Regenerating Pulmonary Vascular Endothelium of Tissue-Engineered Lungs using Endothelial Progenitor Cells and Acellular Scaffolds

Abstract

Terminal lung diseases damage the organ through substantial irreversible changes to its architecture, leading to a suboptimal level of function. For this patient population, lung transplantation is the primary curative method to re-obtaining their previous quality of life. Relative to other whole organs used for transplant, the acquisition of transplantable lungs is low, resulting in a large deficit of lungs available for patients on the transplant list. Ongoing efforts towards creating functional organs from acellular scaffolds has the potential to address the deficit of lungs. The regeneration of a functional vasculature within acellular organ and tissue scaffolds is a necessity for their overall longevity and function. Current efforts in engineering transplantable lungs with long-term functionality are hindered due to vascular related complications within the newly generated organ. In over to overcome these limitations, improvements in re-endothelialization of scaffolds and acellular tissue are required. The studies described in this thesis provide valuable insight towards characterization of pulmonary endothelial progenitors and cells for re-endothelization, understanding the interaction between endothelial progenitors and vessel-specific extracellular matrices, and the potential for using iPSC-derived angiogenic hemangioblasts for developing vascular endothelium. Within both in vitro culture and acellular murine lung matrices, we have successfully characterized a highly proliferative rat pulmonary endothelial progenitor capable of re-endothelialization of the vasculature without site preference and can be used as a model for identifying progenitors in humans that are suitable for tissue engineering and clinical applications. We continued our efforts by using label-free liquid chromatography-mass spectrometry (LC/MS) analysis of decellularized porcine pulmonary vessels to compare the diversity and abundance of various extracellular matrix (ECM) proteins. The data generated from our LC/MS analysis can be used for future quantitative proteomic analyses. The decellularized ECM from both pulmonary arteries and veins were both used to evaluate their effect on endothelial progenitor maturation, and we were able to detect increase expression of endothelial genes from cells cultured in ECM hydrogels. Finally, we developed a protocol to differentiate human iPSCs into angiogenic hemangioblasts. These differentiated cultures contained sub-populations of CD31+/VEGF2+ and VE-Cadherin+/CD73+ cells. The outcomes from this study will provide guidance for future experiments and insight on characterizing both endothelial progenitors for re-endothelialization and proteomic analysis of pulmonary vasculature ECM.