Proc. Natl. Acad. Sci. USA Vol. 88, pp. 6239-6243, July 1991 Medical Sciences Direct proliferative actions of stem cell factor on murine bone marrow cells in vitro: Effects of combination with colony-stimulating factors (hemopoietic colonies/autoradiography/clonal cultures/clone transfer) D. METCALF* AND N. A. NICOLA The Walter and Eliza Hall Institute of Medical Research, P.O. Royal Melbourne Hospital 3050, Victoria, Australia Contributed by D. Metcalf, April 16, 1991 ABSTRACT Stem cell factor (SCF), the ligand for the c-kit for up to 7 days in a fully humidified atmosphere of 10%o CO2 protooncogene product, was able to stimulate blast cell and in air. The transfer ofintact clones to recipient cultures or the granulocytic colony formation by precursors from normal reculture of resuspended colony cells was performed as murine bone marrow. The blast cell colonies contained a high described (6). Colony formation was scored by using a content of progenitor cells able to form macrophage and/or dissection microscope at 35x magnifications, and cultures granulocyte colonies. Clone transfer studies, the secondary were then fixed by using 1 ml of 2.5% glutaraldehyde. The culture of colony cells, and the culture of populations freed of intact cultures were floated onto slides and, after drying, accessory cells all indicated a direct proliferative action ofSCF. were stained for acetylcholinesterase and then stained with SCF receptors were present in high numbers on blast cells and Luxol fast blue and hematoxylin. in lower numbers on immature granulocytic, monocytic, and Stimuli. All stimuli used were recombinant nonglycosy- eosinophilic cells. Combination ofSCF with granulocyte, gran- lated factors purified after expression in Escherichia coli: ulocyte-macrophage, or multipotential colony-stimulating fac- SCF (rat) and granulocyte colony-stimulating factor (G-CSF; tors, but not macrophage colony-stimulating factor, resulted in human 108 units/mg) respectively supplied by K. Zsebo and enhancement of colony size. Granulocyte colony-stimulating L. Souza (Amgen Biologicals); and recombinant murine factor enhanced cell proliferation initiated by SCF, but not granulocyte-macrophage colony-stimulating factor (GM- vice-versa, and resulted in a 10-fold increase in colony cell CSF), macrophage colony-stimulating factor (M-CSF), and numbers and a 7-fold increase in progenitor cells in blast multipotential colony-stimulating factor (Multi-CSF) pro- colonies. No evidence was obtained that SCF, alone or in duced in this laboratory. After purification, the latter group combination with granulocyte colony-stimulating factor, could of CSFs assayed at 3 x 108, 1 x 108, and 1 x 108 units/mg stimulate self-generation by blast colony-forming cells. of protein, respectively, by the methods for unit estimation described (6). The protooncogene c-kit encodes a membrane receptor, Stem Cell Enrichment. Enrichment by fluorescence- present on hemopoietic and other cells but defective or activated cell sorting (FACS) of stem and progenitor cells absent in the various W (dominant spotting) mutant mice with from normal mouse bone marrow was based on methods defective stem cells and erythropoiesis (1, 2). The ligand for described previously (7). the c-kit product has recently been purified and cloned (3, 4), Autoradiographic Studies. Recombinant rat SCF was ra- and production ofthis molecule is absent or defective in mice diolabeled with 1251 (125I-SCF) by using iodine monochloride with the Steel mutation that also exhibit defective erythro- (8) to a specific radioactivity of 30,000 cpm/ng. BALB/c poiesis. Initial studies documented the ability of the c-kit bone marrow cells (5 x 106) were incubated with 125I-SCF ligand [termed stem cell factor (SCF) or mast cell growth (200,000 cpm) for 45 min at 230C in 60 1.l of RPMI 1640 factor] to stimulate the proliferation of mast cells (3, 4) and medium containing 10%o fetal calf serum and 10 mM Hepes primitive hemopoietic precursor cells (3) and to enhance buffer (pH 7.3). Specificity was determined in parallel tubes erythropoietin-stimulated erythropoiesis and granulocyte- containing 300 ng of unlabeled SCF (3400 + 100 cpm versus macrophage colony formation in the presence of colony- 350 + 40 cpm). Cytocentrifuge cell preparations were fixed stimulating factors (4, 5). with 2.5% glutaraldehyde and then exposed for 21 days with The present studies were undertaken to establish whether Kodak NTB2 emulsion. After development, the preparations or not SCF is a direct proliferative stimulus for hemopoietic were stained with May-Grunewald Giemsa. cells and the nature of the enhanced cell proliferation ob- servable when SCF is used in combination with the various RESULTS colony-stimulating factors. SCF, acting alone in cultures of 75,000 bone marrow cells, stimulated colony formation, but the concentration required MATERIALS AND METHODS was 1000-fold higher than for the colony-stimulating factors Cultures. Primary cultures were performed in 35-mm Petri (Fig. 1). The