tretinoin and 2-amino-4-phosphonobutyric-acid

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.

Topics: Aminobutyrates; Animals; Benzopyrans; Cell Adhesion; Cell Differentiation; Cell Line; Embryonic Stem Cells; Excitatory Amino Acid Antagonists; gamma-Aminobutyric Acid; Glutamate Decarboxylase; Membrane Potentials; Mice; Neurons; Phenotype; Pyridines; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Time Factors; Tretinoin; Tubulin

The mGlu5 receptor is the only metabotropic glutamate receptor subtype expressed by mouse embryonic stem (ES) cells grown under non-differentiating conditions [Cappuccio, I., Spinanti, P. Porcellini, A., Desiderati, F., De Vita, T., Storto, M., Capobianco, L., Battaglia, G., Nicoletti, F., Melchiorri, D., 2005. Endogenous activation of mGlu5 metabotropic glutamate receptors supports self-renewal of cultured mouse embryonic stem cells. Neuropharmacology 1, 196-205]. We now report that ES cells differentiating into embryoid bodies (EBs) progressively lose mGlu5 receptors and begin to express mGlu4 receptors at both mRNA and proteinc level. A 4-day treatment of EBs with the mGlu4 receptor agonist, L-2-amino-4-phosphonobutanoate (L-AP4), increased mRNA levels of the mesoderm marker, brachyury and the endoderm marker, H19, and decreased the expression of the transcript for the primitive ectoderm marker, fibroblast-growth factor-5 (FGF-5). These effects were prevented by the mGlu4 receptor antagonists, alpha-methylserine-O-phosphate (MSOP). Plating of EBs for 4 days in vitro in ITSFn medium induced cell differentiation towards a neural lineage, as reflected by the expression of the intermediate filament protein, nestin, and the homeobox protein, Dlx-2. Pharmacological activation of mGlu4 receptors during cell incubation in ITSFn medium increased the expression of both neural markers. Similar results were obtained when neural differentiation was induced by exposure of EBs to retinoic acid. These data suggest that differentiation of cultured ES cells is associated with changes in the expression pattern of mGlu receptors and that activation of mGlu4 receptors affects cell differentiation in a context-dependent manner.

Topics: Adaptor Proteins, Vesicular Transport; Aminobutyrates; Analysis of Variance; Animals; Cell Differentiation; Cell Line; Drug Interactions; Embryo, Mammalian; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Fetal Proteins; Fibroblast Growth Factor 5; Gene Expression Regulation, Developmental; Glycine; Homeodomain Proteins; Immunohistochemistry; Mice; Phosphoserine; Receptors, Metabotropic Glutamate; Reverse Transcriptase Polymerase Chain Reaction; RNA, Long Noncoding; RNA, Messenger; RNA, Untranslated; Stem Cells; T-Box Domain Proteins; Transcription Factors; Tretinoin