Human Hepatoma Cell Line Conditioned Medium Promotes Migration and Increases Alpha Smooth Muscle Actin Expression in Multipotent Mesenchymal Stromal Cells

Introduction: Multipotent mesenchymal stromal cells (MSC) are currently used clinically as therapy for graft versus host disease. There is however evidence that MSC can be recruited to tumor sites and sustain tumor growth. To investigate the molecular mechanism and factors implicated in MSC migration we used a human hepatoma cell line (Huh-7) conditioned medium (CM). Methods: We analyzed human MSC migration using transwell chamber migration assay. Inflammatory cytokines in Huh-7 CM were detected using an antibody cytokine array. Chemokine receptor expression on MSC was analyzed by RT-PCR. We applied Western Blotting to identify signalling pathways implicated in Huh-7 CM induced migration and further investigated aSMA expression in MSC after exposure to recombinant chemokines identified previously in Huh-7 CM. Results: Huh-7 CM triggered 4-fold increase of MSC migration compared to control medium, whereas platelet derived growth factor BB (PDGF-BB), used as positive control, induced a 4.5-fold increase. Phosphorylation of Erk was induced by PDGF-BB and Huh-7 CM, whereas phosphorylation of FAK was only induced by Huh-7 CM. Inhibition of phosphorylation of Erk correlated with decreased migration. We identified high levels of Angiogenin, GCP2, IGFBP2, MIP3a, Macrophage inhibitory protein 1d (MIP1d) and Macrophage chemoattractant protein-1 (MCP-1) in Huh- 7 CM. Further, expression of chemokine receptor CCR1 and CCR5, CXCR2 was detected in most MSC isolated (6/8) whereas CCR2 and CXCR1 has not been detected. Recombinant MIP1d and MCP-1 did not activate MSC migration in the transwell system. However, an antibody directed against MCP-1 decreased non significantly MSC migration. Finally, expression of aSMA was increased in MSC cultured in Huh-7 CM and recombinant MIP1d compared to control and PDGF-BB conditions. Conclusions: Inflammatory chemokines present in Huh-7 CM activates migration of MSC but also increases aSMA expression after prolonged culture. We identified MCP-1 as a player in MSC migration whereas MIP1d is implicated in inducing a myofibroblast-phenotype.