Umbilical cord blood (CB) from the early gestational human fetus is recognized as a rich source of hematopoietic stem cells. To examine the value of fetal CB for gene therapy of inborn immunohematopoietic disorders, we tested the feasibility of genetic modification of CD34(+) cells from CB at weeks 24 to 34 of pregnancy, using lentiviral vector-mediated transfer of the green fluorescent protein (GFP) gene. The transduction rate of CD34(+) cells was 42 +/- 9%, resulting in GFP expression in 23 +/- 4% of colonies derived from colony-forming units (CFUs) and 11 +/- 1% from primitive long-term culture-initiating cells (LTC-ICs). Cell cycle analysis demonstrated transduction and GFP expression in cells in the G(0) phase, which contains immature hematopoietic progenitors. Transduced fetal CD34(+) cells could be expanded 1000-fold in long-term cultures supplemented with megakaryocyte growth and development factor along with Flt-3 ligand. At week 10, expression of GFP was observed in 40.5 +/- 11.7% of CFU-derived colonies. While prestimulation of CD34(+) cells with cytokines prior to transduction increased the efficiency of GFP transfer 2- to 3-fold, long-term maintenance of GFP-expressing CFUs occurred only in the absence of prestimulation. The GFP gene was found integrated into the genomic DNA of 35% of LTC-IC-derived colonies initiated at week 10, but GFP expression was not detectable, suggesting downregulation of transgene activity during the extended culture period. These results indicate that human fetal CB progenitors are amenable to genetic modification by lentiviral vectors and may serve as a target for gene therapy of hematopoietic disorders by prenatal autologous transplantation.