Original Paper
International Journal of Cell Cloning 1030-86 (1992)
Transforming Growth Factor p Inhibits the Action of Stem Cell Factor on Mouse and Human Hematopoietic Progenitors Ian K. McNiece", Ivan Bertoncellob, Jonathan R. Keller c, Francis W Ruscetti', Cynthia A. Hartley", Krisztina M. Zsebo" 'Arngen Inc., Thousand Oaks, California, USA; bPeter MacCallum Cancer Institute, Melbourne, Victoria, Australia; 'Frederick Cancer Kesearch Facility, Frederick, Maryland, USA. Key Words. Stem cells SCF TGF-P Inhibitors Hernatopoiesis Abstract. In agar culture of post 5-fluorouracil mouse bone marrow cells (FUBM), recombinant rat stem cell factor (rrSCF) synergises with granulocyte colonystimulating factor (G-CSF), interleukin-3 (IL-3) or interleukin-6 (IL-6) to stimulate primitive progenitor cells (HPP-CFCs). The addition of recombinant human transforming growth factor P (rhTGF-P) to cultures of FUBM containing rrSCF plus rhC-CSF, rrSCF plus recombinant murine (rm)IL3, or rrSCF plus rhIL-6 resulted in 100% inhibition of colony formation. Highly enriched populations of primitive bone marrow cells were obtained by isolating lineage negative (Lin -), Sca1-positive (Sca-1+) cells from normal mouse bone marrow. RhTGF-p inhibited 90% of colony formation stimulated by rrSCF plus rmIL-3 in agar culture of the Sca-l+ cells. RhTGF-P also inhibited colony formation in agar culture of post FU human bone marrow cells. The synergistic increase in colony formation obtained with recombinant human SCF (rhSCF) plus rhGMCSF and rhSCF plus rhIL-3 was inhibited by rhTGF-P (approx. 60% and 87% inhibition, respectively). RhTGF-P also totally inhibited the erythroid colony formation stimulated by rhSCF plus recombinant human erythropoietin (rhEpo). These data demonstrate that TGF-P inhibits SCF-stimulated colony formation of mouse and human BM. This inhibition on progenitor cells appears to be a direct action of TGF-P and is consistent with the target cells of SCF being more primitive progenitors than the CFCs stimulated by the CSFs alone.
Correspondence: Dr. Ian K. McNiece, Arngen Inc., Amgen Center, Thousand Oaks, CA 9 1320-1789. USA. Received November 22, 1991; provisionally accepted December 23, 1991; accepted for publication January 10, 1992.
0737- 1454/92/$2.00/0OAlphaMed Press
Introduction Transforming growth factor beta (TGF-P) has been shown to inhibit hematopoietic progenitor cell growth [l, 21. In particular, TGF-p was shown to inhibit the clonal growth of high proliferative potential colony forming cells (HPP-CFCs) [ 2 , 31. HPPCFCs have previously been shown to be a primitive progenitor cell population whose properties correlate closely with those of primitive stem cells [4]. TGF-P is produced by a variety of cells including platelets, bone cells, and T lymphocytes and has been shown by immunohistochemical staining to be locally produced in areas of active hematopoiesis [ 5 ] , suggesting that TGF-P may play a regulatory role in hemopoietic cell growth. Recently a novel growth factor termed stem cell factor (SCF) was isolated from Buffalo rat liver cells (BRL-3A) by its synergistic action on HPP-CFCs [6]. The properties of the recombinant rat and human S C F (rrSCF and rhSCF) suggest that it is a multipotent factor acting on cells of the myeloid, mast and lymphoid lineages [6-lo]. In liquid culture of normal mouse bone marrow cells, rrSCF generates pure populations of mast cells [8], while in semisolid agar culture, rrSCF stimulates mixed colony formation of neutrophil, macrophage and megakaryocyte cells [7]. Interleukin-7 (IL-7) synergises with rrSCF to stimulate pre-B cell colony formation in semisolid agar culture of normal mouse bone marrow cells [6, 91. In agar culture of human bone marrow cells, r h S C F synergises with C S F s and erythropoietin (Epo) to stimulate progenitor cells of the myeloid and erythroid lineages [lo]. These prop-
McNiece/Bertoncello/Keller/Ruscett~a~tleyEsebo.
81
erties demonstrate that SCF is a multilineage factor which may act directly upon a common lymphoidmyeloid stem cell. In this report we examine further the role of TGF-p on early hematopoiesis and demonstrate an inhibitory role of TGF-P on SCF-stimulated proliferation and differentiation of primitive progenitor cells in vitro.
including 714, B220, L3T4, and Lyt-2, and positively labeled cells were removed using magnetic beads (Dynal, Great Neck, NY) [13]. The low density, Lin- cells were then labeled with Sca-1 antibody [14] as previously described [15] and Sca-1 positive cells FACS sorted with an initial gating on the blast cell population [15].
