Platform Effects on Regeneration by Pulmonary Basal Cells as Evaluated by Single-Cell RNA Sequencing

Summary: Cell-based therapies have shown promise for treating myriad chronic pulmonary diseases through direct application of epithelial progenitors or by way of engineered tissue grafts or whole organs. To elucidate environmental effects on epithelial regenerative outcomes in vitro, here, we isolate and culture a population of pharmacologically expanded basal cells (peBCs) from rat tracheas. At peak basal marker expression, we simultaneously split peBCs into four in vitro platforms: organoid, air-liquid interface (ALI), engineered trachea, and engineered lung. Following differentiation, these samples are evaluated using single-cell RNA sequencing (scRNA-seq) and computational pipelines are developed to compare samples both globally and at the population level. A sample of native rat tracheal epithelium is also evaluated by scRNA-seq as a control for engineered epithelium. Overall, this work identifies platform-specific effects that support the use of engineered models to achieve the most physiologic differential outcomes in pulmonary epithelial regenerative applications. : Greaney et al. compare pulmonary epithelial regeneration across multiple modalities in vitro, finding that decellularized scaffolds achieved the most physiologic differentiation over more artificial platforms. scRNA-seq enables high-resolution comparison between engineered and native cell populations, thereby better gauging progress toward the generation of a tissue that may function on implantation. Keywords: Pulmonary Cell Biology, Regenerative Medicine, Tissue Engineering, Primary Epithelial Cells