Investigation of the safety and feasibility of AAV1/SERCA2a gene transfer in patients with chronic heart failure supported with a left ventricular assist device - the SERCA-LVAD TRIAL.

The SERCA-LVAD trial was a phase 2a trial assessing the safety and feasibility of delivering an adeno-associated vector 1 carrying the cardiac isoform of the sarcoplasmic reticulum calcium ATPase (AAV1/SERCA2a) to adult chronic heart failure patients implanted with a left ventricular assist device. The SERCA-LVAD trial was one of a program of AAV1/SERCA2a cardiac gene therapy trials including CUPID1, CUPID 2 and AGENT trials. Enroled subjects were randomised to receive a single intracoronary infusion of 1 × 1013 DNase-resistant AAV1/SERCA2a particles or a placebo solution in a double-blinded design, stratified by presence of neutralising antibodies to AAV. Elective endomyocardial biopsy was performed at 6 months unless the subject had undergone cardiac transplantation, with myocardial samples assessed for the presence of exogenous viral DNA from the treatment vector. Safety assessments including ELISPOT were serially performed. Although designed as a 24 subject trial, recruitment was stopped after five subjects had been randomised and received infusion due to the neutral result from the CUPID 2 trial. Here we describe the results from the 5 patients at 3 years follow up, which confirmed that viral DNA was delivered to the failing human heart in 2 patients receiving gene therapy with vector detectable at follow up endomyocardial biopsy or cardiac transplantation. Absolute levels of detectable transgene DNA were low, and no functional benefit was observed. There were no safety concerns in this small cohort. This trial identified some of the challenges of performing gene therapy trials in this LVAD patient cohort which may help guide future trial design.

Prevalence of AAV1 neutralizing antibodies and consequences for a clinical trial of gene transfer for advanced heart failure.

Adeno-associated virus serotype 1 (AAV1) has many advantages as a gene therapy vector, but the presence of pre-existing neutralizing antibodies (NAbs) is an important limitation. This study was designed to determine: (1) characteristics of AAV NAbs in human subjects, (2) prevalence of AAV1 NAbs in heart failure patients and (3) utility of aggressive immunosuppressive therapy in reducing NAb seroconversion in an animal model. NAb titers were assessed in a cohort of heart failure patients and in patients screened for a clinical trial of gene therapy with AAV1 carrying the sarcoplasmic reticulum calcium ATPase gene (AAV1/SERCA2a). AAV1 NAbs were found in 59.5% of 1552 heart failure patients. NAb prevalence increased with age (P=0.001) and varied geographically. The pattern of NAb titers suggested that exposure is against AAV2, with AAV1 NAb seropositivity due to crossreactivity. The effects of immunosuppression on NAb formation were tested in mini-pigs treated with immunosuppressant therapy before, during and after a single AAV1/SERCA2a infusion. Aggressive immunosuppression did not prevent formation of AAV1 NAbs. We conclude that immunosuppression is unlikely to be a viable solution for repeat AAV1 dosing. Strategies to reduce NAbs in heart failure patients are needed to increase eligibility for gene transfer using AAV vectors.

Antigen-specific cytolysis by neutrophils and NK cells expressing chimeric immune receptors bearing zeta or gamma signaling domains.

TCR- and IgG-binding Fc receptors (Fc gamma R) mediate a variety of critical biologic activities including cytolysis via the structurally related zeta- and gamma-chains. In previous studies, we have described chimeric immune receptors (CIR) in which the ligand-binding domain of a heterologous receptor or Ab is fused directly to the cytoplasmic domain of the TCR zeta-chain. Such zeta-CIRs efficiently trigger cytotoxic function of both T and NK cells in a target-specific manner. In this report, we compared the ability of both zeta- and gamma-CIRs to activate the cytolytic function of two distinct classes of Fc gamma R-bearing effectors, NK cells and neutrophils. Mature neutrophils expressing zeta- and gamma-CIR were generated in vivo from murine hemopoietic stem cells following transplantation of syngeneic mice with retrovirally transduced bone marrow or in vitro from transduced human CD34+ progenitors following differentiation. Both zeta- and gamma-based CIRs were capable of activating target-specific cytolysis by both NK cells and neutrophils, although the zeta-CIR was consistently more efficient. The experimental approach described is a powerful one with which to study the role of nonlymphoid effector cells in the host immune system and permits the rational design of immunotherapeutic strategies that rely on harnessing multiple immune cell functions via CIR-modified hemopoietic stem cells or progenitors.

Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice.

We constructed two megabase-sized YACs containing large contiguous fragments of the human heavy and kappa (kappa) light chain immunoglobulin (Ig) loci in nearly germline configuration, including approximately 66 VH and 32 V kappa genes. We introduced these YACs into Ig-inactivated mice and observed human antibody production which closely resembled that seen in humans in all respects, including gene rearrangement, assembly, and repertoire. Diverse Ig gene usage together with somatic hypermutation enables the mice to generate high affinity fully human antibodies to multiple antigens, including human proteins. Our results underscore the importance of the large Ig fragments with multiple V genes for restoration of a normal humoral immune response. These mice are likely to be a valuable tool for the generation of therapeutic antibodies.

Systemic T cell-independent tumor immunity after transplantation of universal receptor-modified bone marrow into SCID mice.

Gene modification of hematopoietic stem cells (HSC) with antigen-specific, chimeric, or "universal" immune receptors (URs) is a novel but untested form of targeted immunotherapy. A human immunodeficiency virus (HIV) envelope-specific UR consisting of the extracellular domain of human CD4 linked to the zeta chain of the T cell receptor (CD4 zeta) was introduced ex vivo into murine HSC by retroviral transduction. After transplantation into immunodeficient SCID mice, sustained high level expression of CD4 zeta was observed in circulating myeloid and natural killer cells. CD4 zeta-transplanted mice were protected from challenge with a lethal dose of a disseminated human leukemia expressing HIV envelope. These results demonstrate the ability of chimeric receptors bearing zeta-signaling domains to activate non-T cell effector populations in vivo and thereby mediate systemic immunity.

Human thymocyte responsiveness to stem cell factor: synergy with interleukin-2 for the generation of NK/LAK cytotoxicity.

Human recombinant stem cell factor (SCF) increases the viability and cell size of a subset of thymocytes in vitro, but does not independently induce phenotypic changes on thymocytes indicative of T cell differentiation. The SCF-responsive thymocytes have characteristics of large granular cells, that do not express T, B or NK cell-related antigens, and are primarily found in immature thymocyte subsets. These large granular thymocytes do not display cytotoxic activity. However, SCF acts synergistically with IL-2 in the generation of cytotoxic effector cells from thymocyte precursors. Synergy in cytotoxicity is observed to both NK-sensitive and NK-resistant targets. Studies of the SCF receptors on thymocytes show that receptors are expressed on mature 'bright' CD3+ cells, immature 'dim' CD3+ cells as well as CD3- cells. IL-2 increases the frequency of SCF receptor-positive cells in cultured thymocytes, which may explain its synergy with SCF in the generation of NK/LAK cytotoxicity. These data show that SCF enhances the functional development of thymic NK/LAK cells in vitro.

Effects of cyclosporin A and FK-506 on stem cell factor-induced histamine secretion and growth of human mast cells.

Stem cell factor (SCF) is a key regulator of human mast cells (MCs) and a potential mediator of allergy. In this study the effects of cyclosporin A (CSA) and FK-506, two potent immunosuppressive drugs, on SCF-dependent histamine release and growth of human MCs were analyzed. Preincubation of tissue MCs with CSA (3 micrograms/ml) resulted in inhibition of histamine release provoked by either recombinant human (rh) SCF (70.3% +/- 20.6% inhibition, p < 0.001) or anti-IgE (76.7% +/- 21.9%, p < 0.001) or by rhSCF+ anti-IgE (77.4% +/- 13.9%, p < 0.001). Almost the same inhibition was produced by FK-506 (rhSCF: 82.0% +/- 18.9% inhibition, p < 0.001; anti-IgE: 71.5% +/- 16.7%, p < 0.001; rhSCF+ anti-IgE: 70.0% +/- 7.3%, p < 0.001). The effects of CSA and FK-506 on SCF-dependent release of histamine were dose-dependent (IC50: CSA, 1 to 10 ng/ml; FK-506, 0.3 to 3 ng/ml). IC50 values about three to 10 times higher were found for MCs preincubated with rhSCF before anti-IgE activation, compared with anti-IgE or SCF alone. SCF-dependent differentiation of human MCs was analyzed in a long-term suspension culture system (n = 6). Unexpectedly, CSA and FK-506 were unable to suppress, but even enhanced SCF-dependent growth of MCs and formation of MC tryptase in long-term culture. Together, CSA and FK-506 inhibit SCF-dependent release of histamine from human MCs and even augment SCF-dependent growth of human MCs in long-term culture.

