Nowak-Imialek Monika, Kues Wilfried A, Rudolph Cornelia, Schlegelberger Brigitte, Taylor Ulrike, Carnwath Joseph W, Niemann Heiner
Institut of Farm Animal Genetics, Friedrich-Loeffler-Institute, Department of Biotechnology, Mariensee, Neustadt, Germany.
Cell Reprogram. 2010 Feb;12(1):55-65. doi: 10.1089/cell.2009.0045.
Fusion of terminally differentiated somatic cells with pluripotent embryonic stem cells has been proposed as model for reprogramming the somatic cell genome, and may contribute to our understanding of the underlying mechanisms of this epigenetic process. We established an interspecies cell fusion model using murine embryonic stem cells (ESCs) and porcine fibroblasts. These inter-species fusion experiments yielded much lower conversion efficiency rates than murine intraspecies fusion. Nevertheless, two double-resistant mouse-pig hybrid clones could be generated. Reactivation of the porcine OCT4 gene, an essential pluripotent stem cell marker, and demethylation of the porcine OCT4 promoter in hybrid clone 1, suggested successful reprogramming of porcine chromosomes. A rapid loss of porcine chromosomes was observed during the selection phase. Spectral karyotyping (SKY) analysis showed that fusion-hybrid clone 1 carried a tetraploid mouse chromosome complement with only few pig chromosomes and/or chromosomal fragments. Hybrid clone 2 had a diploid set of murine chromosomes complements and also contained an interspecies chromosome fusion product. Interspecies cell fusion results in hybrid cells that retained the complement of mouse chromosomes and preferentially lose porcine chromosomes during colony expansion. Neither species-specific chromosomal segregation nor reprogrammed diploid porcine cells were observed. These findings indicate that generation of reprogrammed pluripotent diploid cells by cell fusion may require additional supporting provisions.
将终末分化的体细胞与多能胚胎干细胞融合，已被提议作为重编程体细胞基因组的模型，这可能有助于我们理解这种表观遗传过程的潜在机制。我们利用小鼠胚胎干细胞（ESCs）和猪成纤维细胞建立了种间细胞融合模型。这些种间融合实验产生的转化效率远低于小鼠种内融合。然而，仍可产生两个双抗性小鼠 - 猪杂交克隆。在杂交克隆1中，猪多能干细胞关键标志物OCT4基因的重新激活以及猪OCT4启动子的去甲基化，表明猪染色体成功重编程。在筛选阶段观察到猪染色体快速丢失。光谱核型分析（SKY）显示，融合杂交克隆1携带四倍体小鼠染色体组，仅含有少数猪染色体和/或染色体片段。杂交克隆2具有二倍体小鼠染色体组，并且还包含一个种间染色体融合产物。种间细胞融合产生的杂交细胞保留了小鼠染色体组，并在集落扩增过程中优先丢失猪染色体。未观察到物种特异性染色体分离，也未观察到重编程的二倍体猪细胞。这些发现表明，通过细胞融合产生重编程的多能二倍体细胞可能需要额外的支持条件。
