From www.bloodjournal.org by guest on December 3, 2014. For personal use only. 1996 88: 1284-1296 Characterization of a bipotent erythro-megakaryocytic progenitor in human bone marrow N Debili, L Coulombel, L Croisille, A Katz, J Guichard, J Breton-Gorius and W Vainchenker Updated information and services can be found at: http://www.bloodjournal.org/content/88/4/1284.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved. From www.bloodjournal.org by guest on December 3, 2014. For personal use only. Characterization of a Bipotent Erythro-Megakaryocytic Progenitor i n Human Bone Marrow By Najet Debili, Laure Coulombel, Laure Croisille, Andre Katz, Josette Guichard, Janine Breton-Gorius, and William Vainchenker The aim of the present study wast o determine if the human ficiency of CFU-MK and ofBFU-E/MK progenitors when comerythroid (E)and megakaryocytic (MK)lineages were closely bined with SCF, IL-3. and Epo. In contrast, when these cullinked to the existence of a bipotent burst-forming unit (BFU) tureswereperformed in the presence of 30% fetal calf E/MK progenitor.In methylcellulose cultures, BFU-E/MK colserum, no BFU-E/MK colonies were observed irrespective onies were observed at day 12 and closely resembled mature of the combination of growth factors used, including the component was BFU-E with the exception that the erythroid presence of MGDF; however, inclusion of the MS-5 cell line surrounded by MK. These colonies were quitedifferent from restored the growth of this bipotent progenitor. In contrast, the colony forming unit (CFU)-GEMM-derived colonies, in cultures performed in the presence of human normal or which were composed of a larger number of erythroblasts aplastic plasma, MS-5 had only a slight effect on the cloning and whichdeveloped later in culture. The existence of these efficiency but improved MKcytoplasmic maturation and MK bilineage colonies composed of 100 t o 1,000 erythroblasts size, suggesting that the maineffect of MS-5 is t o diminish intermingled with a few MK and without granulocytic cells the inhibitoryeffect of the fetalcalf serum on theMK differwas confirmed by theplasma clot technique and immunoentiation. The clonal origin of bipotent BFU-E/MK colonies alkaline phosphatase labeling of the MK. To investigate if was demonstrated in liquid culture of single CD34' CD38'OW this bipotent progenitor belonged t o t h e compartment of cells by immunophenotyping individual clones. At day 12, primitive progenitors, CD34' marrow cells were subfraction30% of the clones contained erythroblasts (glycophorin A') ated according t o expression of the CD38 antigen. The bipoand some MK (CD41') without granulocytes ( G ) or macrotent BFU-E/MK progenitor as well as a large fraction of MK phages (M) (CD14' and CD15'). A t day 20, clones containing in the CD34+ CD38' or in theCD34' progenitors were found erythroblasts and MK were rare (5%). In contrast multilinCD38- cell fractions. Growth of this bipotent BFU-E/MK proeage clones could be frequently detected at this time withgenitor required the combination of stem cell factor (SCF), out passage from BFU-E/MK clones at day 12 t o GEMM at interleukin-3 (IL-3). and Epo in serum free conditions. Addiday 20. These resutts suggest that a bipotent BFU-E/MK tion of IL-6 had only a marginal effect, whereas megakaryoprogenitor may bea nonrandom step in the hierarchical decyte growth and development factor (MGDF) was not an velopment of stem cells. absolute requirement, but slightly increased the plating efC 7996 by The American Society of Hematology. T HE HEMATOPOIETIC system is composed of heterogenous populations of cells that have been schematically divided into three compartments,' ie, a stem cell,a progenitor cell, and a terminally differentiating cell compartment. Regulation of cell proliferation and differentiation in the progenitor compartmentis mainly controlledby identified cytokines, but molecular mechanisms involved in the commitment of a pluripotent stem cell toward one celllineage are presently unknown. Several lines of evidence, most of them based on the analysisof thecomposition of colonyforming unit (CFU)-Sor of the progeny of paired daughter suggest that decisions at the level of early stem cells are stochastic. However, whether the restriction of stem cells potential occurs progressivelythrough the production of intermediate oligopotent progenitors with all the various combinations of potentialities or only after one division with the generation of only monopotent progenitors is still Clonal From INSERM U362, Insritut Gustave Roussy, Villqjuff; and INSERM (191, Hopital Henri Mondor, CrPteil, France. Submitted September IS. 199s; accepted April I I , 1996. Supported by grants from the Institut National de la Sante' et de la Recherche Me'dicale, Association de la recherche contre le cancer. Ligue nationate conare le cancer, and Electricif6 de France. Addressreprintrequests to NajetDebili,PhD, INSERM U.162. Institut Gustave Roussv, Villejuih 94805, France. The publication costs of this article were defrayedin part by page chargepayment. This article must therefore he herebymarked "advertisement" in accordance with 18 U.S.C. section 1734 soiel~to indicate this fact. 0 1996 by The American Society of Hematolog?. 0006-4971/96/8804-0017$3.00/0 1284 transfer experiments have demonstrated that a bipotent neutrophWmacrophage progenitor exists that retains hiresponsiveness toboth granulocyte macrophagecolony stimulating factor (GM-CSF) and M-CSF.7 Theexistence of such a tight linkage between two celllineagescould be anexception along the hematopoietic differentiation pathway, but it cannot be excluded that other types of bipotent progenitors also exist. For several years, numerous similarities between the erythroid (E) and megakaryocytic (MK) celllines have been emphasized. In humans, almost all leukemiccelllines described as erythroleukemic or megakaryoblastic express Eand MK-specific genes and this dual expression is found in the same cell.'" Similar observations have also been made in some acute leukemias."' Indeed, it has been shown that the regulation of E- and MK-specific genes share many features""' and transgenicmice expressing thymidinekinase gene under the control of the GPIIb promoter have defective erythropoiesis and megakaryocytopoiesis." It is thus possible that restriction toward the E or the MK pathways occurs relatively late in the hematopoietic hierarchy and that a bipotent BFU-E/MK can be identified.15 Mixed E-MK colonies have been described by several investigators in both humans and mice,"-"' but no detailedanalysis of their properties have beenprovided. The present