Genetic manipulation of human embryonic stem cells in serum and feeder-free media.
Generic methods for genetic manipulation of human embryonic stem cells (hESCs) are important for both present research and future commercial applications. To date, differences in cell derivation and culture have required independent optimization of transfection and transduction protocols and some lines have remained refractile to all methods. Here we describe a culture protocol that has been extensively tested in 12 different hESC lines (1, 2) and shown to support efficient gene transfer independent of the method of gene delivery or history of the cell line. The system is based on Matrigel monolayer culture and conditioned medium from mouse embryonic feeder cells (MEFs) and entails transient high-density culture followed by rapid adaptation to low density for gene transfer. Under these conditions, plasmid transfection, virus infection, and siRNA transfection are highly effective. Stable genetically modified hESC lines can be generated with plasmid transfection, viral infection, or electroporation without loss of pluripotency or differentiation potential. The majority of lines generated in this system display a normal karyotype.
Braam
Stefan R
SR
Department of Anatomy & Embryology, Leiden University Medical Centre, Leiden, The Netherlands.
Denning
Chris
C
Mummery
Christine L
CL
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D002460:Cell Line; D002469:Cell Separation; D015925:Cryopreservation; D003470:Culture Media; D017077:Culture Media, Conditioned; D018274:Electroporation / Q000379:methods; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism*; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D006801:Humans; D051379:Mice; D010957:Plasmids / Q000235:genetics; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D014161:Transduction, Genetic / Q000379:methods; D014162:Transfection / Q000379:methods
D003470:Culture Media; D017077:Culture Media, Conditioned; D034741:RNA, Small Interfering