Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells
Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells
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DOI：
10.1016/s0960-9822(01)00144-0
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发表时间：
2001-04-03
期刊：
影响因子：
9.2
通讯作者：
Benvenisty, N
中科院分区：
文献类型：
--
作者：
Eiges, R;Schuldiner, M;Benvenisty, N
人胚胎干细胞（ES）是一种多能细胞系，来源于囊胚期胚胎的内细胞团（ICM）[1-3]，其特点是能够在培养中作为正常核型的未分化细胞无限繁殖，并且可以在体外诱导分化成各种细胞类型[1,2,4 -6]，因此，人胚胎干细胞有望作为移植的无限细胞来源。然而，这些独特的细胞系往往在培养中自发分化，因此难以维持。此外，菌落可能包含多种细胞类型，并可能由多能细胞以外的细胞组成[2,6]。为了克服这些困难并建立具有未分化表型的细胞系，我们将一种由胚胎干细胞富集基因的启动子调控的报告基因引入细胞。为了将DNA引入人类胚胎干细胞，我们建立了一种不同于小鼠胚胎干细胞的特定转染方案。在小鼠Rex1启动子的控制下，用增强型绿色荧光蛋白（EGFP）转染人ES细胞。转染的细胞在未分化状态下显示高水平的GFP表达。随着细胞的分化，这种表达在单层培养以及早期（简单）胚状体（EBs）和成熟胚状体（EBs）的原始内胚层中显著降低。使用荧光激活细胞分选器（FACS）可以对表达GFP的未分化细胞进行分析和分类，因此，我们建立了只有未分化细胞荧光的人类胚胎干细胞系，这些细胞可以在培养中进行跟踪和选择。我们还提出，培养的多能性是由同质细胞群体形成EBs的能力所证明的，有效转染人类胚胎干细胞的能力将为研究和操作这些细胞提供手段，用于基础和应用研究。
Human embryonic stem (ES) cells are pluripotent cell lines that have been derived from the inner cell mass (ICM) of blastocyst stage embryos [1-3], They are characterized by their ability to be propagated indefinitely in culture as undifferentiated cells with a normal karyotype and can be induced to differentiate in vitro into various cell types [1, 2, 4-6], Thus, human ES cells promise to serve as an unlimited cell source for transplantation. However, these unique cell lines tend to spontaneously differentiate in culture and therefore are difficult to maintain. Furthermore, colonies may contain several cell types and may be composed of cells other than pluripotent cells II, 2, 6]. In order to overcome these difficulties and establish lines of: cells with an undifferentiated phenotype, we have introduced a reporter gene that is regulated by a promoter of an ES cell-enriched gene into the cells, For the introduction of DNA into human ES cells, we have established a specific transfection protocol that is different from the one used for murine ES cells. Human ES cells were transfected with enhanced green fluorescence protein (EGFP), under the control of murine Rex1 promoter. The transfected cells show high levels of GFP expression when in an undifferentiated state. As the cells differentiate, this expression is dramatically reduced in monolayer cultures as well as in the primitive endoderm of early stage (simple) embryoid bodies (EBs) and in mature EBs, The undifferentiated cells expressing GFP can be analyzed and sorted by using a Fluorescence Activated Cell Sorter (FACS), Thus, we have established lines of human ES cells in which only undifferentiated cells are fluorescent, and these cells can be followed and selected for in culture. We also propose that the pluripotent nature of the culture is made evident by the ability of the homogeneous cell population to form EBs, The ability to efficiently transfect human ES cells will provide the means to study and manipulate these cells for the purpose of basic and applied research.