Materials and Methods
Growth Factors Recombinant factors were prepared as previously described; rat SCF (rrSCF) [7] and human SCF (rhSCF) [7], human granulocyte-macrophage CSF (rhGM-CSF) [ 161, human granulocyte CSF (rhG-CSF) [17], rhIL-3 [18], rhEpo [19], and rhIL-6 [20] were prepared from inclusion bodies formed in E. coli containing the appropriate expression plasmid and purified to greater than 99% purity. rh TGF-p [21] was prepared from the medium of a transfected Chinese Hamster Ovarian cell host (CHO) and purified to greater than 98% purity. Recombinant murine IL-3 (rmlL-3) was purchased from Gibco Laboratories (Grand Island, NY). Recombinant human macrophage CSF (rhCSF-1) was a gift from Cetus Corp. (Emeryville, CA). The factors were used at optimal doses previously determined for GM-CFC colony formation or for synergistic interactions where the factors have no stimulatory effect alone: rrSCF, 100 ng; rhSCF, 100 ng; rhGM-CSF, 62.5 ng; rhG-CSF, 100 ng; rhE-3, 100 ng; rhEpo, 4 units; rhIL-6, 100 ng; rmIL-3, 150 units; and rhCSF-1, 1,600 units per culture.
Mouse Bone Marrow Cells Bone marrow cells were obtained from 2 to 3 month old Balb/c or B6D2F1 (BDF,) mice. Balblc mice were pretreated with 5-fluorouracil (150 mg/ kg) 2 days prior to sacrifice and harvest of bone marrow cells (FUBM). Bone marrow cells from normal BDF, mice were highly purified for primitive progenitor cells as described below. Human Bone Marrow Cells Bone marrow cells from patients treated with 5fluorouracil as part of the bone marrow transplant program at the University of Virginia were kindly supplied by Dr E! Quesenberry (University of Virginia, Charlottesville, VA). Informed consent was obtained under guidelines approved by the University of Virginia Institutional Human Investigations Committee. Low density nonadherent cells were depleted of mature cells as previously described [ 1ll. Agar Culture of Bone Marrow Cells Bone marrow cells were plated in double layer agar cultures in 35 mm dishes as previously described [12]. Alpha modification of Eagle's MEM (Flow Laboratories) supplemented with 20% fetal calf serum (FCS) was used for all cultures. The number of cells plated is presented in the Results section. Growth factors were incorporated in the underlays at a maximum of 13.2% of the total culture volume (1.5 ml) per dish. Cultures were gassed with a 5 % O,, 10% CO,, 85% N, mixture and incubated at 37°C and colonies were scored using a dissecting microscope. Only colonies containing 50 or more cells were scored. HPP-CFC colonies were scored as colonies with a diameter greater than 0.5 mm and containing approximately 50,000 or more cells. Purification of Bone Marrow Cells Bone marrow cells were obtained from normal BDF, mice, and low density, lineage negative cells were isolated as previously described [ 131. Briefly, low density cells were separated using Nycodenz (Nyegaard, Oslo, Norway) gradients. The cells were then labeled with a panel of monoclonal antibodies
Results Inhibition of Mouse Bone Marrow Colony Formation by rhTGF-p rrSCF has been previously shown to stimulate HPP-CFCs in agar culture of bone marrow cells of mice pretreated with 5-fluorouracil [6]. As shown in Table I, rhG-CSF, rmIL-3 and rhIL-6 alone resulted in no significant colony formation, while rrSCF and rhCSF-1 alone stimulated significant colony formation. The combination of rrSCF plus rhG-CSF stimulated a synergistic increase in total colonies and HPP-CFCs, while the combinations of rrSCF plus rmIL-3 and rrSCF plus rhIL-6 stimulated synergistic increases in only total colony numbers (Table I). The addition of rhTGF-P to cultures of rrSCF alone or in combination resulted in total inhibition of colony formation (Table I, Fig. 1). In contrast, the rhCSF-1 stimulated colony formation was not inhibited by rhTGF-p (Table I).
TGF-B inhibition of the action of SCF
82
Table I. Colony formation in agar culture of post FU mouse bone marrow
Factors
- rhTGF-P
+rhTGF-P
0 3.3 i 0.7 0 0.3 f 0.3 0 2.0 f 0.5
0 0 0 0 0
A. Total Colonies
Saline rrSCF rhG-CSF mIL-3 rhIL-6 rhCSF- 1 rrSCF/rhG-CSF diCF/rmIL-3 rrSCF/rhIL-6
2.3 i 0.5 0 0
15.0 i 0.5 ll.Of2.9 10.7 i 0.7
0
B. HPP-CFC 0 0 0 0 0 0
Saline rrSCF rhG-CSF mIL-3 rhIL-6 rhCSF- 1 rrSCF/rhG-CSF ITSCF/ITIIIL-~ rrSCF/rhIL-6
9.0 f 0.9 2.7 2.2
0.3 i 0.3
Bone marrow cells from 2 day post FU Balb/c mice were plated in agar culture at 100,OOO cells and incubated for 14 days. Colony numbers are the means i SE of triplicate dishes. RhTGF-P was added at 10 ng per culture. The data are presented from a single experiment which is representative of four separate experiments.
The dose response range of rhTGF-P inhibition of rrSCF plus rhG-CSF colony formation was determined from 0.15 ng to 10 ng. As shown in Figure 2, a dose of 2.5 ng or higher per culture resulted in total colony inhibition, and half maximal inhibition occurred at approximately 1 ng per culture. To determine if the effect of rhTGF-P was a direct action on rrSCF responsive progenitors, normal mouse bone marrow cells were highly enriched for primitive progenitors as described in the Methods section. As shown in Table 11, rrSCF synergised with rmIL-3 in cultures of unseparated and Lin-/ Sca-I+ cells. Addition of rhTGF-p resulted in 89% inhibition of colony formation by Lin -/Sca-l+ cells (Table 11).
