Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
List of types of proteins wikipedia , lookup
Purinergic signalling wikipedia , lookup
Cell encapsulation wikipedia , lookup
Tissue engineering wikipedia , lookup
Cell culture wikipedia , lookup
Organ-on-a-chip wikipedia , lookup
Signal transduction wikipedia , lookup
Cellular differentiation wikipedia , lookup
From www.bloodjournal.org by guest on August 3, 2017. For personal use only. Expression of Growth Factor Receptors in Unilineage Differentiation Culture of Purified Hematopoietic Progenitors By U. Testa, C. Fossati, P. Samoggia, R. Masciulli, G. Mariani, H.J. Hassan, N.M. Sposi, R. Guerriero, V. Rosato, M. Gabbianelli, E. Pelosi, M. Valtieri, and C. Peschle We have evaluated the expression of growth factor receptors (GFRs) on early hematopoietic progenitor cells IHPCS) purified from human adult peripheral blood and induced in liquid suspensionculture t o unilineagedifferentiationlmaturation through the erythroid (E), granulocytic (GI, megakaryocytic (Mk), or monocytic (Mol lineage. The receptors for basic fibroblast GF (bFGF1, erythropoietin (Epo), thrombopoietin (Tpo), and macrophagecolony-stimulating factor (MCSF) have been only assayed at mRNA level; the majority of GFRs have been evaluated by both mRNA and protein analyses: the expression patterns were consistent at both levels. In quiescent HPCs the receptors for early-acting [fk3 ligand (FL), c-kit ligand (KL), bFGF, interleukin-6 (IL-611 and multilineage [IL-3, granulocyte-macrophageCSF (GM-CSFII HGFs are expressed at significant levels but with different patterns, eg, kit and ftf3 are detected on a majority and minority of HPCs, respectively, whereas IL-3Rs and GMCSFRs are present on almost all HPCs. In the four dfferentiation pathways, expression of early-acting receptors shows a progressive decrease, more rapidly for bFGFR-1 and fct3 than for c-kit; furthermore, c-kit is more slowly downmodulated in the E and Mk than the G and Mo lineages. As a partial exception, IL-6Rs are still detected through the early or late stages of maturation in the Mk and M o lineages, respectively. IL-3R expression is progressively and rapidly downmodulated in both E and Mk pathways, whereas it moderately decreases in the Mo lineage and is sustained in the G series. The expression of GM-CSFR is gradually downmodulated in all differentiation pathways, ie, the receptor density markedly decreases but late erythroblasts are still partially GM-CSFR’ and terminal G, Mk and Mo cells are essentially GM-CSFR+. Expression of receptors for late-acting cytokines is lineage-specific.Thus, EpoR, G-CSFR. TpoR, and M-CSFR exhibit a gradual induction followed by a sustained expression in the E, G, MK, and M o lineages, respectively. In the other differentiation pathways the expression of these receptors is either absent or initially low and thereafter suppressed. These observations are compatible with the following multi-step model. (1) The early-acting GFRs are expressed on quiescent HPCs with different patterns, whereas the multilineage GFRs are present on 290% to 95% HPCs. (2) Multilineage GFs, potentiated by early-acting HGFs, trigger HPCs into cycling. HPC proliferation/differentiation is followed by declining expression of the early-acting GFRs and in part of multilineage GFRs (see above). (3)Multilineage GFs trigger the expression of the unilineage GFRs (see Testa U, et ai: 6/ood81:1442, 1993). Interaction of each unilineageGF with its receptor leads t o sustained expression of the receptor (possibly via transcription factors activating the receptor promoter) and thus mediates differentiation/ maturation through the pertinent lineage. 0 7996 by The American Society of Hematology. H derived growth factor receptor (PDGFR); (2) Rs belonging to the tyrosine kinase superfamily,” namely the receptors for CSF-I (M-CSF) and steel factor, encoded by the c-fms and c-kit proto-oncogenes, respectively, and the $t3/FLK2 receptor; and (3) Rs belonging to the peculiar highly conserved ‘‘cytokines receptor ~uperfamily”’~~’~ which includes IL-2RP chain, IL-4R, ILdR, IL-3Ra chain, GM-CSFRa chain, G-CSFR, and EpoR. Elucidation of HGFR control mechanisms is obviously crucial to unveil the cellular and molecular basis of hematopoiesis, particularly at the level of HSCsMPCs. Our laboratory has shown that purified PB HPCs express high-affinity HGFRs, with prevalent IL-3R, a lower level of IL-6Rs and GM-CSFRs and barely detectable levels of EpoRs.14 Subse- EMATOPOIESIS IS sustained by a pool of hematopoietic stem cells (HSCs) that can extensively self-renew and differentiate into progenitor cells (HPCs).’ HPCs are committed to a specific lineage(s) and are functionally defined as colony- or burst-forming units (CFUs, BFUs), ie, early and late HPCs of the erythroid series (BFU-E and CFU-E, respectively), the megakaryocytic lineage (BFUMK, CFU-MK), the granulocyte-monocytic series (CFUGM, CFU-G, CFU-M), as well as multipotent CFUs for all four lineages (CFU-GEMM). HPCs in turn differentiate into morphologically recognizable precursors that mature to terminal elements circulating in peripheral blood (PB). The survival, growth, and differentiation of hematopoietic cells is at least in part regulated by a network of hematopoietic growth factors (HGFs) termed colony-stimulating factor (CSF) or interleukin (IL).’ Erythropoietin (Epo), G-CSF, thrombopoietin (Tpo), and M-CSF essentially stimulate late unilineage HPCs (CFU-E, CFU-G, CFU-MK, and CFU-M, respectively) and the derived differentiated precursors. Multilineage HGFs, including IL-3 and GM-CSF, affect CFU-GEMM and early unilineage or bilineage HPCs (BFUE, BFU-MK, and CFU-GM).’ Other cytokines Vft3 and ckif ligands (FL, KL), basic fibroblast GF (bFGF), leukemia inhibitory factor (LIF), and IL-61 act on the primitive stages of hematop~iesis.~-~ The availability of recombinant HGFs favored analysis of their cell-surface receptors (Rs). HGFRs have been recently cloned and classified into three different categories: (1) Rs belonging to the Ig superfamily,” eg, IL-1R and platelet- Bfood, Vol88, No 9 (November 1). 1996 pp 3391-3406 From the Department of Hematology-Oncology Istituto Superiore di Sanitci; Blood Transfusion Center, University “La Sapienza, ” Rome, Italy; and Thomas Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, PA. Submitted March 13, 1996; accepted July 4, 1996. Address reprint requests to C. Peschle, MD, Thomas Jefferson University, Thomas Jefferson Cancer Institute, Bluemle Life Sciences Bldg, Room #528-233, S 10th St, Philadelphia, PA 19107-5541. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with I8 U.S.C. section I734 solely to indicate this fact. 0 19% by The American Society of Hematology. OOO6-497I#6/8809-~0$3.OO/O 3391 From www.bloodjournal.org by guest on August 3, 2017. For personal use only. 3392 TESTA ET AL quent studies by double labeling of total human'' or Rhesus monkey bone marrow (BM) ~ e l l s ' ~with ~ ' ' anti-CD34 and either anti-IL-3R or GM-CSFR monoclonal antibodies (MoAbs) confirmed that CD34+ cell subsets express IL-3Rs andor GM-CSFRs. Furthermore, studies on fractionated BM CD34' cells showed that small cells possess a higher IL6R, IL-3R, and GM-CSFR density than large ones.'' These studies did not provide insight into the expression pattern of HGFRs during HPC differentiatiordmaturation along the diverse hematopoietic lineages. We have recently developed methodology that enables stringent purification and abundant recovery of PB HPCS~.'~-'' and their selective unilineage differentiation along the erythroid (E),19-24 granulocytic (G),23.24 megakaryocytic (Mk),*' or monocytic ( M o ) ~ lineage. The availability of these experimental tools allowed us to explore the expression of HGFRs at different stages of HPC differentiatiordmaturation restricted along the E, G, Mk, or Mo pathway. MATERIALS AND METHODS HGFs and Culture Medium Recombinant human IL-3 (rhIL-3; 2 X IO6 Ulmg), rhGM-CSF, and rhIL-6 (2 X 10* Ulmg) were supplied by the Genetics Institute (Cambridge, MA); rhEpo (1.2 x lo5 U/mg) and bovine basic fibroblast GF (rbFGF; 2 x lo7 U/mg) by Amgen (Thousand Oaks, CA); and rhFL (1.9 X lo6 U/mg) and rhKL (1 X 1O5 Ulmg) by Immunex (Seattle, WA). rhG-CSF (1 X 10' Ulmg) and rhM-CSF (6 x lo7U/ mg) were purchased from R&D Systems (Minneapolis, MN), and rhTpo was generously provided by Genentech (San Francisco, CA). Iscove's modified Dulbecco's medium (IMDM; GIBCO, Grand Island, NY) was prepared weekly before each purification experiment. Adult PB HPC Purc9cation Adult PB was obtained from male donors after informed consent as described.' HPCs were purified as reported7.I9and modified.