Fluvastatin Induces Neuroglial but not Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

3-hydroxy-2-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, or statins, have been described as the most effective class of drugs to reduce serum cholesterol levels. It has recently been reported that statins induced up-regulation of bone-morphogenetic protin-2 (BMP-2) expression in osteoblasts and in animal bone defect models. However, the effects of statins on mesenchymal stem cells (MSCs), which are the progenitor cells of osteoblasts, The purpose of this study is to investigate the effects of fluvastatin (Lescol®, a commonly prescribed lipid-lowering agent, on the proliferation and differentiation of MSCs. We hypothesized that fluvastatin increases proliferation and osteogenic differentiation of bone marrow-derived mesenchynmal stem cells. Human bone marrow MSCs were harvested using the methods previously described by the authors. After obtaining enough number of cells for experiments, MSCs were seeded into six-well plates at the density of 4 x 103 cells/cm^2. Fluvastatin (Novartis International AG, Basal, Switzerland) was added into the MSC cultures in seven different concentrations (0.0064 μg/ml, 0.032 μg/ml, 0.16 μg/ml, 0.8 μg/ml, 4 μg/ml, 20 μg/ml, 100 μg/ml) for 7 days. Six replicates were performed for each concentration. MTT assay was used to evaluate the rate of cell proliferation. Reverse transcription polymerase chain reaction (RT-PCR) was performed to look at the expression of osteoblast-related genes such as type I collagen, osteopontin, osteonectin, osteocalcin and BMP-2. Surprisingly, the results showed that change of morphology of the MSCs into neuroglial cell-like was observed in all seven experimental groups 24 hours adding adding fluvastatin into culture. Immunohistochemical stains were performed using antibodies against neuroglial cell-specific antibodies including glial fibrillary acidic protein (GFAP), microtubule-associated protein 2 (MAP2), neuron specific enolase (NSE) and O4. All the immunohistochemical stains showed positive findings after 7 days in all groups, indicating that MSCs were undergoing neuroglial differentiation after being treated with fluvastatin. RT-PCR for MAP2 and GFAP was also performed and expression of MAP2 and GFAP mRNA was noted in all experimental groups. MTT results revealed that after being treated with fluvastatin for seven days, proliferation rate was decreased and this effect was dose-dependent. RT-PCR tests for type I collagen, osteopontin, osteonectin, osteocalcin and BMP-2 showed negative results in all groups. It has been demonstrated in this study that fluvastatin did not induce osteogenic differentiation of MSCs and BMP-2 expression was not up-regulated. However, fluvastatin did induce neuroglial differentiation of MSCs. Besides, fluvastatin inhibited the proliferation of MSCs in a dose-dependent manner. More work is needed to prove the functionality of the neuroglial-differentiated MSCs.

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mesenchymal stem cells ； fluvastatin ； proliferation ； differentiation

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