- TGF - p
+TGF-P
Effect of rhTGF-P on Human Bone Marrow Cells
hibitory effect on colony formation stimulated by rhCM-CSF. RhSCF synergised with rhEpo, rhIL-3.
I
.
cells. Cultures contained 50.000 cells and were incubated for 14 days.
83
McNiece/Bertoncello/Keller/Ruscetti/Hartley/Zsebo. Table 11. Effect of TGF-P on SCF stimulated growth of highly enriched bone marrow cells GROWTH FACTORS
COLONY FORMATION Unseparated Lin - ISca- 1+
ITSCF + rmIL-3 ITSCF + mIL-3 + rhTGF-P
57.0 i 0.9 9.0 i 0.9
47.0 rt 0.5 5.0 i 0.8
84%
89%
Percentage Inhibition
Unseparated bone marrow cells from normal BDF, mice were plated at 50,OOO cells per culture while Lin-/Sca-l+ cells were plated at 2,000 cells per culture. Colony numbers are the means i SE of triplicate dishes. TGF-0 was added at 10 ng per culture. The data presented are from a single experiment which is representative of three separate experiments. 100
-
rhG-CSF and rhGM-CSF (Table 111) as previously reported [7, 101. The addition of rhTGF-P to cultures containing rhSCF plus rhEpo resulted in 100% inhibition of erythroid blast colony formation (Fig. 3). RhTGF-p also inhibited the synergistic increase in colony formation obtained with rhSCF plus rhGMCSF (approx. 60%inhibition) and rhSCF plus rhIL3 (approx. 87% inhibition). No significant inhibition was obtained for the combination of rhSCF plus rhG-CSF by rhTGF-p (Table 111).
Z
f m r
z
0
ce W
0
80-
40Y 80-
20
O
i
0
I
1
2
4
6
I
I
8
10
Discussion
rhTGF Ing]
Dose response curve of rhTGF-P inFig. 2. hibition of colony formation. Cultures contained rrSCF plus rhG-CSF and were incubated for 14 days after which time total colonies (W) and HPP-CFC colonies (A)were scored.
-TGF-P
+TGF-P
Colony formation stimulated by Fig. 3. rhSCF plus rhEpo in the presence and absence of rhTGFP in agar culture of post FU human bone marrow. Cultures contained 10,000 lineage negative cells and were incubated for 14 days.
TGF-P has been shown to be locally produced in areas of active hematopoiesis [ 5 ] and to selectively inhibit primitive hematopoietic progenitor cells [ 2 ] . In this report we have demonstrated the ability of TGF-0 to inhibit colony formation stimulated by SCF on mouse and human progenitor cells. SCF has been described by other groups as a mast cell growth factor [22] and a c-kit ligand [23]. The inhibition by TGF-p of SCF-stimulated HPP-CFC colony formation in cultures of mouse bone marrow is consistent with the role of SCF being on early hematopoietic cells. This is further supported by the stimulation of Sca-l+ cells by SCF and the inhibition of this stimulation by TGF-p. In cultures of post FU human bone marrow, TGF-p had little effect on colony formation stimulated by rhL-3 or rhG-CSF. The colony formation stimulated by rhSCF plus rhEpo, rhGM-CSF or rhIL-3 was greatly inhibited, suggesting that these combinations of factors stimulate more primitive progenitor cells than the progenitor cells stimulated by rhGM-CSF, rhIL-3 or rhG-CSF alone. This is consistent with the studies of Bernstein and colleagues who have demonstrated that rhSCF plus rhIL-3 stimulates a pre-CFC population not stimulated by any factor alone [24]. The majority of progenitor cells stimulated by rhG-CSF plus rhSCF were unaffected by rhTGF-P in contrast to total inhibition of rhTGF-P
TGF-B inhibition of the action of SCF
84 Table 111. Colony formation in agar culture of post FU human bone marrow
Factors Saline rhSCF rhG-CSF rhIL-3 rhGM-CSF rhEpo rhSCF/rhG-CSF rhSCF/rhIL-3 rhSCF/rhGM-CSF rhSCF/rhEpo
-
rhTGF-P
+rhTGF-P
0 0
0 0
17.0 k 1.4 4.3 i 0.7 10.3 i 0.3
20.7 i 1.5
0 33.0 t 2.1 27.7 2 1.8 37.3 i 0.5 64.0 i 4.3
4.3 ? 0.3 6.0 i 1.2 0
27.0 + 2.1 4.0 i 0.5 15.0 i 1.6 0
Bone marrow cells from patients pretreated with 5-FU were depleted of mature cells as described in Methods and plated in agar culture at 10,OOO cells and incubated for 14 days. Colony numbers are the means 2 SE of triplicate dishes. RhTGF-P was added at 10 ng per culture. The data presented are from a single experiment which is representative of two separate experiments. on mouse progenitors stimulated by rhG-CSF plus rrSCF. As the doses of FU administered to mice are approximately 10-fold higher than those administered to humans, the human post FU marrow most likely contains more committed progenitors than the mouse post FU marrow. The effects of SCF are probably broad, stimulating very primitive progenitor cells and committed progenitors. It may be a population of committed progenitors which respond to rhSCF plus rhG-CSF which are not affected by rhTGF-p. The other possibility is that a primitive population of progenitors responsive to rhSCF plus rhG-CSF are resistant to the effects of rhTGF-p. Further studies are required to examine these points. The direct action of rrSCF on highly purified Sca- 1+ cells has been demonstrated at the single cell level in combination with rmIL-3 or rhIL-6 [15, 251. Similarly, the action of TGF-p appears to be direct as shown in Table 11, inhibiting the action of rrSCF plus rmIL-3 in cultures of highly purified progenitor cells. This is consistent with the results of other investigators who have suggested a direct effect by TGF-P [ 2 ] . Steel mice, which have a defective hematopoietic micro-environment associated with a lack of support of stem cells, mast cells, germ cells and melanoblasts, can be cured of their hematopoietic defects by treatment with SCF [26].This along with the growing in vitro data of effects of SCF on primitive populations, demonstrates a key role of SCF in stem cell development. Along with the positive regulators of early events in stem cell development, it has been proposed that negative regulators are involved in the control mechanisms. TGF-P represents
one of the negative regulators of primitive cells; its action appears to be selective, having little effect on more mature progenitors (Tables I, 111 [ 2 ] ) . In recent studies by Jucobsen and colleagues, TGF-p was shown to trans-downmodulate cell surface GM-CSF and IL-3 receptors on factor dependent cell lines [271. In addition, the ED,, (the dose giving 50% of maximal effect) at which TGF-p induced receptor trans-downmodulation was the same as that for growth inhibition. The mechanism by which TGF-P inhibits SCF stimulated colony formation may also be by trans-downmodulation of the SCF receptor. It is generally accepted that stem cells exist in a quiescent state in the microenvironment of the bone marrow. As SCF has been shown to be produced by stromal cells [9], it is possible that TGFp, which is also produced by cells in the bone marrow, maintains stem cells in a quiescent state by trans-downmodulating the SCF receptor.
