Engineering Pluripotent Stem Cells-Derived Inner Ear Organoids With Enhanced Maturation and Reproducibility by Micro-Topographical Cues.

Abstract

Inner ear organoids (IEOs) derived from pluripotent stem cells (PSCs) provide a promising platform for modeling neurosensory disorders and hearing loss; however, conventional systems often exhibit substantial structural variability, incomplete maturation, and limited reproducibility due to insufficient control of early organoid morphogenesis. Here, we demonstrate that micro-topographical cues applied during initial IEO formation enhance the development and functional maturation of PSC-derived IEOs. This microengineering strategy introduces temporally defined microscale geometric confinement to regulate early cell-cell and cell-extracellular matrix (ECM) interactions, thereby promoting epithelial organization and developmental fidelity. Microengineered IEOs (M-IEOs) exhibit improved reproducibility and neurosensory maturation, including increased hair cell-like populations, stereocilia-like structures and kinocilium-like features exhibiting a characteristic (9 × 2) + 2 microtubule organization. Functionally, M-IEOs exhibit enhanced electrophysiological responsiveness, supported by complementary transcriptomic and in situ analyses indicating activation of inner ear lineage maturation pathways. Furthermore, we demonstrate the versatility of M-IEOs by integrating them with a microfluidic vascular system to model vascular-epithelial interactions and inflammatory responses, highlighting its potential for disease modeling and pharmacological screening. Together, these findings establish transient micro-topographical guidance as an instructive regulator of inner ear organoid development and provide a robust, vascular-compatible platform for neurosensory research, disease modeling, and translational screening applications.