A developmental atlas of mouse vestibular-like inner ear organoids

In 2014, a method was introduced to differentiate mouse embryonic stem cells into inner ear vestibular-like sensory epithelia in vitro. The resulting inner ear organoids represent a potentially inexhaustible source of tissues for investigating mechanosensory hair cell development, function, and responses to ototoxic and regenerative treatments. However, there has been limited exploration into how closely the molecular characteristics of organoid-derived hair cells match those that develop in vivo. To address this gap, we have employed single-cell RNA sequencing (scRNA-seq) to generate a comprehensive atlas of inner ear organoid development at six stages, spanning the formation of definitive ectoderm to the emergence of sensory hair cells. Here, we specifically utilize our inner ear organoid atlas to define the dynamic changes in gene expression that occur during otic vesicle cell maturation into sensory epithelial supporting cells and hair cells. Inner ear organoids were generated using Fbxo2-Venus-Hygromycin-Cre mESCs following established protocols with minor modifications. Organoids were dissociated in biological duplicate at six developmental time points: days in vitro (DIV) 3, DIV4, DIV8, DIV11, DIV16, and DIV21. Cells were processed for scRNA-seq using 10X Genomics Chromium Single Cell 3' Reagents v3.1. After sequencing, we used Cell Ranger for read mapping and quantification, followed by further analysis using Seurat.