The in-vitro culture of human embryonic stem and keratinocyte cells has great potential to revolutionise the therapeutics industry. Indeed it is hoped that these cells will provide a superior alternative to current tissue and organ transplantation. However, both of these cell types require animal and/or donor products for their successful maintenance in-vitro. This requirement results in a significant risk of cross contamination from the animal or donor products to either the primary keratinocyte or hES cells. These potentially transplantable cells therefore need to be cultured in an environment free from animal or donor products to remove the risk of contamination to the patient. The ideal growth conditions must comprise of two attributes; firstly they must be free from animal or donor products, and secondly the culture system must be fully defined. Recently, it was discovered that an extra-cellular matrix protein, vitronectin, could be used in conjunction with growth factors and growth factor-binding proteins (VN:GF combination), to promote enhanced cell migration and growth through the co-activation of integrin and growth factor receptors. Given that growth factors and serum are clearly important in supporting the in-vitro cultivation of mammalian cells, and that vitronectin is an abundant protein in serum, I hypothesised that these VN:GF combinations could be translated into a serum-free medium that would support the serial propagation and self renewal of primary keratinocytes and hES cells. As reported in this thesis I have developed a defined, serum-free media for the culture of these cells that incorporates the VN:GF combinations. While the two media differ slightly in their compositions, both support the serial, undifferentiated expansion of their respective cells types. Together, this represents a significant advance that will ultimately facilitate the therapeutic use of these cells. However, the in-vitro expansion of these cells in these new media still required the presence of a feeder cell layer. In view of this I aimed to explore the in-vitro micro-environment of primary keratinocytes using a novel proteomic approach in an attempt to find candidate factors that could be used in conjunction with the VN:GF media to replace both serum and the feeder cells. The proteomic approach adopted examined the secretion of proteins into the defined, minimal protein content VN:GF media when the feeder cells were cultured alone, as well as in co-culture with primary keratinocytes. This strategy allowed assessment of proteins/factors that are secreted in response to both autocrine and paracrine cellular interactions and revealed a number of candidate factors that warrant further investigation. Ultimately this proteomic information and the associated new insights into the keratinocyte in-vitro culture microenvironment may lead to the development of a culture system for these cells that is not reliant on either a feeder cell layer or serum for their successful propagation. Moreover, it is likely that this will also be relevant to the feeder cell-free propagation of hES cells. This has obvious advantages for the culture of primary keratinocytes and hES cells in that it will allow a safe defined culture system for the undifferentiated propagation of these cells. This will facilitate the generation of cells and tissues free from xenogeneic and allogeneic contaminants, thus ensuring any therapeutics developed from these cell types are approved for therapeutic applications and importantly, will minimise risks to patients.
| Identifer | oai:union.ndltd.org:ADTP/265590 |
| Date | January 2007 |
| Creators | Richards, Sean Dennis |
| Publisher | Queensland University of Technology |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
| Rights | Copyright Sean Dennis Richards |
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