Papayannopoulou et al.: Epo and Tpo Synergy Experimental Hematology 24:660-669 (19961 661 @ 1996 International Society for Experimental Hematology Rapid Communication ulation with fluorescence microscopy. Purified subsets were grown in plasma clot and methylcellulose clonal cultures and in suspension cultures using the combinations of cytokines Insights into the cellular mechanisms cadaveric bone marrow cells obtained from Northwest described in the text. Single cells from the different subsets Center, Puget Sound Blood Bank (Seattle, WA), were were. also deposited (by FACS) on 96-well plates containing of erythropoietin-thrombopoietin synergy washed, and incubated overnight in IMDM with 10% medmm and cytokines. Clonal growth from single-cell wells calf serum on tissue culture plates to remove adherent were double-labeled with antiglycophorin A-PE and anti­ Thalia Papayannopoulou, Martha Brice, Denise Farrer, Kenneth Kaushansky From the nonadherent cells, CD34+ cells were isolated CD41- FITC between days 10 and 19. direct immunoadherence on anti-CD34 monoclonal anti­ University of Washington, Department of Medicine, Seattle, WA (mAb)-coated plates, as previously described [15]. Purity Immunocytochemistry Offprint requests to: Thalia Papayannopoulou, MD, DrSci, University of Washington, isolated CD34+ cells ranged from 80 to 96% by this For immunocytochemistry, either plasma clot or cytospin cell Division of Hematology, Box 357710, Seattle, WA 98195-7710 od. Peripheral blood CD34 + cells from granulocyte preparations were used. These were fixed at days 6-7 and (Received 24 January 1996; revised 14 February 1996; accepted 16 February 1996) ulating factor (G-CSF)-mobilized normal donors 12-13 with pH 6.5 Histochoice (Amresco, Solon, OH) and provided by Dr. Scott Rowley from Fred Hutchinson stained at room temperature in the following sequence: anti­ Research Center (Seattle, WA) following approved pro­ CD41 antibody; biotinylated goat Fab' 2 antimouse IgG (Tago, Fetal liver cells were prepared from samples obtained Burlingame, CA); streptavidin-conjugated alkaline phos­ the Central Laboratory for Human Embryology, Depart­ phatase (Vector Laboratories, Burlingame, CA); and alkaline Abstract and for the expression of a well-coordinated of Pediatrics, University of Washington. Nucleated fetal phosphatase substrate kit I (Vector® Red; Vector Laboratories). Using suspension cultures of purified bone marrow CD34+ program in the end-stage, functional cells [1]. The cells were used after the removal of erythroid cells cells, we have analyzed the effects of the combination of ery­ cloned ligand for the c-Mpl receptor, Tpo, appears and red cells) by direct immunoadherence using Suspension cultures anti-erythroid mAb 23.6 [16]. thropoietin (Epo) and thrombopoietin (Tpo) on the in vitro the role of a lineage-specific cytokine for the mE'l?:akar Liquid cultures of CD34+ purified cells from marrow or from differentiation toward erythropoiesis and thrombopoiesis. lineage [2-6]. Its pivotal role in m<~ga.ka.rvc>cvtOJJOie~ fetal liver were carried out for up to 3 weeks. CD34+ purified The number of CD41+ cells that accumulated over 2 weeks of platelet production is supported by several in vitro cells were inoculated at 104-105/mL. Medium for suspension culture, as well as the number of globin+ cells in the same cul­ vivo studies [7,8], especially those with c-Mpl-null anti-CD34 antibodies were used: QBEND/10 (AMAC, cultures consisted of IMDM with 1o/o bovine serum albumin tures, was found to be significantly higher with the Epo+ Tpo and more recently with Tpo-null mice [10]. The effects ME); HPCA-2 (Becton Dickinson Immunocytome­ (BSA), 10% normal human plasma or serum, 10-4 M 2-mer­ combination compared to either cytokine alone. No evidence on in vitro megakaryocytopoiesis are amplified Jose, CA); and mAb 12.8 [17], kindly donated by Dr. captoethanol (Eastman Kodak, Rochester, NY), and a comple­ was found that Tpo affected the differentiative action of Epo. combined with other cytokines in a synergistic (KL FHCRC. Two antiplatelet antibodies against ment of desired cytokines. IL-3 (Genetics Institute, Cam­ Instead, there was a significant expansion of erythroid pro­ additive (IL-3) fashion [11], but its exact role in (CD41) were used: 13.1 (kindly provided by Dr. D. bridge, MA) was used at 10 U/mL, and KL (SCF, Amgen, genitors, both erythroid colony-forming and burst-forming from megakaryocytes remains to be clarified. and Tab [18] (a generous gift from Dr. Roger McEver, Thousand Oaks, CA) was used at 50 ng/mL. Epo (Genetics units (CFU-E and BFU-E), by 7 days in culture, suggesting a the detailed spectrum of progenitors on which of Oklahoma, Oklahoma City, OK). A directly con- Institute, Cambridge, MA) was used at concentrations from proliferative effect of Tpo on erythroid cells in vitro. To deter­ effects and their composite phenotype have not 1-FITC was purchased from Immunotech (Miami, 0.2 to 10 U/mL. Human recombinant Tpo was used at 1200 mine the phenotypic features of erythroid progenitor cells defined. As a late-acting cytokine, Tpo's in purchased antibodies included FITC-conjugated units/mL (optimal concentration determined in preliminary which were targets of Tpo's action, and specifically to inquire early-acting cytokines (IL-3 or KL) is not exrJecitedN 'z antimouse IgG secondary antibody (American experiments; 50 units was defined as the amount supporting whether the effect was directed mainly toward bipotent ery­ significant synergy with Epo, another lin La Mirada, CA), biotinylated secondary antibody half-maximal proliferation in a BaF3/mpl MIT assay) [20] or throid/megakaryocytic (E+Mk) progenitors, we isolated sub­ cytokine, observed both in in vitro and in Foster City, CA, or Tago, Burlingame, CA), irrele­ at 15 ng of recombinant protein per mL (R&D Systems, Min­ sets enriched for both erythroid and megakaryocytic pro­ [12, 13], is of special interest and has relied on of the same isotype as test antibodies (Caltag Labo­ neapolis, MN). Cultures were fed every 2-3 days by replacing genitors from CD34+ cells. We found that 1) BFU-E and undocumented but likely-expression of a South San Francisco, CA), anti-CD45 RA (AMAC), 80% of the medium, diluting the suspension, or removing CFU-Mk co-segregate in the subset of CD34+ cells that is nega­ receptor by megakaryocytic progenitors, as · (PE)-conjugated streptavidin (Biomeda). cells when necessary to keep the cell concentration below tive for the phosphatase isoform CD45RA; 2) the presence of megakaryocytes [14]. antibodies (anti-'{, 51.7; anti-[3, 16.2 and [337) were 106/mL. Aliquots were removed at days 6-7 and 12-13 for CD41 on this subset appears to segregate late erythroid and The focus of the present work was to analyze in our laboratory and have been described previous­ replating and evaluation. late CFU-Mk from early erythroid and early CFU-Mk, which on erythroid differentiation in vitro. Using Anti-glycophorin A-PE conjugated to PE was pur­ Dako (Carpinteria, CA). are CD41-negative; 3) bipotent erythroid/Mk progenitors, tures of purified human bone marrow CD34+ Semisolid assays studied by single-cell culture assays, were found mainly in the ed a detailed quantitative assessment of the Cells were cultured using the methylcellulose method to evalu­ CD41+ and rarely in the CD4r subsets, which included more Epo+ Tpo combination on both megakaryocytic labeling and FACS sorting ate BFU-E, CFU-GM, and CFU-Mix (mixed erythroid/myeloid/ multipotent progenitors; 4) by comparing the frequencies of differentiation over time within the same ' ulluJ'"" were assayed for the presence of develop­ macrophage) colonies and the plasma clot technique to evalu­ pure erythroid or pure megakaryocytic progenitors to that of that Tpo has a bidirectional effect on both by indirect live cell immunofluorescence ate either CFU-E or colonies derived from CFU-Mk. Methylcel­ bipotent E+Mk progenitors, we conclude that the erythroid­ megakaryocytopoiesis, as erythroid and m anti-CD41 antibodies (13.1, Tab), followed by lulose medium was as previously described [14]; plasma clot enhancing effect of Tpo is directed mainly toward pure ery­ progeny are enhanced in its presence. Tpo's antimouse IgG. Analysis was carried out with composition was adopted from Mazur and South [20] for CFU­ throid progenitors expressing CD41 and Mpl, as suggested by thropoiesis do not result from an enhancing microscopy (Zeiss Universal) or by flow cytometry Mk and included 1o/o BSA, 10% normal human serum, 10-4 M independent experiments employing anti-Mpl antibody, ferentiative effect of Epo, but are mediated FACStar; Cell Analysis Facility, Department 2-mercaptoethanol, selected combinations of cytokines, 10% rather than only on bipotent E+Mk progenitors. tive effects on early and late erythroid •"''"'c;u<·•~ , University of Washington). Purified bone bovine citrated plasma, thrombin 0.25 U/mL, fibrinogen 500 were labeled with anti-CD34 (QBEND/10 or these cultures. Using purified CD34+ subsets ]lg/mL, and 2 ]1M CaCl2 • All cultures were set up in duplicate or Key words: Erythropoietin-Thrombopoietin-Bipotent cultures, we concluded that the great -CD45RA (all directly conjugated), and subsets triplicate plates, incubated in a fully humidified atmosphere +/CD45RN or CD34+/CD45RK were sorted erythroid/Mk progenitors-CFU-MK and throid progenitors that are targets for Tpo's with 5% C02 in air, and studied after 1 or 2 weeks. Mk colonies BFU-E purification effects are not bipotent (E+Mk), but are rather con~itions using appropriate