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 erythropoiesis in more primitive stem cells, we assessed the influence of retrovirus-mediated gene transfer of human (h) EpoR complementary DNA (cDNA) into murine embryonic stem (ES) cells on erythroid differentiation of these cells. The hEpoR cDNA was efficiently transduced into ES cells, forming hEpoR that stably expressed ES (ES-hEpoR) cells. Expression of hEpoR cDNA was confirmed in ES-hEpoR cells by reverse transcriptase-polymerase chain reaction and Northern blot analysis. Colony assays demonstrated that definitive erythroid and primitive erythroid colonies were significantly increased from ES-hEpoR cells, when compared with mock virus-transduced ES (ES-Neo) cells, during the time course of differentiation induced by withdrawal of leukemia inhibitory factor, in either the presence or the absence of Epo. Multipotential colony-forming units (CFU-Mix) were also increased in ES-hEpoR cells at different stages of differentiation, but no changes were detected for CFU-granulocyte-macrophage colonies (CFU-GM). Time course studies by Northern blot analysis demonstrated elevated levels of expression of beta-H1 and beta-Major globin genes in embryoid bodies derived from ES-hEpoR cells stimulated with Epo, when compared with similar expression from ES-Neo cells. Expression 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 definitive erythropoiesis in differentiating embryoid bodies can be enhanced by retrovirus-mediated gene transfer of an hEpoR gene.
Department of Microbiology/Immunology, The Walther Oncology Center, Indiana University School of Medicine and the Walther Cancer Institute, Indianapolis 46202-5254, USA.
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Broxmeyer
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D000818:Animals; D015152:Blotting, Northern; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics*; D004622:Embryo, Mammalian / Q000166:cytology*; D004627:Embryonic Induction / Q000187:drug effects; D015672:Erythroid Precursor Cells / Q000166:cytology / Q000187:drug effects; D004920:Erythropoiesis / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D005914:Globins / Q000187:drug effects / Q000235:genetics; D006801:Humans; D051379:Mice; D017467:Receptors, Erythropoietin / Q000235:genetics* / Q000502:physiology; D012190:Retroviridae; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D014157:Transcription Factors / Q000187:drug effects / Q000235:genetics
D018076:DNA, Complementary; D017467:Receptors, Erythropoietin; D014157:Transcription Factors; D005914:Globins