A versatile method for differentiation of multiple neuronal subtypes from mouse embryonic stem cells

Embryonic Stem (ES) cells have recently evoked a great scientific interest, being valuable both for the study of early mammalian development and for regenerative medicine. In this respect, several methods have been recently described to achieve neuronal differentiation of ES cells; however, most of them either require extended in vitro culture or give rise to lineage-restricted neuronal differentiation. To overcome these limitations, we have developed an improved method for preparing mouse ES cell aggregates for in vitro neurogenesis studies. This protocol relies on the generation of size-controlled Embryoid Bodies (EBs) in a chemically-defined serum free medium without the addition of any specific factors. Neuronal differentiation is assessed by both RT-PCR and immunofluorescence analysis. The expression profile of multiple neuronal-subtype specific markers is analyzed, thus showing that ES-derived EBs undergo a rapid and progressive differentiation toward both glial and neuronal lineages, giving rise to a wide range of neuronal subtypes (i.e. dopaminergic, serotonergic and cholinergic neurons). Indeed, a marked temporal correlation in the onset of gene expression is observed between ES cell differentiation and mouse development. This method provides an efficient platform for studying the molecular mechanisms underlying the events of multiple neuronal subtype specification in ES cells