| Summary: | Despite the risk of malignancy associated with hES cells, their indefinite growth in culture has provided an opportunity to manipulate their fate. This thesis has investigated the possibility of using the exogenous cell surface markers; the Galα1-3Galβ1-4GIcNAc-R (α-gal) epitope and the murine MHC class I molecule H2-K<sup>k</sup>, under the transcription control of the human telomerase reverse transcriptase (hTERT) promoter to identify undifferentiated hES cells for selective elimination. The use of the H2-K<sup>k</sup> gene as a potential lysis epitope was unsuccessful. Despite integration and transcription of the H2-K<sup>k</sup> gene, cell surface expression of the H2-K<sup>k</sup> epitope was not achieved, and proof of protein translation could not be found. However, genetic manipulation of undifferentiated hES cells with the α1,3galactosyltransferase (α1,3Gal) gene, the catalyst for production of α-gal epitopes, resulted in cell surface expression on transgenic cell lines (M2 and F11) comparable with the endogenous α-gal expressing ovine foetal fibroblast cell line (PDFF). Expression of the α-gal epitope on undifferentiated hES cells elicits a severe, yet highly specific cytolytic response; on average 95% of the transgenic hES cells die compared with just 8-12% of wild type non-expressing H9 cells, when exposed to human serum containing active-complement. In addition, when transgenic M2-hES cells were differentiated the α-gal epitope was down regulated, in the same manner as established markers of undifferentiated hES cells (Tra-1-81 and SSEA-4). Following differentiation the transgenic M2-hES derivatives survived exposure to active serum-complement. This novel system for selective ablation could potentially provide natural immune protection, through the presence of circulating antibodies to α-gal that would protect graft-recipients against the presence of, or de-differentiation of, hES-cell derivatives following engraftment.
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