maximum number of colonies developing was dishes usually containing 75,000 bone marrow cells from only two-thirds of that stimulated by GM-CSF but twice the 8-week-old C57BL/6/WEHI mice in a 1-ml volume of Dul- number stimulated by G-CSF. After 7 days of culture, becco's modified Eagle's medium with a final concentration SCF-stimulated colonies had a distinctive morphology. The of 20% (vol/vol) fetal calf serum and 0.3% agar (6). Stimuli in a volume of 0.1 ml were added prior to the addition of the Abbreviations: SCF, stem cell factor; CSF, colony-stimulating fac- cells in agar-medium. After gelling, cultures were incubated tor; G-CSF, granulocyte colony-stimulating factor; GM-CSF, gran- ulocyte-macrophage colony-stimulating factor; M-CSF, macro- phage colony-stimulating factor; Multi-CSF, multipotential colony- The publication costs of this article were defrayed in part by page charge stimulating factor; IL-4, IL-5, and IL-6, interleukins 4, 5, and 6; payment. This article must therefore be hereby marked "advertisement" FACS, fluorescence-activated cell sorting. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 6239 Downloaded by guest on September 28, 2021 6240 Medical Sciences: Metcalf and Nicola Proc. Natl. Acad. Sci. USA 88 (1991) 800 > 600 80 - /C~~GSF ~~~~~~~~SCF k 400 40 200 40 0.015 0.25 4 62 1000 Concentration ng/rrd 400 -~~~~~ I FIG. 1. Stimulation of colony formation by SCF, G-CSF, or . GM-CSF in cultures of 75,000 C57BL/6/WEHI bone marrow cells. 200 _ p/G~S~F Cultures scored at day 7 ofincubation and data points represent mean / colony counts from replicate cultures. | | |oc colonies were small and of three major types-multicentric colonies containing blast cells, compact colonies usually 12.5 50 100 200 Cels per dish x 10-3 containing immature granulocytes, and colonies of mature granulocytic cells similar to those stimulated by G-CSF. The FIG. 2. Linearity of colony formation stimulated by SCF or cultures also contained a few small macrophage or granulo- G-CSF in cultures of varying numbers of C57BL/6/WEHI bone cyte-macrophage colonies but no eosinophil, megakaryo- marrow cells. Note the progressive increase in mean colony size with cytic, erythroid, or multipotential colonies (Table 1). increasing numbers of cultured cells. Mean data are from duplicate SCF-stimulated colony formation was linear with respect cultures and pools of 50 colonies (where possible). to cultured cell numbers (Fig. 2) but, as cultured cell numbers were increased, there was a progressive rise in mean colony with size enhancement increasing progressively with increas- cell numbers (up to 10-fold), a phenomenon seen also in ing SCF concentrations (Fig. 4). A similar result was ob- G-CSF-stimulated cultures. The increased colony size in- served with the reverse combination using increasing con- volved both blast cell and immature granulocytic colonies. centrations of G-CSF. No change occurred in the relative frequencies ofthe various Direct Prolfferative Actions of SCF. The linearity of colony colony types but, in cultures ofhigh cell numbers, a few small formation with cultured cell numbers suggested a direct megakaryocytic colonies were sometimes present. action of SCF, but further evidence for this was sought by In cultures containing 100 ng of SCF per ml combined with clone transfer studies and the culture ofFACS-enriched stem 1000 units of colony-stimulating factors per ml (a 4-fold and progenitor cells. supramaximal concentration), there were only additive or The subsequent proliferation of individual day 3 clones, subadditive effects on numbers. with total colony However, initiated by either SCF or G-CSF, was analyzed and then the G-CSF, GM-CSF, or Multi-CSF there was a marked super- clones were transferred to cell-free recipient cultures con- additive effect on colony size that was most evident with the combination ofSCF and G-CSF 3). In cultures with SCF taining no stimulus, SCF, or G-CSF. Clones transferred were (Fig. the 20 from each donor culture and excluded those and G-CSF, there was a >10-fold increase in mean colony largest cell numbers. The sizes of blast cell and immature granulo- already showing evidence of maturation from their dispersed cytic colonies were enhanced, but the cultures still contained small mature granulocytic colonies typical of those seen in cultures stimulated by either SCF or G-CSF alone. Differ- ential colony counts suggested that combination of the two stimuli resulted in conversion of some blast colonies to granulocyte-containing colonies (Table 1). Combination of G-CSF with increasing concentrations of SCF indicated a simple additive effect on colony numbers, Table 1. Colony formation stimulated by SCF alone or in combination with G-CSF Mean Number of colonies Mean cells per Stimulus per ml colonies colony Blast G GM M SCF, 100 ng 81 370 19 51 8 3 G-CSF, 2 ng 43 300 0 31 6 6 20 40 60 80 100 120 140 SCF, 100 ng + N wnbe of coloies G-CSF, 2 ng 121 2740 10 89 16 6 Cultures contained 75,000 C57BL/6/WEHI bone marrow cells FIG.