Comparison of the Effects of Growth Factors on Retroviral Vector-Mediated Gene Transfer and the Proliferative Status of Human Hematopoietic Progenitor Cells

Abstract

We have examined the ability of the recombinant hematopoietic growth factors (HGF) interleukin-3 (IL-3), IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF) to increase retroviral vector-mediated gene transfer into human hematopoietic progenitor cells (HPC). The efficiency of neo gene transfer by the N2 vector into human HPC was enhanced by preculture with either GM-CSF or IL-3 (but not IL-6) and with each combination of the three factors. The combination of IL-3 plus IL-6 consistently produced significantly higher levels of G418-resistant colonies (50-60%) than any of the other combinations of HGF tested. Following preculture with HGF and transduction by N2, marrow was maintained in long-term bone marrow culture (LTBMC) for 2 months. The levels of G418-resistant HPC remained stable, and no apparent depletion of total HPC content resulted from the prior exposure to highly stimulatory doses of factors. The proliferative status of the HPC, following exposure to the HGF, was measured as the percentage of HPC that were inhibited from forming colonies by exposure to the S-phase-specific drug, hydroxyurea. The ability of the different HGF to increase the rate of gene transfer by N2 correlated significantly with the extent to which they stimulated HPC proliferation. These results suggest that the mechanism by which HGF increase rates of gene transfer into HPC is by stimulating cell proliferation. Techniques that produce high rates of gene transfer into long-lived human HPC will facilitate studies to quantitate expression of exogenous genes in hematopoietic cells and may be applicable to clinical gene therapy.

Autocrine transforming growth factor-β regulation of hematopoiesis: many outcomes that depend on the context

“…Similarly, Ho/ Rho low HSC stimulated to undergo cell division and then growth arrested with TGF-b showed markedly reduced engrafting potential (Wiesmann et al, 2000). In human and murine gene transfer experiments, when TGF-b was used to growth arrest the cells after retroviral infection, severely decreased repopulation was seen (Nolta and Kohn, 1990). TGF-b mediated arrested growth at the G 1 /S transition and G 0 quiescence are not the same.…”

True

“…207 Prestimulation of HSC with IL-3 and IL-6 together with ckit ligand (KL) resulted in beneficial effects on stem cell viability and transduction efficiency. [208][209][210][211] However, in vitro stimulation of HSC with these cytokines is associated with cellular differentiation and irreversible lineage commitment. 212,213 Therefore, optimization of the cytokine cocktail, especially with the addition of thrombopoietin (TPO) and Flt-3/Flk-2 ligand (FL) may favor HSC cycling with preservation of stem cell properties including self-renewal and in vivo engraftment capacity.…”

Expression of human Wiskott–Aldrich syndrome protein in patients’ cells leads to partial correction of a phenotypic abnormality of cell surface glycoproteins

“…Lowdensity mononuclear cells were obtained by density gradient centrifugation with Ficoll-Hypaque (Pharmacia, Piscataway, NJ, USA) as described previously. 25 EBVtransformed B-LCL were generated by inoculating 10 7 freshly obtained mononuclear cells with EBV-containing supernatant conditioned by the marmoset line B95-8 (ATCC) in the presence of 1 g/ml cyclosporin A (Pharmacia). These cell lines were propagated in RPMI-1640 medium (Irvine Scientific) with 10% (v/v) heat inactivated fetal bovine serum and supplemented with l-glutamine at 2 mmol/l, penicillin/streptomycin at 100 U/ml (R-10).…”