Date of Award

2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Cellular, Molecular and Biomedical Sciences

First Advisor

Daniel J. Weiss

Abstract

Chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) are chronic lung diseases with significant worldwide impact and poor end-stage prognoses. Despite both diseases differing greatly in pathology, both COPD and IPF are hallmarked by drastic alterations to the extracellular matrix (ECM) within distinct anatomical lung regions. These alterations negatively impact cellular regeneration, and subsequently, lung homeostasis, physiology, and repair following injury. Whole decellularized lungs, stripped of cellular material while retaining lung ECM structure and composition, have previously provided complex models for both high-throughput proteomic evaluation of lung ECM as well as ex vivo culture models to study the impact of lung ECM on cellular function. Herein, we build upon previous proteomic studies by dissecting whole decellularized lungs to isolate specific anatomical regions for proteomic comparison between non-diseased, COPD, and IPF lungs, including alveolar-enriched (aECM), airway (airECM), and vasculature (vECM) regions. We demonstrate that in non-diseased lungs the different regions have distinct proteomic signatures, highlighted by varying amounts of basement membrane and cartilage-associated proteins. Notably, diseased lungs differ in ECM composition in a region-specific manner (i.e. increased type-III collagen in IPF aECM). As there is currently a critical lack of advanced ex vivo models for determining the impact of non-diseased and diseased human lung ECM on appropriate lungs cells, we sought to develop a reproducible protocol for developing ECM hydrogels from regional lung ECM. As such, we began by developing aECM hydrogels with tunable stiffnesses from non-diseased lungs for 3D culture of human induced pluripotent stem cell (iPSC)-derived alveolar type 2 epithelial cells (iAT2s). We demonstrated that iAT2s can be cultured as 3D spheroids (i.e. alveolospheres) in aECM hydrogels, similar to standard Matrigel culture. However, aECM hydrogels additionally promoted morphological differentiation of a subset of iAT2s into elongated ring-like cells reminiscent of alveolar type 1 (AT1) epithelial cells, a natural descendent of AT2s. The emergence of these elongated cells correlated with upregulated gene expression of AT1 and also intermediate transitional differentiation markers. Taken together, this research describes a highly reproducible methodology for characterizing non-diseased and diseased lung ECM composition and suggests a role of non-diseased aECM in cellular differentiation, while providing a promising model to further understand the complex role of regional and diseased lung ECM on cellular function and regeneration.

Language

en

Number of Pages

192 p.

Download

Included in

Cell Biology Commons

Share

COinS