Critical roles of Src family tyrosine kinases in excitatory neuronal differentiation of cultured embryonic stem cells

Embryonic stem (ES) cells have been tested for potential cell transplantation therapy for CNS disorders. Understanding their differentiation mechanism and identifying factors involved in driving excitatory and inhibitory neuron lineages should enhance the efficacy and efficiency of the cell transplantation therapy. We tested the hypothesis that selective expression of Src family tyrosine kinases is required for phenotype-specific differentiation and functional maturation of ES cell derived neurons. Cultured mouse pluripotent ES cells were treated with retinoic acid (RA) to induce neural differentiation. After RA induction, neurons derived from ES cells showed significant neurite growth, increased expression of Src, Fyn and Lck and an extension of Src kinase expression from cell body to neurite processes. ES cell derived neuron-like cells expressed neurofilament, synaptophysin, glutamate receptors, NMDA and kainate currents, became vulnerable to excitotoxicity and formed functional excitatory synapses. These developmental events were blocked or attenuated when cells were grown in the presence of Src family kinase inhibitor PP2. However, there was no change in the expression of GABAergic-specific protein GAD67 during PP2 treatment. Our data suggest that Src tyrosine kinases are involved in the terminal differentiation of excitatory neuronal phenotype during ES cell neural differentiation after RA induction.