Fig. 1

Differentiation of iPSCs into BASCs. a Schema of iPSC differentiation procedure: iPSCs were differentiated for 24 days through hanging-drop-based formation of embryoid bodies (EBs); the BM was supplemented from 0 to 24 days with 20 ng/mL keratinocyte growth factor (KGF) and DCI (① d10–d24 or ② d14–d24). EBs were induced using the hanging-drop method for the first 3 days, and the obtained EBs were transferred at 3 days to super-low-adherent culture dishes and then at 5 days to adherent culture dishes. Cells were cultured until 24 days in the medium. b Pluripotency of undifferentiated iPSCs (0 days) at passage 25. Immunofluorescence labelling of mouse iPSCs for the stem cell markers OCT4, SOX2, and SSEA-1. The GFP gene was knocked-in under the Nanog promoter, which allowed detection of GFP (green) in undifferentiated cells. Scale bar = 100 μm. c Flow cytometry analysis for BASC identification. Comparison of protocols ① d10–d24 DCI and ② d14–d24 DCI revealed that BASC differentiation efficiency did not differ significantly between the protocols (P > 0.05, Student’s t test); horizontal line inside indicates median and whiskers indicate min to max values. DCI, 10 nM dexamethasone plus 0.1 mM 8-bromoadenosine 3′5′-cyclic monophosphate sodium salt and 0.1 mM 3-isobutyl-1-methylxanthine; iPSCs, induced pluripotent stem cells; BASCs, bronchioalveolar stem cells; GFP, green fluorescent protein