In vitro expansion of SP-Cpos/CCSPposprogenitors. Pulmonary epithelial cells from dissociated lung tissues were cultured in vitro on fibronectin-coated plates supplemented with "complete Q286 medium". (A) Lung epithelial cells derived from lamb #1487 and maintained for 2 passages were analyzed by immunostaining for the expression of SP-C (green), CCSP (red) and the co-expression of SP-C and CCSP (400x magnification). Inset: enlargement of double-positive cells. (B) Flow cytometry analysis of a representative animal shows the percentage of SP-Cpos cells and CCSPpos cells above the background fluorescence. The double staining shows an enriched culture of AECIIs with the presence of SP-Cpos/CCSPpos bronchioloalveolar progenitors after dissociation, and at the first and second passage in vitro. For each condition, the left graph (smaller) corresponds to controls and the right graph (larger) presents SP-C and CCSP specific immunostaining.