Growth of human hemopoietic colonies in response to recombinant gibbon interleukin 3: comparison with human recombinant granulocyte and granulocyte-macrophage colony-stimulating factor.

Supernatants of COS-1 cells transfected with gibbon cDNA encoding interleukin 3 (IL-3) with homology to sequences for human IL-3 were tested for ability to promote growth of various human hemopoietic progenitors. The effect of these supernatants as a source of recombinant IL-3 was compared to that of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) as well as to that of medium conditioned by phytohemagglutinin-stimulated leukocytes. The frequency of multilineage colonies, erythroid bursts, and megakaryocyte colonies in cultures containing the COS-1 cell supernatant was equivalent to the frequency observed in the controls and significantly higher than found in cultures plated with recombinant GM-CSF. G-CSF did not support the formation of multilineage colonies, erythroid bursts, and megakaryocyte colonies. In contrast, growth of granulocyte-macrophage colonies was best supported with GM-CSF, while recombinant IL-3 yielded colonies at lower or at best equivalent frequency. The simultaneous addition of higher concentrations of GM-CSF to cultures containing IL-3 in optimal amounts did not enhance the formation of multilineage colonies, erythroid bursts, and megakaryocyte colonies. However, the frequency of such colonies and bursts increased with GM-CSF when cultures were plated with suboptimal concentrations of IL-3. Growth of colonies within the granulocyte-macrophage lineage is optimally supported by GM-CSF and does not increase with further addition of IL-3.     

Interaction of granulocyte-macrophage colony-stimulating factor and interleukin 3 in human long-term bone marrow culture.

Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3), or a combination of both growth factors were added weekly to normal human long-term bone marrow cultures (LTBMC). GM-CSF had a greater effect on the total nonadherent cell population than the committed progenitor cells (granulocyte-macrophage colony-forming units, CFUgm), whereas IL-3 had the opposite effect and stimulated the expansion of greater numbers of CFUgm than GM-CSF. The combination of both factors had an additive effect on CFUgm. The longevity of the growth factor-treated cultures was not reduced. These data indicate that IL-3 stimulates an earlier progenitor cell population than GM-CSF and that a combination of the two factors should be more effective in vivo and could be applied to the expansion of bone marrow progenitor cells in culture before bone marrow transplantation.     

Effects of recombinant murine granulocyte colony-stimulating factor on granulocyte-macrophage and blast colony formation

We performed the present study to define the in vitro hemopoietic activity of murine recombinant (r) granulocyte colony-stimulating factor (G-CSF) using murine hemopoietic culture systems of normal bone marrow cells, fetal liver cells, and spleen cells of 5-fluorouracil (FU)-treated mice. Recombinant G-CSF supported only neutrophil and/or macrophage colony formation by normal bone marrow cells. It did not enhance the formation of erythroid bursts in the fetal liver cell assay, but interleukin-3 (IL-3) did. Paradoxically, rG-CSF could support the colony formation of multilineage colonies as well as blast colonies from the spleen cells of 5-FU-treated mice, while r-granulocyte-macrophage colony-stimulating factor (GM-CSF) and r-erythropoietin (Ep) did not. When blast colonies, formed in the presence of G-CSF, were replated to dishes containing IL-3, they were able to differentiate along multilineage pathways. However, when they were replated to dishes containing rG-CSF, they could differentiate only into neutrophils and macrophages. Single cells transferred from blast colonies formed only neutrophil-macrophage colonies. These data indicate that rG-CSF had a direct effect on the growth and development of GM progenitors at a late stage and a significant effect on multipotential hemopoietic precursors. Although it remains to be clarified how G-CSF acts on multipotential stem cells, this unique effect is important in the understanding of its pluripotent hemopoietic activity in vivo.     