experiments used clonogenic assays and single cell cultures to document the existence of human bone marrow cells able to generateonly erythroblasts and megakaryocytes, to determine their phenotype, relative frequency, andoptimal growth requirements. This burst forming unit (BFU)-E/MK segregated in the CD34'/CD38'"" cell fraction, required 12 days to differentiate and produced exclusively erythroblasts and megakaryocytes. Blood, Vol 88, NO 4 (August 151, 1996: pp 1284-1296 From www.bloodjournal.org by guest on December 3, 2014. For personal use only. BIPOTENTERYTHRO-MEGAKARYOCYTICPROGENITOR 1285 HPCA-2 (CD34) and FITC-IOB6 (CD38). Mer one washing, cells were suspended in IMDM at a concentration of 5 X l@cells/&and Bone Marrow Cells separated by cell sorting. In other experiments, triple staining with RBone marrow was obtained from patients undergoing hip surgery PE-HPCA-2,FITC-anti-CD38,andincubationwithHoechst33342 after informed consent was obtainedin accordance with the institudye (Sigma ata concentration of 5 pg/mL for 90minutes at 37OC’’ was tional guidelines of the Committee on Human Investigation. Cells performed to study the cell cycle status of hematopoietic progenitors. were collected by vigorous shaking of bone fragments in Iscove’s Cells were sorted on anODAM,ATC 3000” cell sorter(ODAM/ modified Dulbecco’s medium (IMDM, GIBCO, Paisley, Scotland), Bruker, Wissembourg, France) or a FacsVantage (Becton Dickinson) supplemented with 100 pg/& of deoxyribonuclease (DNase t y p equipped with two argon ion lasers (INNOVA 70-4 and 90-5; CoherI; Sigma, St Louis, MO), centrifuged once, counted, and separated ent Radiation, Palo Alto, CA) tuned to 488 and 360 nm, respectively, on Ficoll-Hypaque (Seromed, Berlin,Germany).Light-density and operating at 500 mW. Amorphologicgateincluding all the (<1.077 g/mL) marrow cells (LDMC) were recovered and after CD34’ cells wasdeterminedontwo-parameterhistograms(side washing were used either for isolation of CD34+ cellsby the immuof thecellvolume scatter[SSC]versuselectricmeasurement nomagnetic bead technique or flow cytometry. [ODAM] or the forward Scatter [FSC, FacsVantage]). Compensation for two-color labeled samples wasupsetwithsingle stained samples. Negativity for the CD38 among the CD34+ cells was determined Antibodies using control cells labeled with the PE-HPCA-2 and an irrelevant Fl’K-a~~ti-ClXl (anti-GpIIbm Immunotech,Luminy, Fran~e), IgGl monoclonal antibody (MoAb). FITC-anti-CD61 (GpIUa; Dako, Glostrup, Denmark), FITC-antiFor limiting dilution experiments, CD34+ CD38”” cells were diglycophorin A (GPA) (Dako), R-PE anti-GPA (Immunotech), FITCrectly sorted into 96-well tissue culture plates (Fklcon, France) with anti-CD15(Lewis”,granulocyticandmonocyticdifferentiation, an automatic cloning device unit. Dako), FITC anti-CD38 (IOB6) (Immunotech), FITC-CDllb (Mac1, granulocytic and monocytic differentiation, Immunotech), FITCAssessment of Hematopoietic Progenitor Cells anti-HLA-DR (Immunotech), R-PE anti-CD41 (Dako), R-PEHPCA-2 (CD34; Becton Dickinson, Mountain View, CA), and RQuantitationofclonogenicprogenitorsinsemi-solidassays. EryPE anti-CD14 (Gp55, granulocytic and monocytic differentiation, throid(CFU-Eand matureandimmatureBFU-E),granulocytic Becton Dickinson) were used either for cell sorting or analysis by (CFU-GM), megakaryocytic (CFU-MK), and mixed(CFU-GE“) flow cytometry. The unconjugated antibody Y2/51 (anti-CD61) was progenitors were quantified using previously described methylcellua generous gift from Dr D. Mason (Oxford, UK). lose assays?’ Each cellular fraction selected was plated at a concentration of 0.5 to 2 X lo3 cells/mL of complete methylcellulose meIsolation of CD34 by the Immunomagnetic Bead dium (0.8% methylcellulose in IMDM, 30% fetal calf serum FCS], 1% deionizedbovineserumalbumin PSA] and iO-4 m o m 0Technique mercaptoethanol). Cultures were also performed in the presence of CD34+ cells were recovered from LDMC (2 X 10’ cells) by the sera from aplastic patients or in serum-free medium as previously immunomagnetic bead technique using the Dynal kit (Dynabeads rep~rted.’~ Briefly,themediumcontained 1.5%deionized senim M450 CD34) according to the manufacturer’s instructions (Dynal, albumin (Cohn fractionV Sigma ChemicalCO),iron saturated huOslo, Norway). CD34+ cells were detached from the beads using man transferrin (300p g / a , Sigma), calcium chloride (28 ng/&), the DETACHa BEAD solution (Dynal). a mixture of sonicated lipids, 7.5X lO-4 mom, cy-thioglycerol, 100 ng/mL insulin, and 0.8% chemically pure methylcellulose in IMDM. Cell Sorting The lipid mixture was produced by sonicating 7.8 mg cholesterol, Subpopulations of CD34+ cells were sorted according to their expres- 6.14 mg oleic acid, and 7.4 mg dipalmitoyl lecithin (all obtained sion of the CD38 antigen starting from LDMC or magnetic bead isolated fromSigma)in 10 mL ofIMDM(withoutsodiumbicarbonate) CD34+cells.Cellswereincubatedsimultaneouslywith the R-PEcontaining 1%albumin. Aliquots (20 pUmL) of this mixture were MATERIALS AND METHODS Fig 1. BFU-E/MK colonies in methylcellulose and plasma clot cultures. (A) A typical BFU-EIMK colony observed with aninverted microscope in cultures containing 30% FCS and MS-5 cells. This type of colony contained erythroblasts and megakaryocytes. The other cells observed on the figure correspond to MS-5 cells. (B) A similar colony in plasma clot. MK appeared in pink color after immunolabeling with an anti-CD61 MoAb. (C) A typical BFU-E/MK colony in cultures where FCS has been replaced by AP. From www.bloodjournal.org by guest on December 3, 2014. For personal use only. 1286 DEBlLl ET AL Table 1. Numbers of Hematopoietic Progenitor Cells in the Different Cell Fractions Sorted According to Their Expression of CD34 and CD38 Antigens and Plated in Methylcellulose Colony Assays With Either FCS or AP Progenitor Cells per 1,000 Cells ~~ ~ imBFU-E mBFU-E Pure Additive Serum CD34' CD34*/CD38CD34AlCD38"CD34'/CD38* ~~ MS-5 Total Pure Erythroid E/MK Total Erythroid + 34 32 12 12 37 46 121 115 33 31 8 6 30 32 121 115 1 1 4 6 7 14 0 0 6 7 10 0 20 22 12 1 5 6 2 0 18 20 12 1 + AP FCS AP FCS AP FCS AP + + + + + + CFU-GEMM MK col 1 3 8 16 8 10 1 0 CFU-GM 47 41 66 46 95 46 75 76 One thousand cells of the different subfractions of CD34' cells were plated in methycellulose colony assays in the presence of either 30% FCS or 10% AP and SCF + IL-3 + Epo a s growth factorMS-5 cells were added at the concentration of 10,000 cells per dish.ElMK were enumerated in the mBFU-E and scored at day 10 to 12 and CFU-GEMM were enumerated in the iBFU and scored at days 20 to 25. Results are from one experiment. Numbers refer to the meanof two counts obtained in two separate plates. added to the culture medium. Colony-stimulating factors were pro10% vided either as agar-leukocyte conditioned medium (agar-LCM: vollvol) or as recombinant human (rh) growth factors: rhStem cell factor (SCF) (S0 ng/mL). G-CSF (20 nglmL). and megakaryocyte growthanddevelopmentfactor (MGDF) (10 nglmL)alsocalled Mpl-ligand (Mpl-L), or thrombopoietin (TPO) (all three kindly provided by AMGEN, Thousand Oaks, CA), rhIL-6(100 U/mL). rhIL3 (100 UlmL), rhGM-CSF (2 ng/mL) (kindly provided by Immunex. I UlmL;AmerSeattle, WA) andhumanerythropoietin(rhEpo: sham). Petri disheswerecultured at 37°C in a S 8 C0?