*@" Briefly, (IA) PB samples were separated over a Ficoll-Hypaque density gradient (Pharmacia, Piscataway, NJ), and (IB) PB mononuclear cells (PBMCs) resuspended in IMDM containing 20% heat-inactivated fetal calf serum (FCS; GIBCO) for three cycles of plastic adherence. Thereafter, (11) cells were separated by centrifugation on a discontinuous Percoll gradient (Biochrom KG, Berlin, Germany). (111) Step 111 purification was potentiated (Step IIIP) as HPC Clonogenetic Assay In FCS' cultures purified HPCs were seeded ( I X 10' cells/mL/ dish, two or three plates per point) and cultured in 0.9% methylcellulose, 40% FCS in IMDM supplemented with a-thioglycerol mol/L) (Sigma, St Louis, MO), ferric ammonium citrate (10 mg/ mL), pure human transferrin (0.7 mg/mL), and different HGFs as detailed below at 37°C in a 5% COz/5% OZ/90%NZ humidified atmosphere. In FCS- cultures,26FCS was substituted by bovine serum albumin (BSA), pure human transferrin, human low-density lipoproteins, insulin, sodium pyruvate, L-glutamine (2 X lo-' mol/ L), rare inorganic elementsz7 supplemented with iron sulfate (4 X IO-* mom), and nucleosides. Both FCS+ and FCS- cultures were supplemented with FL (100 ng/mL), KL (100 ng), IL-3 (100 U), GM-CSF (10 ng), Epo (3 U), M-CSF (250 U), and G-CSF (500 U). CFU-GEMM, BFU-E, and CFU-GM colonies were scored on day 14-15 and 16-18 in FCS" and FCS- cultures, respectively. Unilineage HPC Liquid Suspension Culture Step IIIP HPCs were seeded ( 5 X IO4 cells/mL) and grown in liquid suspension culture supplemented with the following HGFs: (1) in FCS- erythropoietic culture, very low doses of 1L-3 (0.01 U/ mL) and GM-CSF (0.001 ng) and a saturating level of Epo ( 3 U)"'-''; (2) in FCS- granulopoietic culture, low amounts of IL-3 ( I U) and GM-CSF (0.1 ng) and saturating amounts of G-CSF (500 U)23,'4; (3) in FCS- megakaryocytopoietic culture, saturating doses of Tpo (100 ng)*'; and (4) in FCS+ monocytopoietic culture, saturating amounts of both FL (100 ng) and M-CSF (500 U).' Cells were periodically counted and analyzed for morphology, membrane phenotype, and HGFR expression at both mRNA and protein levels. Unilineuge HPC Clonogenetic Culture HPCs growing in unilineage liquid suspension cultures were assayed in unilineage clonogenetic cultures. These were performed by adding the HGF stimuli used for unilineage liquid suspension cultures (see above) in FCS- (erythropoietic, granulopoietic, megakaryocytopoietic cultures) or FCS' (monocytopoietic culture) medium (see above). Mk colonies were scored on day 10- I2 and other colonies on day 14-16, The scoring threshold was 100 cells/colony for erythroid, granulocytic, and monocytic clones and 3 celldcolony for megakaryocytic ones. Control Cells Normal human adult fibroblasts, PB granulocytes, and monocytes were obtained by standard procedures. Fetal liver erythroblasts were obtained as described." Human TF-1, K562, U937, KG I , HL-60, and HEL leukemic cell lines were grown under standard conditions. Immunojluorescence HPC Analysis MoAbs. Anti-CD34 (8G12 clone), -HLA-DR, -CD38, -CD61, -VLA-4, -CD3 I , -CD58, -CD44, -CD4 (Becton Dickinson, Mountain View, CA), +kit, -gp170 (Immunotech, Marseille, France), and -Thy- I (PharMingen, San Diego, CA) MoAbs directly conjugated with fluorochrome [fluorescein isothiocyanate (FITC) or phycoerythrin (PE)] were used to characterize the membrane phenotype of step IIIP HPC. Labeling procedure. Cells were washed twice in phosphate-buffered saline (PBS) and then incubated for 60 minutes at 4°C in the presence of an appropriate amount of MoAb. After three washes with cold PBS, cells were resuspended in PBS containing 2.5% formaldehyde and then analyzed by FACS (FACScan; Becton Dickinson) for fluorescence intensity with a program suitable for doubleimmunofluorescence analysis. At least 4,000 cells were analyzed for each determination. The gating for analysis of the living cell population was determined by propidium iodide staining.*x Fluorescence Analysis of HGFR Expression Two types of reagents were used: ( I ) anti-HGFR MoAbs either simply purified or fluorochrome-conjugated; and (2) recombinant HGFs either biotin or fluorochrome conjugated. All reagents are listed in Table 1. Anti-IL-3, -c-kit, -jt3 MoAbs. PE-labeled anti IL-3R a-chain MoAb, clone 9G5"' was purchased from PharMingen; PE-labeled anti-c-kit was obtained from Immunotech: PE-labeled anti-jf3, clone SF1.3403" was kindly supplied by Dr 0. Rosnet (Marseille, France). These antibodies were incubated as indicated above for fluorochrome-conjugated antibodies. Control experiments for exogenous ligand versus MoAb competition were performed by incubating the step IIIP cells with a fixed From www.bloodjournal.org by guest on August 3, 2017. For personal use only. GROWTH FACTOR RECEPTORS IN HEMATOPOIESIS 3393 Table 1. Reagents Used for HGFR Expression Analysis on Step lllP HPCs Induced to Unilineage Differentiation Fluorescence Receptor Early-acting HGFs Multilineage HGFs MoAb Ligand bFGFR - - kit f1r3 + [PE-labeled MoAb (Immunotech)] + [PE-labeled M o A ~ ~ ~ I + [Biotin-conjugatedKL (R&D)I IL-6R - IL-3R + [PE-labeledM o A ~ ~ ~ I GM-CSFR Unilineage HGFs RT-PCR (Ref No.) +[MoA~~~I - + [Biotin-conjugatedIL-6 (R&D)I + [PE-labeledGM-CSF (R&D)I - EpoR + [PE-labeled G-CSF (R&D)I G-CSFR M-CSFRlfms TpoRlmpl amount of anti-IL-3R or anti-jf3 MoAb in absence or presence of increasing concentrations of IL-3 (0.01, I, or 100 U/mL) or FL (1, 10, or 100 ng/mL), respectively. No competition was observed for IL-3 at the dosage added in E and G cultures (data not shown). To assay the binding of anti-jt3 MoAb in Mo culture, cells were first (1) washed twice in neutral PBS, treated with 1 mL of cold PBS (titrated to pH 3.0 with HCI) for 1 minute at 4°C to remove the large majority of surface-bound FL, washed once with neutral PBS; and then (2) incubated with PE-labeled anti-jt3 MoAb: this procedure allows to remove greater than 95% surface-bound FL. Control experiments for exogenous ligand versus MoAb competition showed that IT,at the concentration used in the Mo culture medium, causes a 50% reduction of anti-jr3 MoAb binding to the cells (data not shown). Anti-GM-CSFR MoAb. Purified unconjugated anti-GM-CSFR a-chain MoAb, clone 17-A3' was purchased from PharMingen. Cells were first incubated for 60 minutes at 4°C in the presence of 2.5 mg/mL of this antibody, washed with cold PBS, and then incubated for 30 minutes at 4°C with biotin-conjugated sheep anti-mouse IgGs (Cappel, West Chester, PA). After washing with cold PBS, cells were incubated with Quantum Red-labeled streptavidin (Sigma) and then analyzed by fluorescence-activated cell sorting (FACS). Control experiments for exogenous ligand versus MoAb competition were performed by incubating the step IIIP cells in the presence of a fixed amount of anti-GM-CSFR MoAb combined or not with increasing concentrations of GM-CSF (0.001, 0.1, or 100 ng/mL). No competition was observed at the cytokine dosage supplemented in E and G cultures (data not shown). Ligands. Biotin-conjugated c-kit and IL-6 were obtained from R&D Systems. Cells were incubated for 60 minutes at 4°C with this reagent, incubated for an additional 30 minutes at 4°C with PElabeled streptavidin (R&D Systems), and then analyzed by FACS. PE-labeled GM-CSF and G-CSF were obtained from R&D Systems. The cells were incubated for 60 minutes at 4°C with this reagent, washed, and then analyzed by FACS. G-CSF was added at saturating level in G culture. To assay the binding of PE-labeled G-CSF in G culture, cells were first (1) washed twice in neutral PBS, treated with 1 mL of cold PBS (titrated to pH 3.0 with HCl) for 1 minute at 4°C to remove the large majority of surface-bound G-CSF, washed once with neutral PBS; and then (2) incubated with PE-labeled G-CSF this procedure allows removal of greater than 95% surface-bound G-CSF (see Results). As for GMCSF, we previously showed that the GM-CSF concentrations used '~ in E and G cultures cause less than 5% receptor o c c ~ p a n c y(see also Results). The specificity of biotinylated and PE-conjugated ligands (KL, IL-6, GM-CSF, G-CSF) was assessed by using TFI erythroleukemic cells incubated with ligand alone or combined with an appropriate amount of blocking antiligand MoAb: the difference in fluorescence intensity between these two conditions reflects the level of specific binding and the number of receptors (Fig 1). Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)Analysis of HGFR mRNA Total RNA, extracted by the guanidium isothiocyanate method3* in the presence of 12 mg of Escherichia coli rRNA as a carrier, was normalized by dot hybridization with a human rRNA probe." The normalized RNA was reverse transcribed according to the manufacturer's instructions (GIBCO-BRL, Gaithersburg, MD). The RT-PCR was normalized for P2-mi~roglobulin'~ (amplification within the linear range was achieved by 20 PCR cycles: denaturation at 95°C for 30 seconds, annealing at 54°C for 30 seconds, and extension at 72°C for 45 seconds). To evaluate the expression of HGFR genes, an aliquot of RT-RNA (corresponding to -2 ng RNA) was amplified within the linear range by 30 PCR cycles. Each sample was electrophoresed in 2% agarose, transferred to nylon membrane, and hybridized with a specific probe. Positive controls (detailed in Results) were normalized for P2-microglobulin and included in all PCR assays. For negative controls, an aliquot of RNA (-2 ng) from each sample was amplified to exclude the presence of contaminant DNA; a mock reaction was also included. PCR cycles: 95"C/30 seconds, 55"C/30 seconds, 72"C/45 seconds (except when otherwise indicated). Primers and Probes bFGFR-I (see ref 33). Primer 5': AAGACCTGGACCGCATCGT (2342-2360); Primer 3': TTACAGCTGACGGTGGAGT (2581-2599); Probe: AGCTCTACGTGCTCCTCAGGGGAGGA TTCCG (2440-2470). c-kit (see ref 34). Primer 5': CG'ITGACTATACAGTTCAGCGAC (1 180-1201); Primer 3' CTAGGAATGTGTGTAAGTGCCTCC (843-864); Probe: GATCAGCAAATGTCACAACAACCTT GGA (1032-1059). IL-6R (see ref 35). Primer 5': TCTCACTGCCATGCCAGCT (1478-1496); Primer 3': CTGTTGAAAACGACCAGGC (20352053); Probe: TCAGCCTGCTCCAGCTGTTCAGCTGGTT GAGGTITCAA (1565-1603). IL-3R a-chain (see refs 36 and 37). Primer 5': AACGACAAGCTGGTGGTCT (105 1-1069); Primer 3' AGGTCCAGCAGCCAGTCT (1 195-1212); Probe: CTGGTCACTGAAGTACAGGTCGTGCAGA AAACTTGAGAC (1089-1 140). GM-CSFR a-chain (see ref 38). Primer 5': CTACCGCGAAGAGGTCTT ( I 3 13-1331); Primer 3': CCAAGTAGCTGGGATGAT (1638-1655); Robe: GCCAGGCGCGGTGGCTCACGCCTGTAA TCCCAGCA (1479-15 14). Annealing at 53°C. From www.bloodjournal.org by guest on August 3, 2017. For personal use only. TESTA ET AL 3394 labeled Ligand Negative Control "1 1 I J 'oak l 0100W 102 104 L 0100 102 1 1" zk, 0 100 102 , I 100 102 , ~ 104 "I "1 '"h 0100 102 l 0100W 102 104 L Fluorescence ) 104 [k, , 104 c-kit - ( G S ) . [G-CSFR) 1w Fig 1. Flow cytometric analysis of HGFR expression in TF-1 erythroleukemic cells. (Left panels) Cells stained with phycoerythrin-conjugated streptavidin (SA-RPE) (negative control). (Right panels) Cells treated with labeled ligands (PE-labeled GM-CSF and G-CSF, biotinlabeled IL-6 and c-kit) combined with SA-RPE. White area, specific binding; black area, nonspecific binding in the presence of an excess of anticytokine blocking MoAb. The low level of PE-GM-CSF binding is probably due to the saturating GM-CSF level (10 ng/mL) in the culture medium. EpoR (see ref 39). Primer 5': TCATGGACCACCTCGGGGCGT (2-19); Primer 3' TAGCGGATGTGAGACGTCATG (519539); Probe: TCTGGTGTTCGCTGCCTACAGCCGACA CGTCGAGC (314-348). Annealing at 54°C. G-CSFR (see ref 40). Primer 5 ' : TCCCAGCATGTCTATGCC ( I7 12-1729); Primer 3': AGCCATGAGGTGGATGTG ( 19581975); Probe: CACTACACCATCTTCTGGACCAACGTCC AG (1850-1879). Annealing at 50°C. M-CSFRgms (see ref 41). Primer 5': AGAGCATCTTCGACTGTG (3946-3964); Primer 3': GAACTTGGCTGTTCACCAG (405 1-4068); Probe: ACGTCTGGTCCTATGGCATCTTGCTCTGG (3984-4012). Annealing at 50°C. TpoR/mp/ (see ref 42). Primer 5': AGCTGATTGCCACAGAAACC (557-576); Primer 3': ACTTGGGGAGGTCTGCTTTG (665-684); Probe: CCAGTCTCCATGTGCTCAGCCCACAAT GCC (621-650). Annealing at 56°C. RESULTS HPC Purijication and Membrane Phenotype Early HPCs were purified from PB by a methodology previously de~cribed'.'~and recently The step IIIP cell population obtained by the modified purification procedure is characterized by 90% CD34' cells and 80% HPC frequency, coupled with 45% HPC re~overy.~"'~ In the purified HPC populations used here, the HPC clonogenetic assay was optimized by adding to the formerly used HGFs (KL, IL-3, GM-CSF, Epo) saturating dosages of GCSF, M-CSF, and FL: in a series of 12 consecutive experiments the HPC frequency and recovery increased to 90.3 t 2.1 (mean t SEM) and 69.0 t 5.3, respectively (Fig 2), compared with 80.3 t 3.2 and 61.9 t 5.1, respectively, obtained in the same experiments with the formerly used HGFs (not shown). The colony type frequency was: BFUE, 33.5% ? 1.8%, CFU-GM (including CFU-G, -M, -GM), 44.9% t 2.8%, and CFU-GEMM, 11.5% t 0.9%. With respect to evaluation of BFU-MK colonies and different types of GM clones, additional clonogenic experiments involving treatment with the above HGFs combined with Tpo (100 ng/mL) showed: BFU-MK, 1.5% t 0.4% (scored on day 10-12, see Materials and Methods), CFU-G, 19.8% t 0.6%, CFU-M, 7.7% t 1.7%, CFU-GM, 8.8% t 1.6%. Step IIIP cells are intensively CD34' (Fig 3) over the negative background and 5% to 15% of these are positive for Thy-I and gp170 membrane antigens, which are expressed on putative HSCs and primitive HPCS.~'." The large majority of step IIIP HPCs are CD38' and HLA-DR', but 5% to 10% are CD34'/CD38'"" and CD34'kILA-DR""". Only a minority (5% to 10%) of CD34' step IIIP cells are positive for a few lineage-specific differentiation antigens, such as CD4. Finally, step IIIP cells are largely positive for adhesion molecules, including CD31, CD58, CD44, and VLA-4. Control Experiments f o r HGFR Assay Control experiments, particularly with respect to competition at receptor level between endogenous HGF and labeled ligand or MoAb, have been detailed in Materials and Methods. Expression of HGFRs in Quiescent HPCs In a first set of experiments we evaluated HGFR expression in quiescent step IIIP HPCs by immunofluorescence (Fig 4) and RT-PCR (Fig 5A and B). Immunofluorescence studies with PE-labeled anti-Jt3 and anti-c-kif MoAbs showed that 35% and 80% of cells were jt3' and c-kit', respectively. Futhermore, 40% to 45% of cells were labeled by biotin-labeled IL-6. Studies with anti-IL-3R a-chain or anti-GM-CSFR achain MoAbs showed that the large majority (ie, 290% to 95%) of cells were IL-3R' and GM-CSFR'. These observations were confirmed by studies with PE-labeled GM-CSF (see below): however, the percentage of GM-CSFR' cells was slightly lower (>70%) than that obtained using antiGM-CSFR MoAb (see below). Finally, studies with PE-labeled G-CSF showed a reactivity with only a minority of cells. RT-PCR analysis. mRNAs encoding HGFRs for earlyacting (bFGF, KL, and IL-6) and multilineage (IL-3 and From www.bloodjournal.org by guest on August 3, 2017. For personal use only. GROWTH FACTOR RECEPTORS IN HEMATOPOIESIS 3395 10'1 lo, Fig 2. Total cell number, percentage of CD34' cells, HPC frequency, and recovery in the step lllP purification procedure (mean k SE values from 12 separate experiments). (Left) Total cell number was evaluated at different steps of purification, including the Ficoll cut (Step I), Percoll gradient (Step II), and at the end of the sequential passages on magnetic beads (Step IIIP). (Right) The percentage of CD34* cells in step lllP was evaluated by flow cytometry. Step lllP HPC (BFU-E + CFU-GM + CFU-GEMM) frequency was evaluated by addition of IL-3, GM-CSF, Iml I* 1001 Y i : s d 1. stop I step II Epo, KL, FL, M-CSF, and G-CSF (see Results). HPC recovery, percentage of HPCs after step IMP, as compared with their number in PBMCs (Step I). GM-CSF) cytokines werc expressed at significant levels in quiescent HPCs (Fig 5A). In contrast, mRNAs encoding HGFRs for late, unilineage cytokines (Epo, G-CSF, M-CSF, and Tpo) were barely (Fig SB, top panels) or not (Fig SB, bottom panels; for TpoR also top panels) expressed in quiescent HPCs in different experiments (top and bottom panels show two separate representative HPC purification and culture experiments). HGFRs Expression During HPC E, G, Mk, and Mo Differentiation/Matiiratioii The unilineage differentiation culture systems. Experiments were performed to evaluate the expression kinetics of HGFRs during unilineage E, G, Mk, and Mo differentiation/ maturation of step HIP HPCs in liquid suspension culture. The main features of these culture systems have been extensively reported4."." and are further detailed in Table 2. Day 0 step IIIP HPCs, assayed in E, G, Mk, or Mo clonogenetic cultures (ie, in unilineage semisolid cultures treated with the same HGF stimuli of corresponding unilineage liquid cultures), generated only the expected E, G, Mk, or Mo colonies, respectively. The frequency of these colonies varied for the different lineages, ie, 28%, 16%. 7%, and 4% for the E, G, Mk, and Mo lineages, respectively (Table 3). During the first week of E, G,Mk, or Mo liquid culture the HPC number, assayed in secondary unilineage semisolid cultures, showed a gradual increase, ie, an increasing number of selectively E, G,Mk, or Mo colonies, respectively: this indicates unilineage HPC proliferation, ie, growth of E-, G-, Mk-, or Mo-committed HPCs in the corresponding culture4.19.21.25 (and Table 2 and results not shown). Cell proliferation was associated with progressive HPC differentiation, as shown by a gradual decrease of the size of the unilineage colonies generated in secondary semisolid culture (Table 3) and the percentage of CD34' cell^.^.'^.