Boston University and Boston Medical Center 2 articles published in JoVE Developmental Biology Generation of Airway Epithelial Cell Air-Liquid Interface Cultures from Human Pluripotent Stem Cells Andrew Berical1,2, Mary Lou Beermann2, Shingo Suzuki3, Jake LeSuer2, Taylor Matte2, Brian Davis3, Darrell Kotton1,2, Finn Hawkins1,2 1Pulmonary Center, Boston University School of Medicine, 2Center for Regenerative Medicine, Boston University and Boston Medical Center, 3Center for Stem Cell and Regenerative Medicine, Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center Recent advances in human induced pluripotent stem cell differentiation protocols allow for the stepwise derivation of organ-specific cell types. Here, we provide detailed steps for the maintenance and expansion of iPSC-derived airway basal cells and their differentiation into a mucociliary epithelium in air-liquid interface cultures. Developmental Biology Generating 3D Spheres and 2D Air-Liquid Interface Cultures of Human Induced Pluripotent Stem Cell-Derived Type 2 Alveolar Epithelial Cells Rhiannon B. Werder*1,2,3, Jessie Huang*1,2, Kristine M. Abo1,2, Olivia T. Hix1,2, Kasey Minakin1,2, Konstantinos-Dionysios Alysandratos1,2, Carly Merritt1,2, Kayleigh Berthiaume1,2, Andrea B. Alber1,2, Claire L. Burgess1,2, Darrell N. Kotton*1,2, Andrew A. Wilson*1,2 1Center for Regenerative Medicine, Boston University and Boston Medical Center, 2The Pulmonary Center and Department of Medicine, Boston University School of Medicine, 3QIMR Berghofer Medical Research Institute The present protocol describes human induced pluripotent stem cell-derived type 2 alveolar epithelial-like cells (iAT2s). These cells can be cultured as self-renewing spheres in 3D culture or adapted to air-liquid interface (ALI) culture.