Vocal fold epithelial cells (VFEs) serve critical physiologic and immunologic functions at the boundary between the upper and lower airways but are difficult to maintain and expand in primary cultures. This technical challenge has impeded progress in VFE biology as well as cell banking for translational applications. Here, using primary human VFEs, we show that simultaneous inhibition of transforming growth factor β (TGF-β), Rho-associated protein kinase (ROCK), and Notch signaling with a small-molecule inhibitor cocktail enables rapid proliferation, successful passaging, and long-term expansion while preserving the core epithelial phenotype. Under anchorage-independent culture conditions, VFE progenitors generate clonal spheres that can be expanded over multiple generations; sphere-dissociated VFEs then revert toward their original phenotype, which includes the ability to form stratified squamous epithelium in organotypic cocultures. Both pathway-inhibited and sphere-cultured VFEs exhibit mechanistically appropriate remodeling of the cellular proteome. These advances offer a robust toolkit for upper airway mucosal biology and regenerative medicine.

Wide-field (WF) neurons of the tectopulvinar pathway integrate retinal and cortical inputs via large dendritic arbors crucial for rapid visual motion detection. Previous studies identified potential marker genes for mouse WF neurons. Here, we validate CBLN2 as a molecular marker of the tree shrew WF neurons and construct AAVs that exploit CBLN2 promoter to selectively target WF neurons across species. Using intersectional genetics in the tree shrew, we show that WF neuron dendrites receive a distinct pattern of VGluT1+ and VGluT2+ inputs based on their distance from the cell body in the dorsoventral axis of the superior colliculus (SC). This represents the first example of a viral tool derived from the tree shrew genome for cell-type-specific targeting across species. Our results provide a foundation for studying SC circuitry in higher-order mammals and for extending this approach to additional conserved cell types in the SC and other brain regions.