Embryonic Stem Cell-Derived Peripheral Auditory Neurons Form Neural Connections with Mouse Central Auditory Neurons In Vitro via the α2δ1 Receptor

Integration of stem cell-derived neurons into the central nervous system (CNS) remains a challenge. A co-culture system is developed to understand whether mouse embryonic stem cell (ESC)-derived spiral ganglion neuron (SGN)-like cells (ESNs) synapse with native mouse cochlear nucleus (CN) neurons. A Cre system is used to generate Cop-GFP ESCs, which are induced into ESNs expressing features similar to auditory SGNs. Neural connections are observed between ESNs and CN neurons 4–6 days after co-culturing, which is stimulated by thrombospondin-1 (TSP1). Antagonist and loss-of-function small hairpin RNA studies indicate that the α2δ1 receptor is critical for TSP1-induced synaptogenesis effects. Newly generated synapse-like structures express pre- and post-synaptic proteins. Synaptic vesicle recycling, pair recording, and blocker electrophysiology suggest functional synaptic vesicles, transsynaptic activities, and formation of glutamatergic synapses. These results demonstrate the synaptogenesis capability of ESNs, which is important for pluripotent ESC-derived neurons to form functional synaptic connections to CNS neurons.