Murine Bone Marrow Progenitor Research Articles

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.

Production of Genetically Stable High-Titer Retroviral Vectors That Carry a Human γ-Globin Gene Under the Control of the α-Globin Locus Control Region

The ability to generate stable high-titer vectors that give rise to high levels of expression of transduced globin genes in erythroid cells is a prerequisite for effective retroviral-mediated globin gene therapy. The human beta-globin gene with its immediate flanking sequences does not contain all the regulatory elements necessary for regulated high-level and position-independent expression in erythroid cells. The regulatory element known as the beta-globin locus control region (BetaLCR) can provide a linked Beta-globin gene with these properties. However, addition of BetaLCR sequences to a retrovirus carrying a beta-globin gene increases its genetic instability. We have developed a new generation of retroviral vectors in which a human gamma-globin gene is placed under the control of the alphaLCR, the major regulatory element of the alpha-globin gene cluster. We demonstrate that these retroviruses are genetically stable in producer cell lines and can be produced at high titers that exceed 5 x 10(6) colony-forming units (CFU)/mL. In addition, we show that the transduced gamma-globin gene can be expressed in the adult erythroid environment of mouse erythroleukemia (MEL) cells at a level comparable to that of a single endogenous Betamaj-globin gene. These retroviruses can also transduce primary murine bone marrow progenitor cells as efficiently as retroviruses that carry the neomycin resistance (neor) gene. This new generation of globin retroviral vectors may prove useful for gene therapy of human beta-globin gene disorders such as sickle cell disease and beta-thalassemia.

The RAR-RXR as well as the RXR-RXR pathway is involved in signaling growth inhibition of human CD34+ erythroid progenitor cells

Previous studies have shown that retinoic acid (RA), similar to tumor necrosis factor-alpha (TNF-alpha), can act as a bifunctional regulator of the growth of bone marrow progenitors, in that it can stimulate granulocyte-macrophage colony-stimulating factor (GM-CSF)- or interleukin-3 (IL-3)-induced GM colony formation, but potently inhibit G-CSF-induced growth. The present study, using highly enriched human CD34+ as well as Lin- murine bone marrow progenitor cells, demonstrates a potent inhibitory effect of 9-cis-RA on burst-forming unit-erythroid (BFU-E) colony formation regardless of the cytokine stimulating growth. Specifically, 9-cis-RA potently inhibited the growth of BFU-E response to erythropoietin (Epo) (100%), stem cell factor (SCF) + Epo (92%), IL-3 + Epo (97%), IL-4 + Epo (88%), and IL-9 + Epo (100%). Erythroid colony growth was also inhibited when CD34+ progenitors were seeded at one cell per well, suggesting a direct action of RA. Using synthetic ligands to retinoic acid receptors (RARs) and retinoid X receptors (RXRs) that selectively bind and activate RAR-RXR or RXR-RXR dimers, respectively, we dissected the involvement of the two retinoid response pathways in the regulation of normal myeloid and erythroid progenitor cell growth. Transactivation studies showed that both the RAR (Ro 13-7410) and RXR (Ro 25-6603 and Ro 25-7386) ligands were highly selective at 100 nmol/L. At this concentration, Ro 13-7410 potently inhibited G-CSF-stimulated myeloid as well as SCF + Epo-induced erythroid colony growth. At the same concentration, Ro 25-6603 and Ro 25-7386 had little or no effect on G-CSF-induced colony formation, whereas they inhibited 75% and 53%, respectively, of SCF + Epo-stimulated BFU-E colony growth. Thus, the RAR-RXR response pathway can signal growth inhibition of normal bone marrow myeloid and erythroid progenitor cells. In addition, we demonstrate a unique involvement of the RXR-RXR pathway in mediating growth inhibition of erythroid but not myeloid progenitor cells.

