Feeder-free culture of human embryonic stem cells in conditioned medium for efficient genetic modification

被引:57
作者
Braam, Stefan R. [2 ,3 ]
Denning, Chris [1 ]
Matsa, Elena [1 ]
Young, Lorraine E. [1 ]
Passier, Robert [2 ,3 ]
Mummery, Christine L. [2 ,3 ]
机构
[1] Univ Nottingham, Ctr Biomol Sci, Wolfson Ctr Stem Cell Tissue Engn & Modelling STE, Nottingham NG7 2RD, England
[2] Hubrecht Inst, NL-3584 CT Utrecht, Netherlands
[3] Leiden Univ, Med Ctr, Dept Anat & Embryol, NL-2300 RC Leiden, Netherlands
基金
英国医学研究理事会; 英国生物技术与生命科学研究理事会;
关键词
D O I
10.1038/nprot.2008.140
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Realizing the potential of human embryonic stem cells (hESCs) in research and commercial applications requires generic protocols for culture, expansion and genetic modification that function between multiple lines. Here we describe a feeder-free hESC culture protocol that was tested in 13 independent hESC lines derived in five different laboratories. The procedure is based on Matrigel adaptation in mouse embryonic fiboblast conditioned medium (CM) followed by monolayer culture of hESC. When combined, these techniques provide a robust hESC culture platform, suitable for high-efficiency genetic modification via plasmid transfection (using lipofection or electroporation), siRNA knockdown and viral transduction. In contrast to other available protocols, it does not require optimization for individual lines. hESC transiently expressing ectopic genes are obtained within 9 d and stable transgenic lines within 3 weeks.
引用
收藏
页码:1435 / 1443
页数:9
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