^^ In the Mo lineage the decrease of colony size from day 0 to day 7-9 was less rapid than for the other lineages (Table 3): in the second step lllP CDM*C.ll Frequency fmgennw FI.C(WIKY L E2ER I v step lllP culture week the survival of a miniscule CFU-M population (eg. -3% of total cells on day 12) is seemingly related to the capacity of FL to stimulate the proliferation of primitive HPCs and CFU-GM.4 Unilineage cultures were supplemented with an early-acting HGF (FL in Mo culture) or multilineage HGFs (IL-3, GM-CSF in E and G culture, in part Tpo in Mk culture).g6 Addition of these HGFs may allow the proliferation of HPCs not restricted to unilineage differentiation. To investigate this aspect, HPCs were sequentially collected from unilineage cultures and assayed in semisolid medium supplemented with saturating levels of KL, FL, multilineage (IL-3, GMCSF), and unilineage (Epo, G-CSF, Tpo, M-CSF) HGFs: these clonogenetic culture conditions allow to assay the differentiation potential of the HPCs growing in liquid unilineage culture. The results (not shown here, see Discussion) indicate that other categories of HPCs survive in unilineage cultures, ie, HPCs with CFU-GEMM/CFU-GM potential in E culture, with CFU-GEMM/BFU-E potential in G and Mo culture, with CFU-GEMM/BFU-E/CFU-GMpotential in Mk culture: it is crucial that the frequency of these HPCs rapidly declines in the first culture week, ie, these HPCs are either gradually channeled into the unilineage differentiation pathway or undergo apoptosis. During the second week of culture the cell clonogenetic capacity is lost (ie, the size of generated clusters is below the colony scoring threshold, except for a few monocytic colonies in Mo culture, see above): HPCs enter into the maturation compartment and progressively express the morphologic, antigenic, and functional pattern of maturing E, G, Mk, and Mo precursors in the respective cultures. The cell number amplification from day 0 through day 14-16 sharply differs in the four unilineage culture systems: IO'-fold for the E series, 102-fold for the G lineage, 30and 12-fold for the Mo and Mk pathways, respectively. These differences seemingly reflect the frequency and particularly the proliferative potential of the different types of HPCs in step IIIP cells. - - From www.bloodjournal.org by guest on August 3, 2017. For personal use only. 3396 1000 + + 600 U v) Y 200 0 200 ' 600 FSC -b 100 100 1 02 CD34 + 104 ' 1000 100 104 ... - 10- 100 102 CD34 -b 102 CD34 - 102 ' FL1 100 102 104 HLA-DR -+ 'p 100 - 100 0 ' + 102 ' CD34 -b . 100 102 + . 1 104 loo!-- I , , , 1 00 1 02 CD34 -+ 164 loo!, 100 I, ., , 102 CD34 -& I 104 I 104 1 . . I 104 TESTA ET AL 102 CD44 + 104 Expression of Early-Acting HGFRs RT-PCR analysis (Fig 5A) indicated that the mRNA expression of early-acting HGFRs (bFGFR-I, c-kit, and IL-6R) was generally characterized by a progressive decline in both E and G differentiation pathways. bFGFR-I mRNA declined more rapidly during E than G differentiation, whereas the decrease of c-kit mRNA is slower in the E than G pathway. In previous studies' j t 3 mRNA showed an abrupt decrease in the E pathway and a slower decrease in the G series. IL6R mRNA also declined to undetectable or low level in E and G maturation, respectively. Immunofluorescence studies with anti-jt3 or anti-c-kit MoAbs confirmed the pattern observed at mRNA level (see Figs 6A and B, and 7A). Thejt3' cell frequency sharply loo! - I , , , 100 102 CD34 -b I 104 Fig 3. Flow cytometric analysis of step lllP cells with a panel of MoAbs (a representative experiment is shown). Cells were labeled with either a PE- or FITCconjugated anti-CD34 or appropriately labeled anti-Thy-1, -gp170, -CD38, -HLA-DR, -CD4, -CD31, -CD58, -CD44. or - V I A 4 MoAbs. On the top line the first two panels from left to right show cell scatter (iodide propidium staining) and negative control. decreased during E and G differentiation, slightly more rapidly in the former than the latter lineage (Figs 6A and 7A). The number of c-kit+ cells slowly decreased in the E and G series to low levels at day 12 or 9, respectively (representative results in Fig 6B): thus, the decline was less rapid in the E than the G lineage (Fig 7A). Similar results were obtained using biotin-conjugated c-kit (data not shown). Further immunofluorescence experiments were performed on Mk and Mo cultures (Fig 7A): t h e j t 3 and c-kit expression pattern in these lineages was similar to that observed for the E and G series. In Mo culture, however, we cannot exclude downmodulation o f j t 3 by the saturating FL level present in the medium (see below studies on G- From www.bloodjournal.org by guest on August 3, 2017. For personal use only. GROWTH FACTOR RECEPTORS IN HEMATOPOIESIS FLI FSC- t 2 104 104 102 * 102 3397 -b t + 0 100 100 100 100 102 104 CD34 -b Kl 104 104 w 102 w 102 t 2f 9 =? 102 104 CD34 -b 4 ;: $.. ,. 100 100 102 104 CD34 -b x w 102 l w y , .":-;' 100 102 104 CD34 + ;::rl v v5 a. 0 100 I00 level by RT-PCR and in all lineages at protein level by immunofluorescence. RT-PCR analysis. ( I ) As shown in Fig 5A, IL-3R mRNA was briskly downmodulated in the E series to undetectable level at day 9, while it was sustained or slightly decreased in the G pathway; (2) GM-CSFR mRNA was characterized by a gradual decrease to low or undetectable level in advanced maturation in the E pathway, compared with a moderate decrease in the maturing G lineage. Fluorescence studies with anti-GM-CSFR and -1L3R MoAbs and PE-labeled GM-CSF provided results in line with those observed at mRNA level. IL-3R assay. At day 0-2, HPCs differentiating along either the E and G lineages were almost all IL-3R'. Starting from day 5 IL-3R' cells rapidly decreased in E culture, whereas expression in the G lineage was essentially unmodified, as evaluated in terms of IL3R' cell frequency (60% to 70% IL-3R' cells at day 12) and fluorescence intensity. IL3R expression progressively and sharply declined during Mk differentiation, almost as rapidly as in the E lineage. The decrease of IL3R expression was slow in the Mo series and a significant proportion (-50%) of mature monocytes displayed IL-3Rs. GM-CSFR assay. Similar results were obtained using anti-GM-CSF MoAb (Fig 6F and results not shown) and PE-labeled GM-CSF (Fig 7B). However, the MoAb assay was more sensitive: this is seemingly caused by amplification of the MoAb fluorescence signal (see Materials and Methods) rather than by receptor occupancy by exogenous GMCSF in E and G culture (the supplemented cytokine concentration causes only 4% receptor oc~upancy,'~ see above). During the first 5 culture days virtually all cells differentiating along the E and G lineages were GM-CSFR', particularly by MoAb assay. In the second week, E precursors exhibited a gradually reduced reactivity with anti-GMCSFR MoAb, ie, a moderate decrease of percent positive cells combined with a pronounced decline of fluorescence intensity. G precursors remained prevailingly (by ligand assay, Fig 7B) or completely (by MoAb assay, Fig 6F) GMCSFR', but showed a sharp decrease of fluorescence level. Interestingly, the expression patterns of GM-CSFR evaluated by RT-PCR (Fig 5A) and MoAb (Fig 6F) assays are very similar. In the Mk and Mo pathways GM-CSFR expression is characterized by a sustained majority of GM-CSFR' cells, coupled with a gradual decrease of fluorescence intensity, particularly in the Mk pathway (Fig 7B; also results not shown performed by MoAb assay). 102 104 CD34 + 100 100 102 104 CD34 -b Fig 4. Flow cytometric analysis of HGFR expression on quiescent step lllP cells la representative experiment is shown). Cells were labeled with FITC-conjugated anti-CD34 and appropriately labeled anti-flt3 MoAb, anti-c-kit MoAb, biotin-labeled IL-6, anti-IL-3R MoAb, anti-GM-CSFR MoAb, or PE-labeled G-CSF. (Top panels) Cell scatter (iodide propidium staining) and negative control. CSFR in G culture): further studies are in progress to evaluate this aspect. Experiments with biotin-conjugated IL-6 showed that IL6R expression ( I ) rapidly decreased to undetectable levels in the E series, (2) slowly decreased in the G series, (3) was sustained until day 7 and then decreased during Mk differentiation, and (4)increased through day 9 and remained detectable until terminal maturation in Mo lineage (Fig 7A, see also Fig 6C). Expression of Multilineage HGFRs The expression pattern of HGFRs specific for IL-3 and GM-CSF was investigated in the E and G lineages at mRNA Expression of Late-Acting Unilineage HGFRs The expression of EpoR, G-CSFR, TpoR, and M-CSFR was investigated in the four differentiation pathways by RTPCR analysis (Fig 5B). These HGFRs, only scarcely expressed in quiescent HPCs, were gradually induced and then sustainedly expressed in the pertinent lineage. Conversely, they are barely or not expressed in the other lineages: (1) EpoR mRNA was transiently expressed at low level in the G and Mk lineages from From www.bloodjournal.org by guest on August 3, 2017. For personal use only. TESTA ET AL 3398 A ( Day 0 2 5 7 9 12 14 0 2 5 7 9 1 2 1 4 Q Posmm CONTROL 0 bFGFR- 1 Fibroblasts c-Kit K562 ** 11-6R u937 m KGI m HI.