BACKGROUND: Establishment of a transient gene delivery system, such as adenovirus (Ad) vectors, into embryonic stem (ES) cells and their aggregation form, embryoid bodies (EBs), is essential for its application in regenerative medicine because the transgene should not be integrated in the host genome. In this study, we optimized Ad vector-mediated transduction into EBs, and examined whether Ad vector-mediated transduction of adipogenesis-related gene into EBs could promote the adipocyte differentiation. METHODS: We prepared beta-galactosidase-expressing Ad vectors under the control of four different promoters (cytomegalovirus (CMV), rouse sarcoma virus, human elongation factor-1alpha, and CMV enhancer/beta-actin promoter (CA)) to estimate the transduction efficiency. Adipocyte differentiation efficiency by transduction of the PPAR gamma or C/EBP alpha gene into EBs was examined. RESULTS: Of the four promoters tested, the CA promoter exhibited the highest transduction efficiency in the EBs. However, Ad vector-mediated transduction was observed only in the periphery of the EBs. When repeated transduction by Ad vector was performed, gene expression was observed even in the interior of EBs as well. When EB-derived single cells were transduced by an Ad vector containing the CA promoter, more than 90% of the cells were transduced. Furthermore, Ad vector-mediated PPAR gamma gene transduction into EBs led to more efficient differentiation into adipocytes than could untransduced EBs, examined in terms of lipogenic enzyme activities and accumulation of the lipid droplets. CONCLUSIONS: Ad vector-mediated transduction into EBs could be a valuable tool for molecular switching of cell differentiation and could be applied to regenerative medicine.
BACKGROUND: Establishment of a transient gene delivery system, such as adenovirus (Ad) vectors, into embryonic stem (ES) cells and their aggregation form, embryoid bodies (EBs), is essential for its application in regenerative medicine because the transgene should not be integrated in the host genome. In this study, we optimized Ad vector-mediated transduction into EBs, and examined whether Ad vector-mediated transduction of adipogenesis-related gene into EBs could promote the adipocyte differentiation. METHODS: We prepared beta-galactosidase-expressing Ad vectors under the control of four different promoters (cytomegalovirus (CMV), rouse sarcoma virus, human elongation factor-1alpha, and CMV enhancer/beta-actin promoter (CA)) to estimate the transduction efficiency. Adipocyte differentiation efficiency by transduction of the PPAR gamma or C/EBP alpha gene into EBs was examined. RESULTS: Of the four promoters tested, the CA promoter exhibited the highest transduction efficiency in the EBs. However, Ad vector-mediated transduction was observed only in the periphery of the EBs. When repeated transduction by Ad vector was performed, gene expression was observed even in the interior of EBs as well. When EB-derived single cells were transduced by an Ad vector containing the CA promoter, more than 90% of the cells were transduced. Furthermore, Ad vector-mediated PPAR gamma gene transduction into EBs led to more efficient differentiation into adipocytes than could untransduced EBs, examined in terms of lipogenic enzyme activities and accumulation of the lipid droplets. CONCLUSIONS: Ad vector-mediated transduction into EBs could be a valuable tool for molecular switching of cell differentiation and could be applied to regenerative medicine.
Authors: Tim Ahfeldt; Robert T Schinzel; Youn-Kyoung Lee; David Hendrickson; Adam Kaplan; David H Lum; Raymond Camahort; Fang Xia; Jennifer Shay; Eugene P Rhee; Clary B Clish; Rahul C Deo; Tony Shen; Frank H Lau; Alicia Cowley; Greg Mowrer; Heba Al-Siddiqi; Matthias Nahrendorf; Kiran Musunuru; Robert E Gerszten; John L Rinn; Chad A Cowan Journal: Nat Cell Biol Date: 2012-01-15 Impact factor: 28.824