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 positive and nega were identified by immunohistochemical labeling with anti progenitors carrying the c-Mpl receptor and Tnpie-labeling of purified CD34+ cells with anti CD41 antibody. A CFU-Mk colony was defined as a cluster of Introduction showing common surface characteristics of ,. anti-CD41-FITC (13.1), and anti-CD45RA- three or more CD41 + cells. Other colonies were identified by In hematopoiesis, differentiation along specific lineages is are targets for both Epo and Tpo, provide done for sorting into CD41+/CD45RA-, morphologic criteria, direct microscopic observation of live cul under the control of late-acting cytokines responsible for the long-standing relationship between ' or CD41+/CD45RA+ subsets of CD34+ cells. tures, or staining with benzidine and hematoxylin (fixed slide amplification of progenitor and precursor cells of each lineage megakaryocytopoiesis. sorting was verified by examination of each pop- preparations of flattened plasma clots). Experimental Hematology vol. 24 119961 Th Papayannopoulou et al.: Epo and Tpo Synergy 663 662
CD::l-1 POSlfiVf Gl\1 1: Table 1. BM -CD34+ cell suspension cultures: increase in both CD41 + cells and globin• cells with Epo+ Tpo combination
Cytokine Nucleated cells C041 + cells Globin• cells x105 x105 x105
FL-MC Tpo 10.0 3.5 ± 0.24 0 Epo 130.0 3.0 ± 0.4 11.1 ± 2.1 I' Epo+Tpo 195.0 16.0 ± 2.3' 160.0 ± 3.1' BM-CD34• Tpo 5.0 2.4 0.29 Epo 70.0 0.17 ± 0.08 64.0 ± 1.0 Epo+Tpo 106.0 8.3 ± 0.12' 111.0 ± 3.0' .. Lo BM-CD34• Tpo 10.0 .cGl Epo 19.0 0.6 ± 0.07 11 .0 ± 2.0 E Epo+Tpo 80.0 9.9 ± 0.53' 32.0 ± 4.1' :::J PB-CD34+ Tpo 4.2 2.13 ± 0.36 0 z Epo 0.93 0.02 ± 0.002 0.524 ± 0.04 Epo+Tpo 5.5 2.4 ± 0.33 0.69 ± 0.07 PB-C D34+ cells are from a G-CSF-mobilized normal donor, and evaluation was done at day 7. In all other experiments, assessment of C041 + and globin• cells was done at day 12 in culture, where maximal differences are observed. IL-3 Epo KL IL-3 Epo 'p < 0.01. Tpo: + + + + + +
BM-CD34 + Cells FL-MNC Statistical analysis Statistically significant differences among sets of samples were Fig. 2. Generation of C041 + cells from bone marrow determined by Mann-Whitney test. C034+ cells in suspension cultures in which Tpo was used either alone or in combination with one other cytokine (IL- Results 3, KL, or Epo). The Epo+Tpo combination gives similar results to IL-3+Tpo in BM samples, but it appeared superior Generation of CD41+ cells in suspension cultures of CD34+ cells in fetal liver samples (fetal mononuclear cells from which the nucleated erythroid cells were removed by immunoad rith the Epo+Tpo combination herence were used in this experiment). The generation of Gpiib-IIIa (CD41) positive cells from bone marrow CD 34+ cells during culture in the presence of Epo+Tpo was compared to cultures with each cytokine (Epo
or Tpo) alone. The cumulative number of CD41+ cells up to 2 Generation of globin• cells in suspension cultures of CD34+ cells 'I weeks was calculated from the proportion of CD41 • cells and with the Epo+Tpo combination I the total number of nucleated cells at each point (positive In addition to the generation of CD41 + cells, we also wanted cells, usually between 1 and 4% at the first day of culture, to examine whether Tpo had any enhancing effect on the dif were screened with the fluorescence microscope to exclude ferentiative action of Epo in the same cultures. For this pur positivity from platelet adherence to cells). When only Tpo pose, we assessed the generation of globin• cells (by was present, there was an initial decline (by the end of the immunofluorescence of fixed smears). By the end of 2 weeks, first week) in the total nucleated cell number but a progressive no globin• cells were observed when only Tpo was present in increase in the frequency of CD41 + cells, so that by 2 weeks in these cultures. In contrast, as seen in Table 1, the generation culture the cell population consisted of more than 70% of globin• cells was significantly higher when the two CD41• cells (Fig. 1C). The majority of these cells were large cytokines were combined compared to Epo alone. To test ove r 20 micron s in diameter- with a lobulated