Recombinant human granulocyte-macrophage colony-stimulating factor plus recombinant human erythropoietin may improve anemia in selected patients with myelodysplastic syndromes

The purpose of this study was to improve erythropoiesis in patients with anemia due to myelodysplastic syndromes (MDS). We treated 13 patients first with recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) for 6 weeks, then with recombinant human erythropoietin (rhEpo) and rhGM-CSF for the next 12 weeks. Five patients had refractory anemia (RA), 3 refractory anemia with ringed sideroblasts (RAS), and 5 refractory anemia with excess of blasts (RAEB). Ten patients were transfusion-dependent at the time of inclusion. Eleven patients completed this phase II study. Five responded with an increase in hemoglobin level (3 patients) or a reduction in transfusion requirement (2 patients). We registered no response in the remaining 6 patients during treatment. Patients responding to combined treatment had relatively low concentrations of plasma Epo and plasma ferritin before treatment with rhEpo and a normal karyotype throughout the study. Long-term bone marrow cultures did not predict the response. Still, responders seemed to have a higher number of colony-forming progenitors than non-responders. In conclusion, combined therapy with rhGM-CSF and rhEpo may stimulate hematopoiesis and correct or improve anemia in some patients with MDS. © 1993 Wiley-Liss, Inc.     

Immunization with recombinant human granulocyte-macrophage colony-stimulating factor as a vaccine adjuvant elicits both a cellular and humoral response to recombinant human granulocyte-macrophage colony-stimulating factor

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important cytokine for the generation and propagation of antigen-presenting cells and for priming a cellular immune response. We report here that use of recombinant human GM-CSF (rhGM-CSF), administered as an adjuvant in a peptide-based vaccine trial given monthly by intradermal injection, led to the development of a T-cell and antibody response to rhGM-CSF. An antibody response occurred in the majority of patients (72%). This antibody response was not found to be neutralizing. In addition, by 48-hour delayed type hypersensitivity (DTH) skin testing, 17% of patients were shown to have a cellular immune response to the adjuvant rhGM-CSF alone. Thymidine incorporation assays also showed a peripheral blood T-cell response to rhGM-CSF in at least 17% of the patients. The generation of rhGM-CSF-specific T-cell immune responses, elicited in this fashion, is an important observation because rhGM-CSF is being used as a vaccine adjuvant in various vaccine strategies. rhGM-CSF-specific immune responses may be incorrectly interpreted as antigen-specific immunity, particularly when local DTH responses to vaccination are the primary means of immunologic evaluation. We found no evidence of hematologic or infectious complications as a result of the development of rhGM-CSF-specific immune responses.     

Stimulation of human hematopoietic progenitor cell proliferation and differentiation by recombinant human interleukin 3. Comparison and interactions with recombinant human granulocyte-macrophage and granulocyte colony-stimulating factors

Recombinant human interleukin 3 (IL3) produced in Escherichia coli was purified and its activities examined in cultures of highly enriched human bone marrow progenitor cells. Human IL3 stimulated multipotential (CFU-GEMM) and erythroid (BFU-E) progenitor cells, generating 95% more BFU-E than recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF). No further enhancement of BFU-E or CFU-GEMM occurred when IL3 and GM-CSF were used in combination. Human IL3 was more effective than GM-CSF in stimulating granulocyte-macrophage colony-forming cells (CFU-GM) in short-term suspension cultures, but did not induce an increase of CFU-GM, BFU-E, or CFU-GEMM above input levels. IL3 was more active on day-14 (d14) than on d7 CFU-GM, similar to GM-CSF, but generated fewer and smaller CFU-GM-derived clones than either GM-CSF or granulocyte CSF (CI-CSF). The simultaneous addition of plateau levels of IL3 and GM-CSF resulted in an infra-additive augmentation of d7 and d14 CFU-GM-derived clones, whereas IL3 and G-CSF enhanced the number and cellularity predominantly of d14 CFU-GM. In liquid cultures, IL3 induced a greater than 100-fold increase in the number of basophil-mast-like cells and eosinophils and allowed maintenance of these cultures for up to 7 weeks. Human GM-CSF was an almost equally potent, stimulus of eosinophil development but had only a marginal effect on basophilic precursors, whereas G-CSF lacked both activities. Therefore, human IL3 is a multilineage hemopoietic growth factor whose activities appear to encompass and extend beyond those of GM-CSF.     

