Hypoxia for MSC expansion and differentiation: the best way for enhancing TGFß-induced chondrogenesis and preventing calcifications in alginate beads.
Fiche publication
Date publication
avril 2017
Journal
Tissue engineering. Part A
Auteurs
Membres identifiés du Cancéropôle Est :
Pr GILLET Pierre, Pr MAGDALOU Jacques, Dr ROEDER Emilie
Tous les auteurs :
Henrionnet C, Liang G, Roeder E, Dossot M, Wang H, Magdalou J, Gillet P, Pinzano A
Lien Pubmed
Résumé
We examined the respective influence of a sequential or a continuous hypoxia during expansion and TGF-ß1-driven chondrogenic differentiation of human bone marrow mesenchymal stem cells (MSCs). The differentiation was performed within alginate beads, a classical tool for the implantation of MSCs within the joint. The standard normoxic 2D (expansion) and 3D (differentiation) MSCs cultures served as reference. In order to determine the quality of the chondrogenesis, we analyzed typical markers such as type II and X collagens, SOX9, COMP, versican, and aggrecan mRNAs using PCR and we assessed the production of type II collagen and HIF-1α by histological stainings. We simultaneously assessed the expression of osteogenic mRNAs (Alkaline Phosphatase, RUNX2 and Osteocalcin) and the presence of micro-calcifications by alizarin red and Raman spectroscopy. Chondrogenic differentiation is clearly improved by hypoxia in 3D. Best results were obtained when the entire process, i.e. 2D expansion and 3D differentiation, was performed under continuous 5% hypoxic condition. In addition, no calcification (hydroxyapatite, proved by RAMAN) was observed after 2D hypoxic expansion even in the case of a normoxic differentiation, in contrast with controls. Finally, a better chondrogenic differentiation of human MSCs is achieved when a reduced oxygen tension is applied during both expansion and differentiation times, avoiding the in vitro osteogenic commitment of the cells and subsequently the calcification deposition.
Mots clés
alginate, chondrogenesis, hypoxia, mesenchymal stem cells
Référence
Tissue Eng Part A. 2017 Apr;: