Enhancement of proliferation and differentiation of erythroid progenitors by co-transduction of erythropoietin receptor and H-ras cDNAs into single CD34(3+) cord blood cells

Our previous studies have demonstrated that retrovirus-mediated gene transd uction of either the human erythropoietin receptor (EpoR) or H-ras cDNA int o single purified hematopoietic progenitor (HPC), CD34(3+), cells from cord blood (CB) resulted in increased numbers and sizes of erythroid cell conta ining colonies. We therefore evaluated if there were further effects when H -ras and EpoR genes were co-transduced into the same progenitor cells. High ly purified single sorted CD343+ CB cells were transduced with retroviral v ectors encoding EpoR or H-ras cDNA, At the single cell level, and in respon se to stimulation by a combination of growth factors, including Epo, the nu mber of colonies formed by BFU-E and CFU-GEMM was significantly increased i n cells transduced with either single H-ras or EpoR cDNA compared to mock v irus-transduced cells as previously described. Increased numbers of BFU-E, but not CFU-GEMM, colonies were produced from cells simultaneously co-trans duced with both EpoR and H-ras genes. Little or no growth was seen in trans duced cells without exogenously added cytokines, The size of all types of c olonies including CFU-GM was increased in cells transduced with H-ras and/o r EpoR cDNAs, and the greatest increase was noticed in cells co-transduced with both genes. Integration and expression of either gene in individual co lonies as assessed by PCR and RT-PCR analysis were 45-62% and 48-58%, respe ctively, with approximately 31% of the cells containing and expressing both genes. These results add to information suggesting an enhancing interactin g role of H-ras and EpoR in erythroid proliferation/differentiation.