteoarthritic lesions, we developed a new method of human cartilage reconstruction by using collagen sponges, BMP-2 and hypoxic culture conditions. We controlled the quality of the cellular phenotype by using new cartilage markers.Le projet PROMOCART (2007â2010) a Ă©tĂ© retenu suite Ă lâappel 2006 ANR-TecSan. Il a Ă©tĂ© conduit par le laboratoire « Biologie et ingĂ©nierie du cartilage » (Lyon) en partenariat avec les laboratoires « Matrice extracellulaire et pathologie » (Caen), lâinstitut de biologie de Lille, la sociĂ©tĂ© SymatĂšse biomatĂ©riaux (Chaponost) et le laboratoire des substituts cutanĂ©s des hospices civils de Lyon. Le cartilage est un tissu au potentiel de cicatrisation spontanĂ© trĂšs limitĂ©. La transplantation de chondrocytes autologues (TCA) est une technique mondialement utilisĂ©e pour le traitement de lĂ©sions limitĂ©es de cartilage articulaire. Cependant, cette approche implique une amplification des chondrocytes sur plastique, ce qui entraĂźne leur dĂ©diffĂ©renciation. Un premier objectif de PROMOCART Ă©tait de dĂ©terminer si la bone morphogenetic protein (BMP)-2 est capable de favoriser le maintien ou la restauration du phĂ©notype des chondrocytes humains. Dans le but dâĂ©tendre la TCA Ă des lĂ©sions cartilagineuses plus importantes comme celles des arthroses dĂ©butantes, nous avons dĂ©veloppĂ© un procĂ©dĂ© de reconstruction de cartilage humain dans des Ă©ponges de collagĂšne en prĂ©sence de BMP-2 et dans des conditions hypoxiques. Nous avons contrĂŽlĂ© la qualitĂ© du phĂ©notype cellulaire par lâutilisation de nouveaux marqueurs du cartilage