title: Enhanced retroviral transduction of 5-fluorouracil-resistant human bone marrow (stem) cells using a genetically modified packaging cell line.
abstract: Pluripotent hematopoietic stem cells (PHSC) are rare cells capable of multilineage differentiation, long-term reconstituting activity and extensive self-renewal. Such cells are the logical targets for many forms of corrective gene therapy, but are poor targets for retroviral mediated gene transfer owing to their quiescence, as retroviral transduction requires that the target cells be cycling. To try and surmount this problem we have constructed a retroviral producer line that expresses the membrane-bound form of human stem cell factor (SCF) on its cell surface. These cells are capable, therefore, of delivering a growth signal concomitant with recombinant retroviral vector particles. In this report we describe the use of this cell line to transduce a highly quiescent population of cells isolated from adult human bone marrow using the 5-fluorouracil (FU) resistance technique of Berardi et al. Quiescent cells selected using this technique were transduced by cocultivation with retroviral producers expressing surface bound SCF or with the parent cell line that does not. Following coculture, the cells were plated in long-term bone marrow culture for a further 5 weeks, before plating the nonadherent cells in semisolid media. Colonies forming in the semisolid media over the next 14 days were analyzed by polymerase chain reaction for the presence of the retroviral vector genome. Over six experiments, the transduction frequency of the quiescent 5-FU resistant cells using the SCF-expressing producer line averaged about 20%, whereas those transduced using the parent producer line showed evidence of reduced levels or no transduction.
authors.affiliation: Department of Haematology, Imperial College School of Medicine, St Mary's Campus, Norfolk Place, London, UK.
authors.forename: J
authors.initials: J
authors.lastname: Povey
authors.forename: N
authors.initials: N
authors.lastname: Weeratunge
authors.forename: C
authors.initials: C
authors.lastname: Marden
authors.forename: A
authors.initials: A
authors.lastname: Sehgal
authors.forename: A
authors.initials: A
authors.lastname: Thrasher
authors.forename: C
authors.initials: C
authors.lastname: Casimir
mesh_terms: D000328:Adult; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D001854:Bone Marrow Cells / Q000166:cytology / Q000502:physiology*; D019008:Drug Resistance, Neoplasm; D005472:Fluorouracil / Q000494:pharmacology*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D012190:Retroviridae
chemical_list: D000964:Antimetabolites, Antineoplastic; D005472:Fluorouracil