The RAR-RXR as Well as the RXR-RXR Pathway Is Involved in Signaling Growth Inhibition of Human CD34+ Erythroid Progenitor Cells

Abstract Previous studies have shown that retinoic acid (RA), similar to tumor necrosis factor-alpha (TNF-alpha), can act as a bifunctional regulator of the growth of bone marrow progenitors, in that it can stimulate granulocyte-macrophage colony-stimulating factor (GM-CSF)- or interleukin-3 (IL-3)-induced GM colony formation, but potently inhibit G-CSF-induced growth. The present study, using highly enriched human CD34+ as well as Lin- murine bone marrow progenitor cells, demonstrates a potent inhibitory effect of 9-cis-RA on burst-forming unit-erythroid (BFU-E) colony formation regardless of the cytokine stimulating growth. Specifically, 9-cis-RA potently inhibited the growth of BFU-E response to erythropoietin (Epo) (100%), stem cell factor (SCF) + Epo (92%), IL- 3 + Epo (97%), IL-4 + Epo (88%), and IL-9 + Epo (100%). Erythroid colony growth was also inhibited when CD34+ progenitors were seeded at one cell per well, suggesting a direct action of RA. Using synthetic ligands to retinoic acid receptors (RARs) and retinoid X receptors (RXRs) that selectively bind and activate RAR-RXR or RXR-RXR dimers, respectively, we dissected the involvement of the two retinoid response pathways in the regulation of normal myeloid and erythroid progenitor cell growth. Transactivation studies showed that both the RAR (Ro 13– 7410) and RXR (Ro 25–6603 and Ro 25–7386) ligands were highly selective at 100 nmol/L. At this concentration, Ro 13–7410 potently inhibited G- CSF-stimulated myeloid as well as SCF + Epo-induced erythroid colony growth. At the same concentration, Ro 25–6603 and Ro 25–7386 had little or no effect on G-CSF-induced colony formation, whereas they inhibited 75% and 53%, respectively, of SCF + Epo-stimulated BFU-E colony growth. Thus, the RAR-RXR response pathway can signal growth inhibition of normal bone marrow myeloid and erythroid progenitor cells. In addition, we demonstrate a unique involvement of the RXR-RXR pathway in mediating growth inhibition of erythroid but not myeloid progenitor cells.

Transduction of MDR1 into human and mouse haemopoietic progenitor cells: use of rhodamine (Rh123) to determine transduction frequency and in vivo selection.

The MDR1 gene product P-glycoprotein (P-gp) extrudes several anticancer drugs including taxol and fluorescent dyes such as rhodamine (Rh123). Modulation of the level of P-gp expression has the potential of overcoming multidrug resistance. One possible approach is the retroviral transfer of the human MDR1 gene into murine and human bone marrow (BM) progenitor cells. The rationale for this approach is increased chemoprotection, which allows chemotherapy of a greater level of intensity to be delivered. In this study, flow cytometric measurement of Rh123 extrusion was used to test P-gp function in human and mouse haemopoietic progenitor cells, which had been transduced with a virus containing the human MDR1 transcription unit. Human CD34+ selected cells were analysed immediately following transduction. In two successive experiments MDR1 cDNA transduction resulted in a 7% and 11% increase of P-gp expressing Rh123 dull cells. To monitor transduction efficiency over time as well as the possibility of in vivo selection of drug-resistant BM cells in mice treated with increasing numbers of taxol cycles, the assay was also successfully applied to peripheral blood lymphocytes of mice transplanted with MDR1 transduced BM cells, demonstrating increased Rh123 efflux in transduced cells. Analysis of another fluorescence assay using fluorescein di-beta galactopyranoside as a substrate for beta-galactosidase in cells transduced with a MDR1: beta-gal activity. We conclude that the Rh123 efflux assay is a sensitive method to monitor P-gp function in MDR1 cDNA transduced cells, and may be used to enrich transduced cells via flow cytometric cell sorting for Rh123 dull cells.

Retroviral Transduction and Expression of the Human Alkyltransferase cDNA Provides Nitrosourea Resistance to Hematopoietic Cells

Myelosuppression is the dose-limiting toxicity for nitrosourea chemotherapy. This toxicity predominantly involves modification of the O6 position of guanine with an alkyl moiety. The enzyme responsible for repair of O6-alkylguanine adducts, O6-alkylguanine-DNA alkyltransferase (alkyltransferase), is expressed at low levels in bone marrow (BM) cells. High alkyltransferase expression prevents the cytotoxicity and carcinogenicity of nitrosoureas in several transgenic and in vitro gene transfer models. We used gene transfer using a novel myeloproliferative sarcoma virus (MPSV) based retrovirus (vM5MGMT) to express the human alkyltransferase cDNA (MGMT) in human and murine hematopoietic cells. Transduced K562 cells had very high levels of alkyltransferase expression and significantly increased resistance to 1,3-bis (2-chloroethyl) nitrosourea (BCNU) as compared with untransduced K562 cells. Primary murine BM progenitors showed a high transduction efficiency with vM5MGMT and have increased BCNU resistance in vitro. After BM transplantation with vM5MGMT-transduced BM cells and BCNU treatment of these mice, BM, spleen and thymus had a 10- to 40-fold increase in alkyltransferase expression that persisted for at least 23 weeks posttransplantation. Progenitor cells procured from mice expressing high levels of alkyltransferase also had increased resistance to BCNU. Thus, an MPSV-based retroviral vector transduces mouse and human hematopoietic cells at high efficiency and results in high levels of gene expression both in vitro and in vivo. Overexpression of the alkyltransferase protein may protect hematopoietic progenitors from nitrosourea-induced myelosuppression.

Inhibition of stem cell factor-induced proliferation of primitive murine hematopoietic progenitor cells signaled through the 75-kilodalton tumor necrosis factor receptor. Regulation of c-kit and p53 expression.

TNF-alpha is a pleiotropic cytokine with stimulatory as well as inhibitory effects on hematopoiesis. We have previously demonstrated that TNF-alpha directly inhibits CSF-induced proliferation of primitive murine lineage-negative bone marrow progenitors (Lin-) and stem cell antigen-1 hematopoietic progenitors through the 75-kDa TNF receptor (TNF-R2), whereas TNF-alpha-induced inhibition of more committed Lin- progenitors is mediated through the 55-kDa TNF-R (TNF-R1), indicating a differential role of the two TNF-Rs in hematopoiesis. Numerous studies have demonstrated the ability of stem cell factor (SCF), a key regulator of hematopoiesis signaling through c-kit, to synergize with other hematopoietic growth factors, but little is known about cytokines capable of inhibiting hematopoiesis induced by SCF. While TNF-alpha has been demonstrated to enhance SCF-induced proliferation of myeloid leukemia blasts, the present report demonstrates that TNF-alpha, by signaling through TNF-R2, inhibits SCF-induced proliferation of normal murine Lin- and stem cell antigen-1 hematopoietic progenitors. SCF-stimulated proliferation of the hematopoietic cell line FDC-P1 was also potently inhibited by TNF-alpha and was accompanied by down-regulation of c-kit cell surface expression as well as c-kit mRNA levels. Finally, treatment of the FDC-P1 cell line with TNF-alpha resulted in increased levels of the tumor suppressor p53 mRNA, suggesting another mechanism by which hematopoietic effects of TNF-alpha may be mediated.

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.

Retroviral Vector for Gene Therapy of X-Linked Severe Combined Immunodeficiency Syndrome

