Airway progenitor cell development and function : mimicking in vivo behavior in vitro

This thesis presents novel in vitro models to study the differentiation of airway progenitor cells. These models were subsequently used to study the function of SOX2 and SOX21 in the development and regeneration of the airways

Integrated 3D Acid Fracturing Model for Carbonate Reservoir Stimulation

Acid fracturing is one of the stimulation methods used in carbonate formations and has been proved effective and economical. Because of the stochastic nature of acidizing in carbonate formation, designing and optimizing acid fracture treatment today still remain challenging. In the past, a simple acid fracture conductivity correlation was usually considered sufficient to estimate the overall average fracture conductivity in the formation, leading to the computation of the productivity index for fractured well performance. However, the nature of heterogeneity could not be included in the modeling. Understanding the important role of heterogeneity to stimulation performance becomes a crucial step in design and optimization of acid fracture jobs. In order to study the effect of this stochastic nature on acid fracturing, a fully 3D acid reaction model was developed based on the geostatistical parameters of the formation. It is possible to describe local conductivity distribution related to acid transport and reaction process. In this study, we have developed a new interactive workflow allowing the model of the fracture propagation process, the acid etching process and the well production interactively. This thesis presents the novel approach in integrating fracture propagation, acid transport and dissolution, and well performance models in a seamless fashion for acid fracturing design. In this new approach, the fracture geometry data of a hydraulic fracture is first obtained from commercial models of hydraulic fracture propagation, and then the 3D acid fracture model simulates acid etching and transport from the fracture propagation model using the width distribution as the initial condition. We then calculate the fracture conductivity distribution along the created fracture considering the geostatistical parameters such as permeability correlation length and standard deviation in permeability of the formation. The final step of the approach is to predict well performance after stimulation with a reservoir flow simulator. The significant improvements of the new approach are two folds: (1) capturing the geostatistical effect of the formation; and (2) modeling the acid etching and transport more accurately. The thesis explains the methodology and illustrates the application of the approach with examples. The results from this study show that the new model can successfully design and optimize acid fracturing treatments

A novel method for expansion and differentiation of mouse tracheal epithelial cells in culture

Air-liquid interface (ALI) cultures of mouse tracheal epithelial cells (MTEC) are a well-established model to study airway epithelial cells, but current methods require large numbers of animals which is unwanted in view of the 3R principle and introduces variation. Moreover, stringent breeding schemes are frequently needed to generate sufficient numbers of genetically modified animals. Current protocols do not incorporate expansion of MTEC, and therefore we developed a protocol to expand MTEC while maintaining their differentiation capacity. MTEC were isolated and expanded using the ROCK inhibitor Y-27632 in presence or absence of the γ-secretase inhibitor DAPT, a Notch pathway inhibitor. Whereas MTEC proliferated without DAPT, growth rate and cell morphology improved in presence of DAPT. ALI-induced differentiation of expanded MTEC resulted in an altered capacity of basal cells to differentiate into ciliated cells, whereas IL-13-induced goblet cell differentiation remained unaffecte

Generation of a biotinylatable Sox2 mouse model to identify Sox2 complexes in vivo

Sox2 is a Sry-box containing family member of related transcription factors sharing homology in their DNA binding domain. Sox2 is important during different stages of development, and previously we showed that Sox2 plays an important role in branching morphogenesis and epithelial cell differentiation in lung development. The transcriptional activity of Sox2 depends on its interaction with other proteins, leading to ‘complex-specific’ DNA binding and transcriptional regulation. In this study, we generated a mouse model containing a biotinylatable-tag targeted at the translational start site of the endogenous Sox2 gene (bioSox2). This tag was biotinylated by the bacterial birA protein and the resulting bioSox2 protein was used to identify associating partners of Sox2 at different phases of lung development in vivo (the Sox2 interactome). Homozygous bioSox2 mice are viable and fertile irrespective of the biotinylation of the bio tag, indicating that the bioSox2 gene is normally expressed and the protein is functional in all tissues. This suggests that partners of Sox2 are most likely able to associate with the bioSox2 protein. BioSox2 complexes were isolated with high affinity using streptavidin beads and analysed by MALDI-ToF mass spectrometry analysis. Several of the identified binding partners are already shown to have a respiratory phenotype. Two of these partners, Wdr5 and Tcf3, were validated to confirm their association in Sox2 complexes. This bioSox2 mouse model will be a valuable tool for isolating in vivo Sox2 complexes from different tissues

Sox21 modulates sox2-initiated differentiation of epithelial cells in the extrapulmonary airways

SOX2 expression levels are crucial for the balance between maintenance and differentiation of airway progenitor cells during development and regeneration. Here, we describe patterning of the mouse proximal airway epithelium by SOX21, which coincides with high levels of SOX2 during development. Airway progenitor cells in this SOX2+/SOX21+ zone show differentiation to basal cells, specifying cells for the extrapulmonary airways. Loss of SOX21 showed an increased differentiation of SOX2+ progenitor cells to basal and ciliated cells during mouse lung development. We propose a mechanism where SOX21 inhibits differentiation of airway progenitors by antagonizing SOX2-induced expression of specific genes involved in airway differentiation. Additionally, in the adult tracheal epithelium SOX21 inhibits basal to ciliated cell differentiation. This suppressing function of SOX21 on differentiation contrasts SOX2, which mainly drives differentiation of epithelial cells during development and regeneration after injury. Furthermore, using human fetal lung organoids and adult bronchial epithelial cells, we show that SOX2+/SOX21+ regionalization is conserved. Lastly, we show that the interplay between SOX2 and SOX21 is context and concentration dependent leading to regulation of differentiation of the airway epithelium.</p