Reciprocal inhibition of binding between interleukin 3 and granulocyte-macrophage colony-stimulating factor to human eosinophils

125I-labeled recombinant human interleukin 3 (IL-3) bound, at 4 degrees C, to a single class of high-affinity receptors on human eosinophils with an apparent dissociation constant (Kd) of 470 pM, but it did not bind to human neutrophils. 125I-labeled recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) also bound to a single class of high-affinity receptors on eosinophils with an apparent Kd of 44 pM and on neutrophils with an apparent Kd of 70 pM. These binding characteristics were consistent with the biological activities of IL-3 and GM-CSF on eosinophils and with the lack of stimulation of neutrophil function by IL-3. Specificity studies under conditions shown to prevent receptor internalization showed that the binding of 125I-labeled IL-3 to eosinophils was partially inhibited by GM-CSF but not by other cytokines. Reciprocal experiments with 125I-labeled GM-CSF showed that IL-3 but not other cytokines partially inhibited binding to eosinophils. In contrast, the binding of 125I-labeled GM-CSF to neutrophils was not inhibited by IL-3 or other cytokines tested. Quantitative inhibition binding experiments on eosinophils showed that the reciprocal inhibition between IL-3 and GM-CSF was not complete up to a concentration of heterologous ligand of 100 nM. These results show that (i) IL-3 binds to eosinophils but not neutrophils and (ii) IL-3 and GM-CSF specifically interact on the surface of eosinophils, providing a possible mechanism for the overlapping activities of IL-3 and GM-CSF on these cells.     

Induction of mixed erythroid-megakaryocyte colonies and bipotential blast cell colonies by recombinant human erythropoietin in serum-free culture

The effects of recombinant human erythropoietin (rEp) on murine hematopoietic progenitors were studied using a serum-free culture. A high concentration of rEp stimulated the formation of mixed erythroid-megakaryocyte colonies (EM colonies) and blast cell colonies, as well as erythroid colonies, erythroid bursts, and megakaryocyte colonies from normal mouse bone marrow cells. Direct effects of rEp on EM colony, megakaryocyte colony, and erythroid burst formation were confirmed by depletion of accessory cells such as T cells, B cells, and macrophages from crude bone marrow cells, and inhibition of the colonies by the addition of rabbit anti-rEp antibody to the culture in a dose-dependent fashion. Replating experiments were performed to confirm the differentiating ability of blast cell colonies grown in the presence of rEp. Most of the blast cell colonies yielded not only secondary erythroid colonies but also megakaryocyte colonies in the presence of 2 IU/mL rEp. Some of the blast cell colonies produced secondary EM colonies in the presence of 16 IU/ml rEp of 2 IU/mL rEp plus interleukin-3, although no granulocyte-macrophage colonies were found in the secondary culture. These results suggest that Ep acts not only as a late-acting factor that is specific for erythroid progenitors, but also as a bipotential EM-stimulating factor for murine hematopoietic cells.     

Treatment of active Crohn's disease with recombinant human granulocyte-macrophage colony-stimulating factor

Treatment for Crohn's disease is aimed at immunosuppression. Yet inherited disorders associated with defective innate immunity often lead to development of a Crohn's-like disease. We performed an open-label dose-escalation trial (4-8 microg/kg per day) to investigate the safety and possible benefit of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the treatment of 15 patients with moderate to severe Crohn's disease. No patients had worsening of their disease. Adverse events were negligible and included minor injection site reactions and bone pain. Patients had a significant decrease in mean Crohn's disease activity index (CDAI) score during treatment (p<0.0001). After 8 weeks of treatment, mean CDAI had fallen by 190 points. Overall, 12 patients had a decrease in CDAI of more than 100 points, and eight achieved clinical remission. Retreatment was effective, and treatment was associated with increased quality-of-life measures. GM-CSF may offer an alternative to traditional immunosuppression in treatment of Crohn's disease.     

