Biomedical subjects

S D Lyman

Publications and source records attributed to S D Lyman.

At least 55 records · Page 3Linked to original sources

PubMed1996

The flt3 ligand promotes the survival of primitive hemopoietic progenitor cells with myeloid as well as B lymphoid potential. Suppression of apoptosis and counteraction by TNF-alpha and TGF-beta.

The recently cloned fIt3 ligand (FL) potently enhances hemopoietic growth factor-induced growth of primitive hemopoietic progenitors. In agreement with previous reports, we found FL alone to be a weak mitogen for primitive Lin-Sca-1+ murine bone marrow progenitors. Using delayed addition of growth-promoting cytokines, we demonstrate that FL potently promotes the in vitro survival of Lin-Sca-1+ progenitors responsive to a potent myeloid growth factor combination (FL, stem cell factor (SCF), granulocyte CSF (G-CSF), and IL-1 alpha). Whereas no such progenitors survived in cultures supplemented with medium alone, 33% survived in FL compared with 75 and 13% in the presence of SCF and IL-1 alpha, respectively. These results were obtained when cells were plated individually, suggesting that the viability-promoting effect of FL is mediated directly on the progenitors. Whereas SCF was superior to FL in promoting the survival of FL-, SCF-, G-CSF-, and IL-1 alpha-stimulated Lin-Sca-1+ progenitors, FL was more efficient than SCF at promoting the survival of progenitors with a B cell potential, as measured by their ability to produce B220+ cells in response to delayed addition of FL, SCF, and IL-7. Seventy-one percent of the B220+ cell production could be recovered following 40-h incubation with FL compared with 2% in response to SCF. Analysis of day 12 spleen CFU content after 40-h preincubation of Lin-Sca-1+ cells in FL or SCF demonstrated that SCF maintained 64% of the day 12 spleen CFU, whereas only 16% survived in the presence of FL. Finally, there was no significant difference between the ability of FL and SCF to maintain the viability of long-term culture-initiating cells (25 and 32%, respectively). The ability of FL to promote the survival of Lin-Sca-1+ progenitor cells was reflected by the finding that FL also suppressed apoptosis. Finally, TGF-beta abrogated and TNF-alpha potently counteracted the survival-promoting effect of FL. Thus, FL promotes the survival of primitive hemopoietic progenitor cells, in particular those with an inherent B lymphoid potential.

Animals↗

PubMed1996

Hematologic effects of flt3 ligand in vivo in mice.

We have investigated the effects of in vivo treatment with flt3 ligand (FL) on murine hematopoiesis, including mobilization of progenitors into the peripheral blood (PB). Mice were injected once daily with 10 micrograms recombinant human FL for 15 days. On days 3, 5, 8, 10, 15, and 22, mice were killed and analyzed for the number of leukocytes and colony-forming units (CFU) in bone marrow (BM), spleen, and PB. Splenic and PB cellularity increased with time in FL-treated mice. In the spleen, there was an increase in B cells, myeloid cells, and nucleated erythroid cells; in the PB, there was an increase in lymphocytes, granulocytes, and monocytic cells. The maximal number of CFU in the BM was observed after 3 days of FL treatment, giving 3.7- and 7.3-fold increases in CFU-granulocyte-macrophage (CFU-GM) and CFU-granulocyte, erythrocyte, monocyte, megakaryocyte (CFU-GEMM), respectively, compared with mouse serum albumin (MSA)-treated controls. After 8 days of FL treatment, there was a maximal 123- and 108-fold increase in splenic CFU-GM and CFU-GEMM, respectively. The maximal number CFU-GM and CFU-GEMM were seen in PB on day 10, with 537- and 585-fold increases, respectively. Burst-forming units-erythroid (BFU-E) increased in the same time frame as those of CFU-GM and CFU-GEMM in BM, spleen, and PB, although the magnitude was not as great. Primitive day-13 CFU-spleen (CFU-S) and phenotypically defined stem cells were also mobilized into the PB of FL-treated mice with similar kinetics and magnitude to that of CFU-GM and CFU-GEMM. We conclude from these studies that FL, when administered as a single agent, is a potent mobilizer of hematopoietic progenitors into the PB.