X-linked severe combined immunodeficiency syndrome (X-SCID) is a genetic disorder characterized by profound impairment of cell-mediated and humoral immunity. Affected children die of recurrent infections within 2 years of birth unless rescued by allogeneic transplantation from a suitable donor. Recently, the genetic defect responsible for X-linked SCID has been identified as a mutation in the gamma chain of the IL-2 receptor, a protein also shared by the IL-4 and IL-7 receptors and therefore now denoted the common gamma chain (gamma c). We report here the development of a high-titer amphotropic retroviral vector for transfer of gamma c. This vector was used to transfer a copy of the gamma c cDNA to murine 3T3 fibroblasts, CD34-enriched hematopoietic progenitor cells obtained from bone marrow and umbilical cord blood of normal donors, and to transplanted murine bone marrow progenitors. Murine 3T3 cells transduced by the retroviral vector were analyzed by Southern blot hybridization and Western transfer. Southern analysis confirmed the integration of unrearranged proviral DNA, and Western blot analysis demonstrated the expression of gamma c protein. CD34-enriched cells were infected with viral vectors bearing gamma c and grown in methylcellulose media. Individual colonies and pools of cells were analyzed 2 weeks later by polymerase chain reaction assay, which confirmed the proviral marking. The vector was also used to transfer a copy of the gamma c cDNA to murine bone marrow cells in a transplantation model. Infected marrow was transplanted into syngeneic Balb/c mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Hematopoietic colony stimulatory factor formation by murine mesangial cells: Gene expression and biological activity

This study examined the ability of mesangial cells to synthesize colony-stimulating factors (CSF), cytoregulatory peptides associated with the differentiation and proliferation of hematopoietic cells. Conditioned media obtained from SV-40 transformed murine mesangial cells stimulated the growth of murine bone marrow progenitor cells of the myeloid series. Differential analysis of these cells showed the presence of both macrophages and granulocytes. Cellular identification of bone marrow colonies stimulated in response to mesangial cell conditioned media was examined by flow cytometric analysis and revealed the presence of F4 80 antigen positive macrophages (67%) and Gran-1 antigen positive granulocytes (21%). Neutralizing antibodies to macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) but not antibody to interleukin-3 (IL-3), or stem cell factor (SCF) significantly inhibited the growth of the progenitor cells induced by mesangial cell conditioned media. Utilizing Northern blot analysis, murine mesangial cells expressed mRNA transcripts for M-CSF, GM-CSF, and granulocyte colony-stimulating factor (G-CSF). Further studies were performed to determine optimal incubation conditions for mesangial cell CSF gene expression. These studies revealed that both GM-CSF and G-CSF mRNA were maximally expressed at early time points (4 and 8 h of incubation), while M-CSF mRNA expression remained unchanged during the incubation of mesangial cells from 4–48 h. Incubation of mesangial cells with various concentrations of fetal bovine serum (FBS, 0.5–15%) markedly increased the mRNA expression of M-CSF, GM-CSF and G-CSF in a dose-dependent manner. These studies indicated that transformed murine mesangial cells are able to synthesize and secrete biologically active CSF that are associated with the migration and proliferation of circulating mononuclear cells in the glomerulus. Furthermore, observations regarding the role of duration of incubation and the media concentration of FBS on mesangial cell CSF mRNA expression may provide useful data to understand the optimal conditions for studies that examine the gene expression of basal or inducible CSF in mesangial cells.

Stem Cell Factor and Interleukin-7 Synergize to Enhance Early Myelopoiesis In Vitro

Interleukin-7 (IL-7) has been shown to be a critical factor in murine lymphoid development. It stimulates pre-B cells to divide in the absence of stroma cells and it is an important growth regulator of immature and mature T cells. IL-7 has been shown to synergize with stem cell factor (SCF) to provide a potent growth stimulus for pre-B cells. However, the combined effects of IL-7 and SCF on murine primitive hematopoietic cells in vitro have not been established. In the present study, the effects of recombinant rat (rr) SCF and recombinant human (rh) IL-7 on primitive murine bone marrow progenitors (Lin-Sca1+) were investigated in single-cell cloning experiments. rhIL-7 alone had no proliferative effect on Lin-Sca1+ cells, but in a dose-dependent manner directly enhanced rrSCF-induced colony formation, with an average increase in colony numbers of 2.7-fold. Interestingly, the cells formed in response to SCF and IL-7 were predominantly mature granulocytes. Thus, SCF and IL-7 synergize to stimulate early myelopoiesis in vitro.

