Generation of macrophage containing alveolar organoids derived from human pluripotent stem cells for pulmonary fibrosis modeling and drug efficacy testing.

Macrophages are a central immune component in various types of in vitro human organoid systems to recapitulate normal and pathological development. However, to date, generation of human alveolar organoids (AOs) containing macrophages for use as a pulmonary fibrosis (PF) model and drug efficacy evaluation has not been reported. Here, we generated multicellular alveolar organoids (Mac-AOs) containing functional macrophages derived from human pluripotent stem cells based on stepwise direct differentiation by mimicking developmental cues in a temporally controlled manner. Derived Mac-AOs contained the expected range of cell types, including alveolar progenitors, mesenchymal cells, alveolar epithelial cells (type 1 and 2), and macrophages. Treatment with transforming growth factor (TGF-β1) induced inflammation and fibrotic changes in Mac-AOs, offering a PF model for validating the therapeutic potential of new drugs. TGF-β1-induced fibrotic responses and collagen accumulation in these Mac-AOs were effectively ameliorated by treatment with Pirfenidone, Nintedanib, and NP-011 via suppression of extracellular signal-regulated kinase signaling. To the best of our knowledge, this is the first report to provide non-epithelial functional macrophage-containing human AO system, which will better recapitulate the complexity of in vivo alveolar tissues and advance our understanding of the pathogenesis and development of effective therapies for PF.