Efficient detection and selection of immature rhesus monkey and human CD34(+) hematopoietic cells expressing the enhanced green fluorescent protein (EGFP)

The feasibility of using the enhanced green fluorescent protein (EGFP) as a selectable reporter molecule of retroviral-mediated gene transfer in immat ure rhesus monkey and human CD34(+) hematopoietic cells was examined. Retro viral transduction with the MFG-EGFP retroviral vector resulted in readily detectable EGFP expression in 27% of human and 11-35% of rhesus monkey bone marrow cells, and in 17-38% of rhesus monkey peripheral blood cells mobili zed with FLT3 ligand (FL) and granulocyte colony-stimulating factor (G-CSF) . In addition, we used the human CD34(+) KG1A cell line as a model to study viability and growth of successfully transduced cells. Cultures of mock- a nd EGFP-transduced KG1A cells generated equal viable cell numbers for at le ast 1 month, indicating the absence of a cytotoxic effect of EGFP expressio n in these cells. FAGS selection on the basis of EGFP and CD34 expression r esulted in enriched subsets (greater than or equal to 87%) of CD34(+) EGFP- negative and CD34(+) EGFP-positive KG1A, rhesus monkey and human bone marro w cells, demonstrating the potential of obtaining almost pure populations o f transduced immature hematopoietic cells. EGFP expression was also readily demonstrated in erythroid and granulocyte/macrophage colonies derived from the CD34(+) EGFP-positive rhesus monkey and human bone marrow cells by eit her inverted fluorescence microscopy or flow cytometry. Using four-color fl ow cytometry, EGFP expression could also be demonstrated in viable and phen otypically defined immature subpopulations of the CD34(+) cells, ie those e xpressing little or no HLA-DR (rhesus monkey) or CD38 (human) antigens at t he cell surface. These results demonstrate that EGFP is a very useful marke r to monitor gene transfer efficiency in phenotypically defined immature rh esus monkey and human hematopoietic cell types and to select for these cell s by multicolor flow cytometry prior to transplantation.