-9S% air humidified incubator. Hematopoietic progenitors were scored at least twice at days 12 to 14 and 20 to 25. Cultures were also performed by the plasma clot technique using eitherserumfromaplasticpatients (AP)orFCSorserumfrom platelet poor plasma (PPP) and a combination of cytokines." When MGDF became available, cultures were performed by the serumfree fibrin clot.'5 Briefly, ingredients for serum-free cultures were the same as for methylcellulose cultures with the bovine citrated plasmabeingreplaced by bovineplasma fibrinogen ( I mglmL. (6 Sigma), 0.01 m o l L e aminocaproicacid,andhorsethrombin mUlmL,Stago).Cultureswereincubatedasdescribedaboveand 0 3834+ 38+ 38+ Exp 1 BFU-E studied after 12 days. Colonies were quantifiedby an indirect immunophosphatasealkalinelabelingtechniqueusingananti-GplIIa MoAb (CDhl, Y2-S I ). Dishes were scanned completely under an inverted microscope at 40 or 100 X magnification. We have previously reported that addition of murine stromal cells from the MS-S cell line to hematopoietic colony assays selectively stimulates the expression of very early clonogenic progenitor cells (immature RFU-E. CFU-GEMM) in synergy with growth factors.'" The MS-S stromal cell line" was kindly provided by K. Mori and maintained in CYMEMsupplemented with 10% FCS. Its growthpromoting effect was assessed by adding 10.000 MS-S cells to both methylcellulose and plasma clot assays. Single cell cdnrres. In limiting dilution experiments. individual CD34* CD38'"" weresortedinto96-wellplatesusingserum-free medium and a combination of seven cytokines (SCF. IL-3. Epo. IL6 , G-CSF, GM-CSF. and MGDF) or of AP and six cytokines (all except MGDF). Plates were examined at day I I to 13. day 18 to 20. and later after incubation at 37°C in an air atmosphere supplemented with S 8 CO:. Individual clones were cytocentrifuged or divided into two parts for flow cytometric analysis. The first half was doubly labeled with BFU-EIMK CFU-MK 34 + 38+ 385 Exp 2 38 Fig 2. Phenotype of the BFU-E/MK progenitor. This figure represents the results of two representative experiments in which CD34' cells have been subfractionated in CD38'. CD38'. and CD38- subfractions. Cultures were performed in plasma clotin triplicate in the presence ofAP and the combination of SCF, IL-3, and Epo. MK were identified as in Fig 1B. CD38' or CD38- cells (lo3)and CD38' cells (5 x 10') were plated. Results are expressed a s t h enumber of colonies per lo3 plated cells. From www.bloodjournal.org by guest on December 3, 2014. For personal use only. PROGENITOR BIPOTENT ERYTHRO-MEGAKARYOCYTIC 1287 IBFU-E 1 CFU-MK BFU-EIMK loo Fig 3. Kineticsof development ofBFU-E,CFUMK, and BFU-EIMK colonies from different CD34' cell subfractions. Experiments were designed as in Fig 2 and cultures were studied at day 7, 12, and 18. cell tractton W+ RESULTS Ident$cation qf a Bipotent Enthroin-Megnkonoc?.tic (BFU-ELWK) Progenitor Cell In preliminary experimentsaimed at optimizingculture conditions for the growth ofmarrow-derived CD34-/CD38primitive progenitors in methylcellulose, we observed that theadditionofthemurine MS-S stromal cell linetothe Day 20 A% *% / Fig 4. Analysis of the cell compositionof individualclones derived from CD34+ CD38~ow at days 11 to 13 of culture and at day 2o of cultures. The results presented are those of five independent experiments that have been pooled representing the analysis by flow cytometry of 93 and 91 clones,respectively. Culture conditions were serum-free plus a combination of SCF, IL-3, Epo, GM-CSF, G-CSF, IL6, and MGDF in two experiments, and in three others AP plus a combination ofSCF. IL-3,- EDO. .. E;. [D) . . . GM-CSF,G-CSF. and I L - ~ (A) EIMK; (m) EIMKIGM; (E)GMIMK; GM; [.).€/GM. (c) 38f day 7 R-PEanti-CD41aandFlTCanti-GPAMoAborFlTCanti-CD41 and R-PE anti-GPA. The second half was labeled with R-PE antiCD14 and FlTC anti-CD15 or FlTC anti-CD1 lb. Cells were analyzed on a FACS sort. Ulrrustnrcrrrrul srrrdies. Cultured cells were studied by electron microscopy (EM). They were washed twice in Hank's medium at 4"C,fixedwith 1.2S% glutaraldehyde in Gey's buffer for IO minutes." washed, and incubated in diaminobenzidinemedium.'" Cells were then post-fixed with osmium tetroxide. dehydrated.and embedded in epon.Thinsectionswereexamined with a Philips CM IO electron microscope (Philips. Eindoven, Netherlands) after lead citrate staining. Day 13 38+ 38- 34+ 38* 38+ day 12 38- 34+ 38+ 38f 38- day 18 assay synergized with added human cytokines (see below) to promote the development of primitive progenitors." Of note. we observed that at early time points (day 12) during the culture a fraction of small (<l00 cells) hemoglobinized colonies containedMK (Fig 1 A). The erythroid compartment of these colonies was similar in size and time required for maturation to that of mature BFU-E-derived colonies.3" Some typical colonies were studiedby morphology and only contained E and MK cells. These progenitors were easily distinguishedfromstandardCFU-GEMMandprimitive BFU-E both by the size of the colonies generated and by the time (> 18 to 20 days) required for their differentiation. Theseprogenitors were also identifiedinplasma-clot assays(FigIB)andthepresence of MK in these mixed colonieswas confirmed by immunolabeling withan antiCD41 or an anti-CD61MoAb. Usually 2 to 30 MKwere counted together with 500 to 1.000 erythroblasts. No other cell type was detected by morphologic criteria. These colonies were not a simple overlapof two types of colonies since they were still observed at low cell concentration where even a single colony was grown. This result was confirmed by single cell assay (see below). These progenitors were designated BFU-E/MK. In another series of experiments, we precisely defined the properties of this progenitor cell with respect to its CD34/ also tried CD38 phenotype and response to cytokines. We to define its place in the hierarchy of hematopoietic progenitor cells by investigating if this bilineage association was purely