nuclear whether Tpo enhances the differentiative action of Epo in appearance and cytoplasmic maturation of varying degree. In vitro, we compared the proportion of globin• cells not only in the presence of Epo alone, CD41 + cells present at the begin the Epo/Tpo combination, but also in the combinations of ::of the culture quickly declined. In the presence of both Tpo with other cytokines, especially KL, since we had previ Tpo, however, CD41+ cells accumulated in much higher ously shown that globin• cells (presumably proerythroblasts) ~U mbers than with Tpo alone (Table 1 and Fig. 1D). This are present in these cultures in the absence of Epo [14]. As CO+Tpo synergy was observed at all doses of Epo tried seen in Table 2, the relative proportion of globin• cells present rA..'l-10 U/mL, data not shown). To compare the magnitude with either KL alone or IL-3 alone does not change signifi '. er the Epo+ Tpo synergy to that of Tpo combined with anoth cantly in the presence of Tpo. In contrast, the proportion of lll:okme (IL-3 or KL), we made similar assessments of accu globin• cells with the Epo+Tpo combination is much lower d 'th anti· . a stain) B FACS analysis of C034+ cells labele WI ~ ted CD41• cells in Tpo+IL-3 and Tpo+KL cultures. The than with Epo alone, although the total numbers are higher. Fig. 1. A. Purified bone marrow C034+ cells at day 2 of cul~ure (Giems n . C~45RK cells; only 0.07% of the cells were caJl ts :Vith IL-3 +T po in three experiments were not statisti These results suggest that Tpo's enhancing effects are more C041 and anti-C045RA. Note that virtually all C041 cell_s are amo g of Tpo alone. Usually over 70% of the cells are J ~I fferen t from those with Epo+Tpo (data not shown), proliferative than differentiative. Indeed, when the effects of C041./C045RA•. C. Oay-12 C034+ cells in suspension cultures_m the pr~senceltures in the presence of Epo+Tpo. The only pop- . 0 0 12 C034+ cells m suspens1on cu from lttal r th combinations were better than Tpo+KL (Fig. 2) . In the Epo+ Tpo combination were assessed at the progenitor C041+ at different stages of maturation. • ay- . II (G' msa and benzidine stain). E. Plasma clot cultures 1) II d megakaryocytic ce s 1e t' (04 · ~ Ver samples, however, the combination of Tpo+Epo level (BFU-E, CFU-E, and CFU-GM), we found that a combina lations present are erythroblasts, re d ce s, an t (CFU Mk colonies labeled by an I· U . d k ytic colonies were presen · ' the C041./C045RK subset. Only erythroid an mega aryoc ed to be superior to that of Tpo+IL-3 (Fig. 2) . tion of two cytokines had significant effects on the expansion F. Putative bipotent E+Mk-derived colony in plasma clot cultures. Experimental Hematology vol. 24 ( 664 Th Papayannopoulou et al.: Epo and Tpo Synergy 665
Table 2. BM-CD34+ cells in suspension cultures: accumulation o; Table 3. CD34+ cell suspension cultu~es: expansi.on in progenit~r cells (CFU-GM, BFU-E, CFU-E) by day 7 1n culture With Epo+ Tpo corl'l· BFU-E% globin+ cells by day 7 in culture in the presence of IL-3, KL, or Ep CFU-GM% Table 4. Hematopoietic progenitors (BFU-E, CFU-GM, and CFU-Mk) with and without Tpo bination present in different CD34+ subsets
Globin+ cells Cytokine CFU-E x 103 BFU-E x 103 Subset BFU-E CFU-GM CFU-Mk Cytokine• No. x105 Experiment (41/45RA) 0 Percent FL-MC Epo 8.7 ± 1.3 2.0 ± 0.4 /o % % Epo+Tpo 17.2 ± 1.2b 9.9 ± 0.8b +/- (20%) Tpo 0 0 29.9 ± 3.6 0.6 ± O.Ql 1.6 ± 0.5 BM-CD34+ Epo 1.8 16.0±1.9 -1- (48.6%) KL 3.6 ± 0.005 13.7 ± 2.0 13.3 ± 3.5 3.5 ± 0.6 0.3 ± 0.1 3.3 ± 0.2b 37.0 ± 4.9b +!+ (6.1%) KL+Tpo 2.9 ± O.ol 25.4 ± 10.6 Epo+Tpo 0.58 ± 0.04 12.3 ± 1.8 0.2 ± 0.1 2a +/- (17.7%) IL-3 0.9 ± 0.002 7.1±12.3 BM-CD34+ Epo 5.1 ± 0.5 4.0± 0.6 24.36 ± 3.1 0 3.6b (69.4%) IL-3+Tpo 1.3 ± .003 17.7±4.1 Epo+Tpo 9.4 ± 0.7b 16.8 ± 4.2b -1- 9.53 ± 1.0 3.2 ± 0.1 1.5b 52.4 ± 4.3 3a +/- (14.45%) Epo 56.3 ± .05 BM-CD34+ Epo 4.9 ± 1.2 26.4± 1.6 37.9 ± 2.3 0.4± 0.4 7.7 ± 1.3 15.4 ± 2.8b 66.2 ± 4.6b -1- (48.74%) Epo+Tpo 12.5 ± 0.02 69.0 ± 7.9 Epo+Tpo 29.0 ± 3.4 2.0 ± 0.2 0.8 ± 0.3 PB-CD34+ Epo 1.3 ± 0.07 9.2 ± 0.2 •All cytokine combinations were initiated with the same Numbers in parentheses indicate the representation of Epo+Tpo 2.9 ± 0.4b 45.8 ± 4.9b 30 inoculum from the same pool of CD34+ cells. 