van der Valk Wouter H, Nist-Lund Carl, Zhang Jingyuan, Perea Camila, Jin Jiahe, Gim Kelly Y, Steinhart Matthew R, Lee Jiyoon, Koehler Karl R
Department of Otolaryngology, Boston Children's Hospital, Boston, MA, USA.
F.M. Kirby Neurobiology Center, Boston Children's Hospital, Boston, MA, USA.
Nat Protoc. 2025 Jun 2. doi: 10.1038/s41596-025-01191-3.
The inner ear has a pivotal role in auditory and vestibular perception. Despite the vast number of individuals worldwide affected by hearing loss and balance disorders, therapeutic options have been largely limited to technological aids. The recent advent of gene therapies for genetic hearing loss in human patients underscores the urgency of developing scalable platforms to investigate a broader spectrum of inner ear disorders. Although animal models are powerful for assessing auditory and vestibular dysfunction, in vitro human inner ear models have shown promise in disease modeling and as platforms for studying developmental biology. Several studies have demonstrated that stem cells can be guided to differentiate into otic progenitor cells by mimicking environmental cues present during normal fetal inner ear development. Here we present a step-by-step approach to creating inner ear organoids (IEOs), which is an extension of our previous method for skin organoid generation, with which it shares foundational methodology and reagents. We used these organoids to elucidate the subtle signaling cues that govern their developmental trajectories. Generating sensory hair cells takes about 40 d, and cultures can be maintained for up to 150 d to allow further development. Moreover, we outline methods for assessing late-stage organoids, including whole-mount imaging of cleared IEOs, vibratome sectioning of live and fixed IEOs and other endpoint analyses, to study inner ear biology. IEOs are ideal for investigating human inner ear development, studying the mechanisms of inner ear disorders and developing therapeutic strategies. This protocol requires proficiency in basic stem cell culture techniques.
内耳在听觉和前庭感知中起着关键作用。尽管全球有大量个体受听力损失和平衡障碍影响，但治疗选择在很大程度上仅限于技术辅助设备。近期针对人类患者遗传性听力损失的基因疗法的出现，凸显了开发可扩展平台以研究更广泛内耳疾病的紧迫性。虽然动物模型在评估听觉和前庭功能障碍方面很强大，但体外人类内耳模型在疾病建模以及作为研究发育生物学的平台方面已显示出前景。多项研究表明，通过模拟正常胎儿内耳发育过程中存在的环境线索，可引导干细胞分化为耳祖细胞。在此，我们展示一种创建内耳类器官（IEO）的分步方法，这是我们之前生成皮肤类器官方法的扩展，二者共享基础方法和试剂。我们利用这些类器官来阐明控制其发育轨迹的微妙信号线索。生成感觉毛细胞大约需要40天，培养物可维持长达150天以允许进一步发育。此外，我们概述了评估晚期类器官的方法，包括对透明化IEO的整体成像、对活的和固定的IEO进行振动切片以及其他终点分析，以研究内耳生物学。IEO是研究人类内耳发育、研究内耳疾病机制以及制定治疗策略的理想选择。本方案需要熟练掌握基本的干细胞培养技术。