Erythropoietin receptor haploinsufficiency and in vivo interplay with granulocyte-macrophage colony-stimulating factor and interleukin 3

Erythropoietin (EPO) and its receptor (EPOR) are critical for definitive erythropoiesis, as mice lacking either gene product die during embryogenesis with severe anemia. Here we demonstrate that mice expressing just one functional allele of the EpoR have lower hematocrits and die more frequently than do wild-type littermates on anemia induction. Furthermore, EpoR(+/-) erythroid colony-forming unit (CFU-E) progenitors are reduced both in frequency and in responsiveness to EPO stimulation. To evaluate the interaction between EPO and granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin 3 (IL-3), GM-CSF(-/-) or IL-3(-/-) mice were interbred with EpoR(+/)(-) mice. Deletion of either GM-CSF or IL-3 also leads to reduction in CFU-E numbers and hematocrits but does not significantly alter steady-state erythroid burst-forming unit numbers. These results suggest EpoR haploinsufficiency and promotion of in vivo erythropoiesis by GM-CSF and IL-3.     

Biological effects of recombinant erythropoietin, granulocyte-macrophage colony-stimulating factor, interleukin 3, and interleukin 6 on purified rat megakaryocytes

The biological effects of recombinant hematopoietic factors, which are considered to stimulate megakaryocytopoiesis in vitro or in vivo, were studied utilizing purified rat megakaryocytes. Recombinant human erythropoietin (rhEPO), recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF), and recombinant murine interleukin 3 (rmIL-3) stimulated both [3H]thymidine and [3H]leucine incorporation into purified rat megakaryocytes. In contrast, recombinant human interleukin 6 (rhIL-6) did not stimulate [3H]thymidine uptake but did increase [3H]leucine incorporation into purified rat megakaryocytes. These in vitro data suggest that DNA synthesis in megakaryocytes may be regulated by EPO, GM-CSF, and IL-3, and that protein synthesis is stimulated by EPO, GM-CSF, IL-3, and IL-6 in vitro.     

In vivo control of differentiation of myeloid leukemic cells by recombinant granulocyte-macrophage colony-stimulating factor and interleukin 3

The normal myeloid hematopoietic regulatory proteins include one class of proteins that induces viability and multiplication of normal myeloid precursor cells to form colonies (colony-stimulating factors [CSF] and interleukin 3 [IL-3], macrophage and granulocyte inducing proteins, type 7 [MGI-1]) and another class (called MGI-2) that induces differentiation of normal myeloid precursors without inducing cell multiplication. Different clones of myeloid leukemic cells can differ in their response to these regulatory proteins. One type of leukemic clone can be differentiated in vitro to mature cells by incubating with the growth-inducing proteins granulocyte-macrophage (GM) CSF or IL-3, and another type of clone can be differentiated in vitro to mature cells by the differentiation-inducing protein MGI-2. We have now studied the ability of different myeloid regulatory proteins to induce the in vivo differentiation of these different types of mouse myeloid leukemic clones in normal and cyclophosphamide-treated mice. The results show that in both types of mice (a) the in vitro GM-CSF- and IL-3-sensitive leukemic cells were induced to differentiate to mature cells in vivo in mice injected with pure recombinant GM-CSF and IL-3 but not with G-CSF, M-CSF, or MGI-2; (b) the in vitro MGI-2-sensitive leukemic cells differentiated in vivo by injection of MGI-2 and also, presumably indirectly, by GM-CSF and IL-3 but not by M-CSF or G-CSF; (c) in vivo induced differentiation of the leukemic cells was associated with a 20- to 60-fold decrease in the number of blast cells; and (d) all the injected myeloid regulatory proteins stimulated the normal myelopoietic system. Different normal myeloid regulatory proteins can thus induce in vivo terminal differentiation of leukemic cells, and it is suggested that these proteins can have a therapeutic potential for myeloid leukemia in addition to their therapeutic potential in stimulating normal hematopoiesis.     

Recombinant human granulocyte-macrophage colony-stimulating factor promotes megakaryocyte maturation in nonhuman primates

Megakaryocytes are responsive to several nonlineage-specific cytokines in vitro. In this study, we examined the in vivo effects of recombinant human granulocyte-macrophage colony-stimulating factor (rh GM-CSF) on late stages of megakaryocytopoiesis in the rhesus monkey. Four rhesus monkeys were given 10 micrograms/kg body weight/day of rh GM-CSF s.c. in two divided doses daily for 8 days. Megakaryocyte maturation was evaluated serially by measuring nuclear ploidy and cytoplasmic size. GM-CSF-treated monkeys developed significant shifts in ploidy distribution from days 3 through 15 (p less than or equal to 0.001), with increased frequencies of 64N and 128N megakaryocytes. Mean megakaryocyte size increased 92.5% on day 9, paralleling the increase in DNA content, although megakaryocyte size within ploidy groups did not increase. Megakaryocyte number remained unchanged following rh GM-CSF treatment. The platelet count responses were variable, and mean platelet volume did not change. The present study demonstrates that therapeutic doses of rh GM-CSF stimulate megakaryocyte endomitosis and increase mean size. The data indicate that GM-CSF is effective in promoting the maturation stage of megakaryocyte development but does not result in a consistent thrombopoietic response.     