random or reflects a preferential association between E and MK differentiation. Phenotype of the Entkroin-Megnknnoc?.tic Progenitor Cell Expression of CD34 and CD38 antigens on the BFU-E/ MK progenitor was doneby plating different subpopulations of marrow cells in plasma clot and methycellulose assays. When bone marrow cells were simultaneously labeled with anti-CD34 and anti-CD38 antibodies. two discrete DoDulations were clearly identified among CD34' cells: cells 1 L brightly stained with CD38 antibodies (referredto as CD38' Cells). whichrepresent 70% of the CD34'cellpopulation, andcellsexpressinglower levelsofthe CD38 antigen From www.bloodjournal.org by guest on December 3, 2014. For personal use only. DEBlLl ET AL 1288 Fig 5. Flow cytometry analysis of clonescontaining erythroblasts and MK. Dot plot analysis oftwo typical BFU-E/MK clones (A and B). These clonescontained a great majority of GPA' cells and a minority ofCD41'cells. The CD41'cells have very high forward and side scatter properties that distinguish them from erythroblasts. More than 99% of the cells were labeled by both antibodies. CD15 and CD14 labeling was negative on these two colonies. Two isotypes controls llgGl FlTC and lgGl PE) are shown, one on a clone negative for the CD41 marker (C) and the other onan immature erythroid clone expressing low level of GPA (D). (CD38'""). This last subpopulation was very heterogeneous including CD38 negative (CD38-, 10% of the CD34cells) cells that fell within the limits defined by control IgG, and CD38' cells (20% of the CD34' cells), although the limit between these two populations was empirical. These three subpopulations, CD34'/CD38', CD34'/CD38', and CD34'/CD38-, were sorted and analyzed for their content inBFU-EIMK progenitors, simultaneously in plasma clot and methylcellulose assays. As shown in Table 1 and Fig 2, BFU-EIMK identified in plasma clot and methylcellulose assays were present in the CD34'/CD38' or CD34'/CD38- fraction and were not found in the CD38' fraction. This contrasted with the distribution of mature BFU-E, 90% of which were detected in the CD38' fraction, an observation that fits with previous observations showing that expression of CD38 discriminates between mature (CD38-) and immature (CD38'""') progenitor cells." If one considers as a single category colonies hemoglobinized at days 10 to 12, a significant proportion of these (19% to 60%) contained MK, whereas mature BFUE-derived colonies scored in assays initiated with CD38' cells were exclusively erythroid. There was some variability in the frequency of the BFU-EIMK-derived colonies among CD38- and CD38' cells. In some, the BFU-E/MK progenitors were predominantly in the CD38". whereas in others they were found in the CD38- cell fraction (Fig 2). Although immature BFU-E (imBFU-E) were enriched in the CD.38and CD38' cell fraction, approximately 40% of them were contained in the CD38- cell fraction. Therefore, taking into account only the expression of the CD38 antigen, BFU-E/ MK appears more immature than the imBFU-E. In plasma clot and methylcellulose, MK progenitors were also enriched in the CD34' CD38'""' cell population with significant heter- ogeneity in the size of the colonies (from 3 tomorethan 100 MK). In the CD34' CD38- cell fraction, 30% to 50% of the MK colonies were bipotent. In contrast, in the CD34' CD38' cell fraction MK progenitors gave rise to pure MK colonies. Kinetics of development of MK-containingprogenitors were next analyzed in plasma clot assays. As shown in Fig 3, the kineticsof BFU-WMK and CFU-MK were comparable. MK colonies were the first colonies to be recognized. and at day 7 of culture, MK colonies were almost the only type of hematopoietic colonies present with some colonies containing up to 50 MK. CFU-MK were counted at dayI2 and contained up to a few hundred MK. BFU-WMK-derived colonies developed with very similar kinetics: they were first detected at day 7 of culture and their number was maximum at about day 12. Althoughtheabsolutenumber of BFU-WMK-derived colonies was low, these mixedcolonies represented up to 60% of the total number of pooled MK and E colonies. At day 18 of culture, CFU-MK- andBFU-WMK-derivedcolonies werestillpresent but begantolyseandwere not detected after day 20. In contrast. large E colonies that included granulocytes, monocytes, and more rarelyM K were detected at this time. By day 18. in methylcellulose cultures, lysingBFUEIMK colonies and growing CFU-GEMM-derived colonies were observed in the same dish, but they clearly corresponded to two distinct types of colonies. Single Cell Cdtlrres The existence of a bipotent BFU-E/MK progenitor was clearly established in single cell cultures. Furthermore, this approach also allowed us to validate previous observations, which suggests thatevenlate in differentiation the E and MK programs are preferentially associated. CD34'/CD38'"" cells from five different bone marrows were directly sorted into wells of 96-well microtiter plates. After sorting, we checked that a single cell was present in more than 95% of the wells. Five hundred wells were initiated per experiment in IMDM supplemented in three experiments with 10% AP and a combination of six cytokines (SCF, Epo, IL-3, GMCSF, G-CSF, and IL-6) and in the other two in serum-free medium supplemented with the same cytokines plus MGDF. This combination of cytokines was chosen to ensure optimal differentiation in E, MK and granulo/macrophagic pathway. Cultures were studied after I O to 12, 18 to 20, and in two experiments after 30 days at 37°C 5% COz. Clones were selected that contained at least 100 cells and only those were analyzed. Those represented 8% 2 2% and 20% 1- 6% of the initial wells at days 12 and 20, respectively. In a preliminary experiment, individual clones were pipetted off, cytocentrifuged. and morphologically analyzed. However. this procedure resulted in partial loss of MK during cytocentrifugation, and in subsequent experiments clones were analyzed by flow cytometry. Each clone was divided into two equal cell fractions. Part of the cells was labeled simultaneously with antibodies against GPA and either CD61 or CD41 and the other half with antibodies directed against CD14 and either CD15 or CD1 1b. Under these conditions, at day 12. 