Long-term treatment of canine cyclic hematopoiesis with recombinant canine stem cell factor.

Grey collie dogs have cyclic fluctuations in their blood cell counts caused by a regulatory defect of hematopoietic stem cells. To examine the role of stem cell factor (SCF) or its receptor in this disorder, we investigated the stimulatory effects of recombinant canine SCF (rc-SCF) on in vitro marrow cultures, cloned and sequenced the grey collie SCF gene, and treated three grey collies with rc-SCF, either alone or in combination with recombinant canine granulocyte colony-stimulating factor (rcG-CSF). Colony-forming unit granulocyte-macrophage formation from grey collie or normal dog marrow showed similar dose-response curves for rc-SCF. Cloning and sequencing the SCF gene for two grey collies showed no evidence of mutations in the coding region of the SCF gene. Treatment with rc-SCF (10 to 100 micrograms/kg/d) did not induce neutrophilia except at the highest dose (100 micrograms/kg/d), but daily rc-SCF abrogated the neutropenic periods in doses of 20 micrograms/kg/d or greater. Combination of rc-G-CSF (0.5 to 1.0 microgram/kg/d) with rc-SCF treatment (20 to 50 micrograms/kg/d) suggested a synergistic effect, ie, the neutrophil levels on combined therapy were higher than the sum of the levels when these two cytokines were given separately. Long-term treatment of these dogs with rc-SCF in doses of 10 to 30 micrograms/kg/d was generally well tolerated, suggesting that SCF may be useful as a therapy for some chronic hypoproliferative disorders of hematopoiesis.

Cell surface expression of c-kit receptors by childhood acute myeloid leukemia blasts is not of prognostic value: a report from the Childrens Cancer Group.

The prognostic significance of c-kit receptor expression on leukemic blast cells was determined in 122 children with acute myeloid leukemia (AML) entered onto Childrens Cancer Group protocol 213. Clinical and laboratory characteristics as well as outcome were analyzed according to the percentage of blast cells expressing c-kit receptors and the relative number of c-kit receptors per cell as determined by indirect immunofluorescence. c-kit receptor expression was strongly associated with the expression of the CD34 antigen. However, contrary to findings in adult patients with AML, c-kit receptor expression by childhood AML blast cells was not predictive of a poor response to therapy.

Human kit ligand (stem cell factor) modulates platelet activation in vitro.

The human stem cell factor (SCF), also termed c-Kit ligand (KL), is a hematopoietic growth factor produced by mesenchymal cells that induces proliferation of bone marrow progenitor cells, megakaryocytes, and mast cells via interaction with c-Kit, its cognate receptor. Expression of the c-kit gene was identified in human platelets by the polymerase chain reaction technique. The presence of the c-Kit receptor was demonstrated by the specific binding of 125I-KL/SCF to ADP-stimulated platelets. The identity of the c-Kit protein was confirmed by immunoreactivity with an anti-c-Kit-specific antibody and by its characterization as a phosphotyrosine-containing protein. Under constitutive conditions, c-Kit was found to be tyrosine-phosphorylated and was associated with a 85-kDa phosphoprotein that could be a fragment of phosphatidylinositol 3-kinase. These data indicate the presence of a new platelet surface molecule that could function in platelet activation. We demonstrate that the secondary wave of platelet aggregation and serotonin secretion induced by epinephrine and ADP, but not by the thromboxane analog U46619, was augmented by KL/SCF. The effect of KL/SCF on epinephrine/ADP-induced platelet activation appeared to be mediated in part through the thromboxane pathway. These data suggest that KL/SCF could modulate hemostasis via interaction with platelets, particularly in conditions where mesenchymal cells are exposed to circulating blood elements, such as in wound healing or atherosclerosis.

Splenic primitive hematopoietic stem cell (PHSC) activity is enhanced by steel factor because of PHSC proliferation.

