Presence Of SCF Research Articles

Tumor necrosis factor–related apoptosis-inducing ligand induces monocytic maturation of leukemic and normal myeloid precursors through a caspase-dependent pathway

Treatment of the human HL-60 cell line with tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) resulted in rapid (6-24 hours) cytotoxicity associated with progressive maturation of the surviving cells along the monocytic lineage. The occurrence of monocytic maturation was demonstrated by a significant increase of both CD14 and CD11b surface expression, the acquisition of morphologic features typical of mature monocytes, and phagocytic capacity in TRAIL-treated cultures. By using selective pharmacologic inhibitors, it was possible to demonstrate that activation of the caspase cascade played a crucial role in mediating TRAIL cytotoxicity and monocytic maturation of HL-60 cells. Moreover, experiments performed using agonistic polyclonal antibodies, which mimic the interactions between TRAIL and each TRAIL receptor, indicated that TRAIL-R1 was responsible for mediating the TRAIL-induced maturation. Importantly, the maturational effects of TRAIL were observed also in primary normal CD34(+) cells, seeded in serum-free liquid cultures for 4 to 8 days in the presence of SCF + GM-CSF. After treatment with TRAIL for 3 additional days, a significant increase in CD14 and CD11b expression, coupled with an increased number of mature monocytes and macrophages, was noticed in the absence of cytotoxicity. These data disclose a novel role for TRAIL as a positive regulator of myeloid differentiation. Moreover, the dichotomous effect of TRAIL on malignant cells (early induction of apoptosis and monocytic maturation of the surviving cells) might have important therapeutic implications for the treatment of acute myeloid leukemia.

Generation of functional and mature dendritic cells from cord blood and bone marrow CD34 + cells by two-step culture combined with calcium ionophore treatment

The object of this study is to explore a culture method to generate a large number of functional and mature dendritic cells (DC) from human CD34 + hematopoietic progenitor cells. In the present study, we used a two-step method combined with calcium ionophore to induce DC from cord blood (CB) or normal human bone marrow (BM) CD34 + progenitor cells. The two-step method consists of 10 days of first step culture for the expansion and proliferation of CD34 + hematopoietic progenitor cells in the presence of SCF, IL-3, IL-6, G-CSF, and 7–11 days of second step culture for the induction of DC in the presence of GM-CSF, IL-4 and TNF-α. By the two-step culture, total nucleated cells were increased 208±66 (mean±SD, n=13), or 94±29 ( n=5)-fold in the culture of CB or BM cells, respectively, compared with the number of CD34 + cells at the time of starting culture. Out of the total nucleated cells, 23±10.4% of cells in CB cell culture and 25±5% of cells in the BM cell culture acquired DC characteristic phenotypes, which were marked expressions of CD1a, HLA-DR, co-stimulatory molecules such as CD80, CD40, and adhesion molecule such as CD58. In allogeneic mixed leukocyte reaction (MLR), two-step cultured cells showed potent allo-stimulatory capacity. With this two-step culture, the absolute number of CD1a + cells that co-expressed HLA-DR, CD80, CD40 and CD58 was enhanced approximately 3 times in CB cell culture and 1.9 times in BM cell culture, compared with the commonly used one-step culture method for the generation of DC from CD34 + cells using SCF, GM-CSF and TNF-α. However, on these DC generated in the two-step culture, the expressions of co-stimulatory molecule CD86 and mature DC marker CD83 were not sufficient. By the treatment of two-step cultured cells with calcium ionophore agent (A23187), the expression of co-stimulatory molecules such as CD86 and CD80 (especially CD86) was up-regulated. Besides, the expression of mature DC marker CD83 was remarkably induced by treatment with A23187 for a short duration (24 h). Consistent with the up-regulation of surface molecules CD86, CD80 and CD83, the two-step cultured cells treated with A23187 also showed a stronger allo-stimulatory capacity compared with the cells without A23187 treatment. In conclusion, the present study demonstrated that the two-step culture method effectively improved the yield of CD1a + DC generated from CD34 + cells, and the phenotypes and functions of these CD1a + DC could be enhanced efficiently by treatment with a calcium ionophore agent.

