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. 3. Effects of 103 units of CSFs or 100 ng of SCF per ml on and were analyzed on day 7 of incubation. The final concentration of the number and mean size (right column) of colonies developing in stimulus in culture is indicated. Data are from duplicate cultures, and cultures of 75,000 C57BL/6/WEHI bone marrow cells. While com- the frequency of various colonies was determined from stained bination of SCF with CSFs results in subadditive or additive colony preparations. Mean colony size was determined from pools of 50 numbers, mean colony size is increased, particularly with G-CSF. sequentially sampled colonies from replicate cultures. G, granulo- Mean data are from duplicate cultures and pools of 50 sequential cyte; GM, granulocyte-macrophage; M, macrophage. colonies. Downloaded by guest on September 28, 2021 Medical Sciences: Metcalf and Nicola Proc. Natl. Acad. Sci. USA 88 (1991) 6241

SFclones I G-CSF cl s o I SCF G-CSF 0 SCF G-CSF 6

Cy.212 0

18

4

6.6s.6 +Sairne 0

1

6 40 A +Sakne

A A~ 1.6 8.3 25 100 SCF ng/Mf FIG. 4. When 101 units of G-CSF are added to increasing con- centrations ofSCF, the rise in colony numbers is merely additive, but FIG. 5. Size achieved by intact day 3 clones initiated by 100 ng a progressive rise is observed in mean colony cell size in cultures of of SCF or 103 units of G-CSF 4 days after transfer to cell-free 75,000 C57BL/6/WEHI bone marrow cells. Curves labeled "+ recipient cultures containing no stimulus (0), 100 ng of SCF, or 103 saline" represent colony formation in cultures containing SCF + 0.1 units of G-CSF. The mean size of clones at transfer is indicated by ml of saline. Mean data are from duplicate cultures and pools of 50 the horizontal lines. colonies. units of G-CSF per ml, or both were resuspended and then morphology. Most SCF-initiated clones (mean size, 20 cells) recultured in cultures stimulated by G-CSF, SCF, or 0.1 ml failed to survive or proliferate further when transferred to ofpokeweed mitogen-stimulated spleen cell-conditioned me- cultures containing no stimulus (Fig. 5), but many exhibited dium (SCM) (6) [SCM constitutes a combined stimulus con- significant further proliferation in cultures containing SCF, taining GM-CSF, Multi-CSF, and interleukins 4, 5, and 6 representing evidence for a direct action of SCF. In the (IL-4, IL-5, and IL-6]. No G-CSF-stimulated colonies con- converse experiment, most G-CSF-initiated clones (mean tained clonogenic cells detectable with any of the three size, 40 cells) failed to survive on transfer to cultures con- stimuli used in secondary cultures. In SCM-stimulated sec- taining no stimulus, although as previously noted (9), some ondary cultures, clonogenic cells were present in only 10 of colony granulocytes were able to exhibit two to four further 35 SCF-stimulated granulocytic colonies (16 + 16 per colony) cell divisions in unstimulated cultures. Transfer of G-CSF- and in only 16 of 41 compact SCF-stimulated colonies (30 + initiated clones to SCF-containing cultures did not result in 49 per colony). In contrast, 33 of 40 SCF-stimulated multi- any significant enhancement of this proliferation but contin- centric blast colonies contained progenitor cells detectable in ued proliferation of such clones was observed after transfer SCM-stimulated cultures (72 + 79 per colony). In SCF- to G-CSF-containing cultures. A striking difference in this stimulated secondary cultures, 21 of 40 SCF-initiated blast pattern was seen when SCF-initiated clones were transferred cell colonies contained progenitor cells (16 + 10 per colony). to G-CSF-containing cultures. Here, the proliferation of With a mean size of 300 cells for SCF-stimulated blast cell many clones was strongly stimulated, with the formation of colonies, the data indicated that an average of 25% of the granulocytic colonies containing up to 2000 cells. colony cells must have been progenitor cells, and in a few The data indicated that SCF does have a direct prolifera- colonies the frequency approached 100%6. In no instance did tive action on hemopoietic clones and that G-CSF can have blast colonies develop, even in SCF-containing secondary a remarkable enhancing action on some SCF-initiated clones. cultures; with SCM as the stimulus, 44% of secondary This enhancement was unidirectional since the proliferation colonies were macrophage, 12% granulocyte-macrophage of G-CSF-initiated clones was not enhanced