EFFICIENT TRANSFER OF SELECTABLE AND MEMBRANE REPORTER GENES IN HEMATOPOIETIC PROGENITOR AND STEM-CELLS PURIFIED FROM HUMAN PERIPHERAL-BLOOD

We have utilized highly purified hematopoietic progenitor and stem cel ls (HPCs, HSCs) from normal peripheral blood to develop methodology fo r: (a) efficient transfer into HPCs of a non-hematopoietic membrane re porter, i.e., the nerve growth factor receptor complementary DNA; and (b) effective gene transduction of putative HSCs, i.e., cells initiati ng 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 neomyci n resistance (neo') gene. More than 80% of transduced HPCs were resist ant to the toxic G418 level. Thereafter, the HPCs were effectively tra nsduced with the LNSN retroviral vector containing a nerve growth fact or receptor complementary DNA; the nerve growth factor receptor was de tected on greater than or equal to 18% of the transduced HPCs. These e xperiments provide a new tool from which (a) to monitor expression of a transduced membrane reporter 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 HPC s/HSCs grown in Dexter type LTC were transduced at 1 week by exposure to supernatant N2 retroviral particles in the absence of exogenous hem atopoietic growth factors. The procedure, devoid of toxic effects, all owed an efficient neo' transduction into LTC-ICs. Thus, we consistentl y detected neomycin-resistant mRNA in the clonal progeny of HPCs produ ced in LTC at 5-8 weeks in both the nonadherent and adherent fractions ; this timing of expression coincides with that of HPC production by L TC-ICs, thereby indicating the effective transduction of the LTC-ICs. These experiments represent a first step toward development of preclin ical models for gene transfer into human peripheral blood HSCs by comp lex retroviral vectors.