Animals↗

PubMed1996

Combined signaling through interleukin-7 receptors and flt3 but not c-kit potently and selectively promotes B-cell commitment and differentiation from uncommitted murine bone marrow progenitor cells.

Multiple cytokines can synergize to stimulate the in vitro proliferation and exclusive myeloid differentiation of multipotent bone marrow progenitor cells. The ligand for c-kit (stem cell factor [SCF]) plays a key role in stimulating myeloid and erythroid cell production of primitive hematopoietic progenitors. SCF in combination with interleukin-7 (IL-7) can also stimulate the combined myeloid and B-cell differentiation of uncommitted hematopoietic progenitor cells as well as the growth of early B-cell progenitor cells, although the involvement of c-kit in early B lymphopoiesis remains controversial. In the present study, the flt3-ligand (FL), which, in combination with other cytokines, has overlapping activities with SCF on myeloid cell production from uncommitted progenitors, was investigated for its ability to induce selective stroma-independent B-cell commitment from uncommitted Lin-Sca-1+ bone marrow progenitor cells. IL-7 alone did not induce any clonal growth and FL alone gave rise to a few clusters (< 50 cells) but no colonies (> 50 cells), whereas the combined stimulation with FL and IL-7 resulted in clonal growth of 10% of Lin-Sca-1+ bone marrow cells. After 12 days of incubation of Lin-Sca-1+ cells in FL + IL-7, an almost 400-fold increase in cell production was observed. Phenotyping showed that greater than 99% of the cells produced were of the B-cell lineage, in that they expressed B220, but not cell surface markers specific for myeloid, erythroid, or T-cell lineages. Furthermore, the cells did not express cytoplasmic mu-heavy chain (cmu) or surface IgM, but were positive for CD24 (heat stable antigen [HSA]) and CD43 (leukosialin), suggesting that the cells produced were blocked at a late pro-B-cell stage. Interestingly, although all FL + IL-7-responsive Lin-Sca-1+ progenitor cells and the resulting pro-B cells expressed c-kit, FL + IL-7 was much more potent (62-fold) than SCF + IL-7 in stimulating production of cells of the B-cell lineage. In addition, whereas FL + IL-7 selectively stimulated the production of pro-B cells, SCF + IL-7 predominantly stimulated the production of mature granulocytes. Replating studies showed that FL + IL-7-responsive Lin-Sca-1+ progenitors were not committed to the B-cell lineage, because after 2 days of incubation in FL + IL-7, 80% of the progenitors retained a myeloid potential. As much as 27% of the FL + IL-7-responsive progenitors remained uncommitted after 7 days of incubation, but all had committed to the B-cell lineage after 10 days of incubation in FL + IL-7. These results show that FL much more potently and selectively than SCF synergizes with IL-7 to enhance B-cell commitment and development from uncommitted progenitor cells.

Animals↗

PubMed1996

Ability of flt3 ligand to stimulate the in vitro growth of primitive murine hematopoietic progenitors is potently and directly inhibited by transforming growth factor-beta and tumor necrosis factor-alpha.

The recently cloned flt3 ligand (FL) stimulates the growth of primitive hematopoietic progenitor cells through synergistic interactions with multiple other cytokines. The present study is the first demonstrating cytokines capable of inhibiting FL-stimulated hematopoietic cell growth. Tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta 1 (TGF-beta l) potently inhibited the clonal growth of murine Lin-Sca-l+ bone marrow progenitors stimulated by FL alone or in combination with granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), interleukin (IL)-3, IL-6, IL-11, or IL-12. TGF-beta 1 inhibited more than 96% of the myeloid colony formation in response to these cytokine combinations, whereas TNF-alpha reduced the number of colonies by 58% to 96% depending on the cytokine by which FL was combined. In addition, both TNF-alpha and TGF-beta 1 inhibited more than 90% of B220+ cell production from B220- bone marrow cells stimulated by FL + IL-7. The effects of TNF-alpha and TGF-beta 1 appeared to be due to a direct effect and on the early progenitors because the inhibition was observed at the single cell level, and because delayed addition of the two inhibitors for only 48 hours dramatically reduced their inhibitory effects. A neutralizing anti-TGF-beta antibody showed the presence of endogenous TGF-beta in the cultures and potently enhanced the ability of FL to stimulate progenitor cell growth in the absence of other cytokines. Agonistic antibodies specifically activating the p75 TNF receptors were more efficient than wild type murine TNF-alpha in signaling growth inhibition of Lin-Sca-l+ progenitor cells, whereas the p55 agonist had less effect than murine TNF-alpha. Finally, TGF-beta increased the number of FL + IL-11-stimulated Lin-Sca-1+ cells in the G1 phase of the cell cycle with 76%, whereas TNF-alpha only had a marginal effect on cell cycle distribution. Thus, TGF-beta, TNF-alpha, and p75 TNF receptor agonists are potent direct inhibitors of FL-stimulated progenitor cell growth in vitro.

