Stephenson, E.; (2009) Derivation of human embryonic stem cells to study early development and genetic disease. Doctoral thesis , UCL (University College London).

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Abstract

Stem cells are unique cells that have both the capacity for self-renewal and, depending on their origin, the ability to form at least one, and sometimes many, specialised cell types of all three embryonic germ lineages - germ cells (endoderm, mesoderm and ectoderm), extra-embryonic tissue and trophoblast. Since the derivation of the fi�rst human embryonic stem cell (hESC) line in 1998, there has been substantial interest in the potential of these cells both for regenerative medicine and cell therapy, and as disease models for monogenic disorders. Aside from the need to improve derivation efficiency and further the understanding of the basic biology of these cells, the ability to work with hESC opens up three broad research areas. The �first is the development of clinical grade culture systems with the aim of producing cell lines suitable for subsequent manipulation for therapy. The second is the opportunity to use these cells as a tool to study the earliest determinative events in mammalian development, such as the origins of patterning in the mammalian embryo. The third is the use of hESCs carrying clinically relevant genetic mutations as models for disease research and therapeutic target identifi�cation. The development of several methods of embryo manipulation tailored to the morphology of the blastocyst is described here, which resulted in the derivation of seven lines from four di�fferent procedures and provided the tools for subsequent research. Acknowledging that each laboratory in isolation is unlikely to derive sufficient lines to draw signifi�cant conclusions regarding manipulation methodology and culture parameters, an international collaboration was initiated with the aim of standardising the reporting of derivation and thus obtaining the maximum information from the generation of each new hESC line. To address the need for the development of clinical grade culture systems, alternative feeder cells were assessed for their suitability in hESC culture and derivation. Modi�fied human foreskin fi�broblasts and human amniotic epithelial cells (hAECs) were investigated, as both cell types can be fully qualifi�ed and validated. Whilst both were able to support the culture of existing lines, only the hAECs showed promise in supporting derivation. In addition, analysis of in-house and commercially available media showed that neither were physiologically optimal for the growth of inner cell mass (ICM) cells or putative hESC, as metabolite concentrations were in excess and subsequent catabolite levels exceeded known toxic levels. The timing and mechanisms establishing patterning and future polarity in the mammalian embryo remains a subject of intense debate. Here, the potential of single blastomeres to generate hESC was used as an assessment of pluripotency. The determination of the most appropriate day for attempting derivation was performed by assessing blastomere development and pluripotent marker expression, and the predicted success of derivation was considered in the light of division patterns. Putative stem-like cells were visible in several cultures. Furthermore, isolated blastomeres from two-, four-, and eight-cell embryos were analysed for the quantitative expression of multiple target genes known to be associated with lineage formation and the stem cell state. Analysis suggested that broad changes in gene expression were occurring with development stage. However, no consistent grouping structure for cells within embryos was observed, and no convincing pattern was seen when considering the individual embryo variance scores. Several approaches are discussed to diff�erentiate between the biological and methodological variability in this experimental design. The suitability of hESC as models for genetic disease was studied following the derivation of two lines carrying Huntington disease (HD). Subsequent di�fferentiation using a stromal co-culture neural induction protocol resulted in the establishment of a stable, highly proliferative cell population which was simple to culture and bank. The cells were of an astroglial phenotype, and therefore highly suited for subsequent studies regarding HD pathophysiology, as glial cells are severely aff�ected in HD. During diff�erentiation the CAG repeat size increased from 46 to 70, showing the salient feature of somatic instability of the huntingtin gene. Therefore this cell population provides a valuable tool in the study of disease pathogenesis and transmission.

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