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.

上皮再生异常与免疫重塑是特发性肺纤维化（idiopathic pulmonary fibrosis, IPF）、慢性阻塞性肺疾病（chronic obstructive pulmonary disease, COPD）以及病毒感染后综合征（post-viral syndromes）等慢性肺部疾病的标志性特征。然而，细胞微环境如何调控这些病理进程仍未明确。我们构建了一款可调控的原代大鼠肺类器官（primary rat-derived lung organoid）模型，通过系统性改变免疫、上皮及间充质组分，探究仅由细胞组成如何决定上皮细胞可塑性（epithelial plasticity）与巨噬细胞极化（macrophage polarization）。在不同培养条件下，我们观察到疾病相关的过渡细胞状态（transitional cell states）自发出现，包括Sox9阳性应激祖细胞、RAS/AT0样中间细胞以及丘状细胞（hillock-like cells），同时伴随特征鲜明的巨噬细胞激活谱。在富含间充质的培养环境中，上皮、免疫与间充质细胞间的串扰会增强炎症信号通路并维持过渡细胞状态的稳定性；而以免疫组分为主的环境则更倾向于诱导ATI样修复与鳞状上皮重塑。值得注意的是，丘状细胞呈现出环境依赖的极化特征，并表达免疫调控相关基因，提示其作为上皮调控者参与调节再生过程中的炎症应答。连接组学（connectomic）分析显示，再生结局与整合了应激感知、免疫协调及上皮细胞命运调控的动态多细胞信号网络密切相关。该平台为模拟环境依赖的修复与再生机制提供了一个可操作的研究体系，可为靶向重塑慢性肺部疾病中上皮细胞命运的治疗策略提供理论依据。

整体实验设计：将完整的天然大鼠肺脏进行酶解消化，获得包含上皮、免疫及间充质细胞的异质性起始群体（PD_1、PD_2、PD_3）并进行测序。此外，通过支气管肺泡灌洗（bronchoalveolar lavage, BAL）从天然大鼠肺脏中分离的原代免疫与上皮细胞也进行了测序（BAL_1、BAL_2、BAL_3）。将PD和BAL细胞悬液分别接种至类器官培养体系中并扩增10天，共设置三类培养条件：PD_3D（100% PD细胞）、Mixed_3D（PD与BAL细胞按50:50比例混合）以及BAL_3D（100% BAL细胞）。本实验设计旨在探究初始免疫、上皮及间充质组分的差异如何调控下游细胞命运、再生可塑性及多细胞信号通路。