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

摘要

class="head no_bottom_margin" id="sec1title">IntroductionIn the neural system, signals perceived by the peripheral nervous system (PNS) are transferred to the central nervous system (CNS) via their connection nerves. In neurodegenerative diseases such as spinal cord injury () and hearing loss (), the PNS and CNS neurons, as well as their connection nerves, are usually damaged. Since embryonic stem cells (ESCs) are pluripotent and can differentiate into ectoderm, mesoderm, and endoderm, stem cell-based approaches have been studied to replace damaged neurons (, ). However, how to integrate stem cell-derived neurons into native nervous system remains a challenge. In this research, auditory system regeneration was investigated to address this issue.In the auditory system, spiral ganglion neurons (SGNs) are PNS neurons that transfer auditory signals to the cochlear nucleus (CN) in the brainstem (A). These neural components are usually damaged in hearing loss (). In stem cell-based replacement, SGN-like cells have been generated in vitro using ESCs and tissue-specific stem cells (, ). However, for newly generated cells to transfer auditory signals to the brainstem, proper neural connections must be established between new cells and native CN neurons, which at least includes connection, myelination, and tonotopic array of neurite outgrowths. This research focused on the synaptic connections of neurite outgrowths.Establishment and Evaluation of the 4C2 ESC Line(A) Spiral ganglion neurons (SGNs), cochlear nucleus (CN), and their connections.(B) The Cre plasmid for 4C2 ESC generation.(C) Timeline of 4C2 cell generation: Cre recombination, puromycin selection, and 4C2 generation. Differential interference contrast (DIC) and epifluorescence microscopy images demonstrate 4C2 cell line establishment, which includes CE1, Cre recombination, puromycin selection, and 4C2 ESC generation.(D) RT-PCR shows that both CE1 and 4C2 ESCs express Gapdh, Pou5f1, Nanog, Fut4, and Sox2. Gfp is detected in 4C2 cells but not CE1 cells. Original gel image in .(E) Immunofluorescence exhibits expression of OCT4, NANOG, SSEA1, and SOX2 in 4C2 cell colonies.Scale bar: 100 μm in (C); 20 μm in (E).