Current toxicity or pathogenesis studies rely heavily on traditional cell culture methods and animal models. While these systems provide much basic information, a human 3D organ model composed of native cell types interacting in a physiologically relevant way would more closely approximate in vivo conditions. Development of improved in vitro organ models would enable researchers to construct and analyze complex biological systems especially as they relate to organ development, disease pathogenesis, and toxicology and drug discovery. The development of such a model requires identifying the appropriate matrix material, cell types and microenvironment. Our hypothesis is that human embryonic stem or progenitor cells can be cultured on scaffolds to create an in vitro human lung model. It is possible to introduce leukocytes to the system to further mimic the in vivo environment and to study the roles of fibroblasts/myofibroblasts and macrophages/dendritic cells in the development of pulmonary fibrosis. The central hypothesis of this project is that human stem or progenitor cells can be on natural acellular lung or other commercially available scaffolds to create an in vitro human lung model which can be used to study lung disease and the development of fibrosis after microbial infection or exposure to xenobiotic agents. In this project, our aims are to (1) Produce a long term sustainable three dimensional (3D) human pulmonary model and (2) Test the validity of the model using exposure to H5N1, known to cause development of acute conditions and fibrosis in the lung or H1N1 2009 suspected of causing similar but less severe responses. Sham exposure or exposure to circulating strains of influenza A that do not elicit these responses will be used as controls as will addition of neuraminidase inhibitors Tamiflu and Amantidine. In order to produce the best lung model various matrices such as Gelfoam, Matrigel and collagen-IV will be examined for the ability to allow cell adhesion and sustainability. Several matrix materials will be considered, however, acellular (AC) human lung may be the best candidate for a fibrosis model. Recent work in our laboratory has shown this matrix is able to support differentiation of mouse embryonic stem cells toward cells comprising lung tissue with an appropriate morphology. Both AC whole rat trachea-lung and small sections of AC human lung will be tested for efficacy. For a human model, cell sources to be considered include cell lines and primary cells. Among the human cell lines to be trialed are A549 cells (Type II pneumocytes), HUV-EC-C or HUVEC-CS (human umbilical vein endothelial cells developed into a cell line), 13Lu (fetal lung cells) and human embryonic stem cells. Primary and progenitor cells such as HUVEC (primary cells), human amniotic fluid cells, and somatic lung progenitor cells may be used in a supporting role or to populate the matrix. The growth medium will be assessed for efficient proliferation and differentiation of cells into lung tissue.