耳神经元也被称为哺乳动物耳蜗中的螺旋神经节神经元（SGN），它们将感觉毛细胞传输的电信号传递到听觉系统的耳核。SGN对毒性伤害很敏感，易受不可逆损伤并难以在受损后再生，导致持续的感音神经性听力损失。然而，获得用于研究或治疗目的的真实SGN仍然具有挑战性。我们在这里开发了一个协议，从人类多能干细胞（hESCs）中产生人类耳神经器官小体（hONOs），其中hESCs被逐步诱导为相应阶段的SGNs，以符合它们的发育轨迹。hONOs在早期阶段富集了类似于SGN的细胞，并在随后的神经元和星形胶质细胞、施万细胞或支持细胞中富集了这些细胞。在这些hONOs中，我们还确定了典型的I型和II型SGN的存在。成熟的hONOs（分化60天）形成了神经网络，其特点是巨大去极化电位（GDP）样事件和玫瑰花状组织诱发的钙迹。电生理分析确认了这些hONOs中存在谷氨酸敏感的神经元。本研究产生的耳神经器官小体提供了研究SGNs和相关疾病的理想模型，促进了感音神经性听力损失的治疗发展。 ©2023作者。细胞增殖由北京干细胞和再生医学研究所和约翰威利和儿子有限公司出版。

Otic neurons, also known as spiral ganglion neurons (SGNs) in mammalian cochlea, transmit electrical signals from sensory hair cells to cochlear nuclei of the auditory system. SGNs are sensitive to toxic insults, vulnerable to get irreversible damaged and hardly regenerate after damage, causing persistent sensorineural hearing loss. Yet, to get authentic SGNs for research or therapeutic purpose remains challenging. Here we developed a protocol to generate human otic neuronal organoids (hONOs) from human pluripotent stem cells (hESCs), in which hESCs were step-wisely induced to SGNs of the corresponding stages according to their developmental trajectory. The hONOs were enriched for SGN-like cells at early stage, and for both neurons and astrocytes, Schwann cells or supporting cells thereafter. In these hONOs, we also determined the existence of typical Type I and Type II SGNs. Mature hONOs (at differentiation Day 60) formed neural network, featured by giant depolarizing potential (GDP)-like events and rosette-organized regions-elicited calcium traces. Electrophysiological analysis confirmed the existence of glutamate-responsive neurons in these hONOs. The otic neuronal organoids generated in this study provide an ideal model to study SGNs and related disorders, facilitating therapeutic development for sensorineural hearing loss.© 2023 The Authors. Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.