Krythropoietin (Epo) is a principle regulator of erythropoiesis that acts by binding lo the Epo receptor (EpoR). Our previous studies showed thai introduction of human ih) EpoR cDNA into daughter progenitors derived from single cord blood CD34"" cells changed the Epo-stimulated differentiation profile towards the erythroid lineage (Lu L. ei al.. J. Leukot Biol. 63:389,1998). To evaluate the role for EpoR on erythroid commitment m more primitive stem cells, we now report the influence of retroviral mediated gene transfer ol hEpoR cDNA into undifferentiated murine embryonic stern (ES). D3, cells on erythroid differentiation of these cells. The hEpoR cDNA was efficiently transduced into ES cells forming a stable cell line (ES-hEpoR). Expression of hEpoR cDNA was confirmed in undifferentiated ES-hEpoR cells by RT-PCR and Northern analysis. In vitro differentiation of ES cells by withdrawal of LIF was assessed in two assays. ES cells were seeded in semisolid methyl cellulose culture in the presence or absence of murine GM-CSF. LL-3, SLF and human Epo. Embryoid bodies (EBs) with or without blood islands were counted ai day 7 ut differentiation. Second. ES cells were placed in suspension culture in the presence of fcpo. EBs were harvested at different times for RT-PCR analysis, and EB-derived cells were assayed in a secondary methylcellulose colony assay in the presence or absence of GMCSF, IL-3. SLF and Epo. The results showed that introduction of hEpoR cDNA had no effect on the tola] number of EBs at day 7 of differentiation. However, the blood island containing EBs from ES-hEpoR cells were significantly increased respectively either in the absence (51%) or presence (61%) of GM-CSF, IL-3, SLF and Epo, compared with parental ES (28% or 43%) or mock transduced ES-Neo cells (35% or 39%) at day 14 of differentiation. Colony assays showed that definitive erythroid (Ery-D) and multipotential (CPU-Mix) colonies were significantly increased from ES-hEpoR cells compared to parental ES or ES-Neo cells. In the absence of growth factors, primitive erythoid colonies (Ery-P) were generated at an earlier stage of differentiaiion from ES-hEpoR cells (day 6) than from parental ES and ES-Neo cells (day 14), and the number of colonies from EShEpoR cells was also increased. These increases were enhanced by addition of Epo. GMCSF, IL-3 and SLF. No changes were detected for CFU-GM colonies. Time course studies demonstrated elevated levels of expression of βm-, a-, Ç- and PBMJ-. but not e2-, globin genes, in ES-hEpoR cells stimulated with Epo compared wilh parental ES or ES-Neo cells. Expression of the GATA-1. but not NF-E2, EKLF, scl and c-myb, gene was enhanced 2 to 4 fold in ES-hEpoR cells compared with parental ES or ES-Neo cells after day 4 of differentiation. In summary, our data indicates that the commitment of the primitive murine embryonic stem cells to erythroid lineage can be enhanced by retroviral mediated gene transfer of a hEpoR gene.
|Original language||English (US)|
|Number of pages||1|
|State||Published - Dec 1 1998|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology
- Cancer Research