-60 0 11-3R ' 0 - -*- *I* GM-CSFR l32m 0 2 5 7 9 EpoR G-CSFR 4 12 14 0 * m - I 0 2 5 7 9 1214 El POSITIVE CONTROL Erythrobl. 1)1 Granuloc. (I M-CSFR/hTlS Monoc. TpoR/mpl HE1 PosmvE CONTROL Erythrobl. Granuloc. 4 P2m POSINVE CONTROL EpoR G-CSFR Granuloc. 4 * .*.'., Erythrobl. 0 Granutoc. 0 HEL 0 M-CSFR/fms TpoR/mpl hm From www.bloodjournal.org by guest on August 3, 2017. For personal use only. GROWTH FACTOR RECEPTORS IN HEMATOPOIESIS 3399 Fig 5. (A) bFGFR-I, a k i t IL-6R. ILSR, and GM-CSFR mRNA levels during in vitro erythroid (E)or granulocytic (GIdifferentiation of step lllP HPCs, as evaluated In representative experiments by RT-FCR. @2-Microgfobulin(Plmt controls are presented. Positive controls are also prasented. Similar results were obtained in two other experiments. IB) EpoR, G-CSFR, M-CSFR, and TpoR mRNA levels during arythroid (E), granulocytic 101. megakaryocytic IMk), and monocytic (Mo) differentiation of step lllP HPCs, as evaluated by RT-PCR in two repreaentative HPC purification cultura axperiments (top and bottom panels).Ptm controls are presented. Positive controls are also presented. Similar results were obtained in two other experiments. day 2 to 5 and day 9 to 14 respectively, while it was not detected in the Mo series; ( 2 ) G-CSFR mRNA was transiently expressed at low level in day 2-5 E culture, and barely detected in the Mk and Mo series until initial maturation; ( 3 ) M-CSFR was transiently expressed at low level in day 2 E culture, barely detected in G lineage until maturation and absent in the Mk series; and (4) TpoR mRNA was barely or not detected in E, G and Mo cultures. These findings were confirmed by fluorescence studies quantifying the binding of PE-labeled G-CSF to HPCs differentiating along the E, G, Mk, and Mo lineages (Figs 6G and 7B). The G-CSF binding, barely present on quiescent HPCs, was mildly induced in E, Mk,and Mo differentiation at day 2-5, then progressively decreased. In the G lineage the early receptor induction was followed by temporary downmodulation and then a rebound up to the initial induction level. Two series of control experiments indicated that the saturating G-CSF level added in the G culture partially downmodulates membrane G-CSFRs. Thus, (1) step IIIP cells were grown from day 0 to 2 in a modified G culture system, treated with standard IL-3 and GM-CSF concentrations (1 U/mL and 0.1 ng/mL, respectively) combined or not with graded concentrations of G-CSF up to the level in G culture ( 5 , 5 0 , or 500 U/mL); thereafter, G-CSF bound to cell mem- brane was removed (see Materials and Methods) and GCSF binding was measured with PE-labeled G-CSF. Cells cultivated in the presence of 500 U/mL G-CSF displayed a lower G-CSF binding capacity than those grown without GCSF or with 5-50 U/mL G-CSF (Table 3). (2) Unilineage HPC differentiation along the G pathway was induced by treatment with standard IL-3/GM-CSF levels without G-CSF addition (in this modified unilineage culture, G-cell growth is slower than in standard G culture, results not shown): interestingly, granulopoietic cells displayed higher percentages of G-CSFR' cells in G-CSF- modified G culture than G-CSF' standard G culture (Fig 7B). The G-CSFR expression patterns evaluated at protein level in G-CSF- culture (Fig 7B) and at mRNA level in G-CSF' culture (Fig 5B) are strictly consistent. DISCUSSION The analysis of key mechanisms controlling human hematopoiesis has been hindered by lack of (1) methodology for stringent purification and abundant recovery of HPCs and ( 2 ) culture systems for unilineage differentiation/maturation of the purified HPCs. Thus, little is known on HGFR expression in hematopoiesis, particularly at discrete stages of HPC differentiatiodmaturation along the different lineages. We Table 2. Step lllP HPCs Unilineage Culture Systems: Analysis of Cell DifferentiationlMaturation(mean from at least three experiments) Day 0 Assay of HPCs" Colony Type and No./100 Cells Unilineage Culture System Erythroid (El BFU-E CFU-MK CFU-G CFU-M 28 0 0 0 First Unilineage Culture Week: Assay of Differentiating HPCs' Second Unilineage Culture Week Analysis of Maturing Precursors (a) HPC No. ( X l @ ) (b)Colony Size (cells no./colony) Lineage-Specific Maturation Marker (% positive cells) dO (a) + 14 + d7 199 - -, d9 d8 0 - d14-16 Glycophorin A Morphology (% values) 614-16 Polychromatic + orthochromatic erythroblasts Megakaryocytic (Mk) Granulocytic-neutrophilic (GI Monocytic (Mo) 0 0 0 7 0 0 0 16 0 0 0 4 (b) 1,300 (a) (b) 3.5 24 (a) + 400 + 22.8 3 8 + 24 (b) 700 + 250 (a) 2 + 11 (b) 2,000 -t 800 + HPC assay in clonogenic semisolid culture supplemented with unilineage HGF stimulus. t Not evaluated; ie, below colony scoring threshold. + -+ -t + -+ -, + N.E.t 19 + 98 0 N.E.t CD42b 24 + 95 0 CDllb N.E.t 37 <400 23 + Late MK cells + 98 Metamyelocytes granulocytes 98 CD14 16 98 98 Monocytes/ macrophages 94 97 + From www.bloodjournal.org by guest on August 3, 2017. For personal use only. 3400 Table 3. Binding of PE-Conjugated G-CSF to Step IMP Cells Grown for Two Days in Absence or Presence of Graded Concentrations of Exogenous G-CSF TESTA ET AL differentiation: it is suggested that these HPCs are either gradually channeled into the unilineage pathway or undergo apoptosis (see Results). Thus, both selective and inductive phenomena may underlie HPC differentiation in unilineage G-CSF Binding cultures. Accordingly, studies on the differentiation of primiG-CSF Concentration Fluorescence Intensity tive HPCs’ and multilineage GF-dependent cell linesS5sugIUlmL) % Positive Cells (arbitrary units) gest that late-acting HGFs modulate HPC differentiation None 14 43 allowing the survival of selected categories of HPCs. whose 5 71 42 differentiation capacities are intrinsically determined. How50 55 37 ever, the HPC differentiation program, although partially 500 39 31 preprogrammed, is in part induced by HGF(s) through mechanisms other than anti-apoptotic ones.56Furthermore, we observed that the percentage of apoptotic cells is (1) low (ie, < 10%) in E and G cultures, where the initially responsive have recently developed novel culture techniques to selecHPC subpopulation is relatively large, and (2) more elevated tively channel HPC proliferation along the different sefies.4,19-25 On this basis, we have analyzed the expression (ie, 530%) in Mk and Mo cultures, where the initially responsive HPC subpopulation is relatively small (results not of an extensive series of HGFRs on 90% purified HPCs shown). undergoing strictly unilineage E, G, Mk, or Mo differentiaIn the second culture week, we monitored a gradual wave tionlmaturation. of unilineage maturation of morphologically recognizable The HGFR assays provided consistent results. The mRNA precursors along the E, G, Mk, or Mo pathway through levels of c-kit, IL-6R, IL-3R, and GM-CSFR, evaluated by terminal maturation. semiquantitative RT-PCR, were compatible with correExpression of early-acting HGFRs (bFGFR- 1, jt3, c-kif) sponding protein results obtained by MoAb andor labeled consistently decreases during hematopoietic differentiation. ligand assay(s): this suggests that posttranscriptional mechaIn line with these observations, bFGF, FL, and K L act on nisms do not play a major role in the expression of these primitive stages of hematop~iesis?.~: FL amplifies the numHGFRs. ber of putative stem cells (long-term culture initiating cells In protein assays, receptor occupancy by its ligand supple[LTC-ICs]): whereas all of these cytokines potentiate the mented in culture was carefully considered: a series of conaction of other HGFs on primitive HPCs [blast CFU (CFUtrol studies insured that this potential bias did not obscure B), high proliferative potential colony-forming cells (HPPresults and interpretation thereof (see Results). CFC)J and more differentiated HPCS.?-~ c-kit expression deAs previously mentioned, we reported that quiescent creases less rapidly in the E and Mk than in the G and HPCs purified from PB express IL-3Rs at elevated level and Mo lineages: these results are consistent with the prevailing IL-6Rs/GM-CSFRs at moderate level, whereas EpoRs were erythroid differentiation of c-kit’ HPCs in cultures supplebarely dete~tab1e.l~ We confirm these results and further mented with Epo, G-CSF, KL, and multilineage HGFs.” show that receptors for early-acting HGFs (bFGF, flt3, and IL-6R expression similarly declines in the E and C linIU)are clearly expressed on the purified HPCs, whereas the eages, but is maintained until the early or late stages of receptors for other unilineage HGFs (G-CSF, M-CSF, and maturation in the Mk and Mo lineages, respectively. The Tpo) are barely or not expressed. This pattem is in line with persistent expression in the two latter series is consistent BM studies showing that: (1) flt3R is selectively expressed with the two biologic activities of IL-6, which stimulates in on CD34’ cells4’; (2) c-kit is present on early/late HPCS,~~.