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.

We first established co-culture with feeder cells to allow long-term propagation of human DAECs. NIH/3 T3 feeders were transduced with GFP to allow for removal by flow cytometry. The workflow and a colony of human DAECs in co-culture with GFP-expressing feeders are shown in Fig. 1A . Although cells successfully proliferated for 13 population doublings (PDs), growth was not as long-lasting as previously reported [17] . We thus tested a list of small molecules (Suppl. Table 1 ) and growth factors with effects on signaling pathways involved in lung development and/or maintenance of adult epithelial cells. We selected the TGF-β type I receptor inhibitor RepSox (10 μM) and the Notch pathway inhibitor DBZ (100 nM) (Suppl. Table 1 ), but only DBZ was able to maintain exponential growth beyond 130 PD (Fig. 1B) and was used for all subsequent experiments.