title: Transduction of human embryonic stem cells by ecotropic retroviral vectors.
abstract: The steadily increasing availability of human embryonic stem (hES) cell lines has created strong interest in applying available tools for gene transfer in murine cells to human systems. Here we present a method for the transduction of hES cells with ecotropic retroviral vectors. hES cells were transiently transfected with a construct carrying the murine retrovirus receptor mCAT1. Subsequently, the cells were exposed to replication-deficient Moloney murine leukemia virus (MoMuLV) derivatives or pseudotyped lentiviral vectors. With oncoretroviral vectors, this procedure yields overall transduction efficiencies of up to 20% and permits selection of permanently transduced clones with high frequency. Selected clones maintained expression of pluripotency-associated markers and exhibited multi-germ layer differentiation both in vitro and in vivo. HES cell-derived somatic cells including neural progeny maintained high levels of transgene expression. Lentiviral vectors pseudotyped with the MoMuLV envelope could be introduced in the same manner with efficiencies of up to 33%. Transgene expression of lentivirally transduced hES cells remained permanent after differentiation even without selection pressure. Bypassing the regulatory issues associated with the use of amphotropic retroviral systems and exploiting the large pool of existing murine vectors, this method provides a safe and versatile tool for gene transfer and lineage analysis in hES cells and their progeny.
authors.affiliation: Institute of Reconstructive Neurobiology, Life and Brain Center University of Bonn and Hertie Foundation, Bonn, Germany.
authors.forename: Philipp
authors.initials: P
authors.lastname: Koch
authors.forename: Henrike
authors.initials: H
authors.lastname: Siemen
authors.forename: Andrea
authors.initials: A
authors.lastname: Biegler
authors.forename: Joseph
authors.initials: J
authors.lastname: Itskovitz-Eldor
authors.forename: Oliver
authors.initials: O
authors.lastname: Brüstle
mesh_terms: D000818:Animals; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D002498:Centrifugation; D004622:Embryo, Mammalian / Q000166:cytology*; D005822:Genetic Vectors; D005855:Germ Layers / Q000166:cytology; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D008979:Moloney murine leukemia virus / Q000235:genetics; D009474:Neurons / Q000166:cytology; D039904:Pluripotent Stem Cells / Q000378:metabolism; D011991:Receptors, Virus / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*; D050916:TRPV Cation Channels / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic / Q000379:methods*; D019076:Transgenes
chemical_list: D015220:Calcium Channels; D011991:Receptors, Virus; D050916:TRPV Cation Channels; C446923:Trpv6 protein, mouse