Introduction of human erythropoietin receptor complementary DNA by retrovirus-mediated gene transfer into murine embryonic stem cells enhances erythropoiesis in developing embryoid bodies

To evaluate the role of the erythropoietin (Epo) receptor (R) in erythropoi esis in more primitive stem cells, we assessed the influence of retrovirus- mediated gene transfer of human (h) EpoR complementary DNA (cDNA) into muri ne embryonic stem (ES) cells on erythroid differentiation of these cells. T he hEpoR cDNA was efficiently transduced into ES cells, forming hEpoR that stably expressed ES (ES-hEpoR) cells. Expression of hEpoR cDNA was confirme d in ES-hEpoR cells by reverse transcriptase-polymerase chain reaction and Northern blot analysis. Colony assays demonstrated that definitive erythroi d and primitive erythroid colonies were significantly increased from ES-hEp oR cells, when compared with mock virus-transduced ES (ES-Neo) cells, durin g the time course of differentiation induced by withdrawal of leukemia inhi bitory factor, in either the presence or the absence of Epo. Multipotential colony-forming units (CFU-Mix) were also increased in ES-hEpoR cells at di fferent stages of differentiation, but no changes were detected for CFU-gra nulocyte-macrophage colonies (CFU-GM). Time course studies by Northern blot analysis demonstrated elevated levels of expression of beta -H-1 and beta -Major globin genes in embryoid bodies derived from ES-hEpoR cells stimulat ed with Epo, when compared with similar expression from ES-Neo cells. Expre ssion of the GATA-1 gene was enhanced in ES-hEpoR cells, when compared with ES-Neo cells, beginning immediately after initiation of the cultures until 8 days of differentiation. These data indicate that primitive and definiti ve erythropoiesis in differentiating embryoid bodies can be enhanced by ret rovirus-mediated gene transfer of an hEpoR gene.