title: Laminin-5 and type I collagen promote adhesion and osteogenic differentiation of animal serum-free expanded human mesenchymal stromal cells.
abstract: Mesenchymal stromal cells (MSC) are differentiation competent cells and may generate, among others, mature osteoblasts or chondrocytes in vitro and in vivo. Laminin-5 and type I collagen are important components of the extracellular matrix. They are involved in a variety of cellular and extracellular activities including cell attachment and osteogenic differentiation of MSC. MSC were isolated and expanded using media conforming good medical practice (GMP)-regulations for medical products. Cells were characterized according to the defined minimal criteria for multipotent MSC. MTT- and BrdU-assays were performed to evaluate protein-dependent (laminin-5, laminin-1, type I collagen) metabolic activity and proliferation of MSC. MSC-attachment assays were performed using protein-coated culture plates. Osteogenic differentiation of MSC was measured by protein-dependant mineralization and expression of osteogenic marker genes (osteopontin, alkaline phophatase, Runx2) after three, seven and 28 days of differentiation. Marker genes were identified using quantitative reverse-transcription polymerase chain reaction. Expansion of MSC in GMP-conforming media yielded vital cells meeting all minimal criteria for MSC. Attachment assay revealed a favorable binding of MSC to laminin-5 and type I collagen at a protein concentration of 1-5 fmol/µL. Compared to plastic, osteogenic differentiation was significantly increased by laminin-5 after 28 days of culture (P<0.04). No significant differences in gene expression patterns were observed. We conclude that laminin-5 and type I collagen promote attachment, but laminin-1 and laminin-5 promote osteogenic differentiation of MSC. This may influence future clinical applications.
authors.affiliation: Department of Orthopedic Surgery, University Hospital Tübingen;
authors.forename: Falk
authors.initials: F
authors.lastname: Mittag
authors.forename: Eva-Maria
authors.initials: EM
authors.lastname: Falkenberg
authors.forename: Alexandra
authors.initials: A
authors.lastname: Janczyk
authors.forename: Marco
authors.initials: M
authors.lastname: Götze
authors.forename: Tino
authors.initials: T
authors.lastname: Felka
authors.forename: Wilhelm K
authors.initials: WK
authors.lastname: Aicher
authors.forename: Torsten
authors.initials: T
authors.lastname: Kluba
keywords: attachment; laminin-5.; mesenchymal stromal cells; osteogenic differentiation; type I collagen