Engineering Functional Distal Lung Epithelium
Date of Award
Doctor of Philosophy (PhD)
Biomedical Engineering (ENAS)
The potential for lung tissue engineering to generate lung tissue capable of gas exchange ultimately rests upon the ability to reconstitute a functional barrier between the air and blood spaces in the engineered lung alveolus. The success of this endeavor will rely in large part on the recapitulation of an alveolar epithelium, comprising alveolar epithelial type 1 cells (AEC1s) and type 2 cells (AEC2s), which provides a surface for gas diffusion, actively maintains critical features of blood-gas barrier function, and comprises resident AEC2 stem cells for ongoing maintenance and repair of the distal lung. Yet, since the beginning of this field in 2010, the sum of lung tissue engineering efforts has yielded no examples in the literature of engineered alveolar epithelium that recapitulates phenotypically mature and functional AEC2s, or engineered alveolar units comprising AEC2s and squamous, alveolar-lining AEC1s. The work in this thesis seeks to address this critical stumbling block in lung engineering, by developing a strategy to engineer alveolar epithelium using primary AEC2s, the principal stem cell of the distal lung. I investigate the use of 1) stromal cell co-culture, 2) exogenous modulation of key signaling pathways, and 3) mechanical stretch, as tools to first, direct the proliferation of AEC2s within acellular extracellular matrix whole lung scaffolds and second, to promote the differentiation of these reseeded AEC2s to squamous AEC1s, in situ. I also seek to use these engineered lung tissues to gain insight into the diversity of cues supporting AEC2 progenitor functions within the alveolus. Through whole lung scaffold recellularization experiments, I first demonstrate that fibroblasts are critical to support AEC2 growth and phenotypic maintenance, while vascular endothelial cells are insufficient as an independent niche cell. However, endothelial cells synergize with fibroblasts, when in the presence of select exogenous soluble factors, to enhance AEC2 phenotype and nascent alveolus formation within decellularized lung scaffolds. AEC2s cultured with both fibroblasts and endothelial cells exhibit molecular and ultrastructural features appropriate to native AEC2s, and achieve the important functional attribute of surfactant secretion. Using single-cell RNA sequencing and in silico ligand-receptor analysis of engineered co- and tri-culture lungs, I show that endothelial cells serve a dual role within the engineered tri-culture system: endothelial cells mitigate pro-fibrotic signaling in the epithelium and in fibroblasts, while inducing two proposed AEC2 niche-supportive signatures – that of lipofibroblasts and that of mesenchymal alveolar niche cells (MANCs) – in neighboring mesenchyme. To investigate the application of biochemical and/or mechanical cues to drive subsequent AEC2 differentiation within engineered alveolar units, I also develop a small-scale engineered lung tissue platform based on decellularized precision-cut lung slices. I demonstrate that withdrawal of Wnt and FGF pathway agonists induces modest differentiation to AEC1-like cells, but that this differentiation is significantly augmented by the application of tidal-level cyclic or tonic uniaxial strain (2.5%). The resulting engineered lung tissue contains alveoli lined by squamous AEC1s and scattered AEC2s, with similar organization to that of native lung. Taken together, this work represents a critical advance in our efforts to reconstitute functional alveoli for lung cell- and tissue-based regenerative therapies, by presenting a multifaceted strategy to engineer a distal lung epithelium of AEC2s and AEC1s within ex vivo alveolar units. These results additionally provide insight into the interplay of cellular, biochemical, and mechanical stimuli that coordinate to support AEC2 progenitor functions – specifically proliferation and differentiation – within the alveolus. This work should pave the way for more deliberate recellularization strategies in lung engineering, and bring us closer to building a durable, transplantable lung.
Leiby, Katherine, "Engineering Functional Distal Lung Epithelium" (2022). Yale Graduate School of Arts and Sciences Dissertations. 491.