20 10 0 10 20 each subset among the total CD34+ population. Epo+Tpo 30 •The proportions of+!+ in experiments 2 and 3 were 1.7 (delayed)" 2.3 ± 0.1 13.3 ± 1.6 Fig. 3. Colony yield (BFU-E or CFU-GM) from subsets of IL-3 0 29.2 ± 4.0 and 0.07%, respectively. CD34+ cells that are either positive or negative for CD45RA. IL-3+Tpo 0 70.1 ± 4.4b bSingle plate count. Length of each bar indicates number of colonies expressed of these progenitors in culture, but only if Tpo was ~resent KL 1.6 ± 0.2 32.2 ± 1.7 as percent of cells plated. Note the reciprocal enrichment from the outset of culture. Both early and late er:thrmd pr~ KL+Tpo 1.2 ± 0.07 54.8 ± 5.5b of the CD45RK subset in erythroid colonies and of the ------·------~- genitors were significantly amplified after 7 days m culture I~ IL-3 and KL with and without Tpo were included in CD45RA+ subset in CFU-GM colonies. the presence of Epo+ Tpo compared to Epo alo~e (Table 3). It IS experiment for comparison. plating in 96-well plates using the automatic FACS sorting of note that significant differences (p < 0.01) mother types of •Addition of Tpo was delayed by 3 days. device. In the plasma clots, three categories of colonies were progenitors such as CFU-GM, were found in three of four bp < 0.01. ------scored: pure erythroid, pure megakaryocytic, and bipotent experiment; with Epo+ Tpo. In fact, the effect of Tpo on o_ther +subset showed many scattered megakaryocytes but few erythroid/megakaryocytic. The plasma clots were stained with progenitors, both erythroid and nonerythroid, wa~ not umque erived colonies. By contrast, many CFU-Mk anti-CD4I by immunohistochemical means and counter to the Epo+Tpo combination and was observed With IL-3+Tpo among CD34+ cells, subsets that were 41+/45~-, 4 stained to reveal the characteristic morphology of erythroid were generated when cells from the CD4I- subset in and KL+ Tpo (Table 3). Enhancement of CFU-E, h~wever, was or 41-/45RK and subjected them to clonogemc for 7 days were replated (Table 5). colonies (Fig. IE). The frequencies of erythroid colonies in seen only with the Epo/Tpo combination and With none of suspension cultures. In three such experiments, we _ these plasma clot cultures were about I4.6% (Table 6) and also did two experiments in which we made use of an the other combinations. These experiments clearly sho:Ved virtually all BFU-E again resided in the CD45RA polyclonal antibody (kindly provided by Immunex). 22% (data not shown) in two experiments, respectively; the that Tpo affected late stages of erythropo_iesis in vitro mamly whether they were CD4I+ or CD4I- (Table 4). Of 1+-selected cells, erythroid progenitors were found frequencies of pure megakaryocytic colonies were about 1.6% through proliferative effects at the progemtor level. BFU-E present in the CD41+/CD45RA- subset were (Table 6) and 7.7% in the 4I+/45RA- cells (Table 4). As both the positive (Mplhi) and negative (Mpl10) fractions. late or mature type, since less than 10% of the total as with CD41, the c-Mplhi_selected subsets were pure erythroid and pure Mk colonies were enriched in these Enrichment of erythroid and megakaryocytic progenitors in specific were large bursts. In contrast, over a third of_ the plasma clots, there was ample opportunity for an overlap enriched in BFU-E compared with CFU-GM. In CD34+ subsets . . bursts present in the CD41-/CD45RK subset_ m between these two types of colonies in plasma clots. This was 'l"'"H'""'·'' for example, the Mplhi subset had BFU-E = To analyze the phenotypic features of erythroid progemt_ors ment and over 40% in another were categonzed as observed several times (Fig. IE), but CD4I +cells also appeared 5% and GM = 0.3 ± 0.02% (E:GM ratio ~8:1). The on which Tpo exerted its effects, we atte_mpted to ennc~ extra-large bursts. Thus the presence of CD:l to sprout from some erythroid colonies rather than represent had BFU-E 3.0 0.7% and GM 8.1 0.8% either erythroid or megakaryocytic progemtors from _CD34 45RA- cells, which include virtually all erythrmd = ± = ± 1:2.6, a reversal of the Mplhi E:GM ratio). In a sec ing overlapping unipotent colonies (Fig. IF). These colonies cells. Previous experiments [22] showed that erythrm~ pro seems to segregate the early from the late BFU-E. were scored as putative bipotent E+Mk colonies. The frequen the ratio was also high in the Mplhi subset: genitors can be greatly enriched in the CD34~/CD45RA_ sub subsets were examined for the presence of C cies of these colonies in the plasma clot culture were 0.3% 1.77% and CFU-GM = 0.03% (E:GM = 57:I). These set and similar observations have been descnbed prevwusly genitors, it was found that +CFU-}vfk-derived (Table 6) or