To test whether primitive hematopoietic stem cells (PHSCs) are stimulated by Steel (SI) factor (c-kit ligand) in vivo, donor mice were studied after three or seven daily injections of SI factor. PHSC activity was measured as long-term erythroid and lymphoid competitive repopulating ability. Cells to be tested (usually marrow or spleen cells from treated donors) were mixed with untreated competitor marrow that produces erythrocytes and lymphocytes that are genetically distinguishable from the donors by differences in hemoglobin (Hb) and glucosephosphate isomerase (GPI) markers. These cell mixtures were injected into lethally irradiated hosts, and after 111 to 293 days, functional abilities of donor PHSC populations were assessed and expressed as percentages of donor-type Hb and GPI in the host's circulating erythrocytes and lymphocytes, respectively. A striking increase in splenic PHSC activity occurred after seven daily injections of SI factor, with a much smaller increase after three daily injections. Both three and seven daily injections of SI factor slightly reduced marrow PHSC activity. Rapid cycling greatly increases PHSC vulnerability to 5-fluorouracil (5FU). To test whether SI factor stimulates PHSCs into rapid cycling, donor mice were given a dose of 5FU in addition to SI factor. The increase in splenic PHSCs after 7 days of treatment with SI factor occurred to a similar degree whether donors were or were not treated with 5FU on day 8. However, a dose of 5FU on day 4 of the SI factor treatments almost totally prevented the increase in splenic PHSC activity. Apparently this increased activity requires PHSC cycling throughout the period of SI factor treatment.

[Effects of recombinant stem cell factor on the proliferation in vitro of LT12 acute promyelocytic leukemic cell line].

Stem cell factor is a recently identified earliest-acting hematopoietic growth factor and a ligand for the c-kit proto-oncogen. Based on our recent observations that recombinant rat interleukin-3 (IL3), human interleukin-6 (IL6) and murine granulocyte-macrophage colony stimulating factor (GM-CSF) possessed different degrees of suppressive activities on the proliferation of LT 12 cell line derived from BNML rat leukemic model, SCF was evaluated alone and in combination with either IL3, IL6 or GM-CSF for effects on leukemopoiesis in vitro. The results indicated that SCF alone had suppressive effect on DNA synthesis and colony forming unit-leukemic blast (CFU-L) in LT12 cells. 100ng/ml of SCF caused substantial reduction in colony number and 3H-TdR uptake although this suppression was of lower magnitude than those induced by IL3, IL6 or GM-CSF. Enhanced suppression on the proliferation of LT12 cells was observed when SCF was used in combination with one of these three factors. Among these combinations, SCF+GM-CSF or SCF+IL6 resulted in more suppression on LT12 cells than SCF+IL3 did. Combination of SCF with two or three factors produced even more suppression. No apparent effect on the size of leukemic colony was seen. Furthermore, in growth kinetics study of LT12 cells in the presence of SCF production of LT12 cells declined. Thus, SCF appears to have divergent hematopoietic activities on BNML rat model: effective stimulation of granulopoiesis and weak suppression of leukemopoiesis.

Characterization of cultured mast cells derived from Ws/Ws mast cell-deficient rats with a small deletion at tyrosine kinase domain of c-kit.

The Ws mutant allele of rats represents a 12-base deletion at the tyrosine kinase domain of the c-kit gene. Although homozygous Ws/Ws rats were deficient in both connective tissue-type mast cells (CTMC) and mucosal-type mast cells (MMC), mast cells did develop when bone marrow cells of Ws/Ws rats were cultured in the presence of concanavalin A-stimulated spleen cell conditioned medium (ConA-SCM). Although the proliferative response of rat cultured mast cells (RCMC) derived from Ws/Ws rats to ConA-SCM was comparable to that of RCMC derived from control normal (+/+) rats, the proliferative response of Ws/Ws RCMC to rat recombinant stem cell factor (rrSCF; a ligand for the c-kit receptor tyrosine kinase) was much lower than that of +/+ RCMC. However, a slight c-kit kinase activity was detectable in Ws/Ws RCMC, and the proliferation of Ws/Ws RCMC was accelerated when rrSCF was added to ConA-SCM. Because CTMC contain rat mast cell protease-I (RMCP-I) and MMC contain RMCP-II, the phenotype of +/+ and Ws/Ws RCMC in various culture conditions was evaluated by immunohistochemistry of RMCPs. Both +/+ and Ws/Ws RCMC showed the MMC-like phenotype (RMCP-I-/II+) when they were cultured with ConA-SCM alone. Most +/+ RCMC and about half of Ws/Ws RCMC acquired a novel protease (RMCP-I+/II+) phenotype when they were cultured with rrSCF alone. However, because the number of Ws/Ws RCMC dropped to one-tenth in the medium containing rrSCF alone, the absolute number of Ws/Ws RCMC with the RMCP-I+/II+ phenotype did not increase significantly. The effect of rrSCF in inducing the novel phenotype was suppressed when ConA-SCM was added to rrSCF. In contrast, +/+ and Ws/Ws RCMC cocultured with +/+ fibroblasts showed the RMCP-I+/II+ phenotype even in the presence of ConA-SCM. Moreover, a fibroblast cell line derived from SI/SI mouse embryos that did not produce SCF did not support the survival of both +/+ and Ws/Ws RCMC but did induce the RMCP-I+/II+ phenotype in about half of +/+ and Ws/Ws RCMC when their survival was supported by the addition of ConA-SCM. The normal signal transduction through the c-kit receptor did not appear to be prerequisite for the acquisition of the RMCP-I+/II+ phenotype.