‘’ vitro” and i n vivosx megakaryocytopoiesis and potentiates whereas ILdR is present on different HPC subsets including the proliferation of monocytes in clonogenetic5’ and uniline34+/33- and 34+/33+cells5’; (3) M-CSFR,” G-CSFR,” and age Mo culture (our unpublished observations). TpoR” are scarcely expressed on CD34’ cells. However, The expression kinetics of multilineage HGFRs differs in these studies were carried out on BM CD34+ cells displaying the four lineages. The IL-3R is rapidly downmodulated in a low or moderate HPC clonogenetic efficiency (comprised E and Mk differentiation, while the expression slowly debetween 2% and 35%). c-kit was also detected on putative clines in the Mo pathway and is sustained in the G lineage, HSCS.’~ thus leading to significant IL-3R expression on terminal Mo In the unilineage cultures HPC differentiation and precurand particularly G cells. The GM-CSFR, although downsor maturation gradually progress in the first and second modulated in all series with respect to receptor density, is week, r e s p e c t i ~ e l y (see ~ ~ ~also ~ - ~Table ~ 3). still detected by MoAb assay on virtually all terminal G, HPC differentiation in the first week deserves discussion. Mo, and Mk cells. These expression patterns are in line with The E, G, Mk, and Mo culture systems are respectively fed the biologic activity of IL-3 and GM-CSF: (1) both stimulate by BFU-E, CFU-GM, CFU-Mk, and CFU-M. The proliferatthe proliferation of early HPCs and their differentiation along the G, Mo, E, and Mk pathways“; (2) their combined action ing HPCs gradually differentiate along the unilineage pathchannels purified HPCs into G differentiationlmaturation way, as indicated by the progressive decrease of ( I ) their coupled with a 5 5 % to 10% Mo cell component’y~2’~22; (3) proliferative potential and (2) CD34 expression. Early-acting both sustain the functional activity of neutrophils and monoand multilineage HGFs added to these cultures may initially cytes.‘”.’‘ allow the survival of HPCs not restricted to the unilineage From www.bloodjournal.org by guest on August 3, 2017. For personal use only. GROWTH FACTOR RECEPTORS IN HEMATOPOIESIS In the unilineage culture systems the assay of late-acting HGFRs at protein level bears two limitations, caused by the addition of the unilineage HGF at saturating level: thus, (1) the HGF unilineage HGFR interaction may interfere with the receptor assay by ligand or MoAb (see G-CSFR results); more important, (2) the HGF/HGFR interaction causes receptor internalization6’ and may therefore lead to an underestimation of HGFR expression in the unilineage differentiation pathway (as indicated by experiments on G-CSFR expression in G culture supplemented or not with graded GCSF levels; see Results). The expression pattern of unilineage HGFRs (EpoR, GCSFR, TpoR, and M-CSFR) has been extensively analyzed by RT-PCR. This assay allows only a semiquantitative evaluation of HGFR mRNA levels: however, the observed expression patterns are markedly differentiated and permit a comparative stage- and lineage-specific analysis. It is also noteworthy that the expression of E ~ o Rand~ M-CSFRffl ~ genes is largely regulated via transcriptional mechanisms. Our observations indicate that the unilineage HGFRs are gradually induced and then sustainedly expressed in the pertinent lineage (ie, expression of EpoR, G-CSFR, M-CSFR, and TpoR is sustained in the E, G, Mo, and Mk series, respectively). In some cases, they are initially and transiently expressed at low level in other lineages (eg, EpoR in G lineage and G-CSFR in E lineage): hypothetically, this may reflect the initial differentiation of a minority of multipotent/ bipotent HPCs (see above). The low EpoR expression on maturing Mk cells is compatible with studies in the mouse indicating presence of EpoRs on rodent megakaryocyte^.^^ Previous studies have provided limited insight into these phenomena: thus, analysis of total murine BM cells by 1251[G-CSF] autoradiography6’ and human erythroid colonies by ‘2sI-[Ep]s.67indicated that the G-CSFR and EpoR are expressed on G and E lineage cells, respectively. Altogether, these novel observations allow a comprehensive analysis of HGFR expression in discrete hematopoietic cell populations at sequential stages of differentiatiodmaturation along the four main lineages. The findings suggest a two-step model for HGFRs expression during hematopoietic differentiation, which lends further support to the HGFR cascade transactivation hypothesis proposed by our group.14 The first step involves interaction of multilineage HGFs with their receptors on HPCs: this leads to progenitor proliferatioddifferentiation, which is followed by expression of unilineage HGFRs. The second one entails interaction of a unilineage HGF with its receptor, leading to sustained receptor expression and unilineage differentiatiodmaturation. This model is also in line with the GM-CSF induction of E ~ o R . ~ ’ It is generally assumed that each late-acting HGFs downmodulates its own receptor at membrane leve1.6’ However, our results indicate that each unilineage HGFR is sustainedly expressed in the pertinent series. By implication, it is suggested that the unilineage HGF/HGFR interaction triggers molecular mechanisms sustaining the receptor expression, 3401 which override the receptor internalization otherwise induced by HGFmGFR interaction. Accordingly, optimal MCSFR expression in a c - h s + FDC-P1 cell line requires the continuous presence of exogenous M-CSF.~*It is also noteworthy that a combined IL-3/GM-CSF stimulus induces G lineage differentiatiodmaturation coupled with G-CSFR exp r e s ~ i o n ’ ~ ~(and ’ ~ ~present ~’ results): it is apparent, therefore, that unilineage HGF/HGFR interaction is sufficient but not necessary to induce unilineage differentiation and sustained HGFR expression. The expression of unilineage HGFRs may be controlled by lineage-specific transcription factors (TF) activating the receptor promoter. In this regard, (1) the erythroid TF GATA-1, sustainedly expressed following interaction of Epo with its receptor, maintains EpoR tran~cription.~~.~’ (2) A functional linkage between lineage-specific TFs and unilineage HGFRs is supported by the similarity of their expression pattern in unilineage HPC differentiation, eg, both erythroidspecific TFs (GATA-1, NF-E2, TAL-1) and EpoR are sustainedly expressed in the erythroid lineage, but only transiently induced at lower level in the early differentiation stages of the G ~ . e r i e s ~ (and * ~ ’ this ~ ~ ~report). ~ ( 3 ) In the monocytic lineage optimal PU.1 expression is required for MCSFR expression.@Hypothetically, the expression of other late-acting HGFRs may be similarly mediated via lineagespecific TF(s). Altogether, our observations are compatible with the following two-step molecular hypothesis. (1) Multilineage HGFs trigger HPCs into proliferatioddifferentiation: this causes induction of lineage-specific TFs, which in turn induce unilineage HGFR(s); (2) in the unilineage HPC differentiatiodmaturation pathway expression of the pertinent, lineage-specific TF(s) and HGFR is sustained by self-maintaining molecular loop(s) at the level of TF and HGFR promoters. Hematopoietic commitment has been interpreted in terms of stocha~tic,~~-~’ inductive? or hybrid74models. The inductive model proposes that binding of HGF(s) to their receptor(s) induces differentiation, in that it determines lineage choice in multipotent HPCs by activating the transcription of a lineage-specific gene program. The stochastic model proposes that multipotent HPCs do not require exposure to external inducing stimuli to undergo lineage restriction: HGFs simply permit the proliferation of intrinsically committed cells and the subsequent expression of the mature cell phenotype. Our results are in part compatible with both models. Hypothetically, (1) the differential expression of receptors for early-acting HGFs on quiescent HPCs may reflect a stochastic process in early commitment. (2) Consistent expression of multi-lineage HGFRs on quiescent HPCs allows the induction of progenitor cycling by IL-3/GM-CSF. (3) Expression of unilineage HGFR(s) on the proliferating HPCs may be stochastic. (4) The sustained expression of each unilineage HGFR through the corresponding differentiatiodmaturation pathway represents an inductive process, mediated by the pertinent unilineage HGF. The unilineage From www.bloodjournal.org by guest on August 3, 2017. For personal use only. A B I I 100 El El U le Icn 1w Day 0 El Day 2 Day 2 KI Day 5 Day 5 .. t t t 2 Day 7 t Day9 jJ w CD34 ...: Day 9 D a y 12 CDY ---c -e CD34 L: - CD34 IC, ,. - Day 12 I Day 0 C Day 0 I J Day 2 Day 2 Day 5 la t f Day 7 ka KI Day 9 :... -.a'.. Day 9 I. Day 12 CD34 4 CD34 4 CD34 Fig 6. - CD34 - Day 12 From www.bloodjournal.org by guest on August 3, 2017. For personal use only. GROWTH FACTOR RECEPTORS IN HEMATOPOIESIS E 3403 Day 0 J El Day 2 Day 5 t Day 2 PI Day 5 Day 7 w c! f, c! Day 7 d d Day 9 Day 9 Day 12 CD34 -D CD34 4 CD34 - Day 12 Day 0 Day l 2 Day 14 CD34 - CD34 - Fig 6. Representative experiments of flow cytometric analysis for fit3 (A), c-kit (6).IL-6R (C), IL-3R (D and E), GM-CSFR IF), and G-CSFR (G) during selective erythroid (E), granulocytic (GI, megakaryocytic (Mk), or monocytic (Mol differentiation of step lllP HPCs. c-kit and GM-CSFR were evaluated by specific MoAbs. flt3, c-kit, GM-CSFR, and G-CSFR expression is shown in E and G lineages, IL-6R expression in Mk and M o lineages, and IL-3R in all four lineages. See also mean ? SEM expression values in Fig 7A and B. From www.bloodjournal.org by guest on August 3, 2017. For personal use only. 3404 TESTA ET AL (Gi IMOi IM,j L 100 8 20 0 3 6 9 1 2 0 3 6 9 12 m B lMOl . m, [GM-CSFR ] 1W + ml 0 3 6 9 wvr 1 2 w 0 3 6 9 bys 1 2 0 3 6 9 DWS HGF prevailing in each microenvironmental niche may channel HPCs expressing its receptor into the COKespondh2 lineage; unipotent HPCs and differentiated precursors growing in the absence of a sufficient level of the pertinent unilineage HGF seemingly undergo apoptosis.' 