title: Epidermal growth factor improves lentivirus vector gene transfer into primary mouse hepatocytes.
abstract: Partial resistance of primary mouse hepatocytes to lentiviral (LV) vector transduction poses a challenge for ex vivo gene therapy protocols in models of monogenetic liver disease. We thus sought to optimize ex vivo LV gene transfer while preserving the hepatocyte integrity for subsequent transplantation into recipient animals. We found that culture media supplemented with epidermal growth factor (EGF) and, to a lesser extent, hepatocyte growth factor (HGF) markedly improved transduction efficacy at various multiplicities of infection. Up to 87% of primary hepatocytes were transduced in the presence of 10 ng EGF, compared with ~30% in standard culture medium (SCMs). The increased number of transgene-expressing cells correlated with increased nuclear import and more integrated pro-viral copies per cell. Higher LV transduction efficacy was not associated with proliferation, as transduction capacity of gammaretroviral vectors remained low (<1%). Finally, we developed an LV transduction protocol for short-term (maximum 24 h) adherent hepatocyte cultures. LV-transduced hepatocytes showed liver repopulation capacities similar to freshly isolated hepatocytes in alb-uPA mouse recipients. Our findings highlight the importance of EGF for efficient LV transduction of primary hepatocytes in culture and should facilitate studies of LV gene transfer in mouse models of monogenetic liver disease.
authors.affiliation: Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Rothe
authors.forename: I
authors.initials: I
authors.lastname: Rittelmeyer
authors.forename: M
authors.initials: M
authors.lastname: Iken
authors.forename: U
authors.initials: U
authors.lastname: Rüdrich
authors.forename: A
authors.initials: A
authors.lastname: Schambach
authors.forename: S
authors.initials: S
authors.lastname: Glage
authors.forename: M P
authors.initials: MP
authors.lastname: Manns
authors.forename: C
authors.initials: C
authors.lastname: Baum
authors.forename: M
authors.initials: M
authors.lastname: Bock
authors.forename: M
authors.initials: M
authors.lastname: Ott
authors.forename: U
authors.initials: U
authors.lastname: Modlich
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D003470:Culture Media; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D017228:Hepatocyte Growth Factor / Q000494:pharmacology; D022781:Hepatocytes / Q000378:metabolism* / Q000637:transplantation; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D014161:Transduction, Genetic
chemical_list: D003470:Culture Media; D004815:Epidermal Growth Factor; D017228:Hepatocyte Growth Factor