In vitro effects of stem-cell factor or interleukin-3 on myelosuppression associated with AIDS.

OBJECTIVE: To determine whether the early-acting hematopoietic growth factors stem-cell factor (SCF) or interleukin-3 (IL-3), are able to overcome the bone-marrow suppressive effects of cytokines or drugs involved in the hematologic abnormalities that accompany HIV-1 infection. DESIGN: In vitro colony formation assays of normal human bone-marrow cells exposed to the myelosuppressive drugs, zidovudine, interferon-alpha (IFN-alpha) and ganciclovir, or the myelosuppressive cytokines, tumor necrosis factor-alpha (TNF-alpha) or transforming growth factor-beta (TGF-beta), implicated in HIV dysmyelopoiesis. RESULTS: SCF (10 ng/ml) enhanced the numbers of erythroid (BFU-E) colonies in the presence of zidovudine or ganciclovir (P < 0.05) and myeloid [colony-forming unit granulocyte macrophage (CFU-GM)] colonies in the presence of ganciclovir or IFN-alpha (P < 0.05) relative to controls. IL-3 (10 ng/ml) also improved erythroid colony numbers in the presence of zidovudine (P < 0.05) and CFU-GM in the presence of IFN-alpha (P < 0.05). Neither factor consistently altered the inhibition of TGF-beta or TNF-alpha. The 50% inhibitory concentration (IC50) of the myelosuppressive agents was altered in only one setting, using IL-3 in the presence of zidovudine. CONCLUSIONS: These data suggest that SCF or IL-3 may have a therapeutic application in overcoming hematopoietic abnormalities associated with drugs commonly used in the care of AIDS patients. However, they may have less capacity to overcome the bone-marrow inhibitory effects of the endogenous cytokines TNF-alpha and TGF-beta.

The in vivo effects of steel factor on natural killer lineage cells in murine spleen and bone marrow.

Steel factor (S1F), also known as stem cell factor, is a potent growth stimulator of hemopoietic progenitor cells. In the context of transplantation of hemopoietic cells to irradiated allogeneic hosts, natural killer (NK) cells exert restrictive control on hemopoietic cell proliferation, and are regularly found in elevated concentration in areas of intense hemopoiesis. The present study was designed to examine the effects with time of S1F in vivo on the numbers of NK cells, identified by the presence of the NK 1.1 surface molecule, in the spleen and bone marrow. Throughout the first 3 days of S1F exposure, NK cell numbers, in spite of rapid (1 day) and significant increases in the other hemopoietic cell lineages, did not change in either the spleen or the bone marrow. However, NK cells were increased 2-fold in both organs by 7 days of S1F exposure. At this time, immature granuloid and erythroid cells and the large lymphoid cells in the spleen had more than doubled their respective control numbers and in the bone marrow, immature granuloid cells increased by 47% of control levels. The presence of a late, but not early, influence of S1F on NK cells of the spleen and bone marrow suggests an indirect effect of S1F on this lineage, occurring only when S1F-stimulated hemopoiesis becomes sufficiently intense, providing, thus, an abundance of NK cell targets.

Effects of in vivo administration of stem cell factor on thrombopoiesis in normal and immunodeficient mice.

