Increased Stable Retroviral Gene Transfer in Early Hematopoietic Progenitors Released from Quiescence

Abstract

It has been previously demonstrated that prestimulation with cytokines could improve gene transfer in hematopoietic progenitors. However, we have shown that no combination of cytokines so far tested is able to release rapidly in vitro the stem cell compartment from quiescence unless an autocrine transforming growth factor-β1 (TGF-β1) is blocked by specific oligonucleotide antisense or antiserum (Hatzfeld et al., 1991, J. Exp. Med., 174, 925). We now report that a 10-hr cytokine prestimulation of SBA¯CD34^high human umbilical cord blood progenitors increases retrovirally mediated transfer of the nls-lacZ reporter gene from 1% to 23.8% and addition of anti-TGF-β serum doubles this increase (47.3%). Interestingly, the effect of anti-TGF-β preincubation on gene transfer is most effective on the most immature progenitors, which develop into high proliferative potential mixed colonies with 1–2 × 10⁵ cells. Anti-TGF-β serum pretreatment increases gene transfer in these early colony-forming units granulocyte-erythroid-megakaryocyte-macrophage (CFU-GEMM) from 54.1% to 93.3%. It augments significantly the stability of gene expression in all subpopulations of mixed colonies. Colonies obtained after pretreatment with anti-TGF-β serum are larger and the expression of the stably integrated recombinant provirus does not reduce their size. This prestimulation method provides a substantial improvement for gene transfer efficiency within the quiescent stem cell compartment that is responsible for long-term engraftment. We have shown that no combination of cytokines so far tested is able to release rapidly in vitro the stem cell compartment from quiescence unless autocrine transforming growth factor-β1 (TGF-β1) is blocked by specific oligonucleotide antisense or antiserum. We now report that a 10-hr cytokine prestimulation of SBA¯CD34^high human umbilical cord blood progenitors increases retrovirally mediated transfer of the nls-lacZ reporter gene from 23.8% without anti-TGF-β serum to 47.3% in the presence of anti-TGF-β serum. This effect is greater on the most immature progenitors, the early colony-forming units granulocyte-erythroid-megakaryocyte-macrophage (CFU-GEMM) (93.3%). These results suggest a simple method to increase stable gene transfer in the quiescent stem cell compartment responsible for long-term engraftment.