Acknowledgments The authors would like to thank D K K . Westcotr and L. Bourdrel (Amgen Inc.) for supplying the rhTGF-p and Dr I! Hunt (Amgen Inc.) for critical review of the manuscript.
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McNiece/Bertoncello/eller/Ruscetti/Hartley/Zsebo. etic progenitors and inhibits the growth of the IL-3 dependent myeloid leukemic cell lines. J Exp Med 1988;168:737-750.
85 12 Bradley T,Hodgson G, Rosendaal M. The effect of oxygen tension on hemopoietic and fibroblast cell proliferation in vitro. J Cell Physiol 1978;94:5 17522.
Keller JR,McNiece IK, Sill KT, Ellingsworth LR, Quesenberry PJ, Sing GK, and Ruscetti FW.Transforming growth factor p directly regulates primitive murine hematopoietic cell proliferation. Blood 1990;75:596-602.
13 Bertoncello I, Bradley TR, Watt SM. An improved negative immunomagnetic selection strategy for the purification of primitive hemopoietic cells from normal bone marrow. Exp Hematol 1990;19:95-100.
3 Bradley TR, Hodgson GS. Detection of primitive macrophage progenitor cells in mouse bone marrow. Blood 1979;54:1446-1450.
14 Spangrude GJ, Heimfeld S , Weissman IL. Purification and characterization of mouse hematopoietic stem cells. Science 1988;241:58-62.
McNiece I, Bertoncello I, Kriegler A, Quesenbeny P. Colony forming cells with high proliferative potential. Int J Cell Cloning 1990;8:146-160.
15 Williams N, Bertoncello I, Kavnoudias H, Zsebo K, McNiece I. Recombinant rat stem cell factor stimulates amplification and differentiation of fractionated mouse stem cell populations. Blood 1992;79:58-64.
2
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5 Ellingsworth LR, Brennan JE, Fok K, Rosen DM, Bentz H, Piez KA, Seyedin SM. Antibodies to the N terminal portion of cartilage-inducing factor A and transforming growth factor p. J Biol Chem 261: 12362-I237 1. 6 Zsebo K, Wypych J, McNiece I, Lu H, Smith K, Karkare S, Sachdev R, Yuschenkoff V, Birkett N, Williams L, Satygal V, Tung W, Bosselman B, Mendiaz E, and Langley K. 1990. Identification, purification, and biological characterization of hematopoietic stem cell factor from Buffalo Rat Liver conditioned medium. Cell 1990;63:195-201. 7 Martin F, Suggs S, Langley K, Lu H, Ting J, Okino K, Moms C, McNiece I, Jacobsen F, Mendiaz E, Birkett N, Smith K, Johnson M, Parker V, Flores J, Patel A, Fisher E, Erjavec H, Herrera C, Wypych J, Sachdev R, Pope J, Leslie I, Wen D, Lin C-H, Cupples R, and Zsebo K. Primary structure and functional expression of rat and human stem cell factor DNAs. Cell 1990;63:203-211. 8
Medlock E, Yung Y, McNiece I, Langley K. Mineo Daley C, Dubbell C, Mendiaz B, Sachdev R, Smith K, and Zsebo K. Role of stem cell factor (SCF) in the stimulation of the mast cell lineage. Blood 1990;76 (suppl 10) 1:154a.
9 McNiece I, Langley K, Zsebo K. The role of stem cell factor in B cell differentiation: synergistic interaction with IL-7. J Immunol 1991;146:3785-3790. 10 McNiece IK, Langley KE, Zsebo KM. Recombinant human stem cell factor synergises with GM-CSF, GCSF, IL-3 and Epo to stimulate human progenitor cells of the myeloid and erythroid lineages. Exp Hematol 1991;19:226-23 1. 11 Emerson SG, Sieff CA, Wang EA, Wong GG, Clark SC, Nathan DG. Purification of fetal hematopoietic progenitors and demonstration of recombinant multipotential colony-stimulating activity. J Clin Invest 1985;76: 1286-1290.
