Fig. 2.

(A) Schematic protocol for directed differentiation of iPSCs to ATII and ATI in vitro in 29 days. Cytokines were added at different steps indicated in the panel (B) Phase-contrast images of differentiated cells at day 22, which are termed ATII cells. (C–D) Immunofluorescent staining of alveolar type II markers (C) pro-surfactant protein C (ProSPC) and (D) Surfactant protein A (SPA). (E) Flow cytometry analysis for the percentage of positive cells for alveolar type II marker (NKX2.1, SPC, SPA, CD54) at day 22. (F) qRT-PCR analysis in iPSC-derived ATII cells compared to hATII cells that were derived from fresh human lung. Values from three independent experiments from the triplicate PCR reactions for a gene of interest (SPC, and SPA) were normalized against average GAPDH Ct values from the same cDNA sample. Fold change of GOI transcript levels between iPS derived-ATII and human type II cells equals 2^−ΔΔCt, where ΔCt = Ct_(GOI) – Ct_(GAPDH), and ΔΔCt = ΔCt_(ATII) - DCt_(ATII). (Bar indicate ± SEM and n = 3 independent experiments for qRT-PCR and flow cytometry). Scale bar, 63 µm.