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Rapid expansion of primary human vocal fold epithelial cells via targeted pathway inhibition and anchorage-independent sphere culture

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

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ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

SUBMITTER: Haiyan Lu

LAB HEAD: Lingjun Li

PROVIDER: PXD064066 | Pride | 2025-11-06

REPOSITORIES: Pride

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Project description:Vocal fold (VF) fibrosis, often caused by phonosurgery, radiation, or trauma, leads to irreversible voice dysfunction due to excessive ECM deposition and increased tissue stiffness. VF fibrosis, a significant cause of chronic dysphonia, lacks FDA-approved treatments, highlighting the critical need for novel antifibrotic therapeutic. TGF-β1 is a major driver in the pathological transformation of fibroblasts into contractile myofibroblasts in VF fibrosis via SMAD3, YAP1, and integrin signaling pathways. These pathways lead to increased ACTA2 expression, excessive collagen production, and progressive tissue stiffening. Leveraging the principle that local cells respond to tissue-specific signals, our ECM hydrogel derived from decellularized vocal fold lamina propria (VFLP-ECM) reduced ACTA2 expression in TGF-β1 stimulated vocal fold fibroblasts, showcasing antifibrotic potential. In this study, we evaluated the therapeutic potential of VFLP-ECM hydrogel in a rabbit VF injury model—treatment involved VFLP-ECM or bovine type I collagen injections into VFs 7 days after injury, with evaluations at 28 days post-injury. We compared two VFLP-ECM formulations: a manual process (VFLP (man)) and an accelerated automated method (VFLP (au)). VFLP (man) showed more modulated fibrosis-associated gene expressions when compared to VFLP (au) and collagen. Proteomic analysis revealed that VFLP (man) preserves a broader array of bioactive proteins, such as vitronectin, crucial in TGF-β1 signaling and ECM remodeling. The transcriptomic analysis uncovered a downregulation of key fibrotic markers and potential inhibition of regulators such as SMAD3, YAP1, and MRTFA, alongside the upregulation of SMAD7, an inhibitor of TGF-β signaling. Notably, treatment with VFLP (man) resulted in vocal folds with similar stiffness as uninjured controls, whereas tissues treated with collagen or VFLP (au) remained stiffer, indicating incomplete mechanical recovery. These data provide the first in vivo evidence that VFLP-ECM hydrogel—mainly when produced via manual decellularization—attenuates fibrosis by disrupting biochemical and biomechanical drivers of myofibroblast activation.

			Action	DRS
	20230731_2D-1_01.index	Other
	20230731_2D-1_01.raw	Raw
	20230731_2D-1_02.index	Other
	20230731_2D-1_02.raw	Raw
	20230731_2D-1_03.index	Other

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Rapid expansion of primary human vocal fold epithelial cells via targeted pathway inhibition and anchorage-independent sphere culture

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