Erythroid differentiation is a dynamic process leading to the production of mature red blood cells. Even small variations in this process may result in severe disease phenotype. To study this process, we used a three-phase erythroid expansion system to expand homogeneous erythroid progenitors (EPs) from peripheral blood mononuclear cells (PB-MNCs) (Bruchova H. et al, 2007, Exp. Hematology, in press). We then characterized the expanded EPs from polycythemia vera (PV) patients and healthy donors at various points of maturation comparing cell proliferation and differentiation stage. EPs from PV patients outgrew controls up to day 14 (∼12 fold for PV and ∼4 fold for control compared to day 1). Differentiation was analyzed using both FACS analysis (with CD71/CD235a staining) and morphological evaluation (Wright-Giemsa staining), and demonstrated a more rapid differentiation of PV EPs when compared to controls up to day 14. We then evaluated apoptosis/cell cycle analysis by propidium iodide staining. Although PV EPs contained larger S phase population (45%) than controls (34%) at day 11, the apoptosis proportion of PV EPs was increased ∼2 fold to control from day 14. To understand the molecular mechanism of these differences between PV and controls, we analyzed the gene expression of several known regulators in erythropoiesis - BCL2, EPOR, cMYB, p27. Two transcripts (EPOR and cMYB) showed unique profiles on PV EPs. The EPOR transcript increased earlier in PV; i.e. from day 7 until day 21 and reached a plateau at day 11, compared to day 9 until day 19 and plateau at day 14 in controls. In addition, PV EPs contained higher levels of EPOR transcripts than control on most of timepoints. Interestingly, cMYB, which is known to augment early progenitor proliferation, was highly expressed from day 7 in PV, through day 11. Control EPs also expressed cMYB from day 9 through day 11; however, cMYB levels from any stages of control EPs were markedly lower than PV EPs at day 7. In this study, we demonstrate that PV erythropoiesis has unique features of hyperproliferation and an accelerated differentiation. These features are associated with earlier and higher expressions of cMYB and EPOR at the early stage of erythropoiesis.
Disclosure: No relevant conflicts of interest to declare
Both authors are equally contributed to this work.