Although severe lung disorders, including cystic fibrosis, asthma, and chronic obstructive pulmonary disease (COPD), represent a significant global disease burden, little is known about the molecular pathways by which the cells of the lung develop, respond to damage, or become diseased. Consequently, there are few treatment options for patients. Improving lung disease outcomes therefore relies on both refining the current understanding of the normal development of the lung epithelium and developing new model systems to provide mechanistic insight into disease biology. In this thesis, I describe a multifaceted approach using both in vivo models and novel mouse and human pluripotent stem cell reporter systems to explore this important topic, focusing primarily on the hypothesis that canonical Wnt signaling is a key stage-dependent inhibitor of proximal lung development. To address this hypothesis, I developed new tools allowing for the precise manipulation of developmental pathways and access to rare cell populations in vitro. This toolkit included both mouse and human pluripotent stem cell (mPSC/hPSC) lines with reporters for specific airway lineages. In parallel, I built on our lab’s previous work in directed differentiation of hPSCs to lung progenitors. I found that canonical Wnt signaling regulates proximodistal epithelial patterning in human NKX2-1+ lung progenitors. While canonical Wnt activation is required for lung specification, withdrawal of Wnt activation leads to emergence of a proximal airway program and loss of distal identity. This finding culminated in the development of a novel protocol to differentiate epithelial-only airway organoids from hPSCs. These organoids are derived from purified NKX2-1+ lung progenitors, contain functional airway cell types including secretory, goblet, and basal cells, and can be further expanded and differentiated to multiciliated epithelia in air-liquid interface culture. To provide a proof of principle for the clinical utility of this platform, I generated airway organoids from cystic fibrosis patient-derived hPSC lines pre- and post-correction of the dF508 mutation in the CFTR gene. These organoids respond in a CFTR-dependent manner to epithelial forskolin swelling assays, highlighting the potential utility of this approach for disease modeling and drug screening for a variety of genetic and acquired airway disorders.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/23391 |
| Date | 10 July 2017 |
| Creators | McCauley, Katherine |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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