6156 of the 93 analyzed clones contained some erythroblasts (ranging from 15% to 100% of the total population) and thus arose From www.bloodjournal.org by guest on December 3, 2014. For personal use only. BIPOTENT ERYTHRO-MEGAKARYOCYTIC PROGENITOR 1289 Table 2. Assessment of Different Progenitor Cell Types in Serum-Free Semi-Solid Clot Assay Supplemented With Different Combinations of Cytokines Progenitor Cells per 1,000 CD34'/CD38" Cytokines EFU-E CFU-MK EFU-E/MK CFU-GM G/MK - 32 2 2 - t 1.5 2 3 0.57 2 3.7 2 0.57 2 3.4 16.3 i 0.57 20 -c 3.6 17.3 11.3 2 2.5 13.327.6 t 4.5 2 2 17.6 t 3 0 - - MGDF E PO MGDF, Epo IL-3, EPO MGDF, IL-3, EPO SCF, Epo MGDF, 12 Epo, SCF IL-3, SCF, EPO MGDF, IL-3, SCF, EPO EPO, IL-3, SCF, IL-6 MGDF, IL-3, EPO, SCF, IL-6 11.3 G-CSF IL-3, IL-6, SCF, EPO, MGDF, IL-3, EPO, SCF, IL-6, G-CSF Cells 1.33 4.3 12.3 13.3 7.3 + 36.3 C 5 3.6 2 0.57 29.3 -t 16 1 2 1 37.6 t 1.66.6 15.3 z 0.57 13.3 25 t 6.5 2 2.5 17.6 t 1.5 12.3 i- 0.57 22.3 t 5 8 6.3 0.66 3 12.3 2 3 t 1.5 2 0.57 2 0 2 4.9 2 2 11.3 ? 3.7 1.6 2 6.6 13 t 4 20 2 3 0.66 2 0.57 2.3 2 1.1 0 2 1.5 6 5 1 12 2 1.1 7 2 3 12.6 2 1.5 5 2 1.7 14 4.5 + 2 0.57 0.6 2 0.57 + CD34+/CD38'OWcells (ie, CD38- CD3V cells)(1.000 per plate) were platedin serum-free semi-solid medium with the indicated combinations of cytokines. Colonies were scored at days 10 t o 12 after immunolabeling of the plates with anti-CD61 antibody as described (see Materials and Methods). Numbers represent the mean c SD of counts performed in three different plates. Results are from one experiment. from a progenitor capable of erythroid differentiation (Fig 4). Twenty-one percent of the clones (pooled experiments) contained only GPA+ cells, while 30% of the clones contained both GPA' cells and cells highly labeled with the antibodies against the CD61 or CD41 antigen (Fig 5). These cells positive for MK markers had high forward and side scatter properties characteristic of cultured MK, which allow easy discrimination between erythroblasts and MK on the basis of light scatter properties. In all these clones, GPA+ cells represent more than 85% and MK 1% to 8% of the events analyzed (Fig 5). None of the cells present in these clones expressed granulocytic markers (CD14 and CD15). Clones containing granulocytes andMK without erythroblasts or granulocytes and erythroblasts without MK were also observed but at a low frequency (3% and 4%, respectively). Since we selected only wells containing over 100 cells, pure small MK clones could not be detected by flow cytometry. However, these were detected by microscopic observation and their MK nature was further documented by CD41 immunolabeling (data not shown). At day 18 to 20 of culture, 20% of the total number of wells contained more than 100 cells and 56% of the 91 analyzed clones contained cells that expressed either CD14 or CD15 or both. A minority of the clones (8%) only contained GPA' cells. BFU-E/MK clones were also uncommon (5%) withveryfewMK (<2%). In contrast, clones containing GPA+, CD14+, CD15+,and CD41+ cells were much more frequent (22%) suggesting that they were derived from CFU-GEMM. These clones contained on average 5-fold more cells than those studied atday 12. Later in culture (around day 30), all the clones analyzed contained CD14' and CD15' cells but no GPA' or CD41+ cells. Two BFU-E/MK clones identified at day 12 were subsequently studied at day 18, and no granulocytic cells could be detected. Dejnition of Optimal Conditions for the Detection of BFU-E/MK Progenitors The BFU-E/MK requirement for cytokines was first assessed in plasma clot cultures, which are optimized for the expression of MK differentiation. Cultures were ended at days 10 to 12 and stained withan anti-CD61 MoAb as Table 3. Effect of the Addition of MS-5 Cells on the Growth of Different Types of Progenitor Cells in MethylcelluloseAssays Progenitor Cells per 1,000 CD34+/CD38'"" Cells mEFU-E iBFU-E Pure Additive A-LCM SCF + EPO + IL-3 + EPO n MS-5 Total Erythroid E/MK 3 5 5 6 - + - 34 2 17 27 5 7 18 2 7 34 t 17 21 2 4 18 7 0 6 2 1 t 23 2 7 18 2 5.5 5 2 1.5 + + 0 Total 3 +3 22 20 2 43.8 31 -c + 2 7 1.7 7 Pure Erythroid 13 2 5 0.2 2 1.7 20 2 4.5 CFU-GEMM 0 7 2 2 11 2 2.5 One thousand CD34'/CD38'OW (ie, CD38CD38' cells) were plated in methycellulose colony assays in the presence of either A-LCM + Epo of cells per dish.E/MK were enumerated or SCF IL-3 + Epo as growth factors and30% FCS. MS-5 cells were added at the concentration10.000 in the mBFU-E and scored at day 12 and CFU-GEMM in the imBFU-E and scored at days 20 t o 25. Duplicate plates were initiated in each condition. Numbers refer to the mean t SEM of counts obtained in the indicated number of experiments (n). + From www.bloodjournal.org by guest on December 3, 2014. For personal use only. DEBlLl ET A1 1290 Fig 6. From www.bloodjournal.org by guest on December 3, 2014. For personal use only. BIPOTENT ERYTHRO-MEGAKARYOCYTIC PROGENITOR 1291 Fig 6. Ultrastructural studies of an MK cultured from CD34' CD38- cells in liquid culture in the presence of MS-5 cells, a combination of SCF, IL-3, and AP. CD34' CD38" cells were grown in liquid culture in the presence of a confluent layer of MS-5. At day 9, a nearly pure MK population was present, whereas later in cultures (day 14 andafter1 other cell types were predominant. (AI A rosette with a large MK surrounded bytwo MS-5 cells. Note thatMK develops blebsin the cdntact with MS-5 cells. Magnification x 2,900. (6)Enlargementof the MK cytoplasm. There isa harmonious distribution of demarcationmembranesand a-granules. Magnification x 19,600. (ClZone of contact between MK and an MS-5 cell. MS-5 cells posess numerous large granules and cisternae of endoplasmic reticulum. The cell membrane extends its pseudopods between thelarge blebs of MK,which are always devoid of organelles. Note the tight contact between the twocells. Magnification x 7,700. described.32In a first set of experiments performed before the cloning of MGDF (MpI-L, TPO), we compared the effect of normal human and aplastic human plasma on the development of CD34+/CD38low (including both CD38- and CD38'). In the presence of normal plasma, the growth of BFU-E/MK and CFU-MK was optimum in the presence of the combination of SCF, IL-3, and Epo with 4 ? 1.4 BFUE N K per 1,000 cells, and 6 5 2.8 CFU-MK per 1,000 cells. Similar results were found with AP, but cloning efficiency was slightly higher ( 5 ? 1.4 BFU-E/MK per 1,000 cells, and 9.5 2 1.9 CFU-MK per 1,000 cells). In a second set of experiments, we tested the effect of MGDF in serum free conditions (Table 2). Best results were obtained when MGDF was added to SCF, IL-3, Epo with 13 to 17 BFU-E/ MK per 1,000 CD34+/CD3g1""(including CD38- + CD38') cells, and 25 to 27 CFU-MK, whereas only six BFU-E/MK progenitors were observed without MGDF. Withdrawal of either IL-3 or SCF even though high concentrations of MGDF were kept constant decreased the number of BFUE/MK progenitors, but not of CFU-MK, which fits well with the known requirement of either IL-3 or SCF for the proliferation of early E cells. Addition of IL-6 either to a combination of three (SCF, IL-3, Epo) or four (the same three + MGDF) did not further increase plating efficiency. Precise quantitation of the E compartment was hampered in plasma clots by the early termination required for MK quantification. Methylcellulose assays were also performed with CD34' CD38'"" cells in the presence of 30% FCS, a concentration that is optimum for E development but selectively inhibits MK d e ~ e l o p m e n t ? Addition ~.