title: Kinetics and effect of integrin expression on human CD34(+) cells during murine leukemia virus-derived retroviral transduction with recombinant fibronectin for stem cell gene therapy.
abstract: The CH-296 recombinant fragment of human fibronectin is essential for murine leukemia virus (MLV)-derived retroviral transduction of CD34(+) cells for the purpose of stem cell gene therapy. Although the major effect of CH-296 is colocalization of the MLV-derived retrovirus and target cells at specific adhesion domains of CH-296 mediated by integrins expressed on CD34(+) cells, the precise roles of the integrins are unclear. We examined the kinetics of integrin expression on CD34(+) cells during the course of MLV-derived retrovirus-mediated gene transduction with CH-296. Flow cytometry revealed that the levels of both very late activation protein (VLA)-4 and VLA-5 on CD34(+) cells freshly isolated from cord blood were insufficient for effective MLV-derived retroviral transduction. However, increases were achieved during culture for preinduction and MLV-derived retrovirus-mediated gene transduction in the presence of a cocktail of cytokines. In addition, we confirmed by using specific antibodies that inhibition of the cell adhesion mediated by the integrins significantly reduced transduction efficiency, indicating that integrin expression is indeed important for CH-296-based MLV-derived retroviral transduction. Only a few cytokines are capable of inducing integrin expression, and stem cell factor plus thrombopoietin was found to be the minimal combination that was sufficient for effective transduction of an MLV-derived retrovirus based on CH-296. Our findings should be useful for improving the culture conditions for CH-296-based MLV-derived retroviral transduction in stem cell gene therapy.
authors.affiliation: Department of Developmental Biology, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan.
authors.forename: Yasuomi
authors.initials: Y
authors.lastname: Horiuchi
authors.forename: Masafumi
authors.initials: M
authors.lastname: Onodera
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Miyagawa
authors.forename: Ban
authors.initials: B
authors.lastname: Sato
authors.forename: Keiko
authors.initials: K
authors.lastname: Onda
authors.forename: Yohko U
authors.initials: YU
authors.lastname: Katagiri
authors.forename: Hajime
authors.initials: H
authors.lastname: Okita
authors.forename: Mayumi
authors.initials: M
authors.lastname: Okada
authors.forename: Makoto
authors.initials: M
authors.lastname: Otsu
authors.forename: Akihiro
authors.initials: A
authors.lastname: Kume
authors.forename: Torayuki
authors.initials: T
authors.lastname: Okuyama
authors.forename: Junichiro
authors.initials: J
authors.lastname: Fujimoto
authors.forename: Tadatoshi
authors.initials: T
authors.lastname: Kuratsuji
authors.forename: Nobutaka
authors.initials: N
authors.lastname: Kiyokawa
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism*; D002448:Cell Adhesion; D003114:Colony-Forming Units Assay; D016207:Cytokines / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism*; D015316:Genetic Therapy; D006801:Humans; D016023:Integrins / Q000378:metabolism*; D007700:Kinetics; D009052:Leukemia Virus, Murine / Q000235:genetics*; D051379:Mice; D011994:Recombinant Proteins / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*; D014161:Transduction, Genetic
chemical_list: D018952:Antigens, CD34; D016207:Cytokines; D005353:Fibronectins; D016023:Integrins; D011994:Recombinant Proteins