Culture Adaptation in Human Embryonic Stem Cell Lines

Human embryonic stem (ES) cells offer considerable opportunities for regenerative medicine, drug discovery and toxicology. To realise this potential, a detailed understanding of their basic biology is required. It has become evident over the past three to four years that human ES cells may undergo genetic and other changes that may enhance their capacity for growth in culture, when they are maintained for extended periods. We have called this process, ‘culture adaptation’. The underlying hypothesis of this proposal is that the mechanisms affected during culture adaptation of ES cells are those that control the balance between self renewal, differentiation and death of these pluripotent stem cells. Understanding the basis for adaptation may not only provide a rational basis for designing improved methods to culture these cells while minimising the appearance of variants, but also may provide insights into the processes that control the self renewal of pluripotent stem cells and suggest approaches for controlling their growth and differentiation. Further, an understanding of culture adaptation of ES cells may provide insights into the mechanisms that drive progression of teratocarcinomas, a rare tumour of young men, but also a paradigm for other stem cell based malignancies.

Human embryonic stem (ES) cells may undergo karyotypic changes upon prolonged culture. These genetic changes often accompany phenotypic changes that render the cells more robust and easier to maintain, a phenomenon termed ?culture adaptation?. This phenomenon is complex and may include alterations in cell fate decisions, as well as changes in proliferation rate, cloning efficiency, and the phenotype of cells with respect to specific markers, differentiation capacity and malignant potential. The karyotypic changes associated with adaptation are non-random, with acquisition of extra copies of chromosomes 17, 12 and X being especially common. These changes parallel similar genomic amplifications in embryonal carcinoma (EC) cells, a cancer counterpart of ES cells, which highlights the potential link of culture adaptation in ES cells to the development of a malignant phenotype. However, little is known about the factors that drive culture adaptation, nor the cellular and genetic mechanisms involved. Nevertheless, the appearance of such adaptive changes in human ES cells is likely to have a substantial impact upon their eventual application, whether in regenerative medicine or drug discovery and toxicology. In this project we will first define the features that characterise the culture adapted phenotype of human ES cells and the extent to which these features occur independently or in a co-ordinated fashion. We will then seek to determine the nature of the selective pressures that drive the appearance of variant ES cells and to establish how interaction of the different features of the culture adapted phenotype with culture and passage conditions provide cells with a selective advantage. Finally we will aim to identify in more detail the specific genetic changes and individual genes that contribute to the culture adapted phenotype of human ES cells.