~~ of A-LCM or the combination of SCF, IL-3, and Epo and even MGDF did not neutralize this inhibition. In contrast, the addition of MS-5 cells tothe assay (independently of the cytokine combination) dramatically counteracted FCS effect both on CFU-MK and BFU-E/MK (Table 3 and Fig IC). Thus, on average eight CFU-MK were scored per 1,000 CD34+/CD38'"" cells (Table l). Most interestingly, even though the total number of small E colonies scored as mBFU-E in the CD38loWfraction did not change when MS-5 cells were added (Table 3), 20% to 25% of these now clearly included MK, which suggests that a proportion of E progenitors were in fact not restricted to the erythroid lineage. The observation that 90% of the mBFU-E are in the CD38+ fraction and generate only E cells independently of the presence of MS-5 cells reinforced the idea that theBFU-E/MK progenitor represents a unique step in the hierarchy of progenitors. However, expression of the MK potential requires specific conditions, and in the absence of MS-5 cells, this progenitor would be scored as a mature BFU-E only capable of differentiating along the E pathway. Of note is the fact that MS-5 cells also increased the size of MKS suggesting thattheymay also act on MK terminal differentiation (see EM studies below). In contrast to their lack of proliferative effect on small E colonies, the number andsize of which did notchange (Table 1 and ref 26), MS-5 cells stimulated the proliferation of immature BFU-E and CFU-GEMM as 20 to 30 of these progenitors were detected per 1,000 CD34+ CD38- cells, but almost none in the absence of MS-5 cells (Table 3). Similar results were observed in plasma clot, after immunolabeling. BFU-E/MK and pure MK colonies were absent when cultures contained 30% FCS. MK differentiation was observed when MS-5 cells were added. Ultrastructural studies confirmed the action of MS-5 cells in promoting terminal MK maturation. For these studies, purified CD34+/CD38Io"cells were cultured for 7 to 10 days in liquid medium in the presence of MS-5 cells plus AP with or without SCF and IL-3 and studied by EM. These cultures were compared with control cultures performed in the absence of MS-5. In 10 distinct experiments, MK differentiation occured in close contact with MS-5 (Fig 6). MK were generally surrounded by MS-5 in a rosette-like shape. In the areas of contact with MS-5. MK developed numerous blebs that were absent outside this zone. Contacts between the two membranes wereverytightbutwithout junction. In some rosettes, MS-5 cells extended their pseudopods between each bleb. The quality of maturation was equivalent to that observed in vivo with the regular distribution of demarcation membranes and a-granules. No such degree of maturity was observed in control cultures (without MS-5). Adhesion between MK and MS-5 cells occurred very early during maturation since small promegakaryoblasts with large nucleoli were associated with MS-S cells. No such intimate contacts were seenwith cells from other lineages, granulocyte or erythroblastic. Cell Cycle Status of the BFU-E/MK Progenitor Results from the experiments described above suggest that BFU-E/MK progenitors are more immature than the CD38+ E-restricted progenitors based on their lack of expression of CD38 and ability to differentiate into two lineages. A third indicator of primitiveness is quiescence.6 In this series of experiments, we determined the cell cycle status of BFU-E/MK by three-color labeling using R-PE anti-CD34 MoAb, FITC anti-CD38 MoAb, and Hoechst dye as shown in Fig 7A and B. In an attempt to increase the sensitivity of the procedure and detect subpopulations within the GO/GI population, which may correspond to more primitive populations, incubation with the Hoechst From www.bloodjournal.org by guest on December 3, 2014. For personal use only. DEBlLl ET AL 1292 A 40 1 5 $ 10 0 BFU-E C M 8 FlTc CN-MK BFU-EIMK Fig 8. Cell cycle status of BFU-EIMK progenitors. CD34'CD38'"" cells were subfractionated according t o the Hoechst staining in a Hoechst'"", GOIGl, and SIGZIM cell subfractions as shown in Fig 6. Cells were grown in fibrin clot in serum-free conditions using a combination of SCF, 11-3, Epo, and MGDF. Results are the number of colonies per 1,000 plated cells and are the average of one experiment made in triplicate. Two other experiments gave similar results. B $1 ss- 2 3 9 9 S/G2 I ' E: 0 0 220 433 m 800 1O M ) HoeBcht Fig 7. Hoechst 3342 staining of CD34'cells.CD34' cells were purified by the immunornagnetic bead technique (Dynal) and subsequentlylabeledby R-PE-anti-CD34, an FITC-anti-CD38, andthe Hoechst dye..(A) A dot plot analysis between the CD34 and CD38 labeling isshown. Only the CD34h"8hcells were analyzed for Hoechst staining and sorted into t w o cell fractions, CD38* and CD38'"". (B) A dot blotanalysis between theCD38 staining and the Hoechst labeling in the CD34highgate is shown. Three populations as shown in the histogram (C) can be observed according to the Hoechst staining: Hoechst'"", GOIG1, and SIGZIM.Of note, cycling cells are essentially in the CD38' cell fractions, whereas Hoechst'"" cells are in theCD38'"" cell fraction. stain was shortened, whereas usually 2 hours is required to reach the equilibrum. With this procedure, three CD34' populations were identified, Hoechst', which may identify more primitive progenitors,'5 Hoechst" (2n), and Hoechst" (S/G2/M) (Fig 7, B and C). There was a clear correlation between the expression of CD38 and the uptake of the Hoechst dye (Fig 7B). Among the CD38'""' subset, all cells were Hoechst' or Hoechst' and only 1% to 4% of the cells were in the Hoechst" (S/G2/M) fraction. In contrast, 10% to 15% of the CD38' cells was in the S/G2/ M phase. Since a similarly low proportion of CD38'"" cells was in the S/G2/M phase when the Hoechst staining period was prolonged for 2 hours, the number of cycling cells was not underestimated. In addition, this result is in agreement with a recent report using propidium iodide to stain DNA." Cell sorting experiments were held in which CD34' CD38- Hoechst'"", CD34' CD38- Hoechst' (GO/ GI), and CD34' CD38- Hoechst" (S/G2/M) cells were