Stem cell factor and interleukin-7 synergize to enhance early myelopoiesis in vitro

Interleukin-7 (IL-7) has been shown to be a critical factor in murine lymphoid development. It stimulates pre-B cells to divide in the absence of stroma cells and it is an important growth regulator of immature and mature T cells. IL-7 has been shown to synergize with stem cell factor (SCF) to provide a potent growth stimulus for pre-B cells. However, the combined effects of IL-7 and SCF on murine primitive hematopoietic cells in vitro have not been established. In the present study, the effects of recombinant rat (rr) SCF and recombinant human (rh) IL-7 on primitive murine bone marrow progenitors (Lin-Sca1+) were investigated in single-cell cloning experiments. rhIL-7 alone had no proliferative effect on Lin-Sca1+ cells, but in a dose-dependent manner directly enhanced rrSCF-induced colony formation, with an average increase in colony numbers of 2.7-fold. Interestingly, the cells formed in response to SCF and IL-7 were predominantly mature granulocytes. Thus, SCF and IL-7 synergize to stimulate early myelopoiesis in vitro.

Nitrobenzyl phosphorodiamidates as potential hypoxia-selective alkylating agents.

A series of novel nitrobenzyltetrakis(chloroethyl)phosphorodiamidates has been prepared, and its cytotoxicity has been evaluated against HT-29 cells under aerobic and hypoxic conditions and against murine bone marrow progenitor cells under aerobic conditions. All compounds were selectively toxic to HT-29 cells under hypoxic conditions, and the selectivity ratios varied from 1.6 to > 90. Analogs lacking either the nitro group or the tetrakis(chloroethyl) moiety were not cytotoxic, confirming that the presence of both nitro and incipient alkylating groups are essential for activity. Surprisingly, some analogs were far more toxic to bone marrow progenitors than to HT-29 cells under aerobic conditions, suggesting that other activation mechanisms must exist in these hematopoietic cells. Cytotoxicity increased with increasing depth in the HT-29 spheroid model, consistent with the preferential hypoxic toxicity of these compounds. Alkaline elution experiments showed a greater number of DNA interstrand cross-links under hypoxic compared to aerobic conditions. The extent of cross-linking in hypoxic cells was essentially identical to that produced by an equitoxic dose of melphalan, suggesting that the cytotoxicity of these compounds results from phosphorodiamidate release and alkylation of DNA.

Progress in the ex vivo expansion of hematopoietic progenitors.

In this review we describe how studies on the cytokine-stimulated growth of murine bone marrow (BM) progenitors have lead to the observations that large increases in progenitor numbers can be achieved in short-term cytokine-stimulated liquid cultures. Transplantation of these ex vivo expanded murine BM cells was shown to decrease the number of BM cells required to confer radioprotection and to increase the recovery rate of both myeloid and erythroid peripheral blood cells. The ex vivo expansion of murine BM cells does not however, markedly diminish stem cells capable of long-term hematopoietic reconstitution. Investigations on the expansion of human BM, peripheral blood, umbilical cord blood and fetal hematopoietic progenitors have demonstrated that clinically useful increases in progenitor numbers from these tissues are possible. Thus, ex vivo progenitor expansion may soon be of use in transplantation protocols to accelerate hematopoietic reconstitution and in gene therapy protocols if hematopoietic stem cells can be maintained during ex vivo culture.

Novel role of interleukin 7 in myelopoiesis: stimulation of primitive murine hematopoietic progenitor cells.

Interleukin 7 (IL-7) has been demonstrated to be an important regulator of the growth of B and T cell precursors as well as mature T cells, whereas IL-7 has been reported to have no direct myeloproliferative effects. Here we show that IL-7 potently and directly enhances colony stimulating factor-induced myeloid colony formation from Lin-Sca-1+ murine bone marrow progenitor cells, increasing the cloning frequency up to ninefold and cell numbers up to 50-fold, without affecting their ability to differentiate along the myeloid lineages In contrast, IL-7 has no effect on proliferation of committed Lin- myeloid progenitors. Thus, in addition to its established lymphopoietic potential, this study implicates a novel role of IL-7 in early myelopoiesis.