16 Boone T,Crandall C, Tsai K, Zsebo K. Purification of recombinant human granulocyte-macrophage colony-stimulating factor from E.coli. J Cell Biochem 19873 lc: l76a. 17 Souza L, Boone T, Gabrilove J, Lai P, Zsebo K, Murdock D, Cahzin V, Bruszewski J, Lu H. Chen K, Barendt J, Platzer E, Moore M, Mertelsmann R, and Welte K. Recombinant granulocyte colony-stimulating factor:effects on normal and leukemic myeloid cells. Science 1986;232:61-65. 18
Yang Y-C. Ciarletta A, Temple P, Chung M, Kovacic S, Witek-Giannotti J, Leary A, Kriz R, Donahue RE, Wong GG, and Clark SC. Human IL-3 (multi-CSF): identification by expression cloning of a novel hematopoietic growth factor related to murine IL-3. Cell 1986;47:3-10.
19 Lin F, Suggs S , Lin C, Browne J, Smalling R, Egrie J, Chen K. Fox G, Martin F, Stabinsky Z, Badrawi S, Lai PH, and Goldwasser E. Cloning and expression of the human erythropoietin gene. Proc Natl Acad Sci USA 1985;82:7580-7584. 20 Wong GG, Witek-Giannotti JG, Temple PA, Knz R, Ferenz C, Hewick RW, Clark SC, Ikebuchi K, Ogawa M. Stimulation of murine hematopoietic colony formation by human interleukin-6 (IL-6). J Immunol 1988;140:3040-3044. 21
Seyedin SM, Thomas AY, Thompson HB, Rosen DM, Piez KA. Purification and characterization of two cartilage-inducing factors from bovine demineralized bone. Proc Natl Acad Sci USA 1985;82:2267-2271.
22
Anderson DM, Lyman SD, Baird A, Wignall JM, Eisenman J, Rauch C, March CJ, Boswell HS, Gimpel SD, Cosman D, and Williams DE. Molecular cloning of mast cell growth factor, a hematopoietin that is active in both membrane bound and soluble forms. Cell 1990;63:235-243.
TGF-B inhibition of the action of SCF
86 23
Huang E, Nocka K, Beier DR, Chu TY, Buck J, Lahm H W ,Wellner I), Leder P,Besmer P. The hematopoietic growth factor KL is encoded by the SI locus and is the ligand of the c-kit receptor, the gene product of the W locus. Cell 1990;63:225-233.
24
Bernstein ID. Andrcws RG, Zsebo Kh4. Recombinant human stem cell factor enhances the formation of colonies by CD34+ and CD34+lin - cells, and the generation of colony-forming cell progeny from CD34+lin - cells cultured with interkukin-3. granulocyte colony-stimulating factor or granulocyte-macrophage colony-stimulating factor. Blood 1991;77:23 16.2321.
Bertoncello I, Williams N. Kavnoudias H, Zsebo K. McNiece I. Demonstration of the direct action of stem cell factor (SCF) in combination with CSFs on Sca-1 positive stem cells at the single cell level. Exp Hematol 1991;19:508a. 26 Suggs S , Williams D, Giessler E, Galli S, Birkett N, Smith K, Hsu R, Takeishi T, Zsebo K. Stem cell factor is the product of the Sl locus in the mouse. Blood 1990;76(suppl 1):167a. 27 Jacobsen SE, Ruscetti FW,Dubois CM, Lee J, Boone TC, Ihle JN, Keller JR. Transforming growth factorp transmodulates the expression of colony stimulating factor receptors on murine hematopoietic progenitor cell lines. Blood 1991;77:1706-1716.
25
International Journal of Cell Cloning 1030-86 (1992)
Transforming Growth Factor p Inhibits the Action of Stem Cell Factor on Mouse and Human Hematopoietic Progenitors Ian K. McNiece", Ivan Bertoncellob, Jonathan R. Keller c, Francis W Ruscetti', Cynthia A. Hartley", Krisztina M. Zsebo" 'Arngen Inc., Thousand Oaks, California, USA; bPeter MacCallum Cancer Institute, Melbourne, Victoria, Australia; 'Frederick Cancer Kesearch Facility, Frederick, Maryland, USA. Key Words. Stem cells SCF TGF-P Inhibitors Hernatopoiesis Abstract. In agar culture of post 5-fluorouracil mouse bone marrow cells (FUBM), recombinant rat stem cell factor (rrSCF) synergises with granulocyte colonystimulating factor (G-CSF), interleukin-3 (IL-3) or interleukin-6 (IL-6) to stimulate primitive progenitor cells (HPP-CFCs). The addition of recombinant human transforming growth factor P (rhTGF-P) to cultures of FUBM containing rrSCF plus rhC-CSF, rrSCF plus recombinant murine (rm)IL3, or rrSCF plus rhIL-6 resulted in 100% inhibition of colony formation. Highly enriched populations of primitive bone marrow cells were obtained by isolating lineage negative (Lin -), Sca1-positive (Sca-1+) cells from normal mouse bone marrow. RhTGF-p inhibited 90% of colony formation stimulated by rrSCF plus rmIL-3 in agar culture of the Sca-l+ cells. RhTGF-P also inhibited colony formation in agar culture of post FU human bone marrow cells. The synergistic increase in colony formation obtained with recombinant human SCF (rhSCF) plus rhGMCSF and rhSCF plus rhIL-3 was inhibited by rhTGF-P (approx. 60% and 87% inhibition, respectively). RhTGF-P also totally inhibited the erythroid colony formation stimulated by rhSCF plus recombinant human erythropoietin (rhEpo). These data demonstrate that TGF-P inhibits SCF-stimulated colony formation of mouse and human BM. This inhibition on progenitor cells appears to be a direct action of TGF-P and is consistent with the target cells of SCF being more primitive progenitors than the CFCs stimulated by the CSFs alone.