assayed for their contentin progenitors. The results showed that most BFU-E/MK progenitors were in the Hoechst' or Hoechst' fractions and only 0.32% and 0.95% of BFU-E/ MK and CFU-MK of the CD38'""' population were in the S/G2/M phase of the cell cycle, respectively. Cloning efficiency of BFU-E/MK was much higher in the CD34' CD38- GO/GI cell population than in the two other cell fractions (1.2% versus 0.28% and 0.25%) (Fig 8). Further functional analysis of these different Hoechst fractions for their content of other clonogenic progenitors of varying maturity showed that 20% of the mBFU-E contained in the CD38' cell fraction were in the S/G2/M phase of the cell cycle. In contrast, only 1.2% of the mBFU-E and less than 0.5% of the CFU-GEMM contained in the CD38- cell fraction were in S phase. DISCUSSION Observations of mixed' MK-E bursts in mice" and also in humans'"have provided the initial basis suggesting the From www.bloodjournal.org by guest on December 3, 2014. For personal use only. BIPOTENT ERYTHRO-MEGAKARYOCYTIC PROGENITOR existence of a bipotent progenitor restricted to both MK and E lineages. Further studies have suggested that divergence of both lineages was a late event in the hierarchy of stem cell differentiation4 although no direct evidence supported this hypothesis. Since then, considerable evidence in support of such a common pathway has been obtained from studies that showed that several transcription factors such as GATA1, Tal- 1 , and NFE-2 are shared by both lineages. l4,l6 Detailed data on the phenotype and growth properties of this bipotent progenitor are lacking partly because most of the studies were performed before the isolation of the different hematopoietic growth factors and the development of cell purification techniques. Since then further similarities in the response to Epo and MGDF of committed progenitors from both lineages and shared expression of Epo-R also strengthened the hypothesis. In this study we provide strong evidence that a bipotent MK-E burst exists in human marrow, which can be distinguished from its more mature progeny, CFUMK and mature BFU-E and also from its immature ancestor CFU-GEMM, by its phenotype, cell-cycle status, and cytokine response. If one assumes that colony size and time taken togenerate erythroblasts and mature MK both reflect the state of differentiation of the progenitor, BFU-E/MK appeared to becloser to mature BFU-E than to immature BFU-E or CFU-GEMM, based on the fact that mixed colonies were small containing at most 1,000 erythroblasts and a variable number ofMK from 2 to 30."' The number of BFU-E/MK was maximal after a period of 12 days in culture and colonies lysed a few days later. In contrast, 16 to 18 days must elapse before primitive BFU-E and CFU-GEMM can express their full potential and complete differentiation. If the kinetics of colony formation classifies BFU-E/MK as close to mBFU-E, they differ from these both by their phenotype and cell cycle status. In contrast to day 12 BFU-E, 90% of which express both CD34 and CD38 antigens, BFU-E/MKwere in the CD34' CD38'"" (either CD38' or CD38-) fraction and the great majority ( S O % ) of BFU-E/MK were in GO/G1 phase of the cell cycle, two properties thatusually characterize immature progenitors.".." Noteworthy, this was also true for a fraction of CFU-MK, therefore suggesting that CD38 may not be expressed by cells of the megakaryocytic lineage to the same extent as on other myeloid precursors. Definite proof that the potential of BFU-E/MK was restricted to E and MK lineages only was obtained in single cell experiments. The frequency of BFU-E/MK was estimated from the proportion of wells that contained more than 100 cells and only GPA' and CD41' cells after 12 days in culture. An estimated 2% to 3% of total wells initiated with CD34' CD38' contained BFU-E/MK based onthese criteria, which represented 0.3% to 1% of normal marrow CD34' cells. By comparison, the frequency of clones containing at least three lineages and thought to originate from standard CFU-GEMM was 4.4% in the CD34' CD38' and 1% in the initial CD34' cell fraction. Despite the fact that these numbers might be underestimated, as only clones containing >l00 cells were analyzed by flow cytometry at day 12, they were in agreement with frequencies of BFU-E/MK calculated from colony assays. Finally, it remains to be determined 1293 whether some lineage markers (especially CD41 as suggested by a recent report15) could be expressed on E/MK progenitors. Our preliminary results do not favor this hypothesis as the CD34'CD41'cell population was essentially enriched in MK progenitor^.^^ In vitro appraisal of the real potential of progenitors is hampered by two difficulties. The first pertains to the fact that specific culture conditions and cytokine combinations that optimally support the differentiation of progenitor cells committed to one lineage may be inhibitory for progenitor cells committed to other lineages. Thus, myeloidand B lymphoid differentiations in long-term cultures require conditions that are mutually exclusive, but this is also true in colony assays where IL-3 has recently been shown to inhibit the emergence of B220+ cells in colonies." In this study, bipotent E/MK progenitors were notdetected in the standard conditions of methylcellulose colony assays optimized for the growth of primitive BFU-E, ie, 30% FCS plus SCF, IL3, and Epo. In the presence of FCS, the addition of MS-5 stromal cell line was absolutely required for the growth of E/MK progenitor and CFU-MK and no combination of cytokines could replace these stromal cells. In contrast, when normal plasma or AP were substituted for FCS, MS-5 cells improved terminal MK differentiation but had no significant effect on the number of MK andE/MK colonies. The inhibitory activity present in serum that inhibits MK gr~wth",".