| Engineered lung tissue could potentially facilitate tissue augmentation strategies in pediatric and adult pulmonary medicine. In addition, engineered lung tissue could provide models for in vitro studies on pulmonary cell/developmental biology, pathobiology and pharmacology. This thesis describes in vitro engineering of organotypic fetal lung tissue by harnessing specific effects of exogenously supplemented fibroblast growth factors (FGFs) on embryonic day 17.5 murine fetal pulmonary cells (FPC) cultured in 3-D collagen gels. FGF10 and FGF7 promoted epithelial proliferation and branching responses, while FGF2 induced mesenchymal proliferation and vascular network formation. The cocktail of FGF10/7/2 synergistically combined these effects to produce constructs with optimal epithelial growth/branching, controlled mesenchymal proliferation, vascular network formation and epithelial-endothelial interfacing. Loss-of-function studies using a soluble vascular endothelial growth factor receptor (sVEGFR) revealed that endogenous VEGF-A is required for vascular network formation. Loss-of-function studies were performed targeting 2 additional molecules known to play key roles in lung development in vivo, the extracellular matrix protein tenascin-C (TN-C) and the morphogen sonic hedgehog (SHH). Treatment with TN-C neutralizing antibodies, as well as inhibition of SHH signaling with cyclopamine, decreased endothelial elongation and interconnectivity, but did not completely ablate network formation, as was the case with sVEGFR treatment. Interestingly, it was found that exogenous SHH alone induces vascular network formation; however, the morphology and secondary sprouting behavior of SHH-induced vessels was markedly different than for FGF-induced vessels. These findings demonstrate the ability of the tissue engineer to "fine tune" vascular formation in engineered tissues in vitro by manipulating multiple morphogenic signaling pathways and extracellular matrix. The functionality of nascent vascular structures within VIII engineered fetal lung tissue was tested in 2 in vivo models: the Matrigel plug model using freshly isolated FPC, and the renal capsule model using constructs cultured in vitro for 1 week. Both studies showed that epithelial structures maintain differentiation in vivo, and that donor-derived vascular structures contribute to the establishment of a perfused vasculature in vivo. Taken together, the findings of this thesis suggest that in vitro engineered lung tissue will provide novel venues for lung biology research and with further advances revolutionize pulmonary regenerative medicine. |
