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Efficient Transfer of Selectable and Membrane Reporter Genes in Hematopoietic Progenitor and Stem Cells Purified from Human Peripheral Blood¹

Thomas Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107; Department of Hematology and Oncology, Istituto Superiore di Sanità, Rome, Italy; Pediatric Clinic, University of Milan, S. Gerardo Hospital, Monza, Italy

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 (neo^r) 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 18% of the transduced HPCs. These experiments 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 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 neo^r 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.

¹ This study was supported in part by Thomas Jefferson University, Philadelphia, PA; Fondazione Tettamanti, CNR (P.F. ACRO), Rome; Progetto Terapia dei Tumori, Istituto Superiore di Sanità, Rome.

² To whom requests for reprints should be addressed, at Thomas Jefferson Cancer Institute, Thomas Jefferson University, Bluemle Life Sciences Building, Room 528, 233 S. 10th St., Philadelphia, PA 19107.

Received 3/11/94. Accepted 6/13/94.

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