Table 5. Suspension cultures initiated from CD34+ subsets: accumu 1atron • o f CD41+ cells and globin+ cells by 2 weeks in culture, and BFU-t and< CFU-Mk expansion after 1 week in culture --acc•rwp of Epo, we entertained the following possibilities: that Tpo enhances the differentiative action of Epo; sec tive for these two molecules were found) were indeed present 5 Subset Cells x10 Expansion that Tpo has only a proliferative effect on all erythroid among both CD41+ and CD4r subsets, albeit with different Experiment (41/45RA) Cell no. Globin+ BFU-E CFU-Mk -~~a,,,,.,..,,<. and third, that Tpo mainly affects bipotent ery- frequencies and different characteristics. Truly bipotent E+Mk progenitors were more frequent in the CD41 + subset. The +/ 108.0 6.1 ± 0.74 [58.0 ± 6.2]" 2.4 X 11.7 X . megakaryocytic progenitors (E+Mk) present in these CD41 + subset also contained pure erythroid, pure megakary -/- 192.0 22.0 ± 1.5 [23.0 ± 1.6]" 29.2 X 25.4 X . Combinations of two or three of these possibilities ocytic, and rare or no CFU-Mix that included megakaryocytes, 2 +/- 4.8 0.31 ± 0.05 2.6 ± 0.74 0.7 X 1.36 X also considered. We found that enhancement of the dif -1- 10.0 1.3 ± 0.3 3.4 ± 0.5 5.4 X 2.65 X action of Epo by Tpo was unlikely on the basis of even when analysis was carried out after 17 days in culture. In +/+ 2.6 0.05 ± O.ol 0.5 ± 0.07 data presented in Table 2. Tpo alone generates no globin+ contrast, the CD4r subset contained fewer true E+Mk and 3 +/- 6.9 0.68 ± 0.08 5.34 ± 0.13 0.71 X 1.6 X and does not enhance the proportion of globin+ cells more CFU-Mix with or without Mk and included earlier pro -1- 58.0 1.67 ± 0.26 24.7 ± 0.2 8.8 X 105.0 X in the presence of KL (or IL-3) but in the absence of genitors with high proliferative capacity. Since only single-cell growths of more than 100-200 cells were analyzed, a bias • IL 3 IL 6 KL and Tpo but not Epo. The initial inoculum in experi- . Most important, the proportion of globin+ "ln this experiment the cytokines included in s~spensron ":ere . .' . ; 3 ~ x 104 cells· it was the same for all the subset cultures. ment 1 was 3 x 104 cells, in experiment 2, 1 x 10 cells, and rn experrmen , , with the Epo+Tpo combination was much lower than directed against the analysis of progenitors giving rise to small Epo only, and a delayed addition of Tpo abrogated much clonal growths is undoubtedly present. This bias likely con effect. When we tested the effect of Tpo on the expan- cerns late pure CFU-Mk or late pure erythroid progenitors, of erythroid progenitors (CFU-E and BFU-E), however, we and it is unlikely that it is directed toward E+Mk progenitors. (56 and 36o/o in 41+ compared to 53 and_ 42o/o in 4r),_ there scription factors (e.g., GATA-1, ets, NFE2) [25,26] or a significant enhancement in the presence of Tpo. Because the frequency of truly bipotent E+Mk progenitors were more cells in each well in the CD41 subset than m the antigens or receptors (e.g., presen~e o_f Ep? rece data confirm previous in vitro and in vivo data in the was very low compared to pure erythroid, and because a CD41+ subset. Because of this, more wells from CD4:- were megakaryocytic cells) [14], and certam signalmg model [11] and recent observations with human cells significant proportion of the erythroid progenitors displayed evaluable. In general, wells in which both eryt?rmd and (e.g., JAK2 kinase) [27]. Also, it is not a random nnPnnm Further, we were interested to know whether the ery Mpl, we believe that the proliferative effect of Tpo is directed CD41 + cells (derived from bipotent E+Mk progemtors) were that virtually all erythroleukemia cell lines that have progenitors on which Tpo exerts its effect display