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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.
The permanent turnover of cells derived from the haemopoietic system is the crucial phenomenon which allows the renewal of blood cells. In adult humans the mean daily production of erythrocytes is estimated to be 2 × 1011 ( Erslev, 1983). Active erythropoiesis occurs after erythroid commitment of undifferentiated pluripotent stem cells which provide cells able to terminally differentiate to mature red blood cells. Erythropoietin (Epo) is the specific cytokine physiologically required for red cell production. However, the commitment of stem cells to both early and late erythroid progenitors (BFU-Es and CFU-Es) appears to be a stochastic process independent of the presence of Epo ( Ogawa, 1993; Papayannopoulou et al, 1993 ; Wu et al, 1995b ; Socolovsky et al, 1997 ), and red cell terminal differentiation itself can be induced in vitro independently of the presence of Epo ( Sui et al, 1996 ; Goldsmith et al, 1998 ). A variety of cytokines have been reported to stimulate primitive erythropoiesis, including stem cell factor (SCF) ( Dai et al, 1991 ; Papayannopoulou et al, 1993 ; Muta et al, 1995 ; Wu et al, 1995a , 1997; Broudy, 1997) and interleukin-6 (IL-6) ( Sui et al, 1996 ). Other synergistically acting cytokines such as IL-1 and IL-3 also seem to play a role in the generation and/or amplification of erythroid progenitors ( Brugger et al, 1993 ; Papayannopoulou et al, 1993 ).
The study of the molecular mechanisms leading to erythroid cell proliferation and differentiation in normal individuals is hampered by difficulties in obtaining sufficient numbers of purified erythroid progenitors. Indeed, biochemical and molecular studies require high numbers of cells whereas erythroid progenitors in bone marrow and in peripheral blood are scarce. Furthermore, no specific surface markers are currently available to enable easy immune selection of immature red cell progenitors.
Other purification techniques requiring complex negative selections and serial depletions of non-erythroid cells ( Fibach et al, 1989 ; Sawada et al, 1989 ; Giampaolo et al, 1994 ; Muta et al, 1995 ; Miller et al, 1999 ; Oda et al, 1998 ; Dai et al, 1998 ) have been described for obtaining cell populations enriched in erythroid progenitors. Finally, fluorescent cell sorting using combinations of antibodies have defined lineage-specific subpopulations of cells among which CD34+/CD45RA−/CD71high were reported to contain enriched erythroid progenitor cells ( Sauvageau et al, 1994 ; Lessard et al, 1998 ). However, these procedures are very expensive and time-consuming and cannot lead to purification of high numbers of erythroid progenitors.
We have developed a new method, based on a three-step purification and in vitro amplification technique, which produces high enrichment and proliferation in liquid culture of large numbers of normal human erythroid progenitors. Serum-free cultures of CD34+ cells obtained from cord blood or adult peripheral blood and grown with the cytokine combination: SCF, IL-6 and IL-3 resulted in the dramatic expansion of cultured cells and the appearance of high proportions of CD36+ cells. CD36 is a multifunctional glycoprotein receptor present on platelets, monocytes, mast cells, erythroid precursors, and some endothelial cells ( Van Schravendijk et al, 1992 ). It is involved in the adhesion of platelets, red blood cells infected by Plasmodium falciparum, and monocytes ( Kieffer et al, 1989 ; Oquendo et al, 1989 ; Huh et al, 1995 ). It is considered to be a receptor for thrombospondin, collagen, low-density lipoproteins, and phosphatidylserine ( Kieffer et al, 1989 ; Oquendo et al, 1989 ; Rigotti et al, 1995 ; Huh et al, 1996 ). In the present work our interest in the CD36 antigen was justified by the timing of its appearance during haemopoietic differentiation: indeed, CD36 is detected early on erythroid progenitors whereas it is a late marker of megakaryocytic and monocytic cells ( Edelman et al, 1986 ; Okumura et al, 1992 ; De Wolf et al, 1994 ; Nakahata & Okumura, 1994). CD36+ cells obtained after 7 d in our liquid culture conditions were isolated, and comprised a pure population of late BFU-Es and CFU-Es.