Megakaryocyte differentiation and reconstitution in nod/scid mice of CD34 cord blood cells after extensive ex vivo expansion

Abstract Safe use of myeloablative regimes is dependent on the availability of grafts containing sufficient number of haemopoietic stem cells to guarantee a durable reconstitution of the entire hematopoietic system. Much interest has been paid to alternative sources of hematopoietic stem cells that could be expanded ex vivo prior to being transplanted. In our hands, cord blood (CB) CD34 + cells can be triggered to undergo extensive proliferation and self-renewal processes, without losing, but, rather, amplifying their in vivo repopulating capacity. A central issue remains whether CD34 + cells, after several weeks of expansion, still retain not only their self-renewal characteristics, but also the same proliferation and differentiation potential towards all of the haemopoietic lineages, among all the megakaryocytic. We compared the proliferation and differentiation capacity toward the Mk lineage, as well as the Mk reconstitution in NOD/SCID mice of fresh, unmanipulated CD34 + cells derived from several cord blood samples and those recovered after extensive expansion in stroma-free cultures supported by FL, TPO and SCF. Unmanipulated CD34 + CB cells were grown in suspension cultures + either 10% FCS or 10% human plasma (HP) with: 1) TPO; 20 TPO + SCF; 3) IL3 + SCF + IL6; 4) IL3 + SCF + IL6 + TPO for 7, 14, 21 and 28 days. Cell production, appearance of CD41 + , CD34 + , CD34 + /CD41 + cells, CFU-Mk output as well as Mk repopulation in NOD/SCID mice were monitored and the indicated time points. The same studies were performed on CD34 + and CD34 − cells obtained by separation procedures (MiniMACS columns or FACS sorting) from CB cells which had been expanded for at least four weeks in stroma-free cultures in the presence of FL, TPO and SCF. In either case cells were cultured in IMDM+ 10% FCS or HP with the 4 described growth factor combinations. TPO by itself triggered maximum Mk production (/up to 500 -fold) in fresh but not in expanded cells, for whom the additional presence of SCF and IL6 was important to generate not only mature Mk, but also more primitive unipotent Mk or multipotent CFU-GEMM progenitors. NOD/SCID mice, studies indicate a normal Mk engraftment of expanded cells. The role of accessory CD34 − cells being investigated).

Proliferation and functional characterization of human CD34+ bone marrow cells cultured in simulated microgravity

The ability of the rotating wall vessel (RWV) bioreactor, developed by NASA for benchtop simulation of microgravity (μ-G), to support expansion of primitive human hematopoietic progenitor cells (HPC) was assessed during short-term cultures. Human bone marrow (BM) enriched for CD34+ cells (mean purity = 85%, n = 3) was seeded at 1.1 to 2.0 × 10e5 cells/mL in 10 mL in RWV, and incubated in the presence of SCF, IL-3, IL-6, GM-CSF, Flt-3L, and MGDF in either simulated μ-G or earth gravity (1-G). Proliferation of these cells after 5 days of culture averaged 11± 10-fold in μ-G, compared to 4.1 ± 3-fold in 1-G control cultures. Expansion of CD34+ cells was also greater in μ-G cultures compared to 1-G (2.6 ± 2.6 vs. 1.0 ± 0.8, respectively), as well as expansion of more primitive CD34+ CD38−/lo cells (1.9 ± 1.4 vs. 0.8 ± 0.6, respectively). To further examine the hematopoietic potential of CD34+ cells propagated in μ-G, long-term repopulating potential was assessed in NOD/SCID recipients. Conditioned NOD/SCID mice were transplanted with either fresh day 0 CD34-enriched BM cells (570K to 990K/mouse), or progeny of equivalent cell numbers expanded in μ-G (2.5 to 11.4 × 10e6/mouse) or 1-G (1.5 to 3.9 × 10e6/mouse). Fresh CD34+ cells provided 39 ± 14% chimerism at 8 weeks of post-transplantation. Despite possessing in μ-G were deficient in NOD/SCID-repopulating potential compared to 1-G controls (2.1 ± 1.4, n = 4 vs. 14.8 ± 13, n = 4, respectively). To examine whether diminished homing may contribute to the reduced engraftment of μ-G cells, adhesion molecule expression was analyzed. While no differences were found between μ-G and 1-G CD34+ cells in their adhesion molecule repertoire, these cells expressed considerably different levels of CD11a, CD43, CD44, CD49d, CD49e, CD62L, and CXCR4 in comparison to day 0 CD34+ cells, possibly contributing to their reduced engraftment potential. These results in simulated μ-G shed further light on stem cell biology, and may begin to explain some of the hematologic changes occurring in humans exposed to true microgravity.

