The in vitro effect of syndecan-3 gene knockout on bone marrow-derived mesenchymal stem cells' properties

Stefan, A.; Kay, A. G.; Forsyth, N.; Jones, F. K.; Pisconti, A.; Kehoe, O

Abstract

Introduction: Inflammation is a central feature of rheumatoid arthritis that affects around 1% of the population and can result in disability and morbidity. The immunomodulatory effects of bone marrow-derived mesenchymal stem cells (MSCs) have been widely studied, and the recent observations that syndecan-3 (SDC3) is selectively pro-inflammatory in the joint led us to hypothesise that SDC3 might play an important role in MSC biology.

Materials and Methods: MSCs were isolated from bone marrow of C57Bl/6 WT (n = 6) and Sdc3−/− (n = 6) mice and used to assess the following: flow cytometry (immunophenotype, size and complexity analysis); population doubling time; colony-forming units; osteogenic, adipogenic and chondrogenic differentiation; adhesion properties to type II collagen, fibronectin and laminin; and migration properties. Western blotting was used to investigate which signalling pathways are affected by SDC3 loss in mMSCs.

Results: Immunophenotypic analysis indicated similar surface marker expression pattern for both WT and Sdc3−/− mMSCs. While both cell types show similar FSC values, the cell complexity in WT mMSCs showed significantly higher values than the Sdc3−/−. The spread cell surface area of Sdc3−/− mMSCs was dramatically lower as compared to WT. The differentiation potential was similar for both WT and Sdc3−/− mMSCs. Collagen and fibronectin significantly improved the adhesion of Sdc3−/−, but not WT, mMSCs. Also, collagen significantly increases the number of Sdc3−/− mMSCs when compared with WT cells. Laminin proved to have no effect. The wound healing assay showed no significant difference between the two cell types. More WT MSCs migrated towards serum or pleiotrophin (PTN) in the transwell assay. Interestingly, Sdc3−/− mMSCs cultured on collagen showed a dramatic increase in AKT phosphorylation accompanied by a decrease in ERK1/2 phosphorylation compared with WT controls.

Discussion: The significantly reduced complexity expressed by the Sdc3−/− mMSCs might be due to the fact that the cytoplasmic domains of syndecans are commonly involved in cytoskeletal regulation; thus, the ablation of Sdc3−/− may trigger cellular morphological changes. These morphological changes were confirmed by the significant reduced cell surface spread of Sdc3−/− mMSC and appeared to be driven by hyperactivation of the PI3K/AKT pathway at the expense of the ERK1/2 pathway.

• Journal Article Type: Conference Paper
• Conference Name: Matrix Biology Europe – July 2018 Meeting Celebrating 50 years of Federation of European Connective Tissue Societies Meetings
• Online Publication Date: Feb 22, 2019
• Publication Date: Feb 22, 2019
• Deposit Date: Jun 28, 2023
• Journal: INTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY
• Print ISSN: 0959-9673
• Publisher: Wiley
• Peer Reviewed: Peer Reviewed
• Volume: 99
• Issue: 6