Kang Mi Lan, Kwon Jin Seon, Kim Moon Suk
Department of Molecular Science and Technology, Ajou University, Suwon 443-759, Korea.
Int J Mol Sci. 2013 Mar 25;14(4):6614-23. doi: 10.3390/ijms14046614.
Several studies have demonstrated that basic fibroblast growth factor (bFGF) can induce neural differentiation of mesenchymal stem cells. In this study, we investigated the neural differentiation of muscle-derived stem cells (MDSCs) following treatment with bFGF and ethosuximide, a small molecule used as an anticonvulsant in humans. Stem cells isolated from rat skeletal muscle (rMDSCs) were pre-induced by culturing with 25 ng/mL bFGF for 24 h and then were transferred to a medium supplemented with or without 4 mM ethosuximide. Neuronal differentiation was assessed by immunocytochemical and western blotting analyses of marker expression. Immunocytochemistry of rMDSCs treated with bFGF and ethosuximide identified abundant cells expressing neuronal markers (TuJ1, neuron-specific class III β-tubulin; NeuN, neuronal nuclear antigen; and NF-MH; neurofilament M and H). Olig2 (oligodendrocyte transcription factor 2)-positive cells were also observed, indicating the presence of oligodendrocyte lineage cells. These findings were substantiated by western blotting analysis of marker proteins. In particular, the expression of NeuN and TuJ1 was significantly higher in rMDSCs treated with ethosuximide and bFGF than in cells stimulated with bFGF alone (NeuN, p < 0.05 and TuJ1, p < 0.001). Expression of the astrocyte marker GFAP (glial fibrillary acidic protein) was not detected in this study. Collectively, the results showed that treatment with bFGF and ethosuximide induced effective transdifferentiation of rMDSCs into cells with a neural-like phenotype. Notably, rMDSCs treated with a combination of bFGF plus ethosuximide showed enhanced differentiation compared with cells treated with bFGF alone, implying that ethosuximide may stimulate neuronal differentiation.
多项研究表明，碱性成纤维细胞生长因子（bFGF）可诱导间充质干细胞向神经细胞分化。在本研究中，我们探究了经bFGF和乙琥胺（一种用于人类的抗惊厥小分子）处理后肌肉来源干细胞（MDSCs）的神经分化情况。从大鼠骨骼肌中分离出的干细胞（rMDSCs）先用25 ng/mL bFGF培养24小时进行预诱导，然后转移至添加或不添加4 mM乙琥胺的培养基中。通过免疫细胞化学和蛋白质印迹分析标记物表达来评估神经元分化。对经bFGF和乙琥胺处理的rMDSCs进行免疫细胞化学分析，发现大量表达神经元标记物的细胞（TuJ1，神经元特异性III类β-微管蛋白；NeuN，神经元核抗原；以及NF-MH，神经丝M和H）。还观察到Olig2（少突胶质细胞转录因子2）阳性细胞，表明存在少突胶质细胞系细胞。这些发现通过标记蛋白的蛋白质印迹分析得到证实。特别是，经乙琥胺和bFGF处理的rMDSCs中NeuN和TuJ1的表达显著高于仅用bFGF刺激的细胞（NeuN，p < 0.05；TuJ1，p < 0.001）。本研究未检测到星形胶质细胞标记物GFAP（胶质纤维酸性蛋白）的表达。总体而言，结果表明bFGF和乙琥胺处理可有效诱导rMDSCs转分化为具有神经样表型的细胞。值得注意的是，与仅用bFGF处理的细胞相比，经bFGF加乙琥胺联合处理的rMDSCs分化增强，这意味着乙琥胺可能刺激神经元分化。