Differential expansion of human primitive hematopoietic stem cell subsets: Correlation between phenotype, cell division history and frequency scid repopulating cells

CD34+ cells were isolated from G-CSF mobilized normal volunteers and cultured for 4 days in serum-free medium (X-VIVO 15) in the presence of SCF (20 ng/ml) + TPO (50 ng/ml) + FLT-3L (50 ng/ml). The phenotype (CD38, Thy-1, c-kit, HLA-DR) and cell division history (CFSE labeling) of CD34+ cells were examined. The frequency of LTC-IC and SCID repopulating cells (SRC) was measured by limiting dilution assays. After 4 days, the total number of CD34+ cells was maintained but a 25% reduction in the frequency of CFU and a 5-fold increase in LTC-IC were observed. The percentage of CD34+CD38− and CD34+DR− cells increased 57 and 5 fold, respectively, as > 85% of the CD38− and DR− cells divided 5 to 6 times from Day 0 to Day 4. The percentage of CD34+/c-kit+ was maintained after 4 days of culture even though > 90% of the cells had divided 2 to 5 times. In contrast, there was a 6-fold reduction in Thy-1+ cells while 60% of the Thy-1+ cells detected at Day 4 had undergone 0 to 2 divisions. Cultures of sorted CD34+Thy-1+ cells indicated that 3 or more cell divisions resulted in a gradual loss of Thy-1 expression. This data indicates that the combination of SCF+TPO+FLT-3L preferentially expands CD34+CD38− and DR− subsets. Data correlating CD34+ subsets, number of cell divisions and frequency of LTC-IC and SRC will be presented.

Retroviral-mediated transfer and expression of the multidrug resistance protein 1 gene (MRP1) protect human hematopoietic cells from antineoplastic drugs.

Multidrug resistance protein (MRP1) is a member of the ATP-binding cassette (ABC) transmembrane transporter superfamily that confers multidrug resistance. The transfer and expression of the MRP1 gene in human hematopoietic stem cells may be a useful alternative to multidrug resistance (MDR1) gene transfer for protection from the myelosuppressive effects of chemotherapy in cancer patients. We constructed a gibbon ape leukemia virus packaging cell line (PG13) using the human MRP1 cDNA in a Moloney murine leukemia virus (MoMuLV) backbone containing a modified LTR. This PG13-based cell line, designated MRP1-PG13, produces retroviral vectors bearing the MRP1 gene at a titer of 1.7x10(5) viral particles/ml. Transduction of the human leukemic cell line K562 showed that viral MRP1-PG13 supernatants routinely transfer the MRP1 gene to approximately 35% of target K562 cells, of which at least one third are capable of proliferating in the presence of otherwise toxic concentrations of etoposide. Southern blot analyses indicated that most clones had only one proviral integration. Northern blot analysis of expanded K562 clones showed the presence of a major full-length approximately 8-kb MRP1 transcript as well as a minor approximately 6-kb transcript in all clones. Flow cytometric analysis of the producer cells and clones of transduced K562 cells demonstrated significantly increased MRP1 expression in these cells (approximately 30-fold increase). Human bone marrow mononuclear cells and CD34+ cells were also transduced with MRP1-PG13 supernatants on fibronectin-coated culture flasks in the presence of SCF, IL-3, and IL-6. PCR analysis of individual hematopoietic colonies in methylcellulose cultures demonstrated proviral DNA in approximately 10% of unselected human hematopoietic progenitor cells cultured from nonsorted mononuclear cell samples and in up to approximately 75% of progenitors when CD34-enriched cell populations were targeted. To assess functional MRP1 gene expression, normal human hematopoietic progenitors and K562 cells were cultured in methylcellulose assays containing vincristine or etoposide. All transduced samples gave rise to approximately 10% drug-resistant colonies, which were shown to be provirus-positive by PCR. Our studies document the development of an amphotropic MRP1 retroviral vector producer cell line and pave the way for large animal and preclinical studies of chemoprotection by MRP1 gene transfer.