Animals↗

PubMed1996

The genes encoding the eph-related receptor tyrosine kinase ligands LERK-1 (EPLG1, Epl1), LERK-3 (EPLG3, Epl3), and LERK-4 (EPLG4, Epl4) are clustered on human chromosome 1 and mouse chromosome 3.

Hek and elk are members of the eph-related family of receptor tyrosine kinases. Recently, we isolated five cDNAs encoding membrane-bound ligands to hek and elk. Because of the promiscuous nature of their binding, we have termed these proteins ligands of the eph-related kinases or LERKs. The LERKs can be divided into two subgroups by virtue of their sequence identity, binding properties, and mode of cell membrane attachment. For example, LERK-2 (EPLG2, Epl2) and LERK-5 (EPLG5, Epl5) are type 1 transmembrane proteins, while LERK-1 (EPLG1, Epl1), LERK-3 (EPLG3, Epl3), and LERK-4 (EPLG4, Epl4) are anchored to the membrane by glycosyl-phosphatidylinositol (GPI) linkage. Using Southern hybridization analysis of human x rodent somatic cell hybrid DNAs, we have assigned the genes that encode the GPI-anchored LERKs (EPLG1, EPLG3, and EPLG4) to human chromosome 1. Fluorescence in situ hybridization to metaphase chromosome preparations using genomic clones from each locus refined this localization to chromosome 1, bands q21-q22. In addition, Southern blot analysis of DNA from interspecific backcross mice indicated that the mouse homologues Epl1, Epl3, and Epl4 map to a homologous region on mouse chromosome 3.

Animals↗

PubMed1996

Accelerated cell-cycling of hematopoietic progenitors by the flt3 ligand that is modulated by transforming growth factor-beta.

Although hematopoietic growth factors have been extensively studied as to their roles in recruitment of hematopoietic progenitors from quiescence state to cell division state, little is known of their effects on cell-cycling of progenitors that have already transited from quiescence into active cell-cycling. We examined the effects of the flt3 ligand (FL) on cell-cycling of hematopoietic progenitors in serum-free culture. Results from our serial observations of colony formation and replating experiments suggest that FL enhances the rate of growth of interleukin-3 (IL-3)-dependent colonies by shortening the time for each progenitor in the colonies to divide. Cell-cycle analysis showed that shortening of cell-cycle time induced by FL is mainly because of alteration in the G1 phase that hematopoietic progenitors go through. We next investigated the role of transforming growth factor-beta (TGF-beta) in cell-cycling of progenitors, using TGF-beta protein and TGF-beta antisense oligonucleotides, because mRNA of TGF-beta was detected by reverse transcriptase polymerase reaction in blast cells that we used as a source of progenitors. TGF-beta lengthened the time required for IL-3-dependent progenitors to become two daughter cells, whereas the effects of TGF-beta antisense oligonucleotides were opposite to those of TGF-beta. The addition of TGF-beta neutralizing monoclonal antibodies to the cultures resulted in effects similar to those seen with TGF-beta antisense oligonucleotides. DNA studies indicated that both TBF-beta and TGF-beta antisense oligonucleotides change the length of G1 phase of the cell-cycle. TGF-beta abrogated the effects of FL on the growth rate of hematopoietic progenitors, whereas the combination of FL with TGF-beta antisense oligonucleotides exerted additive effects. These data show that FL has the potential to accelerate cell-cycling of hematopoietic progenitors, which is susceptible to the modulation by TGF-beta.

