In Vitro Transformation of Mouse Testis Cells by Oncogene Transfection
In Vitro Transformation of Mouse Testis Cells by Oncogene Transfection
复制标题
DOI：
10.1095/biolreprod.111.095307
复制
发表时间：
2012-05-01
影响因子：
3.6
通讯作者：
Shinohara, Takashi
中科院分区：
文献类型：
--
作者：
Morimoto, Hiroko;Lee, Jiyoung;Shinohara, Takashi
生殖细胞肿瘤(GCTS)具有独特的生物学特性和病理特征。尽管有几种体内GCT模型可用，但GCTS的研究受到阻碍，因为在体内开发GCTS非常耗时，并且无法对转化过程进行详细的分子分析。在这里，我们开发了一种新的策略来在体外转化小鼠睾丸细胞。慢病毒介导的显性阴性Trp53、Myc和激活的Hras1基因导入表达CD9的睾丸细胞，在体外可引起肿瘤转化。虽然这些细胞类似于胚胎干细胞，但它们是非整倍体的，缺乏Nanog的表达，Nanog参与了ES细胞的未分化状态的维持。将Yamanaka因子(Pou5f1、Myc、Klf4和Sox2)导入同一细胞群体，获得整倍体ES样细胞。尽管这些细胞表达Nanog，但它们与ES细胞的不同之处在于它们表达CD44，一种癌症干细胞抗原。这两种处理在印记基因的不同甲基化区域引起了相似的DNA甲基化模式的变化。此外，尽管它们的表型和核型不同，但这两种类型的细胞在移植时都产生了类似的混合GCT，包括畸胎瘤、精原细胞瘤和胚胎癌。因此，体外睾丸细胞转化有助于分析GCT的形成过程，我们的结果也表明GCT形成和重新编程之间具有密切的相似性。
Germ cell tumors (GCTs) are unique in that they exhibit diverse biological characteristics and pathological features. Although several in vivo GCT models are available, studies on GCTs are hampered because in vivo development of GCTs is time consuming and prevents a detailed molecular analysis of the transformation process. Here we developed a novel strategy to transform mouse testis cells in vitro. Lentivirus-mediated transfection of dominant negative Trp53, Myc, and activated Hras1 into a CD9-expressing testis cells caused tumorigenic conversion in vitro. Although these cells resembled embryonic stem (ES) cells, they were aneuploid and lacked Nanog expression, which is involved in the maintenance of the undifferentiated state in ES cells. Euploid ES-like cells were produced by transfecting the Yamanaka factors (Pou5f1, Myc, Klf4, and Sox2) into the same cell population. Although these cells expressed Nanog, they were distinct from ES cells in that they expressed CD44, a cancer stem cell antigen. Both treatments induced similar changes in the DNA methylation patterns in differentially methylated regions of imprinted genes. Moreover, despite the differences in their phenotype and karyotype, both cell types similarly produced mixed GCTs on transplantation, which were composed of teratomas, seminomas, and embryonal carcinomas. Thus, in vitro testis cell transformation facilitates an analysis of the GCT formation process, and our results also suggest the close similarity between GCT formation and reprogramming.