Pulmonary Organoid Models Demonstrate Compositionally Driven Epithelial Plasticity and Immune Polarization
Aberrant epithelial regeneration and immune remodeling are hallmarks of chronic lung diseases such as idiopathic pulmonary fibrosis (IPF), COPD, and post-viral syndromes. Yet how cellular context shapes these trajectories remains unresolved. We present a tunable, primary rat-derived lung organoid model that systematically varies immune, epithelial, and mesenchymal inputs to reveal how composition alone dictates epithelial plasticity and macrophage polarization. Across conditions, we observed the spontaneous emergence of disease-relevant transitional cell states, including Sox9? stressed progenitors, RAS/AT0-like intermediates, and hillock-like cells, alongside distinct macrophage activation profiles. In mesenchyme-rich contexts, epithelial, immune, and mesenchymal crosstalk appeared to reinforce inflammatory signaling and stabilize transitional persistence, while immune-dominant inputs favored ATI-like repair and squamous remodeling. Notably, hillock-like cells displayed context-specific polarization and expressed immune-regulatory genes, suggesting a role as epithelial orchestrators that help calibrate inflammatory response during regeneration. Connectomic analysis revealed that regenerative outcomes were associated with dynamic multicellular signaling networks that integrate stress sensing, immune coordination, and epithelial fate. This platform provides a tractable system for modeling context-specific repair and regenerative mechanisms and could inform therapeutic strategies aimed at redirecting epithelial fate in chronic lung disease. Overall design: Whole native rat lungs were enzymatically dissociated to generate heterogeneous starting populations containing epithelial, immune, and mesenchymal cells (PD_1, PD_2, PD_3) and sequenced. Additionally, primary immune and epithelial cells isolated via bronchoalveolar lavage (BAL) from native rat lungs were sequenced (BAL_1, BAL_2, BAL_3). Both PD and BAL suspensions were seeded into organoid culture and expanded for 10 days. Three organoid conditions were generated: PD_3D (100% PD), Mixed_3D (50:50 mix of PD and BAL), and BAL_3D (100% BAL). This design was intended to explore how varying the initial immune, epithelial, and mesenchymal composition shapes downstream cell fate, regenerative plasticity, and multicellular signaling.