Mesenchymal stem cells (MSCs) obtained from bone marrow (BM) are currently used as an alternative therapy in amyotrophic lateral sclerosis (ALS) patients. Selection of optimal passages of autologous BM-derived MSCs during long-term in vitro expansion is important for clinical trials in patients with ALS. We isolated and expanded MSCs from the BM of eight ALS patients to analyze the growth kinetics, differentiation potential, cellular surface antigen expression, karyotype modifications and secretion of various cytokines during long-term culture. The morphology and size of the cells changed from small and spindle-like cells to large and polygonal types in later passages. The growth rate of the MSCs was highest in the third passage, followed by a gradual decrease. There were no special modifications of cell surface antigens or the karyotype of the MSCs from the first to the tenth passage. MSCs in the fourth passage were differentiated into adipocytes, osteocytes and chondrocytes. When we analyzed the cultured media of MSCs at the third, fifth, seventh and ninth passages, IL-6, VEGF and IL-8 showed high expression, with more than 50pg/10,000 cells at these passages; however, their expression progressively decreased with additional passages. In addition, secretion of IL-15, GM-CSF, IL-10, PDGF-bb, G-CSF, IL-1beta, basic FGF and IFN-gamma gradually decreased over prolonged culture. We suggest that MSCs at earlier passages are more suitable for stem cell therapy in ALS patients because of their stability and more potent anti-inflammatory and neuroprotective properties.
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