Pluripotent stem cell-derived lung/airway lineage

The incidence of various respiratory diseases, including chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, cystic fibrosis, and congenital pulmonary surfactant deficiency disorders, continues to rise, creating a significant unmet need for clinical treatment and new drug development. However, primary human lung tissue sources are extremely limited, and lung cells rapidly lose their functional characteristics during in vitro culture. In addition, significant species differences exist between human and rodent lung cell lineages and signaling pathways, making it difficult for animal models to accurately recapitulate human airway and alveolar physiological structures and pathological injury features. These limitations greatly restrict the development of in vitro respiratory disease models, high-throughput screening of respiratory toxic drugs, lung tissue engineering, and lung cell replacement therapies.

In 2014, Nature Biotechnology published a landmark standardized induction protocol in the field of lung differentiation, which fully recapitulated the embryonic developmental sequence from human foregut to lung bud formation. By precisely regulating multiple key developmental signaling pathways, including TGF-β, BMP, Wnt, and retinoic acid (RA), this study established a five-step serum-free, directed differentiation system with high standardization and scalability. The protocol first uses Activin to induce highly purified definitive endoderm, followed by a temporally controlled dual-pathway inhibition strategy to precisely specify foregut cell identity. Subsequently, a combination of Wnt agonist, BMP4, RA, and FGF, termed the “ventralization cocktail,” efficiently induces lung bud multipotent progenitors. FOXA2/NKX2.1 double-positive lung progenitor cells can reach up to 86%, demonstrating substantially higher purity compared with traditional approaches available at that time. Lung progenitor cells obtained at day 15 of differentiation possess the potential to generate multiple respiratory lineages. Continued culture to day 48 enables simultaneous generation of six major types of mature human respiratory epithelial cells, including airway basal cells, ciliated cells, club cells, goblet cells, alveolar type I gas-exchange cells, and functional alveolar type II cells. Among them, alveolar type II cells can specifically synthesize, uptake, and release surfactant proteins. Electron microscopy reveals characteristic lamellar bodies, showing high similarity to human fetal alveolar structures. In vivo transplantation studies further demonstrated that these lung progenitor cells can autonomously generate lung-like tissues containing pseudostratified airway structures, alveolar cyst-like structures, smooth muscle, and cartilage mesenchyme after implantation into immunodeficient mice, without generating ectopic organs such as thyroid or intestinal tissues. Meanwhile, this system is compatible with co-culture using decellularized human lung extracellular matrix, allowing seeded cells to stably engraft and mature while expressing alveolar functional markers. The entire differentiation process is compositionally defined and highly reproducible between batches. Based on this standardized human lung cell platform, it can be widely applied to patient-specific in vitro disease modeling of genetic disorders such as cystic fibrosis, high-throughput evaluation of pulmonary drug absorption and airway toxicity for inhaled/systemic drugs, fundamental studies of human lung embryonic development, artificial lung tissue engineering, and preclinical development of alveolar repair cell therapies. This platform provides a high-fidelity, traceable, standardized human lung cell solution for respiratory disease research and regenerative medicine translation.

Differentiation process of pluripotent stem cells into lung/airway lineage. Through a five-stage serum-free directed induction system, cells sequentially undergo definitive endoderm, foregut endoderm, lung bud progenitor, and airway progenitor stages, ultimately generating human lung/airway cells including airway basal cells, ciliated cells, goblet cells, Clara cells, and alveolar type I/type II cells. This system can stably mimic human lung development and provides a high-fidelity cellular platform for respiratory disease modeling, drug screening, lung tissue engineering, and regenerative medicine research.