Long-Term Culture of Distal Airway Epithelial Cells Allows Differentiation Towards Alveolar Epithelial Cells Suited for Influenza Virus Studies

Imai-Matsushima, Aki; Martin-Sancho, Laura; Karlas, Alexander; Imai, Seiichiro; Zoranovic, Tamara; Hocke, Andreas C.; Mollenkopf, Hans-Joachim; Berger, Hilmar; Meyer, Thomas F.

Failure of feeder removal to induce a phenotype towards alveolar epithelial cells implied that additional signals are required. We thus adopted an induction step published by Huang et al. [22] . Here, they used CHIR99021, keratinocyte growth factor, 3-isobutyl-1-methylxanthine (IBMX), 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP), dexamethasone and FGF-10 to induce differentiation of human pluripotent stem cells into airway epithelial cells that express p63 but lack markers of more differentiated cells [16] . We also increased the concentration of DBZ during induction to suppress persistent Notch signaling, which causes differentiation toward airway club cells in mice. After three days of induction, cells were passaged and feeders removed to allow differentiation (Fig. 4A ). This resulted in reduction of SCGB1A1 (Suppl. Fig. 2A ), the goblet cell marker MUC5B, and the ciliated cell marker FOXJ1 (Suppl. Fig. 2B ), and upregulation of AQP5 after 72 h (Suppl. Fig. 2C ). To exclude epithelial to mesenchymal transition (EMT), we measured the expression of the epithelial marker CDH-1, the mesenchymal marker α-SMA, and the EMT regulatory factor Slug, after feeder removal with and without induction medium (Suppl. Fig. 2D ). No substantial reduction of CDH-1 or increase of α-SMA or Slug were recorded, suggesting the epithelial phenotype is maintained.