~' is most likely accounted for by TGF-p.23,40.4' Two potential mechanisms, which might act in concert, may be hypothesized to explain the effect of MS-5 cells: (1) adsorption of inhibitory molecules on proteoglycans synthesized by stromal cells may result in their neutralization, a mechanism well documented for TGF-p42;(2) alternatively, stromal cells may secrete cytokines able to counteract this suppressive effect as recently reported for bFGF.43Whether this is accounted for by MGDF is as yet unclear; recent experiments showedthatMS-5,which has reported for other stromal cells,@ synthesizes low amounts of MGDF (F. Wendling, manuscript in preparation). However, exogenous MGDF added to colony assays with 30% FCSin the absence of MS-S supported the growth neither of BFU-ENK nor CFUMK. In serum-free plasma clot assays, MGDF alone optimally recruited CFU-MK but could not replace SCF or IL3 in the E/MK progenitor assay. This, together withthe observation that addition of MGDF to the combination of SCF, IL-3, and Epo increased the cloning efficiency of E/ MK progenitors suggests that optimal development of E/MK progenitors requires combinations of cytokines that probably act sequentially on progenitors at different maturation stages. Further experiments will be required to characterize MS-Sderived molecules that affect MK maturation. Two additional parameters may lead to underestimation of the real potential of progenitors in culture: asynchrony in the acquisition of specific differentiation markers identifying commitment to different lineages during the course of clone formation and discrepancy in the number of cells representative of each lineage.5 For example, emergence of lymphoid cells in colonies grown from iymphohematopoietic progenitor inthe mouse is a very late event.45Three arguments, however, rule out that E M K clones were in factmultilineage From www.bloodjournal.org by guest on December 3, 2014. For personal use only. DEBlLl ET AL 1294 clones: (1) a similar phenotype was observed in clones that were sequentially studied at days 12 and 18; (2) time course studies in methylcellulose colony assays indicated thatE/MK and CFU-GEMM represent two successive nonoverlapping waves of progenitor cells; (3) the sensitivity of immunolabeling of individual colonies, which allows the recognition of one to two MK cells within a colony. Recent advancesin the characterization of molecules associated with commitment to E and MK differentiation also strengthen the hypothesis of atightlinkage between both lineages. Expression of several transcription factors such as GATA-I, Tal-l, and NFE-2 is primarily found in both lineages,l?,14,16.17,4~~U and at least in vitro, GATA-I plays a pivotal role for expressionof both E and MK genes.“ However. recent experiments suggesting that GATA-1 is only required during the late stages of the E differentiation4’ and the lack of a majordefect inmegakaryocytopoiesis in GATA-I knock-out mice,’” question the real requirement of GATA1 expression forearly E and MK development.Whether GATA-I has aninductive role in the commitmentof pluripotent stem cells as recently suggested in an avian cell line” or if its lineage-specific effects may be reiated to its restricted question. Our pattern of e ~ p r e s s i o n ”is~still ~ ~ an ~ ~unsolved ~ results suggest that in the near future, it may be possible to have access to cellular models that directly allow us t o address these questions in specific cell subpopulations. Such an assay, which allows simultaneous identification of a high number of bipotent progenitors as well as their monopotentprogeny, will alsobe useful to characterize growth requirements of each compartment, with particular emphasis on the response to Epo and MGDF. The Eand MK lineages are the only two hematopoietic cell lineages that are regulated by humoral factors (Epo and Mpl-L/TPO/ MGDF), the level of which is regulated by the mature cell mas^.'^-^'' Several lines of evidence suggest that the Epo-R is expressed on M K ” and conversely it cannot be excluded thattheMpl-Rispresent on asubset of E progenitors.” However, preliminary attempts to identify Epo-R and MplR on BFU-EMK progenitors by flow cytometry have failed (data not shown), but this does not exclude the possibility that E M K progenitors express both the Epo-R and Mpl-R at very low, albeit biologically active, levels. NOTEADDED IN PROOF After acceptance of this report, a report also demonstrating the existence of bipotent E and NK progenitors was published.“ ACKNOWLEDGMENT We thank S. Gillis (Immunex, Seattle,WA) for a gift of IL-3, IL6 , and GM-CSF and P. Hunt (Amgen, Thousand Oaks, CA) for the gift of megakaryocyte growth and development factor.We are grateful to F. Lepesteur and B. lzac for technical assistance. The Y2/51 MoAb was a gift from Dr D. Mason (Oxford, UK). We are grateful to the surgeons for providing the bone marrow samples. We also thank S. Burstein for improving the English manuscript. REFERENCES 1. Metcalf D: The Hemopoietic Colony Stimulating Factors. Am- sterdam, The Netherlands, Elsevier. 1984 2. Suda T. Sudd J, Ogawa M: Disparate differentiation In mouse hemopoieticcoloniesderivedfrompairedprogenitors. Proc Natl Acad Sci [JSA X1:2520, 1984 3. Till JE, McCulloch EA, Siminovitch L: A stochastic model of stem cell proliferation based on the growth of spleen colony forming cells. 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Prandini MH, Joulin V, Mignotte V, Prenant M, Vainchenker W, MarguerieG, Uzan G: Megakaryocytic and erythro344:447, cytic lineages share specific transcription factors. Nature I990 15. Tronik-Le Roux D, Roullot V, SchweitzerA,tlerthier R. Marguerie G: Suppression of erythro-megakaryocytopoiesisand the induction of reversible thrombocytopenia in mice transgenic for the thymidine kinase genetargeted by theplateletglycoprotein ullb promoter. J Exp Med 181:2141, 1995 1 h. Orkin SH: GATA-binding transcription factors in hernatopoietic cell.;. Blood 80575, 1992 17. Orkin SH: Transcription factors and hematopoietic development. J Biol Chem 270:4955, 1995 18. Longmore CD, Pharr P, Neumann D, Lodish F: Both megakaryocytopoiesisanderythropoiesisareinducedin mice infected with a retrovirus expressing an oncogenic erythropoietin receptor. Blood 82:2386, 1993 19. McLeod DL, Shreeve MM, Axelrad AA: lnduction of megakaryocyte colonies with platelet formation in vitro. Nature 261:492, 1976 20. 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Debili N, Wendling F, Cosman D, Titeux M, Florindo C, Dusanter I, Methia N, Charon M, Nador R, Bettaieb A, Vainchenker W: The Mpl receptor expression is restricted to the megakaryocytic lineage from late progenitors to platelet.Blood 85:391, 1995 33. Solberg LAJ, Jamal N, Messner HA: Characterization of human megakaryocytic colony formation inhuman plasma. J Cell Physiol 124:67, 1985 34. Vainchenker W, Chapman J, Deschamps JF, Vinci G , Bouget J, Titeux M, Breton-Gorius J: Normal human serum contains a factor(s) capable of inhibiting megakaryocyte colony formation. Exp Hematol 10:650, 1982 35. Chaudhary PM, Roninson IB: Expression and activity of Pglycoprotein, a multidrug efflux pump, in human hematopoietic stem cells. Cell 66235, 1991 36. Reems JA, Torok-Storb B: Cell cycle and functional differences between CD34+/CD38h' and CD34+/CD38'" human marrow cells after in vitro cytokine exposure. Blood 85:1480, 1995 37. 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