Directed Differentiation of Human Pluripotent Stem Cells to Functionally Mature Lung Alveolar Cells.

Gas exchange occurs in the alveolar sacs, composed of alveolar type 1 (AT1) and type 2 (AT2) cells. Bipotent alveolar progenitor cells (BPCs), which give rise to both AT1 and AT2 cells, are disrupted in many lung diseases. Generating mature alveolar cells from human pluripotent stem cells (hPSCs) offers a promising strategy for alveolar epithelium regeneration but remains challenging. We developed a directed differentiation protocol to produce expandable BPCs from hPSCs, which form 3D “alveolospheres” with immature AT1- and AT2-like cells. To induce AT1 cell differentiation, we activated the Hippo-YAP1 pathway using small molecules and co-cultured with fetal lung fibroblasts. Furthermore, to drive exclusive AT1 and AT2 marker expression and physiological cell ratios for maturation, we mimicked mechanical stimulated-breathing forces and air exposure in an organ-on-chip. This approach addresses a key hurdle in generating mature alveolar cells and supports the advancement of stem cell–based therapies for distal lung regeneration.