Cortical excitatory neurons (Cx neurons) are the most dominant neuronal cell type in the cerebral cortex and play a central role in cognition, perception, intellectual behavior, and emotional processing. Robust in vitro induction of Cx neurons may facilitate as a tool for the elucidation of brain development and the pathomechanism of the intractable neurodevelopmental and neurodegenerative disorders, including Alzheimers disease, and thus potentially contribute to drug development. Here, we report a defined method for the efficient induction of Cx neurons from the feeder-free-conditioned human embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells). Using this method, human ES/iPS cells could be differentiated into ~99% MAP2-positive neurons by three weeks, and these induced neurons displayed several characteristics of mature excitatory neurons within 5 weeks, such as strong expression of glutamatergic neuron-specific markers (subunits of AMPA and NDMA receptors and CAMKIIα), highly synchronized spontaneous firing and excitatory postsynaptic current (EPSC). In addition, the Cx neurons showed susceptibility to Aβ oligomer toxicity and excessive glutamate excitotoxicity, which is another advantage for toxicity testing and searching for therapeutic agent discovery. Taken together, this study provides a novel research platform for studying neural development and degeneration based on the feeder-free human ES/iPS cell system.

Synapse formation and functioning are vital for neural network development. Astrocytes ensheathing synapses enable appropriate synapse functioning through glutamate clearance from the synaptic cleft; however, it remains unclear how astrocytic glutamate transporter proteins function at peri-synaptic contact. Here, we report that down syndrome cell adhesion molecule (DSCAM) in Purkinje cells controls climbing fiber (CF) synaptogenesis in the developing cerebellum. Dscam-mutant mice mimicked loss-of-function mutations in the astrocytic glutamate transporter GLAST expressed in Bergmann glia. These mice showed impaired glutamate clearance by the delocalization of GLAST, which complexed with the extracellular domain of DSCAM. Riluzole-induced reduction of free glutamate at the synaptic cleft rescued the impairment of CF synapse formation in Purkinje cell-selective Dscam-deficient mice. DSCAM was required for motor learning, but not gross motor coordination. In conclusion, the intercellular association of synaptic and astrocyte proteins in the tripartite synapse is critical for synapse formation and functioning in neural transmission.