Differentiation of Normal and Cystic Fibrosis Human Lung Epithelial Cells in a Decellularized and Reconstituted Mouse Lung

Engineered lung tissue may eventually address the chronic shortage of transplantable lung tissue and permit modeling of lung disease in a controlled ex vivo environment. However, there are presently no sources of primary lung stem cells which can be expanded at sufficient scale to permit engineering multiple lungs from a single donor. I have developed a method for conditionally reprogramming primary human bronchial epithelial cells in culture to extend their functional lifespan, and have used these cells to reconstitute lung epithelium in a decellularized lung matrix. For conditional reprogramming, I cultured primary human bronchial epithelial cells derived from patients with or without cystic fibrosis with a small molecule Rho-associated coiled kinase inhibitor and co-cultured it with irradiated J2 3T3 fibroblasts. I determined the ability of the human bronchial epithelial cells to differentiate after 40 population doublings by culture at an air liquid interface for 35 days as confirmed by transepithelial electrical resistance measurement, histology, Ussing chamber analysis, and immunofluorescence staining of differentiation factors. I also found that this conditional reprogramming method permits cloning of human bronchial epithelial cells, and that these cells can support genetic modification by CRISPR. Next, I developed a method for decellularizing and reconstituting murine lungs in a bioreactor with vascular perfusion and simulated breathing. Lungs reconstituted with the conditionally reprogrammed human bronchial epithelial cells formed both upper and lower airway structures after only 12 days of culture. I confirmed the formation of a bronchial pseudostratified epithelium and alveolar formation in the reconstituted lungs by histology, western blotting, and immunofluorescence staining. To develop an eventual universal donor paradigm for engineered tissue, I developed an in vivo luciferase rejection screen in mice using luciferase expressing life-extended primary skin fibroblasts transplanted intradermally. The methods developed for long-term culture of primary lung epithelial cells permits rapid scale-up of patient derived human bronchial epithelial cells and clonal selection without the need for genetic manipulation; facilitating the study of lung diseases and optimization of organ reconstitution in tissue engineered models.