Please use this identifier to cite or link to this item: http://scholarbank.nus.edu.sg/handle/10635/32345
Title: HYDROGEN PEROXIDE-INDUCED CYTOTOXICITY AND OXIDATIVE DNA DAMAGE ON HUMAN EMBRYONIC STEM CELLS AND THEIR DIFFERENTIATED FIBROBLAST-LIKE PROGENIES
Authors: LU KAI
Keywords: biocompatibility, cell culture, embryonic stem cell, toxicity, toxicology, translational research
Issue Date: 17-Aug-2011
Source: LU KAI (2011-08-17). HYDROGEN PEROXIDE-INDUCED CYTOTOXICITY AND OXIDATIVE DNA DAMAGE ON HUMAN EMBRYONIC STEM CELLS AND THEIR DIFFERENTIATED FIBROBLAST-LIKE PROGENIES. ScholarBank@NUS Repository.
Abstract: Background: Human embryonic stem cells (hESCs) are pluripotent cells derived from in vitro fertilized (IVF) embryos. Because of their origin in healthy human tissues, self-renewability and differentiate capability, hESCs can potentially be adopted as a biological model for toxicity study. To date, wide use of hESCs in cytotoxicity and DNA damage study has been restricted by technical limitations. One such limitation is from MEF feeder cells. Although MEF help maintain cultured hESCs in there undifferentiated status, they nevertheless become a form of contamination when pure populations of hESCs are intended to be harvested for various analyses. Meanwhile, reactive oxygen species (ROS) are known to play an important role in physiological and pathophysiological processes during embryo development. Hypotheses: Effective cytotoxicity and DNA damage study can be enabled through the development of a feeder-cell-free hESC culture system. Because of the drastically different cell physiology between pluripotent hESCs and differentiated cells, their cytotoxicity and DNA damage responses upon ROS challenge could be distinct and regulated differently by underlying molecular mechanisms. Methods: An autologous feeder-cell-free system was developed for the cultivation of undifferentiated hESCs. Fibroblastic-like cells were derived from hESCs as a model for differentiated cells. Hydrogen peroxide (H2O2) was used as an in vitro ROS generating system. Cytotoxicity and DNA damage of H2O2-treated hESCs and differentiated cells were studied. Results: Extracellular matrix (ECM) was extracted from fibroblast-like cells which were differentiated from hESCs. The ECM coating was capable of supporting undifferentiated growth of hESCs and excluded MEF cells from the culture system. Feeder-free-cultured hESCs and hESC-derived fibroblast-like cells were used to study H2O2-induced cytotoxicity and DNA damage. In comparison with differentiated cells, higher induction of intracellular ROS was observed in H2O2-treated hESCs. DNA synthesis was more intensively inhibited in hESCs, which was also accompanied by higher induction of apoptosis. However, DNA damage was found to be lower in hESCs. Genes involved in DNA damage signaling pathway were more readily expressed in hESCs. Conclusion: The autologous feeder-cell-free hESC cultivation system made the use of hESCs in toxicity study more feasible. Cytotoxicity and DNA damage study of ROS showed that pluripotent hESCs had unique damage responses.
URI: http://scholarbank.nus.edu.sg/handle/10635/32345
Appears in Collections:Ph.D Theses (Open)

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