by SCF. and 44% granulocytic in composition. SCF-stimulated sec- Evidence of the ability of SCF to have direct proliferative ondary colonies were exclusively composed ofgranulocytes. actions on some primitive precursor cells was obtained from The same pattern was observed after recloning of colonies the culture of 200 FACS-enriched stem and progenitor cells. stimulated by a mixture of SCF and G-CSF. The highest As reported (3), few such cells were stimulated to proliferate frequency of progenitor cells was observed in multicentric by SCF alone (0.3 + 0.6 of 200 cells). Nevertheless some blast colonies (mean size, -3000 cells), and in recipient colonies developed, including small blast cell colonies. In cultures containing SCM, 22 of 23 colonies contained 532 + these cultures, G-CSF alone also stimulated little colony 389 progenitor cells per colony, the secondary colonies again formation (0.3 ± 0.6 of 200 cells), but combination ofthe two mainly containing macrophages and/or granulocytes. In sec- stimuli resulted in the formation of21.7 ± 2.5 colonies per 200 ondary cultures containing SCF or G-CSF, smaller numbers cells. of progenitor cells were detected, and these exclusively Clonogenic Cell Content of SCF-Stimulated Colonies. Se- formed granulocytic colonies. The mean number of clono- quential colonies stimulated by 100 ng of SCF per ml, 103 genic cells in the enlarged blast colonies stimulated by the Downloaded by guest on September 28, 2021 6242 Medical Sciences: Metcalf and Nicola Proc. Nad. Acad. Sci. USA 88 (1991) Table 2. 1251-SCF labeling of adult BALB/c mouse bone presence oflarge numbers ofSCF receptors on blast cells (the marrow cells morphological population containing progenitor cells), (it) the Labeled cells % of linearity of SCF-induced colony formation with varying numbers of cultured cells, (iii) the ability of SCF-initiated Mean grain total clones to continue proliferation after transfer to cultures Cell type % count label containing SCF but no other cells, (iv) the ability of SCF to Blast cells 86 130 65 stimulate some colony formation by recultured 7-day colony Promyelocytes/myelocytes 75 9 3 cells, and (v) the ability of SCF to stimulate some colony Metamyelocytes/polymorphs 4 3 1 formation by enriched populations freed of accessory cells. Promonocytes 84 21 10 These are the same criteria used previously to establish direct Monocytes 51 7 3 proliferative actions of other hemopoietic regulators (6). Eosinophilic myelocytes 100 20 4 While the data indicate a direct proliferative action ofSCF, Eosinophils 45 6 1 1000-fold higher concentrations of SCF are required than Lymphocytes 7 47 13 with the CSFs. This is curious in view of the high receptor Nucleated erythroid cells 0 0 0 numbers for SCF on blast cells-the population likely to Autoradiograph exposure time was 21 days. Data represent back- contain the clonogenic cells responding to SCF. The devel- ground-subtracted grain counts over 50-100 sequential cells of each opment of blast cell colonies after SCF stimulation is highly type. Percent contribution to total labeling was calculated from the distinctive and might suggest a capacity of SCF to induce differential count of the BALB/c marrow cells used. self-generation by progenitor cells. However, despite the high content of progenitor cells in blast colonies, no blast combination of G-CSF with SCF was increased 7-fold com- colony-forming cells were detected. Therefore, these data pared with that in SCF-stimulated blast cell colonies. Again, provide no evidence for a capacity of SCF to stimulate no blast cell colonies developed in secondary cultures stim- self-generation by blast colony-forming cells. Because many ulated by SCM, SCF, or G-CSF. of the progenitor cells in blast colonies appear to be mac- Distribution of SCF Receptors on Marrow Ceils. Autora- rophage precursors and SCF receptors are present on mono- diographic analysis of normal BALB/c marrow cells prein- cyte-macrophage populations, it is curious why so few cubated with 1251-SCF showed prominent labeled cells, and macrophage-containing colonies develop in SCF-stimulated this labeling was blocked completely by coincubation with a primary cultures. Equally curious is the ability of SCF to 40-fold excess of unlabeled SCF. As shown in Table 2, the stimulate the formation ofmature granulocytic colonies when pattern of labeling was highly distinctive. Most blast cells receptors are not detectable on metamyelocytes