Final Report Summary - LUNG REPAIR MODEL (Modelling lung repair in health and disease) This project was aimed to investigate the role of Wnt11 in the differentiation of human alveolar epithelial cells and human lung Stem Cells (hLSCs). We used a tissue-engineered 3-dimensional (3D) lung microtissue model mimicking epithelial-mesenchymal interactions in human lung tissue for this purpose. Our hypothesis was that Wnt11 promotes ATII and hLSC differentiation via activating the noncanonical pathway. Moreover, we proposed that it may act as a counterbalance of canonical Wnt and TGF-β1 signalling, thus preventing epithelial-mesenchymal transition (EMT) in alveolar epithelium. Based on our preliminary results we suggested that Wnt11 promotes physiological repair mechanisms in the lung preventing pathological tissue remodelling and may therefore have therapeutic potential to promote effective alveolar epithelial repair.During the project, Dr Bartis gained considerable expertise in isolating, culturing and characterising primary epithelial cells from human lung tissue. We found that Wnt11 is able to antagonise several TGF-β1-mediated effects in lung epithelial cells on the level of SMAD2/3 signalling. These effects include the expression of several EMT markers on epithelial cells, such as N-cadherin, alpha-smooth muscle actin and vimentin, epithelial spreading and motility during wound healing in vitro. In our original proposal we listed the characterisation of pluripotent hLSCs in the 3D microtissue co-cultures. Unfortunately we faced hardships with these Because of the futile efforts to culture pluripotent hLSCs, we set new project objectives during the work. Here we report the results of the new project objectives: (1)We found that Wnt11 is also able to antagonise some TGF-β1 effects in human lung fibroblasts, such as proliferation. However, it did not alter significantly transdifferentiation to myofibroblasts. (2)We characterised 3D co-cultures of lung epithelial cells and bone marrow-derived mesenchymal stem cells (BM-MSC). We found that BM-MSCs differentiated into the adipose lineage - but not undifferentiated cells - were able to prevent EMT and stimulate differentiation and surfactant synthesis in co-cultured ATIIs. These effects might be similar than that of pulmonary lipofibroblasts which are fibroblast-like adipose-differentiated mesenchymal cells residing in close proximity to ATII cells in the lung. (3)We also characterised the possible role of Wnt signalling during ATII-to-ATI transdifferentiation using both microarray and PCR approach. We found that high levels of Wnt4 and Wnt7a probably plays a role in alveolar epithelial transdifferentiation, while Fzd2 receptor activity probably contributes to epithelial repair mechanisms. These results might contribute to the better understanding of the molecular regulation of ATII-to-ATI transdifferentiation and alveolar repair. In summary, this project established that Wnt11 is able to antagonise several effects of TGF-β1 on both alveolar epithelial cells and pulmonary fibroblasts by interfering with the SMAD pathway. We have also characterised some novel aspects of epithelial-mesenchymal interactions in the lung involving BM-MSCs and alveolar epithelial cells. Moreover, we highlighted the role of Wnt signalling in ATII-to-ATI transdifferentiation. The significance of our findings are the following: (1) We characterised Wnt11 effects in the lung and showed that Wnt11 interferes with TGF-β1 signalling in several aspects. As TGF-β1 is the one of most important growth factor in the pathogenesis of several chronic respiratory diseases, such as idiopathic lung fibrosis (IPF), asthma, chronic obstructive pulmonary disease (COPD) and acute lung injury (ALI). As Wnt11 is capable of antagonizing a number of effects mediated by TGF-b1, it may have a therapeutic potential. (2) It has been known before that pulmonary lipofibroblasts chaperone ATII cells and are important part of the ATII stem cell niche. However, the factors regulating ATII homeostasis, “stemness”, proliferation and differentiation are yet unknown. Our preliminary experiments with the 3D lung microtissue indicate that this is a viable model system for in vitro studies of human ATII niche. Dr Bartis continues his investigations in this direction back in his home country. (3) Our knowledge of the molecular regulation of ATII-to-ATI transdifferentiation is obscure. We took a microarray approach to look into the gene expression changes during this process. It seems that expression changes in Wnt pathway-associated genes are particularly prominent during transdifferentiation. Based on both microarray and qPCR data we suggest that Wnt ligands Wnt4 and Wnt7a might play an important role in ATII-to-ATI transdifferentiation while Fzd2 receptor is involved in alveolar injury repair. These data will not only contribute to the better understanding of the molecular background of ATII-to-ATI transdifferentiation and alveolar injury repair, but also might have therapeutical implications in the future.