Functional study of mitotic checkpoint in human embryonic stem cells

人类胚胎干细胞（hESC）和iPS细胞可以在长时间培养中获得染色体异常。这些变化包括整个或部分染色体的获得以及结构重排。核型异常的hESC经常表现出肿瘤转化和分化缺陷的迹象，因此它们可能对其在再生医学中的潜在应用构成重大危险，而这些变化可能影响其在其他应用中的使用，包括毒理学，药物发现和疾病建模。这些涉及DNA重排的染色体变化似乎可能是由于细胞周期S期DNA合成过程中的缺陷造成的。然而，许多变化涉及整个染色体的获得，强烈表明这些变化是由有丝分裂缺陷引起的，导致染色体非连接异常和染色体在子细胞中的不均匀分布。胚胎干细胞与体细胞的有丝分裂机制，特别是控制姐妹染色单体平等分离的纺锤体组装检查点可能不同，这反映了早期胚胎的特定要求。据报道，检查点凋亡解耦发生在人和小鼠胚胎干细胞中，但潜在的分子机制尚未探索。在这个项目中，我们将重点研究极光激酶的表达和作用，极光激酶是细胞分裂的关键调节因子。它们通常在癌细胞中高度表达，在我们之前的研究中，我们也发现它们在早期哺乳动物胚胎和胚胎干细胞中也很丰富。这种富集可能与胚胎细胞的快速细胞分裂速率有关，但也可能使这些细胞容易受到有丝分裂检查点的错误调节，从而导致染色体异常的积累。特别是在生殖细胞中正常表达的极光激酶C，在小鼠着床前发育过程中与极光激酶B具有重叠和不同的功能，极光激酶C的存在可能有助于hESCs中有丝分裂检查点的独特特性。我们将采用小分子抑制、siRNA敲低和mRNA过表达等方法破坏Aurora B和C的功能，研究其对hESC凋亡、自我更新和分化的短期和长期影响。该结果将为hESCs中有丝分裂检查点的功能提供见解，并有助于提供减少或预防hESCs核型异常发生和提高其基因组稳定性的方法。

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.