Thrombocytopenia is an important clinical problem for many acquired immunodeficiency syndrome (AIDS) patients. Recently, the utility of recombinant cytokines in alleviating the hematopoietic complications of AIDS and AIDS therapy has been evaluated. The newly cloned cytokine stem cell factor (SCF) has been demonstrated to be a potent regulator of hematopoietic progenitor cell proliferation. Therefore, we evaluated the ability of SCF to alleviate thrombocytopenia caused by infection with LP-BM5 murine leukemia virus (mLV) in a murine model of AIDS (MAIDS). In addition, we evaluated the effects of SCF on previously demonstrated azidothymidine (AZT)-induced elevations of platelet counts. SCF was administered to normal or LP-BMS-infected C57BL/6 mice in combination with oral AZT for up to 1 month and effects on platelet, megakaryocyte (MK), and megakaryocyte colony-forming cell (CFU-MK) numbers were evaluated. SCF alone significantly increased the number of circulating platelets in thrombocytopenic MAIDS mice by 53%. SCF also significantly elevated platelet levels by 29% in normal mice. AZT elevated platelet counts 100% in normal and 50% in MAIDS mice. AZT and SCF increased platelet counts in an additive manner. SCF alone was a potent inducer of splenic CFU-MK in both MAIDS and normal mice, increasing splenic CFU-MK 13- to 15-fold at day 15 as compared with untreated controls. By day 30, however, the numbers of splenic CFU-MK had returned to control levels. In infected mice, AZT alone increased the number of splenic CFU-MK. SCF administered to AZT-treated MAIDS mice did not further enhance these increases. In contrast, in normal mice, AZT decreased splenic CFU-MK numbers. In AZT-treated mice, SCF enhanced the numbers of splenic CFU-MK 90-fold at day 8. In MAIDS mice, the number of bone marrow CFU-MK was significantly increased by SCF treatment at all time points. SCF significantly affected the total number of femoral CFU-MK in AZT-treated mice only at day 15. In normal mice, SCF or SCF and AZT in combination increased the total number of bone marrow CFU-MK five-fold at day 8, but failed to induce changes in the total number of femoral CFU-MK after that. These results indicate that SCF elevates platelet levels in both thrombocytopenic MAIDS and normal mice and profoundly affects CFU-MK proliferation. Combinations of SCF and AZT may be further explored to enhance the therapeutic effectiveness of these two drugs in alleviating thrombocytopenia.

Stem cell factor influences the proliferation and erythroid differentiation of the MB-02 human erythroleukemia cell line by binding to a high-affinity c-kit receptor.

Stem cell factor (SCF) acts in synergy with other growth factors such as erythropoietin (Epo), granulocyte-macrophage colony-stimulating factor (GM-CSF), or interleukin-3 (IL-3), to stimulate the growth of primitive hematopoietic cells. Because of the prominent role of CSF in the maintenance of normal erythropoiesis in vivo, we examined the effects of SCF on the Epo-inducible human erythroleukemia cell line MB-02, and characterized the c-kit receptor in these cells. MB-02 cells cultured in serum-containing media do not survive in the absence of exogenous growth factors, but the addition of SCF, Epo, or IL-3 as a single factor enhanced MB-02 survival. Furthermore, in the presence of Epo, SCF (5 to 25 ng/mL) enhanced MB-02 proliferation in a dose-dependent manner, and increased the relative and absolute number of benzidine-positive cells generated. SCF also enhanced cell proliferation in the presence of either IL-3 or low concentrations of GM-CSF. A neutralizing anti-c-kit receptor monoclonal antibody (SR-1) blocked binding of 125I-SCF to MB-02 cells by 98%, and the effect of SCF on MB-02 growth, c-kit receptor-binding parameters were quantitated by equilibrium-binding experiments with 125I-SCF. MB-02 cells display a single class of high-affinity (50 pmol/L) c-kit receptors, with approximately 8,000 receptors per cell. The molecular weight of the c-kit receptor was determined by affinity cross-linking 125I-SCF to MB-02 cells. 125I-SCF-c-kit receptor complexes of approximately 155,000 and approximately 310,000 daltons were found, likely representing the monomeric and dimeric forms of the c-kit receptor. The binding affinity and molecular weight of the c-kit receptor on MB-02 cells are similar to those of normal human marrow cells. These results suggest that SCF synergizes with Epo to influence not only the proliferation but the erythroid differentiation of MB-02 cells. Thus, the MB-02 cell line may be a useful model in which to investigate the molecular mechanisms of SCF action.