Robust generation of distal respiratory airway organoids by an engineered cuboid chip

Human distal respiratory airway (DRA) cells are located in the smallest conducting airways of the human lung, specifically in the terminal and respiratory bronchioles, and represent a newly discovered cell type. Due to their absence in mice and the limited existing research on these cells, there is an urgent need to establish an in vitro model of human distal respiratory airways to study the role of such cells in respiratory diseases.

Here, we developed a robust differentiation protocol to derive DRA organoids (DRAOs) from human pluripotent stem cells (hPSCs) and investigated their role in the treatment of chronic obstructive pulmonary disease (COPD). We engineered a cuboid chip-based culture platform, where seeding single-cell suspensions mixed with Matrigel onto the chip increased lung progenitor cell (LPC) spheroid yield by 7.5-fold and upregulated NKX2.1 expression nearly 300-fold. Single-cell transcriptomic analysis further demonstrated that the platform enhanced distal lung-related gene expression. Prolonged culture of LPC spheroids yielded expandable hollow lung organoids. The resulting cells exhibited protein and molecular profiles closely resembling native human DRA cells, with over 70% co-expressing SFTPB and SCGB3A2. Using this method, we demonstrate accelerated production of DRAOs with significantly enhanced structural and functional maturity in 30 days. Furthermore, these DRAOs retain the potential to undergo alveolar differentiation. By exposing these organoids to cigarette smoke extract (CSE), we established a COPD-like model. In addition, DRAOs can survive in COPD mice, repair alveolar structural damage, and alleviate COPD symptoms.

Our method will facilitate increasingly widespread culture and differentiation of organoids, paving the way for constructing more accurate models of complex organs that mimic in vivo structures and functions. Furthermore, the favorable applicability of DRAOs in cell therapy studies using a mouse model of COPD-related alveolar injury provides critical insights for cell therapy in respiratory diseases.