Dexamethasone inhibition of confluence-induced apoptosis in human mesenchymal stem cells

Abstract

Human mesenchymal stem cells (MSCs) have been extensively characterized with respect to their in vitro expansion and differentiation potential, especially with respect to osteogenesis. Dexamethasone (Dex) is a well-known inducer of osteogenic differentiation of MSCs, but little is known about the effect of Dex treatment on apoptosis in MSCs. In this study, apoptosis in MSCs was examined with respect to cell density and Dex supplementation, using DAPI staining and DNA fragmentation ELISA Assay. In MSC cultures initiated at 1.0, 3.0, and 9.0 × 103 cells per cm2, it was found that higher MSC density correlated with increased apoptosis and that this apoptotic effect was diminished in cultures containing 100 nM Dex. MSCs and fibroblasts were co-cultured, along with empty insert controls, and assayed for apoptosis by ELISA and DAPI counts to determine if soluble factors accounted for the cell density-related apoptosis. No difference was seen between MSCs cultured with inserts containing either MSCs, fibroblasts, or empty control. To determine cell contact effects, BrdU-labeled MSCs were cultured alone or with unlabeled chondrocytes at 2x and 8x the number of MSCs, with and without Dex, and apoptosis levels quantified. The results showed increased apoptosis at greater cell densities, and that the amount of apoptosis was greatly diminished in cultures containing Dex. These results show that apoptosis in MSCs is cell density-related, requires direct cell contact, and that Dex treatment reduces or eliminates this density-related apoptosis. These results may impact how MSCs should be cultured for clinical applications. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.