Functional study of mitotic checkpoint in human embryonic stem cells

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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.