The Notch/Jagged pathway inhibits proliferation of human hematopoietic progenitors in vitro.

The cell surface receptor Notch1 is expressed on CD34+ hematopoietic precursors, whereas one of its ligands, Jagged1, is expressed on bone marrow stromal cells. To examine the role of Notch signaling in early hematopoiesis, human CD34+ cells were cultured in the presence or absence of exogenous cytokines on feeder layers that either did or did not express Jagged1. In the absence of recombinant growth factors, Jagged1 decreased myeloid colony formation by CD34+ cells, as well as 3H-thymidine incorporation and entry into S phase. In the presence of a strong cytokine signal to proliferate and mature, (interleukin 3 [IL-3] and IL-6, stem cell factor [SCF], and G-CSF), Jagged1 did not significantly alter either the fold expansion or the types of colonies formed by CD34+ cells. However, in the presence of SCF alone, Jagged1 increased erythroid colony formation twofold. These results demonstrate that Notch can modulate a growth factor signal, and that in the absence of growth factor stimulation, the Jagged1-Notch pathway preserves CD34+ cells in an immature state.

Further characterization of CD34-low/negative mouse hematopoietic stem cells.

We have previously reported that in adult mouse bone marrow, CD34low/- c-kit+ Sca-1+ lineage markers negative (Lin-) (CD34-KSL) cells represent hematopoietic stem cells with long-term marrow repopulating ability whereas CD34+ c-kit+ Sca-1+ Lin- (CD34+KSL) cells are progenitors with short-term reconstitution capacity. To further characterize cells in those two populations, relative expression of various genes were examined by reverse transcriptase polymerase chain reaction (RT-PCR). In CD34-KSL cells, none of the genes studied was found to be expressed with the exception of GATA-2, IL-1R alpha, IL-2R gamma, AIC-2B, c-kit, EPO-R, and c-mpl. In contrast, expression of GATA-1 and all cytokine receptor genes examined except IL-2R beta, IL-7R alpha and IL-9R alpha were found in CD34+KSL. The difference between these two populations was also shown in single cell culture analysis of these cells. When cells were clone-sorted and cultured in the presence of SCF, IL-3 and EPO, CD34-KSL cells required much more time to undergo the first cell division than CD34+KSL cells. Dormancy and random fashion of cell division by CD34-KSL cells were also evident by the analysis of the second cell division, which was found to be delayed and unsynchronous compared with CD34+KSL cells. Clonal culture analysis showed that CD34-KSL cells were more potent in proliferation and multilineage differentiation capacities than CD34+KSL cells. In a paired-daughter cell experiment, 75% of CD34-KSL and 50% of CD34+KSL paired-daughter-derived colonies were nonidentical with wide variety of lineage combinations. Taken together, these data support our previous notion that CD34-KSL cells are at higher rank in hematopoietic hierarchy than CD34+KSL cells. In addition, our results using highly enriched stem cell population directly obtained from mouse bone marrow support the proposed stochastic nature of lineage commitment.