Animals↗

PubMed1996

The FLT3 ligand is a direct and potent stimulator of the growth of primitive and committed human CD34+ bone marrow progenitor cells in vitro.

The present studies investigated the effects of the recently cloned flt3 ligand (FL) on the in vitro growth and differentiation of primitive and committed subsets of human CD34+ bone marrow (BM) progenitor cells. FL alone was a weak growth stimulator of CD34+ BM cells, but synergistically and directly enhanced colony formation in combination with interleukin (IL) 3, granulocyte colony-stimulating factor (G-CSF), CSF-1, granulocyte macrophage (GM) CSF stem cell factor (SCF), and IL-6. FL and SCF were equally effective in stimulating colony formation in combination with IL-3. However, the tri-factor combination of FL + IL-3 + SCF stimulated 2.3-fold and 2.5-fold more colonies than FL + IL-3 and SCF + IL-3, respectively. These additional recruited progenitors appeared to be predominantly located in a primitive (CD71-) subset of the CD34+ progenitors, as 4.5-fold more colonies were formed by CD34+CD71- cells in response to FL + IL-3 + SCF than to FL + IL-3 or SCF + IL-3. Similar findings were observed in serum-containing and serum-deprived cultures. Whereas FL did not enhance burst-forming unit-erythroid (BFU-E) colony formation of CD34+ BM cells in the presence of serum, a low number of BFU-E colonies were formed in response to FL plus erythropoietin (Epo) under serum-deprived conditions. In addition, FL both in serum-containing and serum-deprived cultures stimulated colony formation of more committed myeloid progenitors in CD34+CD71+ BM cells. Thus, FL potently stimulates the growth of primitive and more committed human BM progenitor cells.

Adult↗

PubMed1996

Flt3 ligand supports the differentiation of early B cell progenitors in the presence of interleukin-11 and interleukin-7.

B cell development is influenced by interactions between B cell progenitors and stromal cells. The precise mechanisms by which these interactions regulate B cell differentiation are currently unknown. Flt3 ligand (FL) is a growth factor which stimulates the proliferation of stem cells and early progenitors. Mice deficient for the FLT3 receptor exhibit severe reductions in early B lymphoid progenitors. We have previously described a clonal assay in vitro which allows us to follow the entire B cell differentiation pathway from uncommitted progenitors to mature, immunoglobulin-secreting plasma cells. The growth factor combination of interleukin (IL)-11, mast cell growth factor (MGF) and IL-7 was shown to maintain the differentiation of these hematopoietic precursors into B cell progenitors capable of giving rise to functionally mature B cells in secondary cultures. Here, we show that FL in combination with IL-11 and IL-7 is sufficient to support the differentiation of uncommitted progenitors from day 10 yolk sac (AA4.1+) or day 12 fetal liver (AA4.1+ B220- Mac-1- Sca-1+) into the B lineage. The frequency of B cell progenitors obtained in these conditions was similar, if not better, than the frequency of B cell precursors that arose when cultured in IL-11+MGF+IL-7. Furthermore, the growth factor combination of IL-11+FL+ IL-7 was able to maintain the potential of bipotent precursors giving rise to both the B and myeloid lineages in secondary cultures. We also show that FL synergizes with IL-7 in the proliferation of committed B220+ pro-B cells and may contribute to the maintenance of an earlier pro-B cell population. Together, these results show that FL is important in supporting the differentiation and proliferation of early B cell progenitors in vitro.

Animals↗

PubMed1996

Flt3 ligand prolongs survival of CD34++ + human umbilical cord blood myeloid progenitors in serum-depleted culture medium.

Flt3 Ligand (L), a newly identified stimulating/co-stimulating cytokine for hematopoietic stem and progenitor cells, was assessed for its capacity, in comparison to that of steel factor, to prolong the survival in culture of hematopoietic stem and progenitor cells from CD34++ + human cord blood. The addition of Flt3-L to serum-depleted cultures significantly prolonged the survival of high-proliferative-potential colony-forming cells (HPP-CFC) as well as that of multipotential (CFU-GEMM) and granulocyte-macrophage (CFU-GM) progenitor cells. The concentrations of Flt3-L necessary for these effects were similar to those of SLF, and low concentrations of Flt3-L and SLF which were inactive when used alone were active in these effects when used together. These results may be of practical value for short-term storage, treatment and/or transport of purified populations of cord blood stem and progenitor cells.

