Effect of recombinant erythropoietin in interaction with stromal factors on cord blood hematopoiesis

To investigate the effect of recombinant erythropoietin (Epo) on primitive human hematopoietic progenitor cells, we cultured cord blood mononuclear cells (CBMNC) and CB CD34+ cells in a Dexter-type long-term culture system (LTC), to which various concentrations of Epo were added at day 0 or 7, with or without direct contact with irradiated allogeneic human marrow stromal layers. In regular stroma-contact cultures, when CBMNC were inoculated, the addition of Epo at 1 to 10 U/mL induced a significant increase in LTC-initiating cells (LTC-IC), particularly in the myeloid component, compared with the control without Epo. Significantly more LTC-IC were generated by the delayed addition of Epo on day 7 than on day 0. On the other hand, when CD34+ cells were inoculated, physiologic concentrations of Epo (0.1 U/mL) induced a more than twofold increase in LTC-IC, which was attributed equally to both the myeloid and erythroid lineages, only when added on day 0. In stroma-noncontact cultures, which were created using a Transwell 0.4-micron microporous membrane filter, dose-dependent suppression of the myeloid component of LTC-IC was observed with a higher concentration of Epo (1 to 100 U/mL) when CBMNC was inoculated. On the other hand, without Epo, fourfold more LTC-IC was generated from CD34+ cells in stroma-noncontact than in stroma-contact cultures, which was then significantly augmented by the addition of Epo (0.1 or 10 U/mL) on day 0. This increase was due to both the myeloid and erythroid lineages. A higher concentration of Epo (100 U/mL) resulted in a decrease in LTC-IC, mainly in myeloid progeny, in all of the culture conditions. Hence, Epo may play an important physiologic role in the maintenance and proliferation of immature stem/progenitor cells, in close interaction with factors from marrow stromal cells.