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.

Bone morphogenetic protein‐5, a key molecule that mediates differentiation in MC3T3E1 osteoblast cell line

“…(iv) BMP‐5 expression declines at late stages of differentiation. COLIA1 is a principal organic component of the bone required for carrying non‐collagenous proteins, deposition of hydroxyapatite in extracellular matrix (ECM) and maintaining osteoblast integrity, attachment, spreading, and orientation . It is also an early marker of osteoblast differentiation.…”

Whole-Exome Sequencing Suggests LAMB3 as a Susceptibility Gene for Morbid Obesity

“…LAMB3 forms one subunit of the Laminin 5 (also called Laminin-332) heterotrimer, which is a component of the extracellular matrix and thought to mediate the organization of cells into tissues (18). Laminin 5 is typically found in the dermoepidermal junction of the skin (19).…”

“…Mutations in each of the three subunits of Laminin 5 are present in patients with junctional epidermolysis bullosa, an autosomal-recessive disease (20,21). Moreover, Laminin 5 induces osteogenic gene expression and promotes osteogenic differentiation of MSCs (18,22). Laminin 5 is also important for b-cell function (23) and has a role in various types of cancer (24)(25)(26).…”

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“…5 ). This was likely due to the deposition of fibronectin and type I collagen on the Ti surface in the P-CM groups as both type I collagen and laminin-5 promote adhesion of hMSCs [ 55 ]. Further, fibronectin is also known to promote cell adhesion.…”