Human Embryonic Stem (hES) cells will potentially have important applications in regenerative medicine, drug discovery and therapeutics. However, the current methods of hES cell culture limit their use in future applications due mostly to the inability to culture undifferentiated hES cells in sufficient numbers and the concern of animal derived products used in culture methods potentially implementing an immune rejection in transplantiation recipients. This project investigated two approaches for improving the culture of undifferentiated hES cells for future application in bioreactor systems. Firstly, proteins from the conditioned medium of three hES cell supportive fibroblast lines (human fetal, human neonatal and mouse embryonic fibroblasts) were identified using two-dimensional liquid chromatography, tandem mass spectrometry. This proteomic analysis identified 175 unique proteins including those from key pathways already implicated in the maintenance of human embryonic stem cell pluripotency including the Wnt, BMP/TGF-β1, activin/inhibin and insulin like growth factor-1 pathways. Identification of proteins in fibroblast conditioned medium will aid the development of a defined medium for undifferentiated hES cell culture. The second approach examined culture of hES cells in hypoxic conditions (5% O2) compared to standard normoxic conditions in CO2 incubators (20% O2). Feeder layer and feeder free culture conditions were examined in the two O2 concentrations. hES cell colonies grown in hypoxic culture had improved percentages of pluripotent cells (judged by increases in cells expressing SSEA-4, Tra-1-60 and nanog and reduction of BMP-4), stable karyotypes and increased proliferation. The maintenance of pluripotency, apoptosis and proliferation was complicated by the detection of the cell surface marker CD30. Proteomic identification and investigation into oxidative stress indicated the presence of antioxidant enzymes such as superoxide dismutases, catalases and peroxiredoxins in human fetal fibroblast conditioned medium and their regulation of redox homeostasis may play a significant role in the maintenance of hES cell pluripotency, apoptosis and proliferation. Together these investigations significantly contribute to the current body of knowledge for the undifferentiated culture of hES cells. Identification of conditioned medium proteins and the benefits of hypoxic culture of hES cells will aid the development of a defined, reproducible method of hES cell scale-up in bioreactor systems.
| Identifer | oai:union.ndltd.org:ADTP/242167 |
| Date | January 2008 |
| Creators | Prowse, Andrew Benjamin James, Biotechnology & Biomolecular Sciences, Faculty of Science, UNSW |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://unsworks.unsw.edu.au/copyright, http://unsworks.unsw.edu.au/copyright |
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