Epo-Independent Proliferation and Differentiation of Genetically Modified Erythroid Progenitor Cells Using a Chemical Inducer of Dimerization.

Recombinant human erythropoietin (Epo) revolutionized the care of anemia in cancer. In light of emerging (albeit controversial) roles for Epo in supporting tumor growth, it may be useful to develop Epo-independent methods for regulating red blood cell production. Previous studies in this laboratory established that marrow transduced with conditional derivatives of fibroblast growth factor receptor-1 (F36VFGFR1) and the thrombopoietin receptor (F36VMpl) can support the chemical inducer of dimerization (CID)-dependent production of erythroid cells in transplanted mice. In the current study, we used human CD34⁺ cord blood (CB) cells to test whether CID-regulated red cell production might require collaboration between CID-initiated signals and those provided by Epo. CD34⁺ CB cells cultured in the absence of Epo did not proliferate and retained expression of the myeloid marker CD33. Epo (5U/mL) promoted proliferative expansion (66.2-fold in 12 days) and CD33 expression was lost as the cells differentiated to mature, glycophorin A⁺ erythroid cells (92.9 +/− 1.8%, n=3). Addition of a soluble human Epo receptor extracellular domain (shEpoR) at a concentration of 3ug/mL completely blocked Epo-dependent proliferation and similar to cells cultured without Epo, they retained expression of CD33. Addition of CID (100nM AP20187) to F36VMpl-transduced CD34⁺ CB cells in the absence of Epo promoted proliferative expansion (89.8-fold in 12 days) and differentiation as glycophorin A⁺ erythroid cells (77.2 +/− 4.8%, n=3). These CID responses were not significantly affected by the addition of shEpoR, as CID induced both mitogenic expansion (84.5 fold in 12 days) and differentiation as glycophorin A⁺ erythroid cells (74.2 +/− 6.7%, n=3) in the presence of concentrations of shEpoR that blocked Epo responses. These data suggest that F36VMpl does not require Epo signaling to support the proliferation and differentiation of human erythroid progenitor cells, and further define an Epo-independent method of erythroid cell production.