Stimulation of canine hematopoiesis by recombinant human granulocyte-macrophage colony-stimulating factor

The effect of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on canine hematopoiesis was evaluated. rhGM-CSF stimulated granulocyte-macrophage colony formation of canine marrow depleted of accessory cells up to tenfold. Stimulation of colony formation was abrogated by anti-rhGM-CSF antiserum or heat inactivation. rhGM-CSF also stimulated in vivo canine hematopoiesis both when given as continuous i.v. infusion and as intermittent s.c. injections. Neutrophil, monocyte, and lymphocyte counts were increased three- to eightfold above controls, whereas values for eosinophils, reticulocytes, and hematocrits were not changed. Bone marrow histology after 2 weeks of treatment with rhGM-CSF showed hypercellularity with myeloid hyperplasia and left-shifted granulocytopoiesis. After discontinuation of rhGM-CSF, peripheral leukocyte counts returned to control level within 3-7 days. Platelet counts decreased rapidly after starting rhGM-CSF, to 5000-15,000 platelets/mm3, and increased within 24 h after stopping rhGM-CSF treatment, whereas marrow histology after 2 weeks of rhGM-CSF application showed the normal number and morphology of megakaryocytes.     

Genomic cloning, characterization, and multilineage growth-promoting activity of human granulocyte-macrophage colony-stimulating factor.

Through the use of long single-sequence oligonucleotide probes, the complete gene for human granulocyte-macrophage colony-stimulating factor (hGM-CSF) has been cloned from a human genomic library. The gene is 2.5 kilobases in length, contains three introns, and is present as a single copy in the human genome. When subcloned into the mammalian expression vector pD3, the gene directs the synthesis of authentic hGM-CSF. In addition to its stimulation of in vitro granulopoiesis and monopoiesis, recombinant hGM-CSF stimulates in vitro erythropoiesis and megakaryopoiesis.     

Recombinant gibbon interleukin-3 acts synergistically with recombinant human G-CSF and GM-CSF in vitro

Recombinant gibbon interleukin-3 (IL-3) is a multilineage hematopoietic colony-stimulating factor (CSF) that recently was cloned and found to be highly homologous with human IL-3. Gibbon IL-3, as well as human granulocyte-CSF (G-CSF) and human granulocyte-macrophage CSF (GM-CSF), stimulated normal human bone marrow cells to form myeloid colonies in soft agar in a sigmoidal dose-response manner. When IL-3 was added to increasing concentrations of G-CSF or GM-CSF, synergistic colony formation occurred as compared with the effects of each CSF alone. Synergism was also noted when G-CSF was added with GM-CSF and when all the CSFs were added simultaneously. The combination of IL-3 and GM-CSF was less stimulatory than all the other CSF combinations. At day 11 of culture, IL-3 induced granulocyte-macrophage (38%), eosinophil (30%), granulocyte (18%), and macrophage (14%) colony formation. In summary, gibbon IL-3 is a growth factor that can synergize with other CSFs to enhance proliferation of myeloid-committed progenitors, suggesting that combinations of CSFs may have clinical utility in patients with neutropenia of various etiologies.     