Antigens, CD34↗

PubMed1996

Stem cell factor, but not flt3 ligand, induces differentiation and activation of human mast cells.

We have examined the effect of human ligand for the flt3/flk2 tyrosine kinase receptor on the differentiation of human mast cells in suspension cultures. We also explored the effect of flt3 ligand (FL) on the human mast cell line HMC-1 and mRNA expression of flt3/flk2 on in vitro developed human mast cells and HMC-1. The growth of cord blood mononuclear cells in suspension cultures was increased when cells were cultured in the presence of FL compared with cells cultured in the presence of stem cell factor (SCF). When SCF and FL were combined, the total cell growth was increased further. Our data show that Fl by itself neither induced differentiation of mast cells nor acted in the SCF-dependent differentiation of human cord blood-derived mast cells (CBMC). Furthermore, no effects of FL were found on the proliferation of HMC-1 cells or the induction of early-immediate response genes in HMC-1 cells. In addition, neither HMC-1 cells nor CBMC expressed mRNA for flt3. As has been shown before, SCF and FL have little biological effect on their own but synergize well with a number of other hematopoietic growth factors. This study shows that a major difference between FL and SCF is that only SCF affects the differentiation and activation of mast cells.

Base Sequence↗

PubMed1996

Isolation and characterization of primitive hematopoietic progenitors of murine fetal liver.

We have isolated hematopoietic progenitors from mouse fetal liver using a sequential protocol of density gradient centrifugation, panning, and cell sorting. Isolated AA4.1+Ly-6A/E+CD43++ cells with density ranging from 1.0631 to 1.0770 g/cm3 were 480- to 600-fold enriched for multipotential progenitors relative to unfractionated cells and showed 40 to 60% colony-forming efficiency. We then examined the effects of various cytokines on the colony formation from enriched fetal liver cells. Steel factor (SF), interleukin-3 (IL-3), IL-4, IL-6, IL-11, and granulocyte colony-stimulating factor (G-CSF) as single agents supported formation of significant numbers of colonies, but flt3/flk-2 ligand (FL) and IL-12 did not. When the cytokines were combined, FL, SF, IL-3, and IL-4 each synergized individually with IL-6, IL-11, IL-12, or G-CSF to support formation of various types of colonies. Next we analyzed the growth factor requirements for proliferation and differentiation of lymphohematopoietic progenitors by using the two-step methylcellulose culture assay we established recently. None of the early-acting factors were effective as a single agent, but combinations of SF or FL with IL-6, IL-11, or G-CSF were effective in supporting B cell potential of the primary colonies. Of these, the combination of FL plus IL-11 appeared to be the most effective.

Animals↗

PubMed1996

Effects of Flt3 ligand and interleukin-7 on in vitro growth of acute lymphoblastic leukemia cells.

We investigated the effects of Flt3/Flk-2 ligand (FL) and interleukin-7 (IL-7) on DNA synthesis and proliferation of blast cells from patients with acute lymphoblastic leukemia (ALL). After 7 days of serum-free suspension culture of 19 samples, FL induced maximal DNA synthesis in two cases, whereas the combination of FL and IL-7 did so in another eight samples with a stimulation index (SI) >2. However, the number of viable cells after 7 days of liquid culture decreased in all but one sample. In this case of a pre-pre-B-ALL with a translocation t(4;11), FL induced dose-dependent proliferation (maximal 100 ng/mL) and cells stimulated with FL could be cultured for up to 4 weeks. A homogeneous population with 98% CD19-positive cells was detected before and after culture, and there was no evidence of nonleukemic cell proliferation as determined by immunophenotyping. The flt3 gene was transcribed in all seven cases studied by reverse-transcriptase polymerase chain reaction (RT-PCR). In the ALL cells responsive to FL, the expression of functional Flt3 receptors was confirmed by demonstrating FL-dependent tyrosine phosphorylation of Flt3. Furthermore, FL-dependent tyrosine phosphorylation of cellular proteins of estimated molecular weights of 70, 115, and 140 kD was detectable in these cells. These data demonstrate the functional heterogeneity of ALL samples and show that functional Flt3 receptors capable of mediating FL-dependent mitogenic signaling are expressed in a subset of ALL.