Growth and Differentiation of Human Stem Cell Factor/Erythropoietin-Dependent Erythroid Progenitor Cells In Vitro

AbstractStem cell factor (SCF) and erythropoietin (Epo) effectively support erythroid cell development in vivo and in vitro. We have studied here an SCF/Epo-dependent erythroid progenitor cell from cord blood that can be efficiently amplified in liquid culture to large cell numbers in the presence of SCF, Epo, insulin-like growth factor-1 (IGF-1), dexamethasone, and estrogen. Additionally, by changing the culture conditions and by administration of Epo plus insulin, such progenitor cells effectively undergo terminal differentiation in culture and thereby faithfully recapitulate erythroid cell differentiation in vitro. This SCF/Epo-dependent erythroid progenitor is also present in CD34+ peripheral blood stem cells and human bone marrow and can be isolated, amplified, and differentiated in vitro under the same conditions. Thus, highly homogenous populations of SCF/Epo-dependent erythroid progenitors can be obtained in large cell numbers that are most suitable for further biochemical and molecular studies. We demonstrate that such cells express the recently identified adapter protein p62dok that is involved in signaling downstream of the c-kit/SCF receptor. Additionally, cells express the cyclin-dependent kinase (CDK) inhibitors p21cip1 and p27kip1 that are highly induced when cells differentiate. Thus, the in vitro system described allows the study of molecules and signaling pathways involved in proliferation or differentiation of human erythroid cells.

Flt 3 ligand, MGDF, Epo and G-CSF enhance ex vivo expansion of hematopoietic cell compartments in the presence of SCF, IL-3 and IL-6.

The aim of the study is to define the ability of Flt3 ligand, MGDF, Epo and G-CSF to modulate the expansion of different hematopoietic compartments in association with a basic cocktail of SCF + IL-3 + IL-6 (S36). CD34+ cells from normal bone marrow were cultured in stroma-free, serum-free medium for 10 days. Using various concentrations of cytokines, total cells could be expanded up to 5200-fold, CD34+ cells up to 78-fold, CFU-GM up to 143-fold, BFU-E up to 46-fold, CFU-MK up to six-fold and LTC-IC up to four-fold. The results were assessed by multiparametric analysis of variance. Three factors had a significant stimulatory effect on the late precursor compartment: Epo (P < 10(-5)), G-CSF (P=5 x 10(-3)) and FL (P=10(-5)). Two were critical for CD34+ cell expansion: FL (P=4 x 10(-5)) and Epo (P=6 x 10(-5)), while two were critical for BFU-E expansion: MGDF (P=8 x 10(-4)) and FL (P=0.017). FL strongly stimulated CFU-GM expansion (P < 10(-5)), whereas none of the growth factors studied had any effect on CFU-MK. FL (P=10(-4)) and MGDF (P=0.002) were essential to obtain high levels of expansion of LTC-IC as determined in limiting dilution assays. In the light of the above results showing a preferential effect on the expansion of precursor cells (3080-fold), CD34+ cells (53-fold), CFU-GM (134-fold), BFU-E (46-fold) and LTC-IC (five-fold), the combination SCF, IL-3, IL-6, FL, MGDF, Epo and G-CSF was chosen as a putative cytokine cocktail for further studies on long-term culture. Sustained production of precursor cells, progenitor cells, LTC-IC and E-LTC-IC for up to 100 days reflects the persistence of very primitive stem cells. This suggests that these populations are probably able to undergo self-renewal divisions. The above combination of cytokines meets the required criterion for potential clinical application, which may be defined as an effective capacity to expand all cell compartments, using as the starting material high concentrations of low purity CD34+ cells.

