Sarichelou I, Cappuccio I, Ferranti F, Mosillo P, Ciceroni C, Sale P, Stocchi F, Battaglia G, Nicoletti F, Melchiorri D
Department of Human Physiology and Pharmacology, University of Rome Sapienza, Rome, Italy.
Cell Death Differ. 2008 Apr;15(4):700-7. doi: 10.1038/sj.cdd.4402298. Epub 2008 Jan 4.
Mouse embryonic stem (ES) cells were stimulated to differentiate either as adherent monolayer cultures in DMEM/F12 supplemented with N2/B27, or as floating embryoid bodies (EBs) exposed to 1 microM retinoic acid (RA) for 4 days, starting from 4 DIV, and subsequently re-plated in DMEM/F12 medium. Adherent monolayer cultures of ES cells expressed mGlu5 receptors throughout the entire differentiation period. Selective pharmacological blockade of mGlu5 receptors with methyl-6-(phenylethynyl)-pyridine (MPEP) (1 microM, added once a day) accelerated the appearance of the neuronal marker, beta-tubulin. In addition, treatment with MPEP increased the number of cells expressing glutamate decarboxylase-65/67 (GAD(65/67)), a marker of GABAergic neurons. In floating EBs, mGlu5 receptors are progressively replaced by mGlu4 receptors. The orthosteric mGlu4/6/7/8 receptor agonist, L-2-amino-4-phosphonobutanoate (L-AP4), or the selective mGlu4 receptor enhancer, PHCCC,--both combined with RA at concentrations of 30 microM--increased the expression of both beta-tubulin and GAD(65/67), inducing the appearance of fully differentiated neurons that released GABA in response to membrane depolarization. We conclude that mGlu receptor subtypes regulate neuronal differentiation of ES cells in a context-dependent manner, and that subtype-selective ligands of these receptors might be used for the optimization of in vitro protocols aimed at producing GABAergic neurons from ES cells.
小鼠胚胎干细胞（ES细胞）在添加了N2/B27的DMEM/F12培养基中作为贴壁单层培养物进行诱导分化，或者从第4天开始作为悬浮类胚体（EBs）暴露于1 μM视黄酸（RA）中4天，随后重新接种于DMEM/F12培养基中。ES细胞的贴壁单层培养物在整个分化期均表达代谢型谷氨酸受体5（mGlu5受体）。用甲基-6-（苯乙炔基）-吡啶（MPEP）（1 μM，每天添加一次）对mGlu5受体进行选择性药理阻断，加速了神经元标志物β-微管蛋白的出现。此外，用MPEP处理增加了表达谷氨酸脱羧酶65/67（GAD（65/67））的细胞数量，GAD（65/67）是GABA能神经元的标志物。在悬浮类胚体中，mGlu5受体逐渐被mGlu4受体取代。亲代谢型谷氨酸受体4/6/7/8受体激动剂L-2-氨基-4-膦酸丁酸（L-AP4）或选择性mGlu4受体增强剂PHCCC，二者均与30 μM的RA联合使用，增加了β-微管蛋白和GAD（65/67）的表达，诱导出现了对膜去极化有反应而释放GABA的完全分化的神经元。我们得出结论，代谢型谷氨酸受体亚型以上下文依赖的方式调节ES细胞的神经元分化，并且这些受体的亚型选择性配体可能用于优化旨在从ES细胞产生GABA能神经元的体外方案。