1 2 0 3 6 9 tmyt 1 2 Fig 7. Percentage of f/t3+, c&it+, IL-6R+ (AI, IL-3R+, GMCSFR+, and G-CSFR+ (B) cells during step lllP HPC erythroid (E), granulocytic (GI, megakaryocytic (Mk), or monocytic (Mol differentiation. The receptor density of IL-3R and GM-CSFR is also presented, as evaluated in terms of mean fluorescence intensity (arbiiary units). In the case of G-CSFR, representative data in G-CSF- culture are also shown (see Results). fh3, c-kit, and IL-3R were evaluated by M o w ; lL-6R. OM-CSFR, and GCSFR by labeled ligand. (further details in Materials and Methods). Mean 2 SEM v d u r from three separate experiments. See also representative experiments in Fig 6A-G. REFERENCES 1. Ogawa M: Differentiation and proliferation of hematopoietic stem cells. Blood 81:2844, 1993 2. Wendling F, Vainchenker W: Thrombopoietin and its receptor the proto-oncogene c-mpl. Curr Opin Hematol 2:231, 1995 From www.bloodjournal.org by guest on August 3, 2017. For personal use only. GROWTH FACTOR RECEPTORS IN HEMATOPOIESIS 3. Lyman SD, James L, Bos TV, de Vries P, Brasel K, Gliniak B, Hollingsworth LT, Picha KS, McKenna HJ, Splett RR, Fletcher FA, Maraskovsky E, Farrah T, Foxworthe D, Williams DE, Beckmann MP: Molecular cloning of a ligand for the flt3/flk-2 tyrosine kinase receptor: A proliferative factor for primitive hematopoietic cells. Cell 75: 1157, 1993 4. Gabbianelli M, Pelosi E, Montesoro E, Valtieri M, Luchetti L, Samoggia P, Vitelli L, Barberi T, Testa U, Lyman S , Peschle C: Multi-level effects of flt3 ligand on human hematopoiesis: Expansion of putative stem cells and proliferation of granulomonocytic progenitorsimonocytic precursors. Blood 86: 1661, 1995 5. Bernstein ID, Andrews RG, Zsebo KM: Recombinant human stem cell factor enhancesthe formation of colonies by CD34+ and CD34+ lin- cells, and the generation of colony-forming cell progeny from CD34+ lin- cells cultured with interleukin-3, granulocyte colony-stimulating factor, or granulocyte-macrophage colony-stimulating factor. Blood 77:2316, 1991 6. Peschle C, Gabbianelli M, Testa U, Pelosi E, Barberi T, Fossati C, Valtieri M, Leone L: c-kit ligand reactivates fetal hemoglobin synthesis in serum-free cultures of stringently purified normal adult burst-forming unit-erythroid. Blood 81:328, 1993 7. Gabbianelli M, Sargiacomo M, Pelosi E, Testa U, Isacchi G, Peschle C: “Pure” human hematopoietic progenitors: Permissive action of basic fibroblast growth factor. Science 249:1561, 1990 8. Leary AG, Wong GG,Clark SC, Smith AG, Ogawa M: Leukemia inhibitory factor differentiation-inhibiting activityhuman interleukin for DA cells augments proliferation of human hematopoietic stem cells. Blood 75:1960, 1990 9. Leary AG, Ikebuchi K, Hirai Y, Wong GG,Yang YC, Clark SC, Ogawa M: Synergism between interleukin-6 and interleukin3 in supporting proliferation of human hematopoietic stem cells: Comparison with interleukin-la. Blood 71:1759, 1988 10. Park L, Gillis S, Urdal D: Hematopoietic growth-factor receptors, in Dexter M, Garland J, Testa N (eds): Colony Stimulating Factors. New York, NY, Dekker, 1990, p 39 11. Ullrich A, Schlessinger J: Signal transduction by receptors with tyrosine kinase activity. Cell 61:203, 1990 12. Cosman Y, Lyman SD, Idrerda RL, Backmann MP, Park LS, Goodwin RG, March CJ: A new cytokine receptor superfamily. Trends Biochem Sci 15:265, 1990 13. Bazan JF: Structural design and molecular evolution of a cytokine receptor superfamily. Proc Natl Acad Sci USA 87:6934, 1990 14. Testa U, Pelosi E, Gabbianelli M, Fossati C, Campisi S , Isacchi G, Peschle C: Cascade transactivation of growth factor receptors in early human hematopoiesis. Blood 81:1442, 1993 15. Jubinsky FT,Laurie AS, Nathan DG, Yetz-Adepe J, Sieff CA: Expression and function of the human granulocyte-macrophage colony-stimulating factor receptor a subunit. Blood 84:4174, 1994 16. Wognum AW, Westerman Y,Visser TP, Wagemaker G: Distribution of receptors for granulocyte-macrophagecolony-stimulatingfactor on immature CD34’ bone marrow cells, differentiating monomyeloid progenitors, and mature blood cell subsets. Blood 84:764, 1994 17. Wognum AW, Visser TP, De Jong MO, Egeland T, Wagemaker G Differential expression of receptors for interleukin-3 on subsets of CD34- expressing hematopoietic cells of rhesus monkeys. Blood 86:581, 1995 18. Wagner JE, Collins D, Fuller S , Schain LR, Berson AE, Almici C, Hall MA, Chen KE, Okarma TB, Lebkowski JS: Isolation of small, primitive human hematopoietic stem cells: Distribution of cell surface cytokine receptors and growth in SCID-Hu mice. Blood 86512,1995 19. Sposi NM, Zon LI, Car&A, Valtieri M, Testa U, Gabbianelli M, Mariani G, Bottero L, Mather C, Orkin SH, Peschle C: Cell cycledependent initiation and lineage-dependent abrogation of GATA-I expression in pure differentiating hematopoietic progenitors. Proc Natl Acad Sci USA 89:6353, 1992 3405 20. Labbaye C, Valtieri M, Testa U, Giampaolo A, Meccia E, Sterpetti P, Parolini I, Pelosi E, Bulgarini D, Cayre YE, Peschle C: Retinoic acid downmodulates erythroid differentiation and GATA1 expression in purified adult progenitor culture. Blood 83:651, 1994 21. Giampaolo A, Sterpetti P, Bulgarini D, Samoggia P, Pelosi E, Valtieri M, Peschle C: Key functional role and lineage-specific expression of selected HOXB genes in purified hematopoietic progenitor differentiation. Blood 84:3637, 1994 22. Condorelli GL, Vitelli L, Valtieri M, Marta I, Montesoro E, Lulli V, Baer R, Peschle C: Coordinate expression and developmental role of Id2 protein and TALlE2A heterodimer in eythroid progenitor differentiation. Blood 85: 1181, 1995 23. Labbaye C, Valtieri M, Barberi T, Meccia E, Masella B, Pelosi E, Condorelli G, Testa U, Peschle C: Differential expression and functional role of GATA-2, NF-E2 and GATA-1 in normal adult hematopoiesis. J Clin Invest 95:2346, 1995 24. Condorelli GL, Testa U, Valtieri M, Vitelli L, De Luca A, Barberi T, Montesoro E, Campisi S, Giordano A, Peschle C: Modulation of retinoblastoma gene in normal adult hematopoiesis: peak expression and functional role in advanced erythroid differentiation. Proc Natl Acad Sci USA 92:4808, 1995 25. Guemero R, Testa U, Gabbianelli M, Mania G, Montesoro E, Macioce G, Pace A, Ziegler B, Hassan HJ, Peschle C: Unilineage megakaryocytic proliferation and differentiation of purified hematopoietic progenitors in serum-free liquid culture. Blood 863725, 1995 26. Valtieri M, Gabbianelli M, Pelosi E, Bassano E, Petti S, Russo G, Testa U, Peschle C: Erythropoietin alone induces erythroid burst formation by human embryonic but not adult BFU-E in unicellular serum-free culture. Blood 74:460, 1989 27. Eliason JF:Granulocyte-macrophage colony formation in serum-free culture: Effects of purified colony-stimulating factors and modulation by hydrocortisone. J Cell Physiol 128:231, 1986 28. Sasaki DT, Dumas SE, Engleman EG: Discrimination of viable and non-viable cells using propidium iodide in two color immunofluorescence. Cytometq 8:413, 1987 29. Sun Q, Woodcock J, Goodall G, Stomski F, Simmons P, Bagley C, Smith W, Gamble J, Wadas M, Lopez A: Monoclonal antibody 7G3 recognizes the N-terminal domain of the human interleukin-3 (IL-3) receptor a-chain and functions as a specific IL-3 receptor antagonist. Blood 87:83, 1996 30. Rosnet 0, Buhring HJ, Marchetto S, Rappold S , Lavagna C, Sainty D, Amoulet C, Chabannon C, Kaur L, Hannum C, Bizuaum D: The human flt3iFLK2 receptor tyrosine kinase is expressed at the surface of normal and malignant hematopoietic cells. Leukemia (in press) 31. Nicola N, Wycherley K, Boyd A, Layton J, Cary D, Metcalf D: Neutralizing and nonneutralizing monoclonal antibodies to the human granulocyte-macrophage colony-stimulating factor receptor alpha-chain. Blood 82:1724, 1993 32. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ: Isolation of a biologically active ribonucleic acid from sources enriched in ribonuclease. Biochem 18:5294, 1979 33. Dionne CA, Crumley G, Bellot F, Kaplow JM, Searfoss G, Ruta M, Burgess WH, Jaye M, Schlessinger J: Cloning and expression of two distinct high-affinity receptors cross-reacting with acid and basic fibroblast growth factors. EMBO J 9:2685, 1990 34. Yarden Y, Kuang W-J, Yan-Feng T, Coussens L, Munemitsu S, Dull TJ, Chen E, Schlessinger J, Francke U, Ulrich A: Human proto-oncogene c-kif: A new cell surface receptor tyrosine kinase for an unidentified ligand. EMBO J 6:3341, 1987 35. Yamasaki K, Taga T, Hirata Y, Yawata H, Kawanishi Y, Sefd B, Taniguchi T, Hirano T, Kishimoto T Cloning and expression of the human interleukind (BSF-uIFN b2) receptor. Science 2825, 1988 36. Kitamura T, Sat0 N, Arai K, Miyajima A: Expression cloning of the human IL-3 receptor cDNA reveals a shared p subunit for the human IL-3 and GM-CSF receptors. Cell 66:1165, 1991 From www.bloodjournal.org by guest on August 3, 2017. For personal use only. 3406 37. Hayashida K, Kitamura T, Gorman DM, Arai K-I, Yokota T, Miyajima A: Molecular cloning of a second subunit of the receptor for human granulocyte-macrophage colony-stimulating factor (GMCSF): Reconstitution of a high-affinity GM-CSF receptor. Proc Natl Acad Sci USA 87:9655, 1990 38. Gearing DP, King JA, Gough NM, Nicola NA: Expression cloning of a receptor for human granulocyte-macrophage colonystimulating factor. EMBO J 8:3667, 1989 39. Jones SS, D'Andrea AD, Haines LL, Wong GG: Human erythropoietin receptor: Cloning, expression and biologic characterization. Blood 76:3 I , 1990 40. Fukunaga R, Seto Y, Mizushima S, Nagata S: Three different mRNAs encoding human granulocyte colony-stimulating factor receptor. Proc Natl Acad Sci USA 8723702, 1990 41. Hampe A, Gobert M, Sherr CJ, Galibert F: Nucleotide sequence of the feline retroviral oncogene v-fms shows unexpected homology with oncogenes encoding tyrosine-specific protein kinases. Proc Natl Acad Sci USA 81:85, 1984 42. Vigon I, Momon JP, Cocault L, Mitjavila MT, Tambourin P, Gisselbrecht S, Souyri M: Molecular cloning and characterization of MPL, the human homolog of the v-mpl oncogene: Identification of a member of the hematopoietic growth factor receptor superfamily. Proc Natl Acad Sci USA 895640, 1992 43. Baum CM, Weissman IL, Tsukamoto AS, Buckle AM, Peault B: Isolation of a candidate human hematopoietic stem-cell population. Proc Natl Acad Sci USA 89:2804, 1992 44. Craig W, Kay R, Cutler RL, Lansdorp PM: Expression of Thy-I on human hematopoietic progenitor cells. J Exp Med 177:1331, 1993 45. Cannistra S, Groshek P, Garlick R, Miller T, Griffin J: Regulation of surface expression of the granulocytelmacrophage colonystimulating factor receptor in normal human myeloid cells. Proc Natl Acad Sci USA 87:93, 1990 46. Kaushansky K, Broudy VC, Grossman A, Humes J , Lin N, Ren HP, Bailey MC, Papayannopoulou T, Forstrom JW, Sprugel KH: Thrombopoietin expands erythroid progenitors, increases red cell production and enhances erythroid recovery after myelosuppressive therapy. J Clin Invest 96:1683, 1995 47. Small D, Levenstein M, Kim E, Carow C, Amin S, Rockwell P, Witte L, Burrow C, Ratajczak MZ, Gewirtz AM, Civin CI: STK1, the human homolog of flk2/flt3, is selectively expressed in CD34' human bone marrow cells and is involved in the proliferation of early progenitor/stem cells. Proc Natl Acad Sci USA 91:459, 1994 48. Briddell RA, Broudy VC, Bruno E, Brandt JE, Srour EF, Hoffman R: Further phenotypic characterization and isolation of human hematopoietic progenitor cells using a monoclonal antibody to the c-kit receptor. Blood 79:3159, 1992 49. Gunji Y, Nakamura M, Osaka H, Nagayoshi K, Nakauchi H, Miura Y, Yanagisawa M, Suda T: Human primitive hematopoietic progenitor cells were more enriched in kit"'" than in kithIghcells. Blood 82:3283, 1993 50. Wognum AW, VanGils FCJM, Wagemaker G: Flow cytometric detection of receptors for interleukin-6 on bone marrow and peripheral blood cells of humans and Rhesus monkeys. Blood 81:2036, 1993 51. Voso MT, Burn TC, Wulf G, Lim B, Leone G, Tenen DG: Inhibition of hematopoiesis by competitive binding of transcription factor PU.1. Proc Natl Acad Sci USA 91:7932, 1994 52. Orlic D, Anderson S, Biesecher LG, Sorrentino BP, Bodine DM: Pluripotent hematopoietic stem cells contain high levels of mRNA for c-kit, GATA-2, p45 NF-E2, and c-myb and low levels or no mRNA for c-fms and the receptors for granulocyte colonystimulating factor and interleukins 5 and 7. Proc Natl Acad Sci USA 92:4601, 1995 53. Debili N, Wendling F, Cosman D, Titeux M, Florindo C, Dusanter-Fourt I, Schooley K, Methia N, Charon M, Nador R, Bettaieb TESTA ET AL A, Vainchenker W: The Mpl receptor is expressed in the megakaryocytic lineage from late progenitors to platelets. Blood 85:391, 1995 54. Berardi AC, Wang A, Levine JD, Lopez P, Scadden DT: Functional isolation and characterization of human hematopoietic stem cells. Science 267:104, 1995 55. Fairbairn LJ, Cowling GJ, Reipert BM, Dexter TM: Suppression of apoptosis allows differentiation and development of a multipotent hemopoietic cell line in the absence of added growth factors. Cell 74:823, 1993 56. Rodel JE, Link DC: Suppression of apoptosis during cytokine deprivation of 32D cells is not sufficient to induce complete granulocytic differentiation. Blood 87:858, 1996 57. DeJong MD, Wagemaker G, Wognum AW: Separation of myeloid and erythroid progenitors based on expression of CD34 and c-kit. Blood 86:4076, 1995 58. Kimura H, Ishibashi T, Uchida T, Maruyamd Y, Friese P, Burstein SA: Interleukin 6 is a differentiation factor for human megakaryocytes in vitro. Eur J Immunol 20:1927, 1990 59. Hansen JH, Kluin-Nelemans JC, Van Damme J, Wientjens GJHM, Willemze R, Fibbe WE: Interleukin 6 is a permissive factor for monocytic colony formation by human hematopoietic progenitor cells. J Exp Med 175: 1 15 I , I992 60. Clark SC, Kamen R: The human hematopoietic colony-stimulating factors. Science 236:1229, 1987 61. Khwaja A, Linch DC:Effects of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor on neutrophil formation and function. Curr Opin Hematology 1:2 16, 1994 62. Walker F, Nicola N, Metcalf D, Burgess A: Hierarchical down-modulation of hemopoietic growth factor receptors. Cell 43:269, 1985 63. Chin K, Oda N, Chen K, Noguchi TC: Regulation of transcription of the human erythropoietin receptor gene by proteins binding to GATA-I and Spl motifs. Nucleic Acids Res 23:3041, 1996 64. Zhang DE, Hetherington CJ, Chen HM, Tenen DG: The macrophage transcription factor PU. I directs tissue-specific expression of the macrophage colony-stimulating factor receptor. Mol Cell Biol 14:373, 1994 65. Fraser JK, Tan AS, Lin F-K, Berridge MV: Expression of specific high-affinity binding sites for erythropoietin on rat and mouse megakaryocytes. Exp Hematol 17:10, I989 66. Sawada K, Krantz SB, Sawyer ST, Civin CI: Quantitation of specific binding of erythropoietin to human erythroid colony forming cells. J Cell Physiol 137:337, 1988 67. Sawada K, Krantz SB, Dai CH, Koury ST, Horn ST, Click AD, Civin CI: Purification of human blood burst-forming unitserythroid and demonstration of the evolution of erythropoietin receptors. J Cell Physiol 142:219, 1990 68. Gliniak BC, Rohrschneider LR: Expression of the M-CSF receptor is controlled posttranscriptionally by the dominant actions of GM-CSF or multi-CSF. Cell 63:1073, 1990 69. Orkin SH: GATA-binding transcription factors in hematopoietic cells. Blood 80575, 1992 70. Till JE, McCulloch EA, Siminovitch L: A stochastic model of stem cell proliferation based on the growth of spleen colony forming cells. Proc Natl Acad Sci USA 51:29, 1964 7 I . Kom AP, Henkelman RM, Ottensmeyer FP, Till JE: Investigations of stochastic model of haemopoiesis. Exp Hematol 1 :362, 1973 72. Nakahata T, Gros AJ, Ogawa M: A stochastic model of self renewal and commitment to differentiation of the primitive hemopoietic stem cells in culture. J Cell Physiol 1 13:455, 1982 73. Curry JL, Trentin JJ: Hemopoietic spleen colony-stimulating factors. Science 236: 1229, 1967 74. Just U, Stocking C, Spooncer E, Dexter TM, Ostertag W: Expression of the GM-CSF gene after retroviral transfer in hematopoietic stem cell lines induces synchronous granulocyte-macrophage differentiation. Cell 64: 1163, 1991 From www.bloodjournal.org by guest on August 3, 2017. For personal use only. 1996 88: 3391-3406 Expression of growth factor receptors in unilineage differentiation culture of purified hematopoietic progenitors U Testa, C Fossati, P Samoggia, R Masciulli, G Mariani, HJ Hassan, NM Sposi, R Guerriero, V Rosato, M Gabbianelli, E Pelosi, M Valtieri and C Peschle Updated information and services can be found at: http://www.bloodjournal.org/content/88/9/3391.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.