Qi Quan, Ding Chenhui, Hong Pingping, Yang Gang, Xie Yanxin, Wang Jing, Huang Sunxing, He Ke, Zhou Canquan
Reproductive Medical Center, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong 510080, P.R. China.
Int J Mol Med. 2015 Mar;35(3):569-78. doi: 10.3892/ijmm.2014.2044. Epub 2014 Dec 18.
The present study aimed to investigate the X chromosome inactivation (XCI) status in long-term cultured human parthenogenetic embryonic stem cells. One human embryonic stem (hES) cell line and 2 human parthenogenetic embryonic stem (hPES) cell lines were subjected to long-term culture in vitro (>50 passages). Karyotyping, array-based comparative genomic hybridization (aCGH), X-inactive specific transcript (XIST) RNA, immunofluorescence staining and real-time PCR were used to assess the chromosome karyotypes of these cells and the XCI status. X chromosome microdeletion was observed in the hPES-2 cells following culture for 50 passages. As early as 20 passages, XIST RNA expression was detected in the hPES-2 cells and was followed by low X-linked gene expression. The XIST RNA expression level was higher in the differentiated hPES-2 cells. The hPES-2' cells that were subclones of hPES-2 retained the XCI status, and had low XIST and X-linked gene expression. XIST RNA expression remained at a low level in the differentiated hPES-2' cells. The human biparental embryonic stem (hBES)-1 and hPES-1 cells did not exhibit XCI, and the differentiated hPES-1 cells had high expression levels of XIST RNA. In conclusion, the chromosome karyotypes of some hPES cell lines revealed instabilities. Similar to the hES cells, the hPES cells exhibited 3 XCI statuses. The unstable XCI status of the hPES-2 line may have been related to chromosome instability. These unstable chromosomes renedered these cells susceptible to environmental conditions and freezing processes, which may be the result of environmental adaptations.
本研究旨在调查长期培养的人孤雌生殖胚胎干细胞中的X染色体失活（XCI）状态。将1个人胚胎干细胞（hES）系和2个人孤雌生殖胚胎干细胞（hPES）系进行体外长期培养（>50代）。采用核型分析、基于芯片的比较基因组杂交（aCGH）、X染色体失活特异性转录本（XIST）RNA、免疫荧光染色和实时PCR来评估这些细胞的染色体核型和XCI状态。hPES-2细胞在培养50代后观察到X染色体微缺失。早在20代时，hPES-2细胞中就检测到XIST RNA表达，随后X连锁基因表达降低。在分化的hPES-2细胞中XIST RNA表达水平更高。作为hPES-2亚克隆的hPES-2'细胞保留了XCI状态，且XIST和X连锁基因表达较低。在分化的hPES-2'细胞中XIST RNA表达维持在低水平。人双亲胚胎干细胞（hBES）-1和hPES-1细胞未表现出XCI，且分化的hPES-1细胞中XIST RNA表达水平较高。总之，一些hPES细胞系的染色体核型显示出不稳定性。与hES细胞相似，hPES细胞表现出3种XCI状态。hPES-2系不稳定的XCI状态可能与染色体不稳定性有关。这些不稳定的染色体使这些细胞易受环境条件和冷冻过程的影响，这可能是环境适应的结果。