Correspondence: Dr. Ian K. McNiece, Arngen Inc., Amgen Center, Thousand Oaks, CA 9 1320-1789. USA. Received November 22, 1991; provisionally accepted December 23, 1991; accepted for publication January 10, 1992.
0737- 1454/92/$2.00/0OAlphaMed Press
Introduction Transforming growth factor beta (TGF-P) has been shown to inhibit hematopoietic progenitor cell growth [l, 21. In particular, TGF-p was shown to inhibit the clonal growth of high proliferative potential colony forming cells (HPP-CFCs) [ 2 , 31. HPPCFCs have previously been shown to be a primitive progenitor cell population whose properties correlate closely with those of primitive stem cells [4]. TGF-P is produced by a variety of cells including platelets, bone cells, and T lymphocytes and has been shown by immunohistochemical staining to be locally produced in areas of active hematopoiesis [ 5 ] , suggesting that TGF-P may play a regulatory role in hemopoietic cell growth. Recently a novel growth factor termed stem cell factor (SCF) was isolated from Buffalo rat liver cells (BRL-3A) by its synergistic action on HPP-CFCs [6]. The properties of the recombinant rat and human S C F (rrSCF and rhSCF) suggest that it is a multipotent factor acting on cells of the myeloid, mast and lymphoid lineages [6-lo]. In liquid culture of normal mouse bone marrow cells, rrSCF generates pure populations of mast cells [8], while in semisolid agar culture, rrSCF stimulates mixed colony formation of neutrophil, macrophage and megakaryocyte cells [7]. Interleukin-7 (IL-7) synergises with rrSCF to stimulate pre-B cell colony formation in semisolid agar culture of normal mouse bone marrow cells [6, 91. In agar culture of human bone marrow cells, r h S C F synergises with C S F s and erythropoietin (Epo) to stimulate progenitor cells of the myeloid and erythroid lineages [lo]. These prop-
McNiece/Bertoncello/Keller/Ruscett~a~tleyEsebo.
81
erties demonstrate that SCF is a multilineage factor which may act directly upon a common lymphoidmyeloid stem cell. In this report we examine further the role of TGF-p on early hematopoiesis and demonstrate an inhibitory role of TGF-P on SCF-stimulated proliferation and differentiation of primitive progenitor cells in vitro.
including 714, B220, L3T4, and Lyt-2, and positively labeled cells were removed using magnetic beads (Dynal, Great Neck, NY) [13]. The low density, Lin- cells were then labeled with Sca-1 antibody [14] as previously described [15] and Sca-1 positive cells FACS sorted with an initial gating on the blast cell population [15].
Materials and Methods
Growth Factors Recombinant factors were prepared as previously described; rat SCF (rrSCF) [7] and human SCF (rhSCF) [7], human granulocyte-macrophage CSF (rhGM-CSF) [ 161, human granulocyte CSF (rhG-CSF) [17], rhIL-3 [18], rhEpo [19], and rhIL-6 [20] were prepared from inclusion bodies formed in E. coli containing the appropriate expression plasmid and purified to greater than 99% purity. rh TGF-p [21] was prepared from the medium of a transfected Chinese Hamster Ovarian cell host (CHO) and purified to greater than 98% purity. Recombinant murine IL-3 (rmlL-3) was purchased from Gibco Laboratories (Grand Island, NY). Recombinant human macrophage CSF (rhCSF-1) was a gift from Cetus Corp. (Emeryville, CA). The factors were used at optimal doses previously determined for GM-CFC colony formation or for synergistic interactions where the factors have no stimulatory effect alone: rrSCF, 100 ng; rhSCF, 100 ng; rhGM-CSF, 62.5 ng; rhG-CSF, 100 ng; rhE-3, 100 ng; rhEpo, 4 units; rhIL-6, 100 ng; rmIL-3, 150 units; and rhCSF-1, 1,600 units per culture.
