Engineering High-Fidelity Human Cochlear Organoids

工程高保真人类耳蜗类器官

• 批准号: 10535013
• 负责人: Eri Hashino
• 金额: $ 67.24万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10535013
• 关键词: Affect; Age; Animal Model; Auditory; Auditory system; Biological; Biomimetics; Biopsy; Brain Stem; CRISPR screen; Cell Differentiation process; Cell Maturation; Cell Therapy; Cells; Chemicals; Clustered Regularly Interspaced Short Palindromic Repeats; Cochlea; Custom; Derivation procedure; Development; Disease; Dorsal; Electron Microscopy; Electrophysiology (science); Engineering; Epithelial; Gelatin; Generations; Genes; Genetic Programming; Genetic study; Genome engineering; Hair Cells; Hearing; Human; Hydrogels; Immunofluorescence Immunologic; In Vitro; Laboratories; Labyrinth; Mediating; Microfluidics; Midbrain structure; Modification; Morbidity - disease rate; Morphology; Mus; Neuraxis; Neurons; Organ; Organoids; Outcome Study; Outer Hair Cells; Pathologic; Pattern; Peripheral; Play; Population; Property; Protocols documentation; Publishing; Reporter; Research; Role; Sensorineural Hearing Loss; Sensory; Sensory Hair; Signaling Molecule; Specificity; System; Systems Development; Technology; Testing; Therapeutic; Thyroid Hormones; Tissue Engineering; Tissues; Vestibular Hair Cells; Zinc Fingers; base; cell type; cochlear development; deaf; deafness; hearing impairment; hearing restoration; hindbrain; human embryonic stem cell; human model; human pluripotent stem cell; improved; in vitro Model; inducible gene expression; inner ear development; mechanical properties; microphysiology system; neural circuit; neural network; next generation; novel; patch clamp; permanent hearing loss; relating to nervous system; sensor; single-cell RNA sequencing; small molecule; sound; spatiotemporal; stem cell technology; three dimensional cell culture; tool; transcription factor

PROJECT SUMMARY Loss of sensory hair cells and/or innervating neurons in the cochlea causes irreversible hearing loss in humans. However, progress on research for realizing biological restoration of hearing has been hampered due to the paucity of human cochlear tissues. My laboratory recently developed a novel organoid system to generate inner ear sensory epithelia containing functional sensory hair cells from aggregates of human pluripotent stem cells in 3D culture. While these first-generation organoids are a valuable tool for studying human inner ear development, they only generate hair cells with structural and functional properties of native vestibular hair cells and fail to produce any cochlear cell types. Another limitation with our original system is the lack of central nervous system components. To overcome these limitations, we aim at developing a next- generation human microphysiological system that more faithfully recapitulates development of the auditory periphery and brainstem. In Aim 1, we will carry out genetic programming and a small-scale CRISPR screen to increase the number of outer hair cells arising in cochlear organoids. The identity of derived hair cells will be validated by single-cell electrophysiology, electron microscopy and single-cell RNA-sequencing. Additionally, maturation of derived hair cells will be promoted by thyroid hormone treatments. In Aim 2, we will establish novel human cochlear-hindbrain assembloids and assess afferent neural circuit development in these assembloids. In Aim 3, we will develop dynamic/tunable hydrogels and test if introducing a spatial gradient of stiffness in the microenvironment during organoid formation can affect tissue patterning or cellular differentiation. Spatial gradients of signaling molecules will be also introduced in hydrogels to test if the dorso- ventral axis formation can be recapitulated during organoid differentiation. The outcome of this study will provide a paradigm-changing approach for studying normal and pathological development of cochlear hair cells and their ascending neural circuits.

项目概要 耳蜗中感觉毛细胞和/或支配神经元的丧失会导致不可逆的听力损失 人类。然而，实现听力生物恢复的研究进展却因以下原因而受到阻碍： 人类耳蜗组织的缺乏。我的实验室最近开发了一种新型类器官系统 从人类聚集体中产生含有功能性感觉毛细胞的内耳感觉上皮 3D 培养中的多能干细胞。虽然这些第一代类器官是研究的宝贵工具 人类内耳发育时，它们只产生具有天然结构和功能特性的毛细胞 前庭毛细胞并且无法产生任何耳蜗细胞类型。我们原始系统的另一个限制是 缺乏中枢神经系统成分。为了克服这些限制，我们的目标是开发下一代 新一代人体微生理系统，更忠实地再现听觉的发展 外周和脑干。在目标 1 中，我们将进行基因编程和小规模 CRISPR 筛选，以 增加耳蜗类器官中产生的外毛细胞的数量。衍生毛细胞的身份将是 通过单细胞电生理学、电子显微镜和单细胞 RNA 测序进行验证。此外， 甲状腺激素治疗将促进衍生毛细胞的成熟。在目标 2 中，我们将建立 新型人类耳蜗后脑组合体并评估这些神经回路的发育 装配体。在目标 3 中，我们将开发动态/可调水凝胶并测试是否引入空间梯度 类器官形成过程中微环境的硬度会影响组织模式或细胞 差异化。信号分子的空间梯度也将被引入水凝胶中，以测试背侧是否 腹轴的形成可以在类器官分化过程中重现。这项研究的结果将 为研究耳蜗毛的正常和病理发育提供一种改变范式的方法 细胞及其上行神经回路。