title: Efficient transfer of selectable and membrane reporter genes in hematopoietic progenitor and stem cells purified from human peripheral blood.
abstract: We have utilized highly purified hematopoietic progenitor and stem cells (HPCs, HSCs) from normal peripheral blood to develop methodology for: (a) efficient transfer into HPCs of a non-hematopoietic membrane reporter, i.e., the nerve growth factor receptor complementary DNA; and (b) effective gene transduction of putative HSCs, i.e., cells initiating Dexter-type long-term culture (LTC-ICs). Purified HPCs induced into cycling by growth factors (interleukin 3, interleukin 6, c-kit ligand) were transduced with the N2 retroviral vector containing the neomycin resistance (neor) gene. More than 80% of transduced HPCs were resistant to the toxic G418 level. Thereafter, the HPCs were effectively transduced with the LNSN retroviral vector containing a nerve growth factor receptor complementary DNA; the nerve growth factor receptor was detected on > or = 18% of the transduced HPCs. These experiments provide a new tool from which (a) to monitor expression of a transduced membrane report on hematopoietic cells, particularly at the level of HPCs/HSCs, and (b) to characterize the transduced cells by double- and triple-labeling membrane antigen analysis. Purified HPCs/HSCs grown in Dexter-type LTC were transduced at 1 week by exposure to supernatant N2 retroviral particles in the absence of exogenous hematopoietic growth factors. The procedure, devoid of toxic effects, allowed an efficient neor transduction into LTC-ICs. Thus, we consistently detected neomycin-resistant mRNA in the clonal progeny of HPCs produced in LTC at 5-8 weeks in both the nonadherent and adherent fractions; this timing of expression coincides with that of HPC production by LTC-ICs, thereby indicating the effective transduction of the LTC-ICs. These experiments represent a first step toward development of preclinical models for gene transfer into human peripheral blood HSCs by complex retroviral vectors.
authors.affiliation: Thomas Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
authors.forename: M
authors.initials: M
authors.lastname: Valtieri
authors.forename: R
authors.initials: R
authors.lastname: Schirò
authors.forename: C
authors.initials: C
authors.lastname: Chelucci
authors.forename: B
authors.initials: B
authors.lastname: Masella
authors.forename: U
authors.initials: U
authors.lastname: Testa
authors.forename: I
authors.initials: I
authors.lastname: Casella
authors.forename: E
authors.initials: E
authors.lastname: Montesoro
authors.forename: G
authors.initials: G
authors.lastname: Mariani
authors.forename: H J
authors.initials: HJ
authors.lastname: Hassan
authors.forename: C
authors.initials: C
authors.lastname: Peschle
mesh_terms: D000328:Adult; D004351:Drug Resistance; D017930:Genes, Reporter / Q000235:genetics*; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells; D006801:Humans; D008297:Male; D009355:Neomycin; D017475:Receptors, Nerve Growth Factor / Q000235:genetics; D014162:Transfection / Q000379:methods*
chemical_list: D017475:Receptors, Nerve Growth Factor; D009355:Neomycin