Supplementary Material for: Regulation of Multilineage Gene Expression and Apoptosis during in vitro Expansion of Human Bone Marrow Stromal Cells with Different Cell Culture Media

The aim of the present study was to analyze the effect of 3 different expansion media on the expression of marker genes of mesenchymal differentiation (bone, cartilage, fat) as well as apoptosis and senescence during repeated passaging in human bone marrow stromal cells (hBMSCs) in order to identify potential expansion strategies for the use of these cells into tissue-engineered growth of bone. Medium 1 (EGF, PDGF, low Glc, 2% FCS) was associated with the highest proliferation rate compared to medium 2 (β-mercaptoethanol, high Glc DMEM, 15% FCS) and medium 3 (low Glc DMEM, 10% FCS). Real time RT-PCR indicated the lowest levels of expression of osteonectin, core binding factor-α 1, lipoprotein lipase and cartilage oligo matrix protein in medium-1 cultures as compared to media 2 and 3. Early passages expressed higher levels of peroxisome proliferator-activator receptor-γ2 in medium 1 than in media 2 and 3, whereas no difference of Sox-9 expression was noticed among the 3 media. Expression of apoptosis- and senescence-related genes (Bax, BCL-2 and P16INK4a) exhibited the lowest level of Bax/BCL-2 ratio and P16INK4a gene expression of hBMSC in medium 1. In conclusion, the replacement of FCS by recombinant EGF and PDGF promoted rapid proliferation of hBMSCs without inducing differentiation of hBMSCs. It also inhibited expression of apoptosis-related genes and limited replicative senescence during repeated passaging. Media with the lowest possible FCS content and replacement by EGF and PDGF thus should be used for 2D culturing during expansion of hBMSCs, whereas β-mercaptoethanol and high concentrations of FCS can help to commence osteogenic differentiation.