Erythroid progenitor cells expanded from peripheral blood without mobilization or preselection: molecular characteristics and functional competence.

Abstract

BACKGROUND: Continued development of in-vitro procedures for expansion and differentiation of erythroid progenitor cells (EPC) is essential not only in hematology and stem cell research but also virology, in light of the strict erythrotropism of the clinically important human parvovirus B19. METHODOLOGY/PRINCIPAL FINDINGS: We cultured EPC directly from ordinary blood samples, without ex vivo stem cell mobilization or CD34+ cell in vitro preselection. Profound increase in the absolute cell number and clustering activity were observed during culture. The cells obtained expressed the EPC marker combination CD36, CD71 and glycophorin, but none of the lymphocyte, monocyte or NK markers. The functionality of the generated EPC was examined by an in vitro infection assay with human parvovirus B19, tropic for BFU-E and CFU-E cells. Following infection (i) viral DNA replication and mRNA production were confirmed by quantitative PCR, and (ii) structural and nonstructural proteins were expressed in >50% of the cells. As the overall cell number increased 100-200 fold, and the proportion of competent EPC (CD34+ to CD36+) rose from 50%, the in vitro culture procedure generated the EPC at an efficiency of >10,000-fold. Comparative culturing of unselected PBMC and ex vivo-preselected CD34+ cells produced qualitatively and quantitatively similar yields of EPC. CONCLUSIONS/SIGNIFICANCE: This approach yielding EPC directly from unmanipulated peripheral blood is gratifyingly robust and will facilitate the study of myeloid infectious agents such as the B19 virus, as well as the examination of erythropoiesis and its cellular and molecular mechanisms.