Abstract

Polycythemia vera (PV) is an acquired myeloproliferative clonal stem cell disorder characterized by cytokine hypersensitivity. The erythroid colony forming assay has been useful in PV diagnosis. However, studies aiming to elucidate molecular mechanism in PV pathogenesis often require large numbers of cells from a specific erythroid stage. For this purpose, we had adapted the mouse expansion assay described by Karur et al (Blood, 2006 in E-Pub) and differential display method described by

Zhang et al (
Blood
,
2003
,
102
;
3938
)
to develop a human erythroid expansion protocol. In this study, we used peripheral blood mononuclear cells (PB-MNCs) from PV patients and healthy donors, and expanded them along erythroid lineage in 21 day culture. Through the culture, we took samples at 8 timepoints (days 1, 7, 9, 11, 14, 16, 19, 21) to evaluate differentiation patterns. We had generated 5 different regions using CD71 (transferrin receptor) and CD235a (glycophorin A) and characterized these regions by standard morphology analysis. This protocol allowed differentiation of PB-MNCs to all erythroid stages ending with late normoblast, reticulocyte, and mature erythrocytes (which do not survive well in this culture environment).

The erythroid differentiation and proliferation patterns were different between PV and normal. In the first week of culture, there was no significant proliferation difference observed between PV and normal. In contrast, the differentiation progressed more rapidly in PV, likely reflecting the differentiation of Epo independent PV population (since the first week culture contains no Epo). In the second week of culture (Epo present), there was a markedly increased expansion of PV erythroid cells. However, the differentiation pattern of PV resembled that of normal.

In conclusion, we demonstrated that our in vitro expansion method allows expansion of PB-MNCs along erythroid lineage with 60~80% stage homogeneity as to the stage of differentiation in both PV and normal. The differences of differentiation and proliferation pattern between PV and normal reflected the expected biological behavior of erythroid progenitors. Thus, the expansion protocol is useful to study molecular mechanism of PV pathogenesis, which requires large number of erythroid progenitors of various stages.

Disclosure: No relevant conflicts of interest to declare.

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Both authors contributed equally to this work.