Retroviral transfer of the recombinant human erythropoietin receptor gene into single hematopoietic stem/progenitor cells from human cord blood increases the number of erythropoietin-dependent erythroid colonies

To test whether an enforced expression of a lineage-specific cytokine receptor would influence the proliferation/differentiation of hematopoietic stem/progenitor cells, retroviral vectors containing the human erythropoietin receptor (hEpoR) gene were used to transduce the hEpoR gene into phenotypically sorted subsets of cells. CD34⁺⁺⁺, CD34⁺⁺CD33^-, and CD34⁺⁺CD33⁺ populations of human cord blood, highly enriched for hematopoietic stem/progenitor cells, were sorted and plated as single cells per well in methylcellulose culture medium containing early acting growth factors in the presence or absence of Epo. The hEpoR gene was efficiently transduced into single high proliferative potential colony-forming cells (HPP-CFC) and multipotential (colony-forming unit granulocyte, erythroid, monocyte, megakaryocyte [CFU-GEMM]), erythroid (burst-forming unit-erythroid [BFU-E]), and granulocyte-macrophage (colony-forming unit-granulocyte-macrophage [CFU-GM]) progenitor cells. As expected in cultures grown in the absence of Epo, no BFU-E or CFU-GEMM colonies grew. In the presence of Epo, the hEpoR-gene transduced cells formed significantly more CFU-GEMM and BFU-E colonies than did the controls. A significant decrease in HPP-CFC colonies was also observed under these conditions. Little or no effect of hEpoR gene transduction was apparent in the numbers of CFU-GM colonies formed in the presence or absence of Epo. All of the above results were similar whether the cell populations assessed were CD34⁺⁺⁺ or their CD33^- or CD33⁺ subsets plated in the presence of growth factors at 200 cells/ mL or after limiting dilution at 2 cells/well. These results suggest that the profile of detectable stem/progenitors can be altered by retrovirus-mediated expression of the hEpoR gene.