A standardized procedure to obtain mesenchymal stem/stromal cells from minimally manipulated dental pulp and Wharton’s 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 “Good Manufacturing Practices” (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 “minimally” and “more than minimally” manipulated samples, in function of the use or not of procedures “that might alter the biological features of the cells”. 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 Wharton’s 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

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-β 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-β3 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-β3 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-β3 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

The canine model to test functional tissue-engineered cartilage

International audienc

Reconstruction du cartilage nasal par ingénierie tissulaire à base de polyéthylène de haute densité et d’un hydrogel

International audienc

Réponse des chondrocytes humains à la bone morphogenetic protein-2 après leur dédifférenciation in vitro : utilisation potentielle de la bone morphogenetic protein-2 pour la thérapie cellulaire du cartilage

International audienc

Décryptage des signalisations moléculaires contrôlant la différenciation des chondrocytes : retombées pour l’ingénierie tissulaire du cartilage : le projet ANR-TecSan PROMOCART

International audienceProject PROMOCART (2007–2010) 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, Symatèse Biomatériaux 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 (2007–2010) a été retenu suite à l’appel 2006 ANR-TecSan. Il a été conduit par le laboratoire « Biologie et ingénierie du cartilage » (Lyon) en partenariat avec les laboratoires « Matrice extracellulaire et pathologie » (Caen), l’institut de biologie de Lille, la société Symatèse biomatériaux (Chaponost) et le laboratoire des substituts cutanés des hospices civils de Lyon. Le cartilage est un tissu au potentiel de cicatrisation spontané très limité. La transplantation de chondrocytes autologues (TCA) est une technique mondialement utilisée pour le traitement de lésions limitées de cartilage articulaire. Cependant, cette approche implique une amplification des chondrocytes sur plastique, ce qui entraîne leur dédifférenciation. Un premier objectif de PROMOCART était de déterminer si la bone morphogenetic protein (BMP)-2 est capable de favoriser le maintien ou la restauration du phénotype des chondrocytes humains. Dans le but d’étendre la TCA à des lésions cartilagineuses plus importantes comme celles des arthroses débutantes, nous avons développé un procédé de reconstruction de cartilage humain dans des éponges de collagène en présence de BMP-2 et dans des conditions hypoxiques. Nous avons contrôlé la qualité du phénotype cellulaire par l’utilisation de nouveaux marqueurs du cartilage