Adolescent↗

PubMed1996

Effects of flt3 ligand on acute myeloid and lymphocytic leukemic blast cells from children.

A ligand for the flt3 tyrosine kinase receptor (flt3R) has recently been cloned. Forty-three cases of childhood acute myeloid leukemia (AML) and 27 cases of childhood acute lymphocytic leukemia (ALL) were examined by flow cytometric analysis for cell-surface flt3R and proliferative response in vitro to flt3 ligand (flt3L). Flt3R was commonly expressed on the cell surface of leukemic cells from all AML subclasses and B-ALL, but we did not detect cell-surface flt3R on T-ALL. Flt3L alone induced the proliferation of the monocytic AML-M5 cells and some erythroleukemic AML-M6 cells. Some isolated instances of weak proliferative responses were also noted in other AML subclasses. Interleukin-4 (IL-4) alone inhibited the proliferation of a group of AML-M5 cells and, when combined with flt3L, suppressed the proliferative effect of flt3L. In general, B-ALL and T-ALL cells failed to respond to flt3L alone or in the presence of combinations of IL-2, IL-3, or IL-7.

Acute Disease↗

PubMed1996

Flt3-ligand production by human bone marrow stromal cells.

Bone marrow stromal cells are important sources of cytokines and growth factors which participate in regulation of proliferation and differentiation of hematopoietic stem and progenitor cells. Recently flt3/flk-2-ligand (flt3-L), a new growth factor which uses a membrane tyrosine kinase receptor, was cloned. It is expressed in transmembrane and soluble forms and stimulates/co-stimulates proliferation and colony formation of hematopoietic stem/progenitor cells. It has not been reported whether flt3-L is produced by cells of the hematopoietic bone marrow microenvironment. We demonstrate the expression of flt3-L in bone marrow fibroblasts (BMF) and in stromal cells of adherent layers of long-term bone marrow cultures by RT-PCR, Northern blot, immunocytochemistry and FACS analysis. The latter two methods localized flt3-L intracellularly and on cell membranes. Treatment with interleukin-1 alpha increased the expression of flt3-L in BMF. This demonstrates production and modulation of flt3-L in stromal cells of human bone marrow.

Base Sequence↗

PubMed1995

Plasma/serum levels of flt3 ligand are low in normal individuals and highly elevated in patients with Fanconi anemia and acquired aplastic anemia.

The flt3 ligand is a growth factor that stimulates the proliferation of hematopoietic progenitor and stem cells. We established a sensitive enzyme-linked immunosorbent assay (ELISA) to measure the concentration of flt3 ligand in plasma or serum from normal individuals, as well as in patients with hematopoietic disorders. Concentrations of flt3 ligand in plasma or serum from normal individuals were quite low: only 12% (7 of 60) of normal individuals had flt3 ligand levels above 100 pg/mL (the limit of detection). In contrast, 86% (19 of 22) of samples from patients with Fanconi anemia and 100% (eight of eight) of samples from patients with acquired aplastic anemia had plasma or serum levels above 100 pg/mL. Mean plasma or serum concentrations (calculated by assigning a value of 0 pg/mL to any sample reading below the level of detection) were as follows: normal volunteers, 14 pg/mL; patients with Fanconi anemia, 1,331 pg/mL; and patients with acquired aplastic anemia, 460 pg/mL. Concentrations of flt3 ligand in blood are, therefore, specifically elevated to a level that may be physiologically relevant in hematopoietic disorders with a suspected stem cell component. The elevated flt3 ligand concentrations in these individuals may be part of a compensatory hematopoietic response to boost the level of progenitor cells.

Anemia, Aplastic↗

PubMed1995

Effect of flt3 ligand on the ex vivo expansion of human CD34+ hematopoietic progenitor cells.

