核移植来源的胚胎干细胞(NTES cells)在以干细胞为基础的细胞治疗中扮演着非常重要的角色，得到全能性良好且表观遗传修饰正常的核移植胚胎干细胞是解决治疗性克隆安全问题的重要前提。DNA甲基化修饰在基因表达和印迹基因的表达中起非常重要的作用，两步法克隆可能存在的不完全重编程问题很可能存在于印迹基因的甲基化水平上，并可能遗传到子代细胞中。因此，研究核移植胚胎干细胞的印迹基因甲基化状态及其与全能性的关系对于干细胞依赖的细胞治疗的临床应用具有重要理论和实际上的指导意义。
在本研究中，作者采用了具有不同遗传背景(C57/DBA, C57/129)的小鼠体细胞核移植胚胎干细胞和正常来源的胚胎干细胞作为研究对象，首先用四倍体囊胚重建的方法鉴定了六株胚胎干细胞的全能性。然后利用Bisulfite Genomic Sequencing并结合COBRA方法，对于六株干细胞系的三个印迹基因(H19, Peg1, Peg3)进行分析。研究发现在长时间体外培养(p20)的四株核移植胚胎干细胞中均存在着不同程度的甲基化异常现象。其中H19表现出了高度动态的甲基化模式，而Peg3则具有相对稳定的甲基化模式。虽然核移植来源的胚胎干细胞具有很好的全能性，但是潜在的印迹异常将成为其在细胞治疗中的潜在危险，筛选具有正常表观遗传修饰的核移植胚胎干细胞系对于干细胞的临床应用将是不可或缺得。
常港(中科院动物研究所联合培养博士生)为本文的第一作者；参与此项工作的还有，刘胜(技术员)，王凤超博士，张郁(技术员)，寇朝辉(技术员)；北京生命科学研究所的高绍荣博士和中科院动物研究所的陈大元研究员是本文的共同通讯作者。
此项研究为科技部863和北京市科委资助课题，在北京生命科学研究所高绍荣实验室完成。
Genomics, In Press, Corrected Proof, Available online 13 November 2008
Differential methylation status of imprinted genes in nuclear transfer derived ES (NT-ES) cells
Gang Chang, Sheng Liu, Fengchao Wang, Yu Zhang, Zhaohui Kou, Dayuan Chen, Shaorong Gao
Compared with fertilized embryo derived ES (F-ES) cells, somatic cell nuclear transfer (SCNT) produced ES (NT-ES) cells were proposed appropriate for cell transplantation based therapies. Although previous studies indicated that NT-ES cells and F-ES cells were transcriptionally and functionally indistinguishable, characterization of DNA methylation patterns of imprinted genes in NT-ES cells is lacking. Here, we show that DNA methylation patterns in the differentially methylated region (DMR) of paternally imprinted gene, H19, displayed distinct abnormalities in certain NT-ES and F-ES cell lines after long-term culture in vitro. DNA methylation profiles of H19 appeared very dynamic in most ES cell lines examined, either hypermethylation or hypomethylation could be observed in specific ES cell lines. In contrast to H19, maternally imprinted genes, Mest and Peg3, showed relatively stable methylation patterns in ES cells, especially Peg3, which displayed better capability in enduring long-term culture in vitro. Our results indicate that abnormal methylation profiles of certain imprinted genes could be observed in both NT-ES and F-ES cell lines after long-term culture in vitro although these cell lines were proved to be pluripotent with germline transmission competent. Stringent screening of epigenetically normal NT-ES cells might be potentially necessary for further therapeutic application of these cells.
