Airway branching morphogenesis in three dimensional culture

Abstract

BackgroundLungs develop from the fetal digestive tract where epithelium invades the vascular rich stroma in a process called branching morphogenesis. In organogenesis, endothelial cells have been shown to be important for morphogenesis and the maintenance of organ structure. The aim of this study was to recapitulate human lung morphogenesis in vitro by establishing a three dimensional (3D) co-culture model where lung epithelial cells were cultured in endothelial-rich stroma.MethodsWe used a human bronchial epithelial cell line (VA10) recently developed in our laboratory. This cell line cell line maintains a predominant basal cell phenotype, expressing p63 and other basal markers such as cytokeratin-5 and -14. Here, we cultured VA10 with human umbilical vein endothelial cells (HUVECs), to mimic the close interaction between these cell types during lung development. Morphogenesis and differentiation was monitored by phase contrast microscopy, immunostainings and confocal imaging.ResultsWe found that in co-culture with endothelial cells, the VA10 cells generated bronchioalveolar like structures, suggesting that lung epithelial branching is facilitated by the presence of endothelial cells. The VA10 derived epithelial structures display various complex patterns of branching and show partial alveolar type-II differentiation with pro-Surfactant-C expression. The epithelial origin of the branching VA10 colonies was confirmed by immunostaining. These bronchioalveolar-like structures were polarized with respect to integrin expression at the cell-matrix interface. The endothelial-induced branching was mediated by soluble factors. Furthermore, fibroblast growth factor receptor-2 (FGFR-2) and sprouty-2 were expressed at the growing tips of the branching structures and the branching was inhibited by the FGFR-small molecule inhibitor SU5402.DiscussionIn this study we show that a human lung epithelial cell line can be induced by endothelial cells to form branching bronchioalveolar-like structures in 3-D culture. This novel model of human airway morphogenesis can be used to study critical events in human lung development and suggests a supportive role for the endothelium in promoting branching of airway epithelium.

Basal cells of the human airways acquire mesenchymal traits in idiopathic pulmonary fibrosis and in culture

“…3D cultures were performed as previously described. 26 Production of Lentivirus and Cell Transduction Lentiviral transduction was performed as previously described. 28 In short, plasmids containing scrambled hairpin (pLKO.1 shSCR; Addgene plasmid 17920) 29 or shRNA against p63 (shp63alpha pLKO.1 puro; Addgene plasmid 19120) 30 were used with packaging plasmids (psPAX2 and pMD2.G) (Addgene plasmids 12260 and 12259, respectively) to generate viral titer in HEK-293 T cells using Arrest-in (Open Biosystems).…”

“…25 In addition, this cell line forms bronchio-alveolarlike structures in three-dimensional co-culture with endothelial cells. 25,26 In this study, we focus on the p63-positive lung epithelial basal cell as one potential progenitor cell for the myofibroblast in IPF.…”

Bronchioalveolar morphogenesis of human bronchial epithelial cells depending upon hepatocyte growth factor

“…1A), although these colonies were not accompanied by a branching structure. VA10 cells form a branching structure in rBM when co‐cultured with HUVECs 18, but we did not observe branching colonies of HBE135 cells in co‐culture with HUVECs (Fig. 1B).…”

“…reported that the human BEC line VA‐10 forms polarized spherical colonies when grown in 3D culture 18. We first cultured HBE135 cells alone in rBM at the same concentration (333 cells per 100 μl rBM), but the HBE135 cells did not form colonies (Fig.…”

“…Because we did not observe expression of the ATI cell‐specific protein AQP‐5 in HBE135 cells, it would be interesting to verify whether HBE135 cell colonies with complex branching structures express AQP‐5 when co‐cultured with certain types of endothelial cells. However, VA10 cells form a complex branching structure in co‐culture with HUVECs 18. Although this discrepancy is not well understood, VA10 and HBE135 cells might be different cell types of the bronchiole or they might respond differently to the amount of HGF expressed by HUVECs.…”