Browsing by Subject "Cell Culture Techniques"
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Item Differentiation of Normal and Cystic Fibrosis Human Lung Epithelial Cells in a Decellularized and Reconstituted Mouse Lung(2017-04-14) LaRanger, Ryan; Hsieh, Jenny; Garcia, Christine K.; Pasare, Chandrashekhar; Lu, Christopher Y.; Shay, Jerry W.; Wright, Woodring E.Engineered lung tissue may eventually address the chronic shortage of transplantable lung tissue and permit modeling of lung disease in a controlled ex vivo environment. However, there are presently no sources of primary lung stem cells which can be expanded at sufficient scale to permit engineering multiple lungs from a single donor. I have developed a method for conditionally reprogramming primary human bronchial epithelial cells in culture to extend their functional lifespan, and have used these cells to reconstitute lung epithelium in a decellularized lung matrix. For conditional reprogramming, I cultured primary human bronchial epithelial cells derived from patients with or without cystic fibrosis with a small molecule Rho-associated coiled kinase inhibitor and co-cultured it with irradiated J2 3T3 fibroblasts. I determined the ability of the human bronchial epithelial cells to differentiate after 40 population doublings by culture at an air liquid interface for 35 days as confirmed by transepithelial electrical resistance measurement, histology, Ussing chamber analysis, and immunofluorescence staining of differentiation factors. I also found that this conditional reprogramming method permits cloning of human bronchial epithelial cells, and that these cells can support genetic modification by CRISPR. Next, I developed a method for decellularizing and reconstituting murine lungs in a bioreactor with vascular perfusion and simulated breathing. Lungs reconstituted with the conditionally reprogrammed human bronchial epithelial cells formed both upper and lower airway structures after only 12 days of culture. I confirmed the formation of a bronchial pseudostratified epithelium and alveolar formation in the reconstituted lungs by histology, western blotting, and immunofluorescence staining. To develop an eventual universal donor paradigm for engineered tissue, I developed an in vivo luciferase rejection screen in mice using luciferase expressing life-extended primary skin fibroblasts transplanted intradermally. The methods developed for long-term culture of primary lung epithelial cells permits rapid scale-up of patient derived human bronchial epithelial cells and clonal selection without the need for genetic manipulation; facilitating the study of lung diseases and optimization of organ reconstitution in tissue engineered models.Item Targeting Distinct Tau Strains and Tau Aggregate Sizes with Heparin and Heparinoids to Explore Differential Inhibition of Cell Uptake and Seeding(2017-01-17) Prueitt, William; Stopschinski, Barbara; Diamond, MarcBACKGROUND: Tauopathies (including Alzheimer's Disease) are incurable, progressive neurodegenerative diseases caused by tau protein aggregation. Evidence suggests that tau aggregates spread pathology as do prions, infectious proteins that transmit a pathologic conformation to native proteins via strains--disease-specific conformers that propagate indefinitely in living systems. Like prion protein, tau also forms strains. It is unknown whether each binds the cell surface heparan sulfate proteoglycans in a unique or generic fashion to trigger uptake. METHODS: I used a "biosensor" cell line responsive to tau aggregates that scores induction of intracellular aggregation based on FRET flow cytometry. I tested the ability of different heparin-like molecules to block tau aggregate uptake and seeding. I measured tau uptake and induction of intracellular aggregation of a reporter. RESULTS: Tau seeding was comparably inhibited by heparin regardless of aggregate size. Data from testing two strains (Clone 9 & 10) for heparin inhibition of cell seeding suggested that they have differential sensitivity (DS9: IC50 = 335.9nM & DS10: IC50 = 2.1uM). Testing 9 heparinoids for tau seeding inhibition indicated that they had highly variable inhibition, some having no effect and some having an effect nearly as strong as heparin. CONCLUSIONS: This data suggests that (1) tau seeding is similarly inhibited by heparin regardless of tau aggregate size, and that (2) seeding of different strains of tau may be variably inhibited by heparin, hinting that specificity and avidity may differ by strain. If true, this knowledge will be applicable across many tauopathies and may influence diagnosis (because tau strains can differentiate pathology) and treatment (strain-specific therapies may be required). This data also indicated that certain size and sulfation patterns of heparin affect seeding inhibition. This matches other data produced in the lab using genetic knockouts, and supports the idea that crucial binding domains on heparin are necessary for pathologic tau spread between cells.