Lavagnolli Thais M C, Fonseca Simone A S, Serafim Rui C, Pereira Virgínia S, Santos Enrico J C, Abdelmassih Soraya, Kerkis Alexandre, Kerkis Irina
Department of Genetics and Morphology, Federal University of São Paulo, São Paulo-SP, Brazil.
Differentiation. 2009 Sep-Oct;78(2-3):124-30. doi: 10.1016/j.diff.2009.07.003. Epub 2009 Jul 28.
The fusion of embryonic stem (ES) cells with differentiated somatic cells is an approach that reverses a somatic cell nucleus to a state of pluripotency. The resulting ES-somatic cell hybrids (ES-SCH) retain most of the properties of ES cells: differentiate into multiple cell types and have the ability to produce embryoid bodies (EB) and chimeras. However, it is still unknown whether ES-SCH will be able to complete the differentiation into germ cells (GC) in vitro similar to ES cells. Here, we show that near diploid ES-SCH, obtained by the fusion of mouse ES and spleen cells, were able to differentiate in vitro into presumptive GC. Differentiation of ES-SCH was induced through EB formation and by the addition of retinoic acid. Presumptive GC obtained reacted positively with anti-EMA, Vasa, Fragilis and Dazl antibodies and expressed GC-specific genes, such as Vasa, Stella, Dazl, Piwil 2, Tex14, Bmp8b, Tdrd1 and Rnf17. Fluorescent in situ hybridization analysis indicates chromosome reduction in the GC-like cells. Expression of meiotic and postmeiotic GC-specific genes such as Haprin, Acrosin, Scyp1, Scyp3 and Stra-8 were also detected. Transmission electron microscopy confirmed ES-SCH differentiation into presumptive GC. The presence of several autosomes and the X chromosome originated from the "somatic" partner did not prevent ES-SCH differentiation towards presumptive GC. Overall our study suggests an interesting in vitro model, which allows the study GC differentiation in reprogrammed somatic cells.
胚胎干细胞（ES）与分化的体细胞融合是一种可将体细胞细胞核逆转至多能性状态的方法。由此产生的ES-体细胞杂种（ES-SCH）保留了ES细胞的大部分特性：可分化为多种细胞类型，并具有产生胚状体（EB）和嵌合体的能力。然而，ES-SCH是否能够在体外像ES细胞一样完成向生殖细胞（GC）的分化仍不清楚。在此，我们表明，通过小鼠ES细胞与脾细胞融合获得的近二倍体ES-SCH能够在体外分化为假定的GC。ES-SCH的分化通过EB形成和添加视黄酸来诱导。获得的假定GC与抗EMA、Vasa、Fragilis和Dazl抗体呈阳性反应，并表达GC特异性基因，如Vasa、Stella、Dazl、Piwil 2、Tex14、Bmp8b、Tdrd1和Rnf17。荧光原位杂交分析表明类GC细胞中染色体减少。还检测到减数分裂和减数分裂后GC特异性基因如Haprin、Acrosin、Scyp1、Scyp3和Stra-8的表达。透射电子显微镜证实ES-SCH分化为假定的GC。源自“体细胞”伙伴的几条常染色体和X染色体的存在并不妨碍ES-SCH向假定GC的分化。总体而言，我们的研究提出了一个有趣的体外模型，该模型可用于研究重编程体细胞中的GC分化。
