Metabotropic glutamate receptors regulate differentiation of embryonic stem cells into GABAergic neurons.
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.
Sarichelou
I
I
Department of Human Physiology and Pharmacology, University of Rome Sapienza, Rome, Italy.
Cappuccio
I
I
Ferranti
F
F
Mosillo
P
P
Ciceroni
C
C
Sale
P
P
Stocchi
F
F
Battaglia
G
G
Nicoletti
F
F
Melchiorri
D
D
D000613:Aminobutyrates / Q000494:pharmacology; D000818:Animals; D001578:Benzopyrans / Q000494:pharmacology; D002448:Cell Adhesion; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005968:Glutamate Decarboxylase / Q000378:metabolism; D008564:Membrane Potentials; D051379:Mice; D009474:Neurons / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D010641:Phenotype; D011725:Pyridines / Q000494:pharmacology; D064529:Receptor, Metabotropic Glutamate 5; D018094:Receptors, Metabotropic Glutamate / Q000187:drug effects / Q000378:metabolism*; D013997:Time Factors; D014212:Tretinoin / Q000494:pharmacology; D014404:Tubulin / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D000613:Aminobutyrates; D001578:Benzopyrans; D018691:Excitatory Amino Acid Antagonists; C442478:N-phenyl-7-(hydroxyimino)cyclopropa(b)chromen-1a-carboxamide; D011725:Pyridines; D064529:Receptor, Metabotropic Glutamate 5; D018094:Receptors, Metabotropic Glutamate; D014404:Tubulin; D005680:gamma-Aminobutyric Acid; D014212:Tretinoin; C121465:6-methyl-2-(phenylethynyl)pyridine; D005968:Glutamate Decarboxylase; C402565:glutamate decarboxylase 1; C401141:glutamate decarboxylase 2; C012729:2-amino-4-phosphonobutyric acid; C108823:metabotropic glutamate receptor 4