Campos Priscila B, Sartore Rafaela C, Abdalla Stacie N, Rehen Stevens K
Institute of Biomedical Sciences, Federal University of Rio De Janeiro-UFRJ.
J Vis Exp. 2009 Sep 4(31):1512. doi: 10.3791/1512.
Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used, while manual dissection of colonies for passaging retains a stable karyotype. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis. Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies.
尽管人类胚胎干细胞（hESC）已被证明具有稳定的二倍体核型，但许多研究报告称，根据培养条件的不同，它们容易出现染色体异常，例如整条或部分染色体的添加。事实上，在长期培养过程中，当使用酶解或化学解离方法时会观察到核型改变，而手动切割集落进行传代则能保持稳定的核型。此外，环境变化，如去除饲养层细胞，似乎也会损害hESC的遗传完整性。一旦染色体改变可能影响细胞生理功能，鉴于hESC是胚胎发生研究和药物测试的重要工具，体外hESC遗传完整性的表征至关重要。此外，对于未来的治疗目的而言，染色体变化是一个实际问题，因为它常常与致癌作用相关。在这里，我们展示了一种简单且有用的方法，可获得高质量的染色体铺片，以便随后通过G显带、荧光原位杂交（FISH）、光谱核型分析（SKY）或比较基因组杂交（CGH）技术对染色体组进行分析。我们建议每隔5代定期检查染色体状态，以监测易位和非整倍体的出现。