Induction of macrophagic and granulocytic differentiation of murine bone marrow progenitor cells by 1,25-dihydroxyvitamin D3.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) was recently shown to promote maturation of 5-fluorouracil (5FU)-treated bone marrow cells by up-regulating macrophage-colony stimulating factor (M-CSF) receptors in the presence of interleukin 1 alpha (IL-1 alpha). In order to reveal how 1,25(OH)2D3 interacts with colony-stimulating factors and regulates the differentiation of bone marrow progenitor cell populations, in the present study, natural bone marrow cells were isolated from untreated mice and used in alpha-minimum essential medium supplemented with 20% heat-inactivated horse serum without added appropriate cytokines. Under the conditions, cells spontaneously differentiated gradually with days of culture, as assessed by expression of macrophage differentiation antigens such as Mac-1 (CD11b) and F4/80. Both M-CSF and granulocyte macrophage-colony stimulating factor (GM-CSF) induced only Mac-1 antigen expression. Simultaneous treatment with M-CSF and 1,25(OH)2D3 enhanced the M-CSF's effect on expression of both antigens, although 1,25(OH)2D3 per se has no effect on the expression for up to 11 days. In addition, successive treatment with 1,25(OH)2D3 and M-CSF or GM-CSF dramatically enhanced expression of both antigens or Mac-1 antigen, respectively. Similarly, both simultaneous and successive treatment with 1,25(OH)2D3 and M-CSF significantly enhanced phagocytic activity and H2O2 production, whereas successive treatment with 1,25(OH)2D3 and GM-CSF significantly enhanced only phagocytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Bone marrow transplantation with interleukin-1 plus kit-ligand ex vivo expanded bone marrow accelerates hematopoietic reconstitution in mice without the loss of stem cell lineage and proliferative potential

Cytokine combinations were tested for their ability to expand murine bone marrow (BM) progenitors in short-term suspension cultures (delta- cultures) with the aim of providing an enriched source of progenitors for BM transplantation (BMT). In a comparison of the efficacy of the combinations interleukin-1 (IL-1) + IL-3, IL-1 + kit-ligand (KL), and IL-1 + IL-6 + KL, BMT with IL-1 + KL expanded progenitors was found to be most effective in accelerating the recovery of peripheral blood leukocytes, platelets, and erythrocytes in lethally irradiated mice. The ex vivo expansion of BM in IL-1 + KL-stimulated delta-cultures also greatly reduced the number of transplanted cells needed to provide radioprotection. All mice survived at least 30 days when receiving 5 x 10(3) delta-cultured d1 5-fluorouracil (5-FU) BM cells (BM cells harvested 1 day after 5-FU administration), whereas complete survival of mice receiving fresh d1 5-FU BM required BMT with a 200-fold greater number of cells. BMT with expanded BM lead to predominantly donor- derived hematopoietic reconstitution for 280 days postprimary BMT and another 71 days after secondary BMT. The expansion of BM did not adversely effect the proliferative capacity and lineage potential of the stem cell compartment.

Bone Marrow Transplantation With Interleukin-1 Plus Kit-Ligand Ex Vivo Expanded Bone Marrow Accelerates Hematopoietic Reconstitution in Mice Without the Loss of Stem Cell Lineage and Proliferative Potential

AbstractCytokine combinations were tested for their ability to expand murine bone marrow (BM) progenitors in short-term suspension cultures (∆-cultures) with the aim of providing an enriched source of progenitors for BM transplantation (BMT). In a comparison of the efficacy of the combinations interleukin-1 (IL-1) + IL-3, IL-1 + Kit-ligand (KL), and IL-1 + IL-6 + KL, BMT with IL-1 + KL expanded progenitors was found to be most effective in accelerating the recovery of peripheral blood leukocytes, platelets, and erythrocytes in lethally irradiated mice. The ex vivo expansion of BM in IL-1 + KL-stimulated ∆-cultures also greatly reduced the number of transplanted cells needed to provide radioprotection. All mice survived at least 30 days when receiving 5 × 103 ∆-cultured d1 5-fluorouracil (5-FU) BM cells (BM cells harvested 1 day after 5-FU administration), whereas complete survival of mice receiving fresh d1 5-FU BM required BMT with a 200-fold greater number of cells. BMT with expanded BM lead to predominantly donor-derived hematopoietic reconstitution for 280 days postprimary BMT and another 71 days after secondary BMT. The expansion of BM did not adversely effect the proliferative capacity and lineage potential of the stem cell compartment.

