Dominance of parental genomes in embryonic stem cell/fibroblast hybrid cells depends on the ploidy of the somatic partner
Dominance of parental genomes in embryonic stem cell/fibroblast hybrid cells depends on the ploidy of the somatic partner
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DOI：
10.1007/s00441-010-0987-3
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发表时间：
2010-06-01
影响因子：
3.6
通讯作者：
Serov, Oleg L.
中科院分区：
文献类型：
--
作者：
Kruglova, Anna A.;Matveeva, Natalia M.;Serov, Oleg L.
将绿色荧光蛋白（GFP）阳性的二倍体胚胎干细胞（ES细胞）与来自DD/c和C57 BL-1（I）1 RK小鼠的四倍体成纤维细胞融合，产生24个杂交克隆。细胞遗传学分析表明，大多数细胞从这些杂交克隆含有近六倍体染色体组。此外，通过多态性微卫星的聚合酶链反应（PCR）分析证实了来自两个亲本细胞的染色体的存在。所有杂交细胞均为GFP阳性，并表现出生长特征和成纤维细胞样形态。此外，大多数杂交细胞的I型胶原，纤连蛋白和核纤层蛋白A/C呈阳性，但Oct 4和Nanog蛋白呈阴性。通过亚硫酸氢盐基因组测序分析评估Oct 4和Nanog基因启动子的甲基化状态。Oct 4和Nanog基因启动子的甲基化位点（CpG位点）在杂交细胞中高度甲基化，而CpG位点在亲本ES细胞中未甲基化。因此，在二倍体ES细胞/四倍体成纤维细胞杂交细胞中，成纤维细胞基因组支配ES基因组。多能性和成纤维细胞标记物的免疫荧光分析表明，融合后不久发生成纤维细胞表型的建立，并且成纤维细胞表型进一步保持在杂交细胞中。四倍体成纤维细胞的核质和胞质与整个ES细胞的融合表明，核质能够建立重建细胞的成纤维细胞表型，而不是成纤维细胞胞质。因此，这些数据表明，在ES细胞/体细胞类型的杂交细胞中，亲本基因组的优势取决于体细胞伴侣的倍性。
Two dozen hybrid clones were produced by fusion of diploid embryonic stem (ES) cells positive for green fluorescent protein (GFP) with tetraploid fibroblasts derived from DD/c and C57BL-I(I)1RK mice. Cytogenetic analysis demonstrated that most cells from these hybrid clones contained near-hexaploid chromosome sets. Additionally, the presence of chromosomes derived from both parental cells was confirmed by polymerase chain reaction (PCR) analysis of polymorphic microsatellites. All hybrid cells were positive for GFP and demonstrated growth characteristics and fibroblast-like morphology. In addition, most hybrid cells were positive for collagen type I, fibronectin, and lamin A/C but were negative for Oct4 and Nanog proteins. Methylation status of the Oct4 and Nanog gene promoters was evaluated by bisulfite genomic sequencing analysis. The methylation sites (CpG-sites) of the Oct4 and Nanog gene promoters were highly methylated in hybrid cells, whereas the CpG-sites were unmethylated in the parental ES cells. Thus, the fibroblast genome dominated the ES genome in the diploid ES cell/tetraploid fibroblast hybrid cells. Immunofluorescent analysis of the pluripotent and fibroblast markers demonstrated that establishment of the fibroblast phenotype occurred shortly after fusion and that the fibroblast phenotype was further maintained in the hybrid cells. Fusion of karyoplasts and cytoplast derived from tetraploid fibroblasts with whole ES cells demonstrated that karyoplasts were able to establish the fibroblast phenotype of the reconstructed cells but not fibroblast cytoplasts. Thus, these data suggest that the dominance of parental genomes in hybrid cells of ES cell/somatic cell type depends on the ploidy of the somatic partner.