Clonal analysis of human embryonic stem cell differentiation into teratomas
Clonal analysis of human embryonic stem cell differentiation into teratomas
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DOI：
10.1634/stemcells.2007-0073
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发表时间：
2007-08-01
期刊：
影响因子：
5.2
通讯作者：
Benvenisty, Nissim
中科院分区：
文献类型：
--
作者：
Blum, Barak;Benvenisty, Nissim
人胚胎干细胞(HESCs)的分化可以通过诱导免疫缺陷小鼠的畸胎瘤在体内进行研究。畸胎瘤内的细胞分化成所有三个胚胎胚层。然而，畸胎瘤中hESCs的增殖和分化的确切性质尚不完全清楚，这种分化是细胞自主的还是受邻近细胞的影响尚不清楚。在这里，我们建立了一种遗传学方法来研究畸胎瘤分化的克隆性，使用混合的HESC系。我们首先通过5-溴-2‘-脱氧尿苷掺入的方法证明，细胞增殖发生在整个畸胎瘤中，并且没有未分化的增殖细胞群。利用激光捕获显微解剖和DNA指纹分析相结合的方法，我们证明不同的细胞系相互作用于相同的独特组织结构。性染色体的荧光原位杂交分析进一步支持了畸胎瘤内的非克隆分化。因此，我们认为人胚胎干细胞在体内的分化是多克隆的，因此可能不是细胞自主的，强调需要三维生长才能实现人胚胎干细胞的复杂分化。
Differentiation of human embryonic stem cells (HESCs) can be studied in vivo through the induction of teratomas in immune-deficient mice. Cells within the teratomas differentiate into all three embryonic germ layers. However, the exact nature of the proliferation and differentiation of HESCs within the teratoma is not fully characterized, and it is not clear whether the differentiation is cell autonomous or affected by neighboring cells. Here, we establish a genetic approach to study the clonality of differentiation in teratomas using a mixture of HESC lines. We first demonstrate, by means of 5-bromo-2'-deoxyuridine incorporation, that cell proliferation occurs throughout the teratoma, and that there are no clusters of undifferentiated-proliferating cells. Using a combination of laser capture microdissection and DNA fingerprinting analysis, we show that different cell lines contribute mutually to the same distinctive tissue structures. Further support for the nonclonal differentiation within the teratoma was achieved by fluorescence in situ hybridization analysis of sex chromosomes. We therefore suggest that in vivo differentiation of HESCs is polyclonal and, thus, may not be cell autonomous, stressing the need for a three-dimensional growth in order to achieve complex differentiation of HESCs.