Figure 3–
a) Quantification of octamer-binding transcription factor (OCT)-4, isoforms A and B, and NANOG transcripts in control cells (no small airway growth medium (SAGM)) and in HUES-3 alveolar type II (ATII) cells after 3, 5 and 8 days of SAGM. The fold increase of each transcript is calculated compared with control cells: p≤0.05 after 3 days, p≤0.02 after 5 days and p≤0.04 after 8 days for OCT-4; p≤0.01 after 3 days, p≤0.02 after 5 days and p≤0.03 after 8 days for NANOG. All values obtained are the mean of at least three independent experiments performed in triplicate. b) Fluorescence-activated cell sorting (FACS) analysis using fluorescein isothiocyanate (FITC)-conjugated stage-specific embryonic antigen (SSEA)-4 stem cell marker on HUES-3-ATII cells after 3, 5 and 8 days of SAGM. The control is the isotype control. c–e) FACS analysis of HUES-3-ATII cells using FITC-conjugated c) monoclonal CD105 antibody (a mesenchymal stem cell marker), d) CD34 antibody (an endothelial marker) and e) thyroid transcription factor (TTF)1 rabbit monoclonal antibody (for type II epithelial cells), after 3 days of SAGM. The data indicated a mean±sem of c) 2.6±0.01%, d) 2.3±0.01% and e) 94.8±0.02% of cells positive within the differentiated cell population. f) Surfactant protein (SP)-B and SP-C (117 and 134 isoforms) expression in HUES-3-ATII cells after 3, 5 and 8 days of SAGM. The fold increase of each transcript is calculated compared with control cells (no SAGM): p≤0.02 after 3 days, p≤0.03 after 5 days and p≤0.05 after 8 days for SP-B; p≤0.01 after 3 days, p≤0.03 after 5 days and p≤0.04 after 8 days for SP-C-117, p≤0.05 after 3 days, p≤0.02 after 5 days and p≤0.04 after 8 days for SP-C-134. g) Quantitative analysis of CXC chemokine receptor (CXCR)4 expression from embryoid bodies (EBs), from formation to their adhesion and until 3 days of SAGM treatment. The fold increase of each transcript is calculated compared with control cells (no SAGM): p≤0.01 for cells after 3 days with SAGM.