toward these erythroid progenitors. Both megakaryocytic and observed were much less frequent (~16-fold) than the pu~e made today also display megakaryocytic features markers, specifically c-Mpl or CD41. When erythroid cells were increased in the same cultures in vitro, erythroid. More bipotent E+Mk progenitors :vere obs~rved m Papayannopoulou, unpublished data]. Furthermore, CD34+ subsets (34+/45RA-) that were highly and the progeny of both lineages was increased in vivo with the CD41+ population (6.2 and 5.4o/o) than m CD41 (0 _and it was shown that Gplla-targeted suicide genes in BFU-E with anti-CD41, we found that virtually all out any apparent competition. This fact can be explained by 2.8%), indicating that this bipotent progenitor comes mt~ influence both erythroid and megakaryocytic cells cells were included in this subset (Fig. 1B). As expected, the action of Tpo on independent progenitors, rather than on play more frequently after the appearance of the C~41 anti [35]. Erythropoietin was known to enhance CFU-Mk were also enriched for megakaryocytic progenitors. As the same progenitor (bipotent E+Mk). On the basis of our in gen. In all bipotent E+Mk cells, there was a predommance of growth [36], a finding confirmed when . . of progenitors present in these CD41 + subsets vitro data, one can suggest that all three types of progenitors erythroid cells compared to CD41 + cells (the latter ranged became available [11]. Additional in vivo data either m erythroid, with characteristics of late BFU-E, this (pure erythroid, pure CFU-Mk, and E+Mk) exist in vivo and are targets for Tpo's action. from 1 to 15o/o of total cells). More of the clonal growths _co~ after treatment with high doses of Epo [37] or in formally demonstrates that these erythroid pro taining megakaryocytic cells (CD41+) were part of CFU-Mix m mals [38] also show effects in both lineages. Of display CD41 on their surface. Megakaryocytic pro The notion that bipotent erythroid/megakaryocytic pro the CD4r subset, in contrast to the CD41+ subset. thropoietin and thrombopoietin share structural highly enriched in the same subset genitors exist is not new [41-45], but attempts to demonstrate and qualitatively similar biologic acti~i~ies ~7]. ~~·-rvnn-), were much less frequent than erythroid their frequency or enrichment under a phenotypic class of Discussion . . Our present studies, showing a ~Iduectwn~l +effect and were also mostly of late type, giving rise to progenitors have not been made previously. Furthermore, it The existence of a close relationship between erythropOiesis on the accumulation of both CD41 and globm cells colonies than those present in the CD41- subset. Sev was unclear whether this bipotent progenitor was just an and megakaryocytopoiesis rests on numerous experime~tal pension cultures in the presen~e.of E?o, extend these pieces of evidence support the notion that late example of a progressive loss of potentialities from a pluripo observations spanning more than 15 years. Thus, ~rogemtor observations. To explain the biduectwnal effect of Tpo and late megakaryocytic progenitors were present in tent cell or something beyond this concept. Our present find cells from both lineages share common features, either tran- generation of CD41 + and the increase of globin+ 1+ compared to CD41- subset. Suspension cultures ings, showing that the bipotent E+Mk progenitor copurifies with CD41 + or CD41- cells showed greater prolifera with the subset (CD41 +) that contains less multi potent pro of total nucleated cells, higher accumulation genitors, are certainly compatible with the view that such a E-Mix) derived colonies observed in clot cultures and cultures of different CD34' and globin+ cells, and greater expansion of BFU-E progenitor is located downstream of the pluripotent cell in the hematopoietic hierarchy. However, its special relationship Plasma clot cultures -Mk in the CD41- subset compared to CD41 + (Table fact that rather late CFU-Mk are present among with erythroid progenitors rather than a random association Experiment Subset E% Mk% E+Mk% cells was also suggested by some previous stud- with both erythroid and granulocytic progenitors (since no 41+/45RA- 2012" 