IN-VITRO DIFFERENTIATION OF EMBRYONIC STEM-CELLS INTO GLIAL-CELLS ANDFUNCTIONAL-NEURONS

Mouse embryonic stem cells were induced to differentiate in culture wi th retinoic acid. Putative precursors of neurons and glial cells (nest in-positive cells) were clearly identified as early as three days afte r the onset of differentiation, At day 6, neuron-like cells could be c learly identified, either as isolated cells or as cellular networks. S ome of these cells were positive for astrocyte- or oligodendrocyte-spe cific antigens (GFAP or O4 antigens, respectively). Other cells were p ositive for neuron-specific antigens (cytoskeleton proteins MAP2, MAP5 and NF200, as well as synaptophysin), Some neuronal-like cells were a lso positive for acetylcholinesterase activity or glutamic acid decarb oxylase expression, indicating that ES cells could differentiate into GABAergic and possibly cholinergic neurons, Electrophysiological analy ses performed in voltage clamp conditions showed that cell membranes c ontained voltage-dependent channels. Overshooting action potentials co uld be triggered by current injection. Taken together, these data prov ide evidence that embryonic stem cells can differentiate first into ne uron-glia progenitors, and later into glial cells and functional neuro ns, in vitro. This technique provides an unique system to study early steps of neuronal differentiation in vitro.