Differentially expressed genes during in vitro differentiation of murine embryonic stem cells transduced with a human erythropoietin receptor cDNA

Our previous study demonstrated that transduction of murine embryonic stem (ES) cells with a human erythropoietin (Epo) receptor (R) cDNA resulted in enhanced erythropoiesis in developing embryonic bodies (EBs). To address po ssible mechanisms of gene regulation, we compared gene expression between h EpoR cDNA-transduced ES (ES-hEpoR) cells and parental ES cells during in vi tro differentiation induced by withdrawal of leukemia inhibitory factor (LI F) and cultured in the absence of Epo using differential display reverse tr anscriptase-polymerase chain reaction (DDRT-PCR). A total of 48 differentia lly expressed cDNA fragments were found; 12 were sequenced and five were co nfirmed by Northern blot analysis to be up- or down-regulated in ES-hEpoR c ells during differentiation compared to parental ES cells. In a GenBank sea rch of the five putatively regulated cDNA fragments, two fragments shared h igh sequence homology to two known genes: the Surf-6 gene and the gene for calcyclin binding protein. Northern blot analysis demonstrated that 2.5-kb and 0.3-kb transcripts of the Surf-6 gene were expressed in undifferentiate d ES-hEpoR and parental ES cells at a low level, but this expression was en hanced from day 2 to 14 of differentiation after withdrawal of LIF and cult ure in the presence of Epo. Furthermore, the enhanced expression of these t wo transcripts was also noticed in EML-C1 cells, a murine multipotential he matopoietic cell line that has erythroid differentiation potential in respo nse to Epo. In summary, our results demonstrate that Surf-6 gene expression is regulated during differentiation of hematopoietic stem/progenitor cells in response to Epo, suggesting a possible role for Surf-6 gene in erythrop oiesis.