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Please use this identifier to cite or link to this item: http://hdl.handle.net/1860/3083

Title: Effect of varying oxygen levels on murine embryonic stem cell differentiation into endoderm cells
Authors: Sheth, Nidhi
Keywords: Biomedical engineering;Embryonic stem cells;Cell differentiation
Issue Date: 13-Aug-2009
Abstract: Derivation of endoderm lineages from embryonic stem (ES) cells is receiving immense attention owing to the need for alternative therapies for debilitating diseases like lung cancer, COPD, emphysema, as well as type II diabetes. Some recent examples are derivation of hepatic cells, pancreatic islet-like structures, as well as alveolar cells from ES cells, making ES cell research a very exciting field. However, progress in the field of lung tissue engineering and pulmonary regenerative medicine has been fairly slow, mainly because of (1) lung’s structural complexity, (2) cellular heterogeneity, and (3) the low turnover rate of its epithelia. In addition, endodermal differentiation efficiency based on current approaches is still low, and new methods to improve efficiency should be considered. Stromal cell-to-cell contacts, extracellular matrix proteins, temperature, and oxygen (O2) levels in the immediate microenvironment can influence stem cell function and differentiation. Specifically, reduced oxygen tension serves as an important physiological and developmental cue for ES cell differentiation. Low oxygen levels (hypoxia) occur in a number of physiological and patho-physiological settings, particularly when rapid tissue growth exceeds blood supply. Embryogenesis occurs in a physiologically “hypoxic” environment (3%–8% O2). Previous studies have shown that lung epithelial branching is significantly enhanced under 3% O2, in comparison to ambient air. Based on these findings, we hypothesized that mimicking the embryonic microenvironment will increase endodermal differentiation yield. At the time of this writing, there have been no published papers highlighting the use of reduced oxygen tension to enhance murine embryonic stem (mES) cell differentiation towards the endoderm. The goal of this project was to derive endoderm cells from mES cells using oxygen tension as a modulatory parameter. ES cell differentiation is accompanied by apoptosis upon LIF withdrawal. In addition, reduced oxygen tension is accompanied by an increase in cell death due to an increase in reactive oxygen species (ROS). Anti-oxidants like beta-mercaptoethanol (BME) and vitamin-E can act as ROS scavengers, and can improve cell survival. This work was based on the hypothesis that reduced oxygen tension and antioxidants enhance mES cell differentiation towards endoderm. mES cell line ES-D3 was cultured under varying oxygen tensions (1%, 3%, 8%, and 21%) in medium containing 10% fetal bovine serum (FBS) with or without BME, as well as 5% FBS with or without BME for 8 days. Our results, specific to ES-D3 cells, show that cell survival was enhanced in medium containing BME under both ambient (21% O2) and reduced oxygen levels (3%, 8%). Endodermal differentiation markers Sox17 and FoxA2, as assessed by quantitative real-time polymerase chain reaction (Q-PCR), showed highest differentiation in medium supplemented with 5% serum and 0.1mM BME under 21% O2 (P < 0.01 as compared to cells differentiated under reduced oxygen tension). Immunofluorescence confirmed presence of epithelial-cell marker pan-cytokeratin, as well as endothelial cell-marker Griffonia simplicifolia lectin I-isolectinB4, in addition to endodermal markers Sox17 and FoxA2. In contrast to cultures at 21% O2, expression ofboth endodermal markers decreased as oxygen tension decreased to 3% and 8%. All cells died under 1% O2. In conclusion, this work shows that anti-oxidants enhance cell survival under both, normoxic and hypoxic (3% O2) conditions. Reduced oxygen tension (1%, 3%, and 8% O2) suppresses endodermal differentiation of ES-D3 cells in serum-supplemented medium. A combination of reduced oxygen tension and anti-oxidants does not enhance endodermal differentiation, as compared to normoxic controls.
URI: http://hdl.handle.net/1860/3083
Appears in Collections:Drexel Theses and Dissertations

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