Diethyldithiocarbamate Induction of Cytokine Release in Human Long-Term Bone Marrow Cultures

Abstract Diethyldithiocarbamate (DDTC) is a biochemical modulating agent that protects murine bone marrow progenitor cells from the cytotoxicity of a variety of cancer chemotherapeutic agents. However, the mechanism of this protection is not well understood. Long-term human bone marrow cultures (LTBMC) were established and at day 17 treated with 30 mumol/L DDTC for 1 hour, after which DDTC was removed and replaced with complete medium. Conditioned medium was then collected 6, 12, 24, and 48 hours later and analyzed for the presence of cytokines. A time- dependent increase in granulocyte-macrophage colony-stimulating factor (GM-CSF) (12-fold), granulocyte-CSF (G-CSF) (66-fold), interleukin (IL)- 6, (three-fold), IL-1 beta (161-fold), and tumor necrosis factor (TNF)- alpha (25-fold) was observed. The maximum increase for the factors other than TNF-alpha was at 24 to 48 hours posttreatment. However, TNF- alpha peaked as early as 6 hours post-DDTC. When conditioned medium from these cultures was tested in a granulocyte-macrophage progenitor cell (GM-CFC) assay, an increase in colony formation was observed that correlated with the increased levels of cytokines in the medium. The specificity of this effect was confirmed by the fact that the closely related congener bis(hydroxyethyl)dithiocarbamate was devoid of colony- stimulating activity. The addition of antibodies for TNF-alpha and/or IL-1 alpha following DDTC treatment did not inhibit the release of GM- CSF, G-CSF, or IL-6 from the LTBMC. These results suggest that DDTC accelerates bone marrow recovery following myelotoxic drug treatment via increased production of cytokines that are known to be essential for hematopoiesis.

Diethyldithiocarbamate induction of cytokine release in human long-term bone marrow cultures

Diethyldithiocarbamate (DDTC) is a biochemical modulating agent that protects murine bone marrow progenitor cells from the cytotoxicity of a variety of cancer chemotherapeutic agents. However, the mechanism of this protection is not well understood. Long-term human bone marrow cultures (LTBMC) were established and at day 17 treated with 30 mumol/L DDTC for 1 hour, after which DDTC was removed and replaced with complete medium. Conditioned medium was then collected 6, 12, 24, and 48 hours later and analyzed for the presence of cytokines. A time-dependent increase in granulocyte-macrophage colony-stimulating factor (GM-CSF) (12-fold), granulocyte-CSF (G-CSF) (66-fold), interleukin (IL)-6, (three-fold), IL-1 beta (161-fold), and tumor necrosis factor (TNF)-alpha (25-fold) was observed. The maximum increase for the factors other than TNF-alpha was at 24 to 48 hours posttreatment. However, TNF-alpha peaked as early as 6 hours post-DDTC. When conditioned medium from these cultures was tested in a granulocyte-macrophage progenitor cell (GM-CFC) assay, an increase in colony formation was observed that correlated with the increased levels of cytokines in the medium. The specificity of this effect was confirmed by the fact that the closely related congener bis(hydroxyethyl)dithiocarbamate was devoid of colony-stimulating activity. The addition of antibodies for TNF-alpha and/or IL-1 alpha following DDTC treatment did not inhibit the release of GM-CSF, G-CSF, or IL-6 from the LTBMC. These results suggest that DDTC accelerates bone marrow recovery following myelotoxic drug treatment via increased production of cytokines that are known to be essential for hematopoiesis.