(A) The expression of the distal progenitor marker SOX9 remained unchanged over time and expression of the AECI marker PDPN was low in HLO cultures. (B) The majority of SFTPC+ cells (green, left panel) co-expressed SOX9 (red). Similarly, many cells expressing the AECI early marker HOPX+ (green, right panel) co-expressed SOX9 (red). Few, scattered cells expressed the late AECII marker SFTPB (white, second panel) or the AECI marker, PDPN (third panel, white). Few PDPN+ cells also showed elongated, squamous morphology seen in the adult lung. (C) Human lung AECII cells labeled with SFTPC (green, left panel) did not co-express SOX9. SFTPB+ cells (white, second panel) in the adult human lung have similar morphology to SFTPB+ cells in HLOs. Human lung AECI cells expressed PDPN (white, third panel), and show characteristic AECI cell shape. Human AECI cells express HOPX (green, right panel), but did not co-express SOX9. (B–C) Scale bar in lower magnification images in B (upper panel) represent 50 µM and the scale bars in higher magnification images in B, C (lower panel) represent 10 µM. (D) D50 HLOs contain lamellar bodies which are organelles specific to AECII cells. Scale bars represent 500 nm.