North East Stem Cell Institute, Newcastle University, International Centre for Life, NE1 3BZ, United Kingdom.
Stem Cells. 2010 Jan;28(1):84-92. doi: 10.1002/stem.263.
The isolation of significant numbers of human primordial germ cells at several developmental stages is important for investigations of the mechanisms by which they are able to undergo epigenetic reprogramming. Only small numbers of these cells can be obtained from embryos of appropriate developmental stages, so the differentiation of human embryonic stem cells is essential to obtain sufficient numbers of primordial germ cells to permit epigenetic examination. Despite progress in the enrichment of human primordial germ cells using fluorescence-activated cell sorting (FACS), there is still no definitive marker of the germ cell phenotype. Expression of the widely conserved RNA helicase VASA is restricted to germline cells, but in contrast to species such as Mus musculus in which reporter constructs expressing green fluorescent protein (GFP) under the control of a Vasa promoter have been developed, such reporter systems are lacking in human in vitro models. We report here the generation and characterization of human embryonic stem cell lines stably carrying a VASA-pEGFP-1 reporter construct that expresses GFP in a population of differentiating human embryonic stem cells that show expression of characteristic markers of primordial germ cells. This population shows a different pattern of chromatin modifications to those obtained by FACS enrichment of Stage Specific Antigen one expressing cells in our previous publication.
在几个发育阶段分离大量人类原始生殖细胞对于研究它们能够经历表观遗传重编程的机制非常重要。只有从适当发育阶段的胚胎中才能获得少量这些细胞，因此分化人类胚胎干细胞对于获得足够数量的原始生殖细胞以进行表观遗传检查至关重要。尽管使用荧光激活细胞分选（FACS）富集人类原始生殖细胞方面取得了进展，但仍然没有明确的生殖细胞表型标志物。广泛保守的 RNA 解旋酶 VASA 的表达仅限于生殖细胞，但与 Mus musculus 等物种不同，在这些物种中，已经开发出了在 Vasa 启动子控制下表达绿色荧光蛋白（GFP）的报告基因构建体，而在人类体外模型中缺乏这样的报告系统。我们在这里报告了稳定携带 VASA-pEGFP-1 报告基因构建体的人类胚胎干细胞系的产生和特征，该构建体在表现出原始生殖细胞特征标志物的分化人类胚胎干细胞群体中表达 GFP。与我们之前出版物中通过 FACS 富集表达阶段特异性抗原 1 的细胞获得的染色质修饰模式不同，该群体表现出不同的模式。