Recombinant and native human urinary colony-stimulating factor directly augments granulocytic and granulocyte-macrophage colony-stimulating factor production of human peripheral blood monocytes

Colony-stimulating factor from human urine (CSF-HU) has been purified to a homogeneous protein, and its complementary DNA (cDNA) has been cloned. Recombinant CSF-HU was prepared from a serum-free medium conditioned by Chinese hamster ovary cells transfected with the cDNA and purified by the same method as that for the native protein. Purified CSF-HU stimulated human bone marrow cells to form macrophage colonies. It also stimulated human mature monocytes prepared from peripheral blood of healthy volunteers to produce human active colony-stimulating activity that stimulates human bone marrow cells to form granulocyte and macrophage colonies. This activity was partially neutralized by the addition of both polyclonal antibodies against human granulocyte colony-stimulating factor and against human granulocyte-macrophage CSF, respectively. The stimulation of monocytes by CSF-HU was not inhibited by the addition of polymyxin-B, which is known as a potent inhibitor of endotoxin. On the other hand, CSF-HU did not stimulate monocyte production of interleukin-1 and interferon. These results indicate that recombinant and native CSF-HU stimulates immature cells as well as mature cells in the human monocyte lineage.     

Effects of bacterially synthesized recombinant human granulocyte-macrophage colony-stimulating factor in patients with advanced malignancy

STUDY OBJECTIVE: To define the clinical and hematologic effects of subcutaneously administered bacterially synthesized recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF). DESIGN: Single arm nonrandomized dose escalation study. PATIENTS: Twenty-one patients with advanced malignancy who were not receiving concurrent myelosuppressive therapy. INTERVENTIONS: Subcutaneous administration of rhGM-CSF by once-daily injection to groups of two to four patients at doses of 0.3 to 30 micrograms/kg body weight.d for 10 consecutive days. Some patients received a second 10-day period of daily rhGM-CSF treatment after a 10-day nontreatment interval followed by alternate-day treatment. Clinical status and hematologic values were monitored frequently. MEASUREMENTS AND MAIN RESULTS: All doses of rhGM-CSF caused an immediate transient fall of 84% to 99% in circulating neutrophils, eosinophils, and monocytes. Continued daily dosing caused a leukocytosis of up to 10-fold with increases in numbers of circulating neutrophils, eosinophils, monocytes, and lymphocytes. There appeared to be a plateau in the increase in neutrophils in the dose range 3 to 15 micrograms/kg.d. Marrow aspirates showed increased proportions of promyelocytes and myelocytes. Alternate-day injection of 15 micrograms/kg maintained a leukocytosis. At doses up to 15 micrograms/kg.d, rhGM-CSF was well tolerated but adverse effects included bone pains, myalgias, rashes, and liver dysfunction. At doses exceeding 15 micrograms/kg.d, pericarditis was a dose-limiting toxicity. Idiopathic thrombocytopenic purpura was reactivated by rhGM-CSF in one patient. CONCLUSIONS: Bacterially synthesized rhGM-CSF induces a leukocytosis in the dose range of 3 to 15 micrograms/kg.d. These doses are appropriate for phase II studies.     

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 interleukin-3, granulocyte colony-stimulating factor, or granulocyte-macrophage colony-stimulating factor.

We tested the ability of recombinant human stem cell factor (SCF) to stimulate isolated marrow precursor cells to form colonies in semisolid media and to generate colony-forming cells (CFC) in liquid culture. SCF, in combination with interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte colony-stimulating factor (G-CSF) caused CD34+ cells to form increased numbers of granulocyte-macrophage colonies (CFU-GM), and to form macroscopic erythroid burst-forming units (BFU-E) in the presence of IL-3, erythropoietin (Epo), and SCF. We tested isolated CD34+lin- cells, a minor subset of CD34+ cells that did not display antigens associated with lymphoid or myeloid lineages, and CD34+lin+ cells, which contain the vast majority of CFC, and found that the enhanced colony growth was most dramatic within the CD34+lin- population. CD34+lin- cells cultured in liquid medium containing SCF combined with IL-3, GM-CSF, or G-CSF gave rise to increased numbers of CFC. Maximal numbers of CFU-GM were generated from CD34+lin- cells after 7 to 21 days of culture, and required the presence of SCF from the initiation of liquid culture. The addition of SCF to IL-3 and/or G-CSF in cultures of single CD34+lin- cells resulted in increased numbers of CFC due to the proliferation of otherwise quiescent precursors and an increase in the numbers of CFC generated from individual precursors. These studies demonstrate the potent synergistic interaction between SCF and other hematopoietic growth factors on a highly immature population of CD34+lin- precursor cells.