Bidirectional reprogramming of mouse embryonic stem cell/fibroblast hybrid cells is initiated at the heterokaryon stage
Bidirectional reprogramming of mouse embryonic stem cell/fibroblast hybrid cells is initiated at the heterokaryon stage
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DOI：
10.1007/s00441-010-1085-2
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发表时间：
2010-12-01
影响因子：
3.6
通讯作者：
Serov, Oleg L.
中科院分区：
文献类型：
--
作者：
Gridina, Maria M.;Serov, Oleg L.
应用免疫荧光分析技术对绿色荧光蛋白标记的二倍体胚胎干细胞与蓝色荧光标记的二倍体成纤维细胞融合后的异核体和杂种细胞进行了研究。通过分析亲本线粒体DNA鉴定异核体。融合后20小时内，大多数异核体（高达80%）具有成纤维细胞样表型，对典型的成纤维细胞标志物（I型胶原、纤连蛋白、层粘连蛋白A/C）和标记失活X染色体的修饰me 3 H3 K27染色质呈阳性，但对Oct 4和Nanog呈阴性。大约20%的异核体具有替代ES样表型，对Oct 4和Nanog呈阳性，具有先前失活的X染色体重新激活的迹象，但对成纤维细胞标记物呈阴性。具有替代表型的杂交细胞在24-48 h内易于鉴定。ES样杂交细胞中成纤维细胞Oct 4等位基因启动子的DNA甲基化水平在第4天与ES细胞相似，但同时，两个亲本Oct 4等位基因在成纤维细胞样杂交细胞中高度甲基化。因此，在异核体阶段启动的双向重编程似乎导致形成两种类型的杂交细胞，其具有亲本基因组的替代优势。然而，两种类型的杂交细胞的进一步命运是不同的：ES样杂交细胞在4-6天形成集落，而成纤维细胞样杂交细胞没有集落。后者作为分离的单细胞生长，并且不能形成集落，如小鼠胚胎成纤维细胞。
Immunofluorescent analysis of markers specific for parental genomes was used to study heterokaryons and hybrid cells soon after the fusion of diploid embryonic stem (ES) cells marked with green fluorescent protein and diploid fibroblasts labeled by blue fluorescent beads. Heterokaryons were identified by an analysis of parental mitochondrial DNAs. Within 20 h after fusion, most heterokaryons (up to 80%) had a fibroblast-like phenotype, being positive for typical fibroblast markers (collagen type I, fibronectin, lamin A/C) and for the modification me3H3K27 chromatin marking the inactive X chromosome but being negative for Oct4 and Nanog. Approximately 20% of heterokaryons had an alternative ES-like phenotype being positive for Oct4 and Nanog, with signs of reactivation of the previously inactive X-chromosome but negative for fibroblast markers. Hybrid cells having alternative phenotypes were easily identified from 24-48 h. The level of DNA methylation at the promoter of the fibroblast Oct4 allele in the ES-like hybrid cells at day 4 was similar to that of ES cells but at the same time, both parental Oct4 alleles were heavily methylated in fibroblast-like hybrid cells. Thus, bidirectional reprogramming initiated at the heterokaryon stage seems to lead to the formation of two types of hybrid cells with alternative dominance of the parental genomes. However, the further fates of two types of hybrid cells are different: ES-like hybrid cells form colonies at 4-6 days but no colonies are derived from the fibroblast-like hybrid cells. The latter grow as disconnected single cells and are unable to form colonies, like mouse embryonic fibroblasts.