Generation of monoclonal antibodies specific for cell surface molecules expressed on early mouse endoderm.
Generation of monoclonal antibodies specific for cell surface molecules expressed on early mouse endoderm.
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DOI：
10.1002/stem.147
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发表时间：
2009-09
期刊：
影响因子：
5.2
通讯作者：
Keller, Gordon M.
中科院分区：
文献类型：
--
作者：
Gadue, Paul;Gouon-Evans, Valerie;Cheng, Xin;Wandzioch, Ewa;Zaret, Kenneth S.;Grompe, Markus;Streeter, Philip R.;Keller, Gordon M.
从胚胎干细胞（ES细胞）分化出功能性细胞群，如肝细胞和胰腺β细胞，依赖于在分化过程早期对定型内胚层的有效诱导。为了以定量的方式监测小鼠ES细胞分化培养中定型内胚层的形成，我们构建了一个报告细胞系，它从Foxa3基因座表达人CD25，从Foxa2基因座表达人CD4。用高浓度的激活素A（Activin）诱导这些报告ES细胞，在培养4到5天内会产生CD25 - Foxa3⁺CD4 - Foxa2⁺细胞群。对该细胞群的分离和特性分析表明，它主要由定型内胚层组成，在适当条件下能够进行肝脏特化。为了开发可用于研究未处理的ES细胞、诱导多能干细胞（iPS细胞）以及小鼠胚胎内胚层发育的试剂，我们制备了针对CD25 - Foxa3⁺CD4 - Foxa2⁺细胞群的单克隆抗体。通过这种方法，我们鉴定出两种抗体，它们能特异性地与ES细胞培养物以及早期胚胎的内胚层发生反应。这些抗体的特异性使人们能够定量监测ES细胞分化培养中的内胚层发育，研究胚胎中的内胚层形成，并分离出培养物或胚胎来源的纯内胚层细胞群。
The development of functional cell populations such hepatocytes and pancreatic beta cells from embryonic stem (ES) cells is dependent on the efficient induction of definitive endoderm early in the differentiation process. To monitor definitive endoderm formation in mouse ES cell differentiation cultures in a quantitative fashion, we generated a reporter cell line that expresses human CD25 from the Foxa3 locus and human CD4 from the Foxa2 locus. Induction of these reporter ES cells with high concentrations of Activin A (activin) led to the development of a CD25-Foxa3+CD4-Foxa2+ population within four to five days of culture. Isolation and characterization of this population revealed that it consists predominantly of definitive endoderm that is able to undergo hepatic specification under the appropriate conditions. To develop reagents that can be used for studies on endoderm development from un-manipulated ES cells, from induced pluripotent stem (iPS) cells, and from the mouse embryo we generated monoclonal antibodies against the CD25-Foxa3+CD4-Foxa2+ population. With this approach, we identified two antibodies that react specifically with endoderm from ES cell cultures as well as from the early embryo. The specificity of these antibodies enables one to quantitatively monitor endoderm development in ES cell differentiation cultures, to study endoderm formation in the embryo and to isolate pure populations of culture- or embryo-derived endodermal cells.
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