and poly- were labeled and exhibited an unusually high number of morphs. grains per cell. In the granulocytic series, most promyelo- Combination of SCF with G-CSF resulted in merely an cytes and myelocytes were also labeled, but grain counts additive effect on colony numbers, suggesting some indepen- were very low; few metamyelocytes and no polymorphs were dence of action, perhaps based on the existence of differing labeled. Monocytic cells were usually more heavily labeled subsets of progenitor cells able to respond initially to each than granulocytic cells of comparable maturation stage, and stimulus. However, combination of SCF with G-CSF must this was reflected in the labeling of =50% of mature mono- also result in a synergistic interaction on many individual cytes, although the grain counts on these cells were low. clones, since these exhibited a major increase in their size and Eosinophilic myelocytes and mature eosinophils exhibited content of progenitor cells. This synergistic interaction ap- the same labeling pattern as monocytic cells. No labeling was peared to be restricted to clonogenic cells able to be initiated observed of nucleated erythroid cells. Most lymphocytes by SCF, since transfer of G-CSF-initiated clones to SCF did exhibited no labeling, but 7% of the cells were labeled, often not result in a significant increase in their further prolifera- with the same high grain counts as observed on blast cells. tion. These cells had the morphology of small or medium lympho- In the presence of SCF, G-CSF is able to strongly amplify cytes. Despite their low frequency (4% of the total popula- progenitor cell generation and the formation oflarge numbers tion), the blast cells accounted for two-thirds of the total of maturing granulocyte progeny-actions not observable in binding of 1251-SCF to marrow cells. cultures stimulated by G-CSF alone. This phenomenon may help to explain the obvious quantitative discrepancy that DISCUSSION exists between the weak proliferative action ofG-CSF acting alone in vitro (6), and the effects of G-CSF in vivo, where SCF was purified on the basis of its capacity to stimulate the major rises in neutrophil levels are readily inducible by formation of hemopoietic colonies by primitive hemopoietic G-CSF injection (10, 11). It is ofinterest that G-CSF has only precursors from bone marrow and the proliferation of mast a very weak capacity to elevate neutrophil levels in W and cell lines (3, 4). Because ofthe striking synergy evident in this Steel mutant mice (12), suggesting that the strong response proliferation ofmarrow cells when colony-stimulating factors elicited by G-CSF in normal mice and most humans may or IL-6 was present, SCF has been regarded as possibly being depend on an enhancement interaction with SCF. mainly an enhancing factor, amplifying the action of other growth factors. We thank Dr. K. Zsebo and Dr. L. Souza of Amgen (Thousand The present experiments were designed to establish (i) Oaks, CA) for generously supplying, respectively, the SCF and whether or not SCF is a direct proliferative stimulus for G-CSF used in these studies; Mrs. L. Di Rago and Miss S. Mifsud normal murine marrow cells and (ii) the nature of the en- for technical assistance; and Dr. C. Li for supplying the FACS- hancement evident when SCF is used in combination with the purified mouse bone marrow stem cells used. This work was sup- CSFs. This latter study concentrated on the SCF-G-CSF ported by the Carden Fellowship Fund ofthe Anti-Cancer Council of combination, since this was quantitatively the most striking Victoria; the National Health and Medical Research Council, Can- and involved a CSF with an action relatively restricted to berra, and the National Institutes of Health Grant 22556. granulocyte precursor cells (6). 1. Charbot, B., Stephenson, D. A., Chapman, V. M., Besmer, P. The data indicate strongly that SCF is a direct-acting & Bernstein, A. (1988) Nature (London) 335, 88-89. proliferative stimulus inducing the formation of multicentric 2. Geissler, E. N., Ryan, M. A. & Housman, D. E. (1988) Cell 55, blast cell colonies and immature or maturing granulocytic 185-192. colonies. The evidence supporting this conclusion is (i) the 3. Zsebo, K. M., Wypych, J., McNiece, I. K., Lu, H. S., Smith, Downloaded by guest on September 28, 2021 Medical Sciences: Metcalf and Nicola Proc. Nati. Acad. Sci. USA 88 (1991) 6243

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