Exploring the Blood-Labyrinthine Barrier: A Novel Approach with hiPSC-Derived Spheroids and Assembloids - Project Summary/Abstract Objectives: Blood-Labyrinthine Barrier (BLB) dysfunction is implicated in a range of inner ear disorders (BLB- IEDs), including Meniere's disease, autoimmune inner ear disease, and sensorineural hearing loss. These conditions disrupt inner ear fluid homeostasis, leading to vertigo, hearing loss, tinnitus, and imbalance. The BLB comprises microvascular endothelial cells, pericytes, and perivascular-resident macrophage-like melanocytes (PVM/Ms), which are essential for auditory and vestibular function. This project aims to investigate the role of the BLB in inner ear homeostasis and disease pathophysiology while developing hiPSC-based models to identify therapeutic targets. Research Design: Using human induced pluripotent stem cell (hiPSC) technology, we will model BLB-specific microvascular interactions and explore novel therapeutic interventions. Our approach focuses on generating hiPSC-derived BLB pericyte spheroids by directing neural crest stem cells toward a pericyte fate using vestibular neuronal spheroid-conditioned medium (VNS-CM). Additionally, we will develop hiPSC-derived PVM/M spheroids by differentiating yolk sac macrophage-like cells into BLB-specific PVM/Ms. These models will be integrated into advanced microfluidic devices to create physiologically relevant 3D BLB spheroids, laying the groundwork for BLB assembloid development in future R01 studies. Methodology: We will characterize BLB-specific structural, molecular, and functional properties of hiPSC- derived pericytes and PVM/Ms using advanced imaging, molecular biology, and functional assays. Structural characterization will involve transmission electron microscopy (TEM) to examine ultrastructural features. Molecular profiling will be conducted through immunocytochemistry and RT-PCR to confirm BLB-specific gene and protein expression. Functional validation will include transepithelial electrical resistance (TEER) and dextran permeability assays to assess barrier integrity, as well as cytokine response assays to evaluate BLB-selective properties under inflammatory conditions. By integrating stem cell engineering and microfluidic technologies, we will construct 3D spheroids that replicate BLB molecular and functional characteristics, providing a robust platform for disease modeling, mechanistic studies, and therapeutic screening for BLB-IEDs. Clinical Relevance: By addressing a critical gap in BLB research, this project will advance our understanding of BLB dysfunction across multiple inner ear disorders. Our hiPSC-derived models will facilitate drug screening for patient-specific responses to treatments such as diuretics, histamine modulators, and corticosteroids, reducing the current trial-and-error approach. Additionally, these assembloids will enable disease modeling of BLB-IEDs, offering new insights into disease mechanisms and therapeutic development. This research aligns directly with the NIDCD's mission to support biomedical and behavioral research in hearing and balance disorders, ultimately improving public health and quality of life.