EFFICIENCY OF RETROVIRAL TRANSDUCTION OF CD 34+ CELLS FROM UMBILICAL CORD BLOOD DEPENDS ON GROWTH FACTOR STIMULATION. 134

The feasibility of gene marking or gene therapy protocols making use of purified CD34+ cells greatly depends on the efficiency of stable transduction. The great potentiality of umbilical cord blood CD34+ cells prompted us to evaluate which factor(s) play a major role(s) in influencing the transduction efficiency of retroviral vectors. CD34+ cells, isolated with an immunomagnetic device (MiniMacs, Miltenyi Biotech, Germany), 90-95% pure as assessed by cytofluorimetric analysis, were transduced with the LXSN retroviral vector carrying the neomicine resistance gene. Infection was made with virus-containing supernatant of Am12 env+ packaging cell line, having a titer of 2×106 PFU/ml. All the infections were performed using a multiplicity of infection of 10 PFU/cell. Multiple or single infections were performed with the following schedules: i) up to 6 infection lasting one hour over 3 days; ii) a 72 hr infections substituting fresh viral supernatant and cytokines every 24 hrs; iii) a single 72 hr infection. In most experiments, CD34+ cells were preincubated with different cytokine cocktails for 24 or 48 hrs before infection. Efficiency of transduction was evaluated both by clonogenic assays and semiquantitative PCR analysis performed either immediately or after 7 day expansion in the presence of cytokines and G-418. G-418-resistant CFU-E, BFU-E and CFU-GM colonies were scored after 7 and 14 days. We provided evidence that the efficiency of transduction was mainly dependent on cell preincubation with growth factors rather than on the number of infections. Precisely, infection performed on freshly isolated CD34+ was almost ineffective (0.4%) while 48 hr preincubation in the presence of SCF*+IL-6+IL-3 allowed to reach a level of transduction up to 40% regardeless of the number of infections. In this respect it is noteworthy that a single infection, lasting as long as a multiple infection course gave identical results. In conclusion the results obtained suggest that high efficiency of transduction of umbilical cord blood CD34+ cells can be achieved in liquid, stroma free, cultures.

Contact- and Growth Factor-Dependent Survival in a Canine Marrow-Derived Stromal Cell Line

Cell-cell interactions and the presence of growth factors such as stem cell factor (SCF; or c-kit ligand) or interleukin-6 (IL-6) are involved in the proliferation and differentiation of the canine marrow-derived stromal cell line DO64. In the presence of SCF, stromal cells are induced to differentiate, but not to proliferate. In contrast, in the presence of IL-6, stromal cells are induced to proliferate rather than to differentiate in culture. Both SCF and IL-6 are produced by the stromal cells themselves and, thus, act as autocrine factors. In addition, DO64 cells also interact physically with each other in culture when grown under optimal culture conditions (70% to 90% cell confluence and in the presence of serum), thereby supporting proliferation and maintaining viability. Under conditions of lower cell density or low serum or growth factor concentrations in culture, DO64 cells tend to aggregate and form clusters. This increase in local cell concentration is associated with preservation of viability, presumably because of the accumulation of autocrine factors. If no signal, neither intercellular nor soluble, is provided, and DO64 cells are not able to reach a critical cell density or to produce sufficient factors in an autocrine fashion, the cells cease to proliferate and eventually die.

Human mast cell heterogeneity.

Mast cell neutral proteases are the most precise markers of heterogeneity among human mast cells. Two types of human mast cells have been recognized. MCTC cells contain tryptase together with chymase, cathepsin-G like protease, and mast cell carboxypeptidase; MCT cells contain tryptase, but lack the other neutral proteases present in MCTC cells. All mast cells develop from hemopoietic stem cells. In vitro procedures for studying mast cell growth have been developed, using the major human mast cell growth factor, stem cell factor (SCF, also called Kit-ligand). Cultures of hemopoietic progenitor cells in the presence of SCF alone result in selective differentiation to mast cells. The same progenitor cells can be induced to differentiate into other lineages when SCF is used with various lineage-specific colony-stimulating factors such as erythropoietin for erythrocytes. Mast cell development from hematopoietic progenitors may represent a "default pathway," occurring optimally in a permissive microenvironment such as skin, bowel, and lung. The presence or absence of certain cytokines in blood and bone marrow may create a non-permissive environment, thus the absence of granulated mast cells in such locations.

Expression of IL-4 and IL-5 mRNAs in human mast cells via FcϵRI in the presence of SCF

Expression of IL-4 and IL-5 mRNAs in human mast cells via FcϵRI in the presence of SCF

Stem cell factor induces mast cell adhesion to fibronectin.