Mouse Bone Marrow Cells Bone marrow cells were obtained from 2 to 3 month old Balb/c or B6D2F1 (BDF,) mice. Balblc mice were pretreated with 5-fluorouracil (150 mg/ kg) 2 days prior to sacrifice and harvest of bone marrow cells (FUBM). Bone marrow cells from normal BDF, mice were highly purified for primitive progenitor cells as described below. Human Bone Marrow Cells Bone marrow cells from patients treated with 5fluorouracil as part of the bone marrow transplant program at the University of Virginia were kindly supplied by Dr E! Quesenberry (University of Virginia, Charlottesville, VA). Informed consent was obtained under guidelines approved by the University of Virginia Institutional Human Investigations Committee. Low density nonadherent cells were depleted of mature cells as previously described [ 1ll. Agar Culture of Bone Marrow Cells Bone marrow cells were plated in double layer agar cultures in 35 mm dishes as previously described [12]. Alpha modification of Eagle's MEM (Flow Laboratories) supplemented with 20% fetal calf serum (FCS) was used for all cultures. The number of cells plated is presented in the Results section. Growth factors were incorporated in the underlays at a maximum of 13.2% of the total culture volume (1.5 ml) per dish. Cultures were gassed with a 5 % O,, 10% CO,, 85% N, mixture and incubated at 37°C and colonies were scored using a dissecting microscope. Only colonies containing 50 or more cells were scored. HPP-CFC colonies were scored as colonies with a diameter greater than 0.5 mm and containing approximately 50,000 or more cells. Purification of Bone Marrow Cells Bone marrow cells were obtained from normal BDF, mice, and low density, lineage negative cells were isolated as previously described [ 131. Briefly, low density cells were separated using Nycodenz (Nyegaard, Oslo, Norway) gradients. The cells were then labeled with a panel of monoclonal antibodies
Results Inhibition of Mouse Bone Marrow Colony Formation by rhTGF-p rrSCF has been previously shown to stimulate HPP-CFCs in agar culture of bone marrow cells of mice pretreated with 5-fluorouracil [6]. As shown in Table I, rhG-CSF, rmIL-3 and rhIL-6 alone resulted in no significant colony formation, while rrSCF and rhCSF-1 alone stimulated significant colony formation. The combination of rrSCF plus rhG-CSF stimulated a synergistic increase in total colonies and HPP-CFCs, while the combinations of rrSCF plus rmIL-3 and rrSCF plus rhIL-6 stimulated synergistic increases in only total colony numbers (Table I). The addition of rhTGF-P to cultures of rrSCF alone or in combination resulted in total inhibition of colony formation (Table I, Fig. 1). In contrast, the rhCSF-1 stimulated colony formation was not inhibited by rhTGF-p (Table I).
TGF-B inhibition of the action of SCF
82
Table I. Colony formation in agar culture of post FU mouse bone marrow
Factors
- rhTGF-P
+rhTGF-P
0 3.3 i 0.7 0 0.3 f 0.3 0 2.0 f 0.5
0 0 0 0 0
A. Total Colonies
Saline rrSCF rhG-CSF mIL-3 rhIL-6 rhCSF- 1 rrSCF/rhG-CSF diCF/rmIL-3 rrSCF/rhIL-6
2.3 i 0.5 0 0
15.0 i 0.5 ll.Of2.9 10.7 i 0.7
0
B. HPP-CFC 0 0 0 0 0 0
Saline rrSCF rhG-CSF mIL-3 rhIL-6 rhCSF- 1 rrSCF/rhG-CSF ITSCF/ITIIIL-~ rrSCF/rhIL-6
9.0 f 0.9 2.7 2.2
0.3 i 0.3
Bone marrow cells from 2 day post FU Balb/c mice were plated in agar culture at 100,OOO cells and incubated for 14 days. Colony numbers are the means i SE of triplicate dishes. RhTGF-P was added at 10 ng per culture. The data are presented from a single experiment which is representative of four separate experiments.
The dose response range of rhTGF-P inhibition of rrSCF plus rhG-CSF colony formation was determined from 0.15 ng to 10 ng. As shown in Figure 2, a dose of 2.5 ng or higher per culture resulted in total colony inhibition, and half maximal inhibition occurred at approximately 1 ng per culture. To determine if the effect of rhTGF-P was a direct action on rrSCF responsive progenitors, normal mouse bone marrow cells were highly enriched for primitive progenitors as described in the Methods section. As shown in Table 11, rrSCF synergised with rmIL-3 in cultures of unseparated and Lin-/ Sca-I+ cells. Addition of rhTGF-p resulted in 89% inhibition of colony formation by Lin -/Sca-l+ cells (Table 11).
- TGF - p
+TGF-P
Effect of rhTGF-P on Human Bone Marrow Cells
hibitory effect on colony formation stimulated by rhCM-CSF. RhSCF synergised with rhEpo, rhIL-3.
I
.
cells. Cultures contained 50.000 cells and were incubated for 14 days.
83
McNiece/Bertoncello/Keller/Ruscetti/Hartley/Zsebo. Table 11. Effect of TGF-P on SCF stimulated growth of highly enriched bone marrow cells GROWTH FACTORS
COLONY FORMATION Unseparated Lin - ISca- 1+
ITSCF + rmIL-3 ITSCF + mIL-3 + rhTGF-P
57.0 i 0.9 9.0 i 0.9
47.0 rt 0.5 5.0 i 0.8
84%
89%
Percentage Inhibition
Unseparated bone marrow cells from normal BDF, mice were plated at 50,OOO cells per culture while Lin-/Sca-l+ cells were plated at 2,000 cells per culture. Colony numbers are the means i SE of triplicate dishes. TGF-0 was added at 10 ng per culture. The data presented are from a single experiment which is representative of three separate experiments. 100
-
rhG-CSF and rhGM-CSF (Table 111) as previously reported [7, 101. The addition of rhTGF-P to cultures containing rhSCF plus rhEpo resulted in 100% inhibition of erythroid blast colony formation (Fig. 3). RhTGF-p also inhibited the synergistic increase in colony formation obtained with rhSCF plus rhGMCSF (approx. 60%inhibition) and rhSCF plus rhIL3 (approx. 87% inhibition). No significant inhibition was obtained for the combination of rhSCF plus rhG-CSF by rhTGF-p (Table 111).