A ligand for the tyrosine kinase receptor flt3/flk-2, referred to here as flt3 ligand (flt3L), was recently cloned. The effect of flt3L on purified human CD34+ progenitor cells was examined. flt3 receptor (flt3R) was detected on the surface of human bone marrow cells that were enriched for CD34 expression. The effects of flt3L and the c-kit ligand Steel factor (SLF) on hematopoietic progenitors were compared in clonal colony assays. Both factors synergized with Pixy321 (interleukin-3 [IL-3]-granulocyte-macrophage colony-stimulating factor fusion protein) to induce granulocytic-monocytic (GM) and high proliferative potential (HPP) colonies and synergized with Pixy321 + erythropoietin (EPO) to induce multipotent granulocytic-erythroid-monocytic-megakaryocytic colonies. Although SLF had a potent effect on colony formation of erythroid restricted progenitor cells (burst-forming unit-erythroid), no effect by flt3L was observed. The addition of flt3L to irradiated long-term marrow cultures seeded with CD34+ cells augmented both total and progenitor cell production. Ex vivo expansion studies with isolated CD34+ bone marrow cells from normal donors showed that flt3L alone supported maintenance of both GM and HPP progenitors for 3 to 4 weeks in vitro. The addition of flt3L to a growth factor combination of IL-1 alpha + IL-3 + IL-6 + EPO resulted in a synergistic effect on progenitor cell expansion comparable to that observed with the addition of SLF to IL-1 alpha + IL-3 + IL-6 + EPO. These data show a function for flt3L in the regulation of both primitive multipotent and lineage-committed hematopoietic progenitor cells.

Antigens, CD34↗

PubMed1995

Structural analysis of human and murine flt3 ligand genomic loci.

Both the murine and human genomic loci that encode flt3 ligand have been cloned. flt3 ligand is a hematopoietic growth factor that stimulates the proliferation of stem and progenitor cells. The portions of the murine and human flt3 ligand genomic loci encompassing the coding region of the protein are approximately 4.0 kb and 5.9 kb, respectively. The human genomic locus is larger as a result of the presence of repeated sequences within introns I, II, IV, V and VI. The transmembrane isoform of the murine and human flt3 ligand proteins are each encoded within seven exons (1-5 + 7 and 8). Analyses of flt3 ligand cDNA clones show that alternative splicing of a putative sixth exon results in the generation of a soluble form of the flt3 ligand protein. The sizes of each of the exons are well conserved between species. Murine and human flt3 genomic loci have a similar exon: intron structure compared to the genomic loci encoding Steel factor and colony stimulating factor 1. These proteins, which appear to be ancestrally related, are hematopoietic growth factors that stimulate cells via specific and structurally related tyrosine kinase receptors on the cell surface.

Alternative Splicing↗

PubMed1995

The FLT3 ligand potently and directly stimulates the growth and expansion of primitive murine bone marrow progenitor cells in vitro: synergistic interactions with interleukin (IL) 11, IL-12, and other hematopoietic growth factors.

The recently cloned murine flt3 ligand (FL) was studied for its ability to stimulate the growth of primitive (Lin-Sca-1+) and more committed (Lin-Sca-1-) murine bone marrow progenitor cells, alone and in combination with other hematopoietic growth factors (HGFs). Whereas FL was a weak proliferative stimulator alone, it potently synergized with a number of other HGFs, including all four colony-stimulating factor (CSF), interleukin (IL) 6, IL-11, IL-12, and stem cell factor (SCF), to promote the colony formation of Lin-Sca-1+, but not Lin-Sca-1- or erythroid progenitor cells. The synergistic activity of FL was concentration dependent, with maximum stimulation occurring at 250 ng/ml, and was observed when cells were plated at a concentration of one cell per culture, suggesting that its effects are directly mediated. 2 wk of treatment with FL in combination with IL-3 or SCF resulted in the production of a high proportion of mature myeloid cells (granulocytes and macrophages), whereas the combination of FL with G-CSF, IL-11, or IL-12 resulted predominantly in the formation of cells with an immature blast cell appearance. Accordingly, FL in combination with G-CSF or IL-11 expanded the number of progenitors more than 40-fold after 2 wk incubation. Thus, FL emerges as a potent synergistic HGF, that in combination with numerous other HGFs, can directly stimulate the proliferation, myeloid differentiation, and expansion of primitive hematopoietic progenitor cells.

Animals↗