Functional study of mitotic checkpoint in human embryonic stem cells

It is well established that human ES cells (hESC), and iPS cells, can gain chromosomal abnormalities during prolonged culture. These changes include the gain of whole or partial chromosomes as well as structure rearrangements. Karyotypically abnormal hESC often show signs of neoplastic transformation and defects in differentiation, so that they may pose significant dangers for their potential use in regenerative medicine, while these changes may affect their use in other applications including toxicology, drug discovery and disease modelling. It seems likely that those chromosomal changes that involve rearrangements of the DNA result from defects during DNA synthesis in S phase of the cell cycle. However, many changes involve the gain of whole chromosomes, strongly suggesting that these changes arise from defects at mitosis resulting in chromosomal non-dysjunction and unequal distributions of chromosome to the daughter cells. It is likely that the mitotic machinery, particularly the spindle assembly checkpoint, which governs the equal separation of the sister chromatids, is different in ES cells from somatic cells, reflecting specific requirements in the early embryo and it is reported that checkpoint-apoptosis uncoupling occurs in human and mouse ESCs, but the underlying molecular mechanism remains unexplored. In this project we will focus on the expression and role of the Aurora kinases which are key regulators of cell division. They are often highly expressed by cancer cells and in our previous studies we also found that they are also enriched in early mammalian embryos and embryonic stem cells. This enrichment may be associated with the fast cell division rate of embryonic cells, but may also make these cells susceptible to the mis-regulation of the mitotic checkpoint resulting in the accumulation of chromosomal abnormality. In particular, Aurora kinase C, an Aurora kinase normally expressed in germ cells, has overlapping as well as distinct functions from Aurora B during mouse preimplantation development and the presence of Aurora C may contribute to the unique properties of mitotic checkpoint in hESCs. We will use small molecule inhibition, siRNA knocking-down and mRNA over expression to disrupt the function of Aurora B and C and study the short-term and long-term effects on hESC apoptosis, self-renewal and differentiation. The results will provide insights into the function of mitotic checkpoints in hESCs and help inform approaches to reduce or prevent the occurrence of karyotype abnormality in hESCs and improve their genome stability.

Human embryonic stem cells (hESCs) have the ability to self-renew indefinitely and differentiate into all types of tissue in the body. Thus they provide an unlimited source for cell transplantation therapy to treat degenerative diseases and for drug discovery. However, these pluripotent stem cells are susceptible to gaining chromosomal abnormalities during in vitro culture, which may make them unsafe to use in regenerative medicine. Aurora kinases are key regulators of cell division and may be highly expressed in many cancer cells. Three mammalian Aurora kinases, including Aurora kinase C, a germline specific Aurora kinase, are expressed in hESCs. Our previous studies in mouse oocytes and embryos showed that over expression of Aurora kinase C promoted securin degradation and chromosome separation, whereas perturbing its function leads to abnormal cell division. In this study, we will use small molecule inhibition, siRNA knocking-down and mRNA over expression to disrupt the function of Aurora B and C and study the short-term and long-term effects on hESC apoptosis, self-renewal and differentiation. The results will provide insights into the function of mitotic checkpoints in hESCs and help inform approaches to reduce or prevent the occurrence of karyotype abnormality in hESCs and improve their genome stability.

The susceptibility of human ES cells (hESC), and likewise induced pluripotent stem cells (iPSC), to 'culture adaptation' and genetic change after prolonged maintenance in culture is of considerable significance for their eventual safe use in regenerative medicine. It could also impact upon their utility for use in vitro systems for toxicology, drug discovery and disease modelling. For companies and researchers developing such translational applications for hESC, it is essential to understand the molecular and cellular mechanisms that underlie the maintenance of the integrity of the hESC genome, and how disruption of these mechanisms can lead to undesired genetic change. Such disruption could be due to the acquisition of mutations that affect components of those mechanisms, or could be due to environmental factors, such as the suboptimal growth conditions under which hESC are maintained. The results obtained from the present study may contribute to the development of culture methods to minimise the impact of such disruptions and help maintain the genomic integrity of cultured hESC and iPSC. The results could also be used to develop new tools to allow early detection of genetic change or of cellular conditions that might promote susceptibility to genetic change. These developments could have an immediate impact on the methodologies being used to prepare hESC or iPSC for applications for regenerative medicine in vivo, or for screening systems in vitro.