Z
f m r
z
0
ce W
0
80-
40Y 80-
20
O
i
0
I
1
2
4
6
I
I
8
10
Discussion
rhTGF Ing]
Dose response curve of rhTGF-P inFig. 2. hibition of colony formation. Cultures contained rrSCF plus rhG-CSF and were incubated for 14 days after which time total colonies (W) and HPP-CFC colonies (A)were scored.
-TGF-P
+TGF-P
Colony formation stimulated by Fig. 3. rhSCF plus rhEpo in the presence and absence of rhTGFP in agar culture of post FU human bone marrow. Cultures contained 10,000 lineage negative cells and were incubated for 14 days.
TGF-P has been shown to be locally produced in areas of active hematopoiesis [ 5 ] and to selectively inhibit primitive hematopoietic progenitor cells [ 2 ] . In this report we have demonstrated the ability of TGF-0 to inhibit colony formation stimulated by SCF on mouse and human progenitor cells. SCF has been described by other groups as a mast cell growth factor [22] and a c-kit ligand [23]. The inhibition by TGF-p of SCF-stimulated HPP-CFC colony formation in cultures of mouse bone marrow is consistent with the role of SCF being on early hematopoietic cells. This is further supported by the stimulation of Sca-l+ cells by SCF and the inhibition of this stimulation by TGF-p. In cultures of post FU human bone marrow, TGF-p had little effect on colony formation stimulated by rhL-3 or rhG-CSF. The colony formation stimulated by rhSCF plus rhEpo, rhGM-CSF or rhIL-3 was greatly inhibited, suggesting that these combinations of factors stimulate more primitive progenitor cells than the progenitor cells stimulated by rhGM-CSF, rhIL-3 or rhG-CSF alone. This is consistent with the studies of Bernstein and colleagues who have demonstrated that rhSCF plus rhIL-3 stimulates a pre-CFC population not stimulated by any factor alone [24]. The majority of progenitor cells stimulated by rhG-CSF plus rhSCF were unaffected by rhTGF-P in contrast to total inhibition of rhTGF-P
TGF-B inhibition of the action of SCF
84 Table 111. Colony formation in agar culture of post FU human bone marrow
Factors Saline rhSCF rhG-CSF rhIL-3 rhGM-CSF rhEpo rhSCF/rhG-CSF rhSCF/rhIL-3 rhSCF/rhGM-CSF rhSCF/rhEpo
-
rhTGF-P
+rhTGF-P
0 0
0 0
17.0 k 1.4 4.3 i 0.7 10.3 i 0.3
20.7 i 1.5
0 33.0 t 2.1 27.7 2 1.8 37.3 i 0.5 64.0 i 4.3
4.3 ? 0.3 6.0 i 1.2 0
27.0 + 2.1 4.0 i 0.5 15.0 i 1.6 0
Bone marrow cells from patients pretreated with 5-FU were depleted of mature cells as described in Methods and plated in agar culture at 10,OOO cells and incubated for 14 days. Colony numbers are the means 2 SE of triplicate dishes. RhTGF-P was added at 10 ng per culture. The data presented are from a single experiment which is representative of two separate experiments. on mouse progenitors stimulated by rhG-CSF plus rrSCF. As the doses of FU administered to mice are approximately 10-fold higher than those administered to humans, the human post FU marrow most likely contains more committed progenitors than the mouse post FU marrow. The effects of SCF are probably broad, stimulating very primitive progenitor cells and committed progenitors. It may be a population of committed progenitors which respond to rhSCF plus rhG-CSF which are not affected by rhTGF-p. The other possibility is that a primitive population of progenitors responsive to rhSCF plus rhG-CSF are resistant to the effects of rhTGF-p. Further studies are required to examine these points. The direct action of rrSCF on highly purified Sca- 1+ cells has been demonstrated at the single cell level in combination with rmIL-3 or rhIL-6 [15, 251. Similarly, the action of TGF-p appears to be direct as shown in Table 11, inhibiting the action of rrSCF plus rmIL-3 in cultures of highly purified progenitor cells. This is consistent with the results of other investigators who have suggested a direct effect by TGF-P [ 2 ] . Steel mice, which have a defective hematopoietic micro-environment associated with a lack of support of stem cells, mast cells, germ cells and melanoblasts, can be cured of their hematopoietic defects by treatment with SCF [26].This along with the growing in vitro data of effects of SCF on primitive populations, demonstrates a key role of SCF in stem cell development. Along with the positive regulators of early events in stem cell development, it has been proposed that negative regulators are involved in the control mechanisms. TGF-P represents
one of the negative regulators of primitive cells; its action appears to be selective, having little effect on more mature progenitors (Tables I, 111 [ 2 ] ) . In recent studies by Jucobsen and colleagues, TGF-p was shown to trans-downmodulate cell surface GM-CSF and IL-3 receptors on factor dependent cell lines [271. In addition, the ED,, (the dose giving 50% of maximal effect) at which TGF-p induced receptor trans-downmodulation was the same as that for growth inhibition. The mechanism by which TGF-P inhibits SCF stimulated colony formation may also be by trans-downmodulation of the SCF receptor. It is generally accepted that stem cells exist in a quiescent state in the microenvironment of the bone marrow. As SCF has been shown to be produced by stromal cells [9], it is possible that TGFp, which is also produced by cells in the bone marrow, maintains stem cells in a quiescent state by trans-downmodulating the SCF receptor.
Acknowledgments The authors would like to thank D K K . Westcotr and L. Bourdrel (Amgen Inc.) for supplying the rhTGF-p and Dr I! Hunt (Amgen Inc.) for critical review of the manuscript.
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24
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