A five stage selection protocol originally applied to mouse embryonic stem cells (mESCs) was examined for the derivation of insulin producing cells from human embryonic stem cells (hESCs). Insulin gene expression was observed and insulin protein was measured by radioimmunoassay. However, the radioimmunoassay results were shown to be susceptible to false positive findings due to the presence of exogenous insulin within differentiation media and it was concluded that this particular strategy was not ideal for the derivation of insulin producing cells from hESCs. An investigation was then undertaken regarding the in vivo differentiation of cells derived from hESCs seeded within 3D scaffolds to determine if this would result in the derivation of insulin producing cells. Within scaffolds there were abundant cells which stained positively for ectoderm lineage markers including nestin. Cells which stained positively for markers of endothelial progenitors representing the mesoderm lineage were also observed and rare cells stained for endoderm markers including insulin. These investigations also demonstrated that transplanting scaffolds seeded with cells derived from hESCs between the liver lobules of immunodeficient mice could lead to the formation of teratomas. Factors that may have influence the formation of teratomas were further investigated and it was demonstrated that teratoma formation was inhibited by altering in vitro treatment of cells. An in vitro investigation was then performed to determine the extracellular matrix (ECM) producing capacity of hESCs and differentiated cells derived from hESCs because ECM proteins are required for the formation of 3D structures similar to pancreatic islets. The results from this investigation indicated that differentiated cells produced multiple ECM proteins at substantially higher levels than hESCs. The ECM producing differentiated cells could be useful in the development of surrogate islet like tissue by supplying a suitable ECM structure within a 3D scaffold environment to aid the function of ??-cell surrogates. Furthermore, these differentiated cells derived from hESCs were shown to produce an adhesive basement membrane in vitro, which is derived from human sources, and could be utilized in the derivation, propagation and differentiation of hESCs.
| Identifer | oai:union.ndltd.org:ADTP/257845 |
| Date | January 2008 |
| Creators | Lees, Justin Guy, Clinical School - Prince of Wales Hospital, Faculty of Medicine, UNSW |
| Publisher | Publisher:University of New South Wales. Clinical School - Prince of Wales Hospital |
| 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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