Master's Thesis Defense - The Effect of Oxygen Tension on Murine Embryonic Stem Cell (mESC) Differentiation towards Endoderm
Date: June 8, 2009
Time: 10:00 AM
Location: Bossone Research Enterprise Center, Room: 709
Advisor: Peter I. Lelkes, Ph.D.
Derivation of endoderm lineages from embryonic stem 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 and pancreatic islet-like structures, as well as alveolar cells, making this a very exciting field. However, progress in the field of 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 is still low, and new methods to improve efficiency should be considered. Expansion of the cell population in vitro has become an essential step in the process of tissue engineering and also the systematic optimization of culture conditions is now a fundamental problem that needs to be addressed. Stromal cell to cell contacts, extracellular matrix proteins, temperature, and oxygen (O2) levels in the immediate microenvironment can influence stem cell function and differentiation. The differentiation and function of stem cells are influenced by these complex signals in the lung microenvironment, including oxygen availability. Low oxygen levels (hypoxia) occur in a number of physiological and pathophysiological settings, particularly when rapid tissue growth exceeds blood supply. Embryogenesis occurs in a physiologic “hypoxic” environment (3%–8% O2). Previous studies have shown that lung epithelial branching is significantly enchanced under 3% O2, in comparison to ambient air. We believe 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 as a modulatory parameter to enhance murine embryonic stem cell (mESC) differentiation towards the endoderm.
The goal of this project was to derive endoderm cells from murine embryonic stem cells using oxygen tension as a modulatory parameter. However, 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 mESC differentiation towards endoderm. mESC 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 0.1mM BME, as well as 5% FBS with or without BME for 8 days. Results show that in 21% O2, endodermal differentiation markers Sox17 and FoxA2, as assessed by reverse transcriptase polymerase chain reaction (RT-PCR), show highest differentiation in medium supplemented with 5% serum and 0.1mM BME. In contrast, expression of both endodermal markers decreased as oxygen tension decreased. In conclusion, this work shows that reduced oxygen tension suppresses endodermal differentiation of ES-D3 cells in serum-supplemented medium.
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