14.6 ± 0.4 1.6 ± 0.3 0.3 ± 0.07 independent experiments in which anti-Mpl was bipotent megakaryocytic/myeloid progenitors were detected) 4r/45RA-1251" 9.96 ± 0.3 0.3 ± 0.07 0.075 ± 0.02 of anti-CD41 showed that Mplhi/45RK cells were supports the notion that an E+Mk bipotent progenitor has a in BFU-E. Unlike anti-CD41, anti-Mpl anti distinct place in hematopoietic differentiation. In fact, on the Single-cell cultures a continuing spectrum of positivity without basis of in vivo data suggesting competition between erythro Experiment Subset Wells with growth/total wells Wells labeled E E-Mix Mk between positive and negative cells. Whether poiesis and megakaryocytopoiesis [46], some investigators suggested that this progenitor may be the most frequent one 41+/45RA- 109/192 48 38 0 ---..•. ,~ .. depends on the specific Abused or reflects present in vivo, especially before the two lineages diverge. 4r/45RA- 101/192 68 40 0 spectrum of Mpl presence among progenitors is Our data do not support such a view. If all erythroid and 2 41+/45RA- 164/485 55 41 our data taken together strongly sug megakaryocytic cells have to go through the stage of a bipo 41-/45RA- 206/485 71 49 0 progenitors share common phenotypic 3' 34+/45RA- 124/192 35 13 5 CFU-Mk progenitors. tent progenitor, competition between the two lineages may 4' 34+/45RA- 95/290 be expected, and the higher levels of one cytokine (Epo or 30 11 5 2 for the presence and frequency of bipo Tpo) present at any given time may drive the cells toward one E = pure erythroid colonies; Mk = pure mega~aryocytic colonies; E+M k = b'1 P0 t en t erythroid/megakaryocytic colonies; nu~gaka.rvc>cvti' progenitors, we analyzed sin mixed colonies with erythroid and nonerythrord components. for the presence of cells with erythroid and or the other pathway. Alternatively, as our data suggest, ery "Total colonies scored. + markers originating from a single cell. In throid and megakaryocytic cells can be generated from both bipotent E+Mk progenitors and unipotent progenitors, and bNumbers in parentheses indicate E+Mix colonies including CD41 cells. I I' bl than the live cell we were able to demonstrate that pro 'Evaluated by immunocytochemical means which, because of cell losses, were ess re ra e giving rise to erythroid only (glycophorin N) and the action of each lineage-specific cytokine (Epo or Tpo) is used in experiments 1 and 2. only (CD41+) progeny (no cells doubly posi- addressed to both pure and bipotent progenitors. Such a view reconciles the contemporaneous enhancement of both lin- Th Fapayannopoulou et al.: Epo and Tpo Synergy 669 668
Martin DIK, Zon LI, Mutter G Orkin SH (1990) E . (c-mplligand) acts synergistically with erythropoietin f . ' xpresswn Marguerie G (1995) Suppression of erythro-megakaryocy o an eryt~rmd transcription factor in megakaryocytic and eages by either Epo or Tpo treatments in vivo under certain stem cell factor to enhance murine megakaryocyte '-v,,vnv 1 t~p~resJs. and the induction of reversible thrombocytope mast celllmeages. Nature 344:344 nia m mrce transgenic for the thymidine kinase gene tar circumstances [12, 13,37]. growth and increases megakaryocyte ploidy in Finally, if pure erythroid and pure CFU-Mk progenitors R~me~ PH, Prandini MH, Joulin V, Mignotte M, Prenant geted by the platelet glycoprotein a!Ib promoter J E Blood 85:1719 Vam~henker G, Marguerie G, Uzan G (1990) Mega Med 181:2141 · xp both express Mpl, it will be intriguing to know how the sig 12. Kaushansky K, Broudy VC, Grossman A, Humes], V:,, karyo.cytic and erythrocytic lineages share specific tran 36. Ishibashi T, Koziol JA ' Burstein SA (1987) H uman recombi- naling pathways in one case (Mpt/erythroid progenitors) Hong P, Sprugel KH, Baily MC, scnptwn factors. Nature 344:447 cause only proliferative effects, and in the second (Mpl+/CFU Forstrom J (1995) Thrombopoietin expands erythroid nant erythropoietin promotes differentiation of murine Mk), induce both proliferative and differentiative effects. Per genitors, increases red cell production and enhances Ih~e JN (1995) Cytokine receptor signalling. Nature megakaryocytes in vitro. J Clin Invest 79:286 37 I :591 haps the different regimen of transcriptional factors present throid recovery following myelosuppressive 37. McDonal? TP, Cottrell MB, Clift RE, Cullen WC, Lin F-K Papayannopoulou T, Nakamoto B, Kurachi S, Tweeddale (1987) Hrgh doses of recombinant erythropoietin stimu in these two types of progenitors dictates the signaling path- Clin Invest 96:1683 M, Messner H (1988) Surface antigenic profile and globin late _Platelet production in mice. Exp Hematol 15:719 ways leading to these changes. 