Abstract
Induction of morphogenesis by competent lung progenitor cells in a 3D environment is a central goal of pulmonary tissue engineering, yet little is known about the microenvironmental signals required to induce de novo assembly of alveolar-like tissue in vitro. In extending our previous reports of alveolar-like tissue formation by fetal pulmonary cells stimulated by exogenous fibroblast growth factors (FGFs), we identified some of the key endogenous mediators of FGF-driven morphogenesis (organoid assembly), for example, epithelial sacculation, endothelial network assembly, and epithelial-endothelial interfacing. Sequestration of endogenously secreted vascular endothelial growth factor-A (VEGF-A) potently inhibited endothelial network formation, with little or no effect on epithelial morphogenesis. Inhibition of endogenous sonic hedgehog (SHH) partially attenuated FGF-driven endothelial network formation, while the addition of exogenous SHH in the absence of FGFs was able to induce epithelial and endothelial morphogenesis, although with distinct morphological characteristics. Notably, SHH-induced endothelial networks exhibited fewer branch points, reduced sprouting behavior, and a periendothelial extracellular matrix (ECM) virtually devoid of tenascin-C (TN-C). By contrast, focal deposition of endogenous TN-C was observed in the ECM-surrounding endothelial networks of FGF-induced organoids, especially around sprouting tips. In the FGF-induced organoids, TN-C was also observed in the clefts of sacculated epithelium and at the epithelial-endothelial interface. In support of a critical role in the formation of alveolar-like tissue in vitro, TN-C blocking inhibited endothelial network formation and epithelial sacculation. Upon engraftment of in-vitro-generated pulmonary organoids beneath the renal capsule of syngeneic mice, robust neovascularization occurred in 5 days with a large contribution of patent vessels from engrafted organoids, providing proof of principle for exploring intrapulmonary engraftment of prevascularized hydrogel constructs. Expression of proSpC, VEGF-A, and TN-C following 1 week in vivo mirrored the patterns observed in vitro. Taken together, these findings advance our understanding of endogenous growth factor and ECM signals important for de novo formation of pulmonary tissue structures in vitro and demonstrate the potential of an organoid-based approach to lung tissue augmentation.