Purification, amplification and characterization of a population of human erythroid progenitors

Abstract

In humans, studies of the erythroid cell lineage are hampered by difficulties in obtaining sufficient numbers of erythroid progenitors. In fact, these progenitors in bone marrow or peripheral blood are scarce and no specific antibodies are available. We describe a new method which allows proliferation in liquid culture of large numbers of pure normal human erythroid progenitors. CD34+ cells were cultured for 7 d in serum-free conditions with the cytokine mixture interleukin (IL)-3/IL-6/stem cell factor (SCF). This resulted in cell expansion and the appearance of a high proportion of CD36+ cells which were purified on day 7. Methylcellulose clones from these cells were composed of 96.6% late BFU-E and 3.4% CFU-GM. These CD36+ cells could be recultured with the same cytokine mixture plus or minus erythropoietin (Epo) for a further 2-7 d. In both conditions further amplification of CD36+ cells was observed, but Epo induced a more dramatic cell expansion. Glycophorin-positive mature cells appeared only in the presence of Epo, and terminal red cell differentiation was observed after 7 d of secondary culture. Cells obtained from adult CD34+ progenitors mostly contained adult haemoglobin, whereas cord blood-derived cells contained equal proportions of adult and fetal haemoglobin. Activation of STAT5 and tyrosine phosphorylation of the Epo receptor and JAK2 were observed after Epo stimulation of these cells. This new method represents a straightforward alternative to the procedures previously described for the purification of normal erythroid progenitors and is useful in the study of erythropoietic regulation.

Identification of CD13+CD36+ cells as a common progenitor for erythroid and myeloid lineages in human bone marrow

“…CD36 has been used as an early erythroid marker [21,22], although it is also expressed on late megakaryocytic and monocytic cells [23]. Indeed, a high proportion of CD36 + progenitors only generated erythroid burstforming unit (BFU-E) and erythroid colony-forming units (CFU-E) [23,24]. In a recent paper, Singh et al [25] also showed that purified subpopulations of CD34 + CD36 + and CD34 − CD36 + cells from baboon bone marrow generated mostly BFU-E and CFU-E colonies.…”

Ex Vivo-Generated CD36 + Erythroid Progenitors Are Highly Permissive to Human Parvovirus B19 Replication

“…The unavailability of an in vitro culture system to productively grow the virus not only limits the source of infectious virus to sera or plasma of viremic donors but also retards the study of the pathogenicity of B19V. Based on recent advances in obtaining a relatively pure population of EPCs (6,8,9,19,20), we sought to determine whether these culture conditions could provide cells that would support productive replication of B19V. In this study, we show that CD36 ϩ EPCs were highly permissive to B19V infection and were able to efficiently produce infectious viral particles after infection with viremic sera or plasma or after transfection with the infectious clone.…”

“…CD34 ϩ cells were cultured in serum-free expansion medium modified from a protocol developed previously by Freyssinier et al (8). Briefly, about 10 4 cells/ml were initiated in expansion medium containing a 1:5 dilution of BIT 9500 (Stem Cell Technologies, Vancouver, British Columbia, Canada) in Alpha minimum essential medium (Mediatech), obtaining a final concentration of 10 mg/ml of bovine serum albumin, 10 g/ml of rhu insulin, and 200 g/ml of iron-saturated human transferrin, and were supplemented with 900 ng/ml of ferrous sulfate (Sigma, St. Louis, MO), 90 ng/ml of ferric nitrate (Sigma), 1 M hydrocortisone (Sigma), 100 ng/ml of rhu stem cell factor (SCF; Stem Cell Technologies), 5 ng/ml of rhu interleukin-3 (IL-3; R&D Systems, Minneapolis, MN), and 3 IU per milliliter of rhu EPO.…”

“…By using recent technical advances to generate large numbers of EPCs from hematopoietic stem cells (HSCs) (8,9), we developed a modified cell culture system that allows the differentiation and expansion of CD34 ϩ HSCs into erythroid progenitors presenting the surface antigen CD36. These CD36 ϩ EPCs expressed the B19V cellular receptor globoside on their cell surfaces and were highly permissive to B19V infection.…”

Unexpected macrophage-independent dyserythropoiesis in Gaucher disease

“…The PBMCs were subjected to Ficoll density gradient separation. The CD34 + cells were isolated by magnetic sorting (Miltenyi Biotec), and an in vitro two-phase liquid culture to allow erythroid differentiation was performed, as described by Freyssinier et al 26 During the first phase, the non-adherent cells were expanded for 7 days in a medium containing 100 ng/mL human recombinant (hr) interleukin (IL)-6, 10 ng/mL hr IL-3 and 50 ng/mL hr stem cell factor (SCF). On day 7, the cells were harvested and cultured for 8 days with the second phase medium (10 ng/mL hr IL-3, 50 ng/mL hr SCF, and 2 U/mL hr erythropoietin (EPO).…”