13. Fib be WE, Heemskerk DPM, Laterveer L, Pruijt JFM, D, Kaushansky K, Willemze R (1995) Accelerated phenoty~e ~f two new human erythroleukemia lines: 38. Sullrvan PS, Jackson CW, McDonald TP (1995) Castration Charactenzatwn and interpretations. Blood 72:1029 decreases thrombocytopoiesis and testosterone restores References tution of platelets and erythrocytes after syngeneic Sato T, Fuse A, Eguchi M, Hayashi Y Sugr'ta K N k platelet production in castrated Balb/c mice: Evidence that 1. Ogawa M (1993) Differentiation and proliferation of plantation of bone marrow cells derived from ,. , , a azawa S, Mmato K, Shima Y, Komori I, Sunami S Ok' y testosterone acts on a bipotential hematopoietic precursor hemopoietic stem cells. Blood 81:2844 bopoietin pretreated donor mice. Blood 86:3308 k ,. H ( , rmoto , Na ·aJrma 1987) Establishment and characterization of cell. J Lab Clin Med 125:326 2. de Sauvage FJ, Hass PE, Spencer SD, Malloy BE, Gurney 14. Fraser JK, Tan AS, Lin F-K, Berridge MV (1989) AL, Spencer SA, Darbonnie WC, Henzel WJ, Wong SC, of a specific high-affinity binding site for ne1mopoletiil a megakaryoblastic cell line (CMK) from a Down's syn 39. Kobayashi M, Laver JH, Kato T, Miyazaki H Ogawa M drome patient with acute megakaryoblastic leukemia. Ex (1995) Recombinant human thrombopoietin (Mplligand) Kuang WJ, Oles KJ, Hultgren B, Solbert LA, Goedde! DV, rat and mouse megakaryocytes. Exp Hematol17:10 Hematol15:495 p enhances proliferation of erythroid progenitors Blood Eaton DL (1994) Stimulation of megakaryocytopoiesis and 15. Papayannopoulou T, Brice M, Blau T (1993) Kit Chiba S, Takaku F, Tange T, Shibuya K, Misawa C, Sasaki 86:2494 . thrombopoiesis by the c-Mplligand. Nature 369:533 synergy with interleukin-3 amplifies the Mlyagawa K, Yazaki Y, Hirai H (1991) Establishment 40. Debili N, Issaad C, Masse JM Guichard J Katz A B t 3. Lok S, Kaushansky K, Holly RD, Kuijper JL, Lofton-Day CE, independent, globin-synthesizing progeny of G · . ' , , re on- erythroid differentiation of a cytokine-dependent onus J, Vamchenker W (1992) Expression of CD34 and Oort P], Grant FJ, Heipel MD, Burhead SK, Kramer JM, Bell human burst-forming units-erythroid in suspension leukemic cell line F-36: A parental line requiring platel~t ~lycoproteins during human megakaryocytic dif LA, Sprecher CA, Blumberg H, johnson R, Prunkard D, tures: Physiological implications. Blood 81:299 cyte-macrophage colony-stimulating factor or ferentiatiOn. Blood 80:3022 Ching AFT, Mathewes SL, Bailey MC, Forstrom JW, Buddie 16. Das Gupta A, Samoszuk M, Papayannopoulou T, -3, and a subline requiring erythropoietin 41. Nicola NA, Johnson GR (1981) The production of commit MM, Osborn SG, Evans SJ, Sheppard PO, Presnell SR, toyannopoulos G (1985) SFL 23.6: A monoclonal 78:2261 . t~d hematopoietic colony-forming cells from multipoten O'Hara PJ, Hagen FS, Roth GJ, Foster DC (1994) Murine reactive with CFU-E, erythroblasts, and "r"cth•·rw• thrombopoietin: Expression cloning, eDNA sequence and DA, Gumucio DL, Brodsky I (1991) Granulocyte tial precursor cells in vivo. Blood 60:1019 Blood 66:522 uu'"'"w~•e c~lony-~timulating factor-dependent growth 42. McLeod DL, Shreeve MM, Axelrad AA (1980) Chromo stimulation of platelet production in vivo. Nature 369:565 17. Andrews RG, Singer JW, Bernstein ID (1986) ~rythropmetm-mduced differentiation of a human some marker evidence for the biopotentiality of BFU-E 4. Wendling F, Maraskovsky E, Debili N, Florindo C, Teepe antibody 12.8 recognizes a 115-kd molecule lme MB-02. Blood 78:2860 Blood 56:318 · M, Tieux M, Methia N, Breton-Gorius J, Cosman D, both unipotent and multipotent hematopoietic M, Morishima Y, Ohno R, Kato Y, Hirabayashi N, 43. Nishi N, Nakahata T, Koike K, Takagi M, Nagamura K, Vainchenker