title: Improved lentiviral gene transfer into human embryonic stem cells grown in co-culture with murine feeder and stroma cells.
abstract: Genetic modification of human embryonic stem cells (hESCs) using biophysical DNA transfection methods are hampered by the very low single cell survival rate and cloning efficiency of hESCs. Lentiviral gene transfer strategies are widely used to genetically modify hESCs but limited transduction efficiencies in the presence of feeder or stroma cells present problems, particularly if vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped viral particles are applied. Here, we investigated whether the recently described semen derived enhancer of virus infection (SEVI) and alternative viral envelope proteins derived from either Gibbon ape leukaemia virus (GALV) or feline leukaemia virus (RD114) are applicable for transducing hESCs during co-culture with feeder or stroma cells. Our first set of experiments demonstrates that SEVI has no toxic effect on murine or hESCs and that exposure to SEVI does not interfere with the pluripotency-associated phenotype. Focusing on hESCs, we were able to further demonstrate that SEVI increases the transduction efficiencies of GALV and RD114 pseudotyped lentiviral vectors. More importantly, aiming at targeted differentiation of hESCs into functional somatic cell types, GALV pseudotyped lentiviral particles could efficiently and exclusively transduce hESCs grown in co-culture with OP9-GFP stroma cells (which were often used to induce differentiation into haematopoietic derivatives).
authors.affiliation: Institute for Transfusion Medicine, Hannover Medical School, D-30625 Hannover, Germany.
authors.forename: Melanie
authors.initials: M
authors.lastname: Wurm
authors.forename: Benjamin
authors.initials: B
authors.lastname: Gross
authors.forename: Malte
authors.initials: M
authors.lastname: Sgodda
authors.forename: Ludger
authors.initials: L
authors.lastname: Ständker
authors.forename: Thomas
authors.initials: T
authors.lastname: Müller
authors.forename: Wolf-Georg
authors.initials: WG
authors.lastname: Forssmann
authors.forename: Peter A
authors.initials: PA
authors.lastname: Horn
authors.forename: Rainer
authors.initials: R
authors.lastname: Blasczyk
authors.forename: Tobias
authors.initials: T
authors.lastname: Cantz
mesh_terms: D000818:Animals; D002460:Cell Line; D018920:Coculture Techniques / Q000379:methods; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D017989:Leukemia Virus, Gibbon Ape / Q000378:metabolism*; D051379:Mice; D017154:Stromal Cells / Q000166:cytology / Q000378:metabolism*; D014161:Transduction, Genetic / Q000379:methods*; D014759:Viral Envelope Proteins / Q000378:metabolism*
chemical_list: D014759:Viral Envelope Proteins