Stem cell factor (SCF) or c-kit ligand is a growth factor cytokine produced by stromal cells that is known to influence mast cell proliferation and differentiation. We hypothesized that SCF may also influence the adhesion of mast cells to connective tissue matrix. To examine this hypothesis, we stimulated MCP5/L mast cells or murine bone marrow-derived cultured mast cells (BMCMC) with either SCF or PMA and observed adhesion to fibronectin (FN). As expected, 80 to 90% of PMA-activated MCP5/L cells or BMCMC adhered to FN. In addition, SCF promoted MCP5/L cell or BMCMC adhesion to FN in a dose-response fashion with 50 to 60% of BMCMC adhering to FN at a concentration 10 ng/ml of SCF. BMCMC adhesion was observed with as little as 200 pg/ml of SCF. Adhesion of SCF stimulated BMCMC to FN did not require IL-3, but was dependent on the concentration of FN used to coat the assay surface. Mast cell adhesion in the presence of SCF appeared to occur through an integrin receptor as adhesion was calcium dependent and could be blocked by an RGD (Ang, Gly, Asp)-containing peptide. SCF did not directly mediate adhesion through interaction with c-kit, as FN-coated surfaces exposed to SCF before initiation of the adhesion assay did not promote adhesion in the absence of soluble SCF. Rather, SCF appeared to stimulate adhesion to FN by activating mast cells through its interaction with c-kit. Thus, antibody to SCF blocked adhesion, and rat and murine SCF stimulated BMCMC adhesion to FN, but human SCF, which does not bind to murine c-kit, did not stimulate adhesion. Genistein, which inhibits tyrosine kinase activity, partially inhibited SCF-induced adhesion. SCF thus stimulates mast cell adhesion and, because SCF is produced normally in tissues, it may be a major factor responsible for the adhesion of mast cells to connective tissue matrix under physiologic conditions.

Monocytes do not make mast cells when cultured in the presence of SCF. Characterization of the circulating mast cell progenitor as a c-kit+, CD34+, Ly-, CD14-, CD17-, colony-forming cell.

Mast cells (MC3) belong to the hemopoietic system and arise from hemopoietic precursor cells. Human MC progenitors can be detected in the bone marrow as well as in the peripheral blood (pb) and are responsive to the mast cell growth factor SCF, the ligand of the c-kit tyrosine kinase receptor. However, little is known about the subsets of cells that become committed to and differentiate into mature human MC. In this study, the identity of the circulating MC progenitor, previously felt to be a monocyte (Mo) or basophil (Ba), was investigated. For this purpose, CD14+ pb monocytes, CD17+ pb basophils and CD34+ cord blood cells were purified to homogeneity (> 95%) from mononuclear cells (normal adult donors, n = 17, cord blood, n = 2) by counter-flow centrifugation followed by cell sorting with mAb. In the presence of rhSCF, MC developed in long term suspension culture from pure CD34+ cells but not from pure Mo, pure Ba, or Ly (MC-tryptase levels on day 42: CD14+ Mo: 3.7 +/- 0.8 vs CD17+ Ba: 3.2 +/- 0.5 vs Ly: 2.0 +/- 1.5 vs control: 196.5 +/- 92.5 ng/ml, p < 0.001). Depletion of CD34+ cells from MNC resulted in a loss of MC in long term suspension culture, whereas depletion of either Mo, Ba, or Ly did not. In methyl-cellulose cultures in the presence of rhSCF, MC and tryptase could be detected in pure (CFU-mast) and mixed (CFU-myeloid/mast) MC colonies. Together, MC do not originate from circulating Mo, Ba, or Ly. The circulating MC progenitor is a CD34+, c-kit+, Ly-, CD14-, CD17- colony-forming cell. This is the first definitive demonstration that mast cells are replenished directly from early hemopoietic progenitors and thus form a unique cell lineage within the hemopoietic system.