W (1994) The Mplligand is a humoral regu forming cells and their precursors. Blood 67:842 H, Sato H (1985) Establishment of a novel human Akabane T (199_0) Induction of mixed erythroid-mega lator of megakaryocytopoiesis. Nature 369:571 18. McEver RP, Baezinger LN, Majerus PW (1980) . blastic leukemia cell line ' MEG-01, wr'th posr-. karyoc~te colomes and bipotential blast cell colonies by 5. Miyazaki H, Kato T, Ogami K, Iwamatsu A, Shimada Y, and quantitation of the platelet membrane Ph Jladelphia chromosome. Blood 66:1384 recombmant human erythropoietin in serum-free culture Souma Y, Akahori H, Horie K, Kokubio A, Kudo Y, Maeda deficient in thrombasthenia using a monoclonal N, Yamamoto M, Fujita H, Miwa A, Hatake K, Blood 76:1330 · E, Kawamura K, Sudo T (1994) Isolation and cloning of a rna antibody. J Clin Invest 66:1311 T, Oka~o H, Katsube T, Fukumaki Y, Sassa S, Miura y 44. Bellucci S, Han ZC, Pidard D, Caen JP (1992) Identification novel human thrombopoietin factor. Exp Hematol 22:838 19. Stamatoyannopoulos G, Farquhar M, Lindsley D, Establishment and characterization of an erythro of a n~rmal human bone marrow cell population co 6. Bartley TD, Bogenberger J, Hunt P, Li YS, Lu HS, Martin F, Papayannopoulou T, Nute PI (1983) Monoclonal -dependent subline, UT-7 /Epo, derived from expressmg megakaryocytic and erythroid markers in cul Chang MS, Sarna! B, Nichol JL, SwiftS, Johnson MJ, Hsu ies specific for globin chains. Blood 61:530 leukemia cell lines, UT-7. Blood 82:456 ture. Eur J Haematol 48:259 RY, Parker VP, Suggs S, Skrine JD, Merewether LA, Logston 20. Kaushansky K, Broudy VC, LinN, Jorgensen MJ, T, T~nge T, Terasawa T, Chiba T, Kuwaki T, 45. Vannucchi AM, Paoletti F, Grossi A (1994) A common C, Hsu E, Hokom MM, Hornkohl A, Choi E, Pangelinan J, Fox N, Zucker-Franklin D, Lofton-Day C (1995) K, Prao Y-F, Miyazono K, Urabe A, Takaku F megakaryocytic and erythrocytic precursor in murine ery M, Sun Y, Mar V, McNinch], Simonet L, Jacobsen F, Xie bopoietin, the Mpl ligand, is essential for full Establishment and characterization of a unique throleukemia (Friend) cells? Exp Hematol 22:110 C, Shutter J, Chute H, Basu R, Selander L, Trollinger D, cyte development. Proc Natl Acad Sci USA 92:3234 cell line that proliferates dependently on GM-CSF 46. McDonald TP, Clift RE, Cottrell MB (1992) Large, chronic Sieu L, Padilla D, Trail G, Elliott G, Izumi R, Covey T, 21. Mazur EM, South K (1985) Human megakaryocyte or erythropoietin. J Cell Physiol 140:323 ' doses of erythropoietin cause thrombocytopenia in mice Crouse J, Garcia A, Xu W, Del Castillo J, Biron J, Cole S, stimulating factor in sera from aplastic dogs: Le Roux D, Roullot V, Schweitzer A, Berthier R, Blood 80:352 . Hu MCT, Pacifici R, Pouting I, Saris C, Wen D, Yung YP, cation, characterization, and determination of Lin H, Bosselman RA (1994) Identification and cloning of poietic cell lineage specificity. Exp Hematol 13: a megakaryocyte growth and development factor that is a 22. Papayannopoulou Th, Brice M, Kaushansky K ligand for the cytokine receptor Mpl. Cell 77:1117 influence of Mpl-ligand on the development 7. Kaushansky K (1995) Thrombopoietin: The primary regu karyocytes from CD34+ cells isolated from ' lator of platelet production. Blood 6:419 peripheral blood and cord blood. Blood 84:324 8. Broudy VC, Kaushansky K (1995) Thrombopoietin, the c 23. Lansdorp PM, Sutherland HJ, Eaves CJ (1990) mpl ligand, is a major regulator of platelet production. J expression of CD45 isoforms on functional Leuk Biol57:719 tions of CD34+ hemopoietic cells from human 9. Gurney AL, Carver-Moore K, de Sauvage FJ, Moore MW row. J Exp Med 172:363 (1994) Thrombocytopenia in c-mpl-deficient mice. Science 24. Bender JG, Unverzagt K, Walker DE, Lee 265:1445 Williams S, Van Epps DE (1994) Phenotypic 10. de Sauvage FJ, Luoh S, Carver-Moore K, Ryan A, Dowd M, characterization of CD34+ cells from normal Eaton DL, Moore MW (1995) Deficiencies in early and late marrow, cord blood, peripheral blood, and stages of megakaryocytopoiesis in Tpo-KO mice. Blood peripheral blood from patients undergoing 84:255 [abstr] cell transplantation. Clin Immunol 11. Broudy VC, Lin NL, Kaushansky K (1995) Thrombopoietin