Cell-Surface Marking of CD34+-Restricted Phenotypes of Human Hematopoietic Progenitor Cells by Retrovirus-Mediated Gene Transfer

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Abstract
Human CD34+ cells lacking detectable levels of HLA-DR antigens (CD34+ DR¯) are highly enriched in hematopoietic pluripotent progenitors with long-term marrow repopulating ability. We investigated the feasibility of transducing and marking CD34+ DR¯ progenitor cells from bone marrow (BM) or mobilized peripheral blood samples (MPB) of 13 patients undergoing BM transplantation with the purpose of developing a protocol for a large-scale clinical application. A new retroviral vector coding for the truncated form (Δ) of the low-affinity nerve growth factor receptor (LNGFR) was used to quantitate the level of gene transfer into CD34+ cells and their progeny by multiparameter cytofluorimetry and immunocytochemistry. Light-density mononuclear cells as well as purified CD34+ cells were transduced either by direct incubation with retroviral supernatants or prestimulated in vitro with various combinations of growth factors prior to transduction. Transduction efficiency, assessed as G418-resistant growth of granulocyte-macrophage colony-forming units (CFU-GM) progenitors from MPB, was 1.7-fold higher (14.9% ± 4.5%) than those from BM (8.5% ± 3.9%) and it was further improved (26.9% ± 3.1%) using a purified CD34+ population as target cells. Three-color fluorescence-activated cell sorting (FACS) analysis demonstrated the presence of transduced ΔLNGFR+ cells within the CD34+ DR¯ subpopulation. In the absence of growth factors, gene transfer into BM or MPB CD34+ DR¯ cells was generally poor, but following a 72-hr prestimulation it peaked at 38% of total CD34+ DR¯ bone marrow (BM) cells in the presence of the c-kit ligand (KL) and at 31% in the presence of IL-3. Furthermore, KL gave, compared to the other cytokines, the highest absolute yield of BM ΔLNGFR+ CD34+ DR¯ cells recovered after transduction (p = 0.05 compared to 24 hr). Gene transfer into in vitro primitive progenitor cells was further confirmed by expression of the ΔLNGFR marker on CD34+ cells and CFU-GM derived from 5-week long-term culture on stroma. We have developed a new retroviral vector encoding a truncated form of the low-affinity nerve growth factor receptor (ΔLNGFR) suitable for a sensitive detection and isolation of transduced hematopoietic progenitor cells. The use of ΔLNGFR cell-surface marker allowed us to study at single-cell level the effect of different combinations of cytokines on gene transfer of restricted phenotypes of hematopoietic progenitor cells from bone marrow and mobilized peripheral blood samples.