We demonstrated by biochemical and immunological analysis that these erythroid progenitors responded to Epo stimulation and behaved normally in semi-solid culture conditions. Furthermore, terminal red cell differentiation could be obtained, and the analysis of cell haemoglobin content revealed that erythroid cells derived from adult CD34+ cells mostly contained adult haemoglobin, whereas cord blood-derived cells exhibited equal percentages of both adult and fetal haemoglobin.
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- MATERIALS AND METHODS
In the present work we have described a method which enabled the purification and in vitro expansion of large numbers of erythroid progenitors from human cord blood or adult peripheral blood CD34+ cells. Importantly, primary cultures were performed in serum-free and Epo-free medium and thus allowed the generation of a population of erythroid progenitors. These cells were mostly composed of late day-10 BFU-Es and CFU-Es which behaved normally in the presence of Epo stimulation.
The method described here represents a useful alternative to some other methods previously reported ( Fibach et al, 1989 ; Wickrema et al, 1992 ; Giampaolo et al, 1994 ; Muta et al, 1995 ). Actually, the previous techniques combined both complex negative selections and culture stages before obtaining satisfactory red cell enrichments. For example, the purification of day-6 erythroid colony-forming cells (ECFC) used in several studies ( Fibach et al, 1989 ; Sawada et al, 1989 ; Dai et al, 1991 , 1998; Wickrema et al, 1992 ; Giampaolo et al, 1994 ; Muta et al, 1995 ) consisted of a multistep procedure comprising, after Ficoll-Hypaque separation, sheep erythrocyte rosetting of lymphoid T cells, overnight adherence for depletion of monocytes, followed by the use of a mixture of four monoclonal antibodies to remove granulocytes, B lymphocytes, NK cells and residual T cells. Thereafter, cells were cultured in methylcellulose in the presence of 30% FCS and Epo for 6 d, then collected from the culture and submitted again to adhesion and Ficoll-Hypaque separation before secondary culture in the presence of FCS, human AB serum and Epo. Moreover, total numbers of erythroid progenitors were low when compared to the numbers reached by the procedure described here ( 3 Table IIIA).
Our data also differ from previous human erythroid cell purification procedures performed in the constant presence of Epo and which lead to the expansion of more mature, mostly GpA-positive cells ( De Wolf et al, 1994 ; Mrug et al, 1997 ; Cippolleschi et al, 1997 ; Malik et al, 1998 ; Oda et al, 1998 ). This point was particularly documented by Oda et al (1998 ), who performed biochemical assays with peripheral blood-derived erythroid cells obtained from day-8 liquid cultures in the presence of both serum and Epo. The cells obtained were 71% GpA+, thereby demonstrating late erythroid differentiation, whereas our method leads to the purification and expansion of a homogenous population of immature erythroid progenitors.
Furthermore, we observed that CD34+ cells originating from both cord blood and adult peripheral blood collected by cytapheresis after stem cell mobilization displayed similar kinetics of culture evolution and cell expansion. This suggests that day-7 CD36+ cell purification from CD34+ cells cultured in IL-3 + IL-6 + SCF is an efficient general method for obtaining high concentrations of human immature erythroid progenitors.
With the above cell numbers, we demonstrated that serum- and cytokine-deprived CD36-positive GpA-negative or -positive erythroid cells displayed Epo responsiveness after 10 min of Epo stimulation. This resulted in the tyrosine phosphorylation of Epo-R, Jak2, SHIP and STAT5. These data confirmed those of Oda et al (1998 ), performed on more mature red cell precursors.
We did not detect any tyrosine phosphorylation of STAT3 in these human progenitors (data not shown). In addition, we recently demonstrated that GAB-1 (Grb2-associated Binder 1), a molecular adaptor whose structure is close to IRS-1/2 is tyrosine phosphorylated in response to Epo in these normal human erythroid progenitors ( Lecoq-Lafon et al 1999 ). This result further assesses the usefulness of our ex vivo purification and expansion technique.
Taken together, our data indicate that the present work allows in vitro expansion of large numbers of erythroid progenitors at mature BFU-E or CFU-E stages. These cell populations exquisitely represent the main Epo targets and are able to fully differentiate in the presence of Epo. Therefore they are adequate reagents for the study of signal transduction events which follow Epo stimulation and thus represent a useful and abundant source of normal cells for in vitro studies of erythropoiesis. Similar studies can be performed with cells from patients suffering from erythroid pathologies and therefore enable more specific analysis of the pathogenesis of acquired red cell lineage disorders.