Dominant myelopoietic effector functions mediated by chemokine receptor CCR1.

Macrophage inflammatory protein (MIP)-1alpha, a CC chemokine, enhances proliferation of mature subsets of myeloid progenitor cells (MPCs), suppresses proliferation of immature MPCs, and mobilizes mature and immature MPCs to the blood. MIP-1alpha binds at least three chemokine receptors. To determine if CCR1 was dominantly mediating the above activities of MIP-1alpha, CCR1-deficient (-/-) mice, produced by targeted gene disruption, were used. MIP-1alpha enhanced colony formation of marrow granulocyte/macrophage colony-forming units (CFU-GM), responsive to stimulation by granulocyte/macrophage colony-stimulating factor (GM-CSF), and CFU-M, responsive to stimulation by M-CSF, from littermate control CCR1(+/+) but not CCR1(-/-) mice. Moreover, MIP-1alpha did not mobilize MPCs to the blood or synergize with G-CSF in this effect in CCR1(-/-) mice. However, CCR1(-/-) mice were increased in sensitivity to MPC mobilizing effects of G-CSF. Multi-growth factor-stimulated MPCs in CCR1(-/-) and CCR1(+/+) marrow were equally sensitive to inhibition by MIP-1alpha. These results implicate CCR1 as a dominant receptor for MIP-1alpha enhancement of proliferation of lineage-committed MPCs and for mobilization of MPCs to the blood. CCR1 is not a dominant receptor for MIP-1alpha suppression of MPC proliferation, but it does negatively impact G-CSF-induced MPC mobilization.

Essential role of signal transducer and activator of transcription (Stat)5a but not Stat5b for Flt3-dependent signaling.

The receptor tyrosine kinase Flt3 plays an important role in proliferation and survival of hematopoietic stem and progenitor cells. Although some post-receptor signaling events of Flt3 have been characterized, the involvement of the Janus kinase/signal transducer and activator of transcription (Jak/Stat) pathway in Flt3 signaling has not been thoroughly evaluated. To this aim, we examined whether Flt3 activates the Jak/Stat pathway in Baf3/Flt3 cells, a line stably expressing human Flt3 receptor. Stat5a, but not Stats 1-4, 5b, or 6, was potently activated by Flt3 ligand (FL) stimulation. Interestingly, FL did not activate any Jaks. Activation of Stat5a required the kinase activity of Flt3. A selective role for Stat5a in the proliferative response of primary hematopoietic progenitor cells to FL was documented, as FL did not act on progenitors from marrows of Stat5a(-/-) mice, but did stimulate/costimulate proliferation of these cells from Stat5a(+/+), Stat5b(-/-), and Stat5b(+/+) mice. Thus, Stat5a is essential for at least certain effects of FL. Moreover, our data confirm that Stat5a and Stat5b are not redundant, but rather are at least partially distinctive in their function.

Abnormal chemokine-induced responses of immature and mature hematopoietic cells from motheaten mice implicate the protein tyrosine phosphatase SHP-1 in chemokine responses.

Chemokines regulate a number of biological processes, including trafficking of diverse leukocytes and proliferation of myeloid progenitor cells. SHP-1 (Src homology 2 domain tyrosine phosphatase 1), a phosphotyrosine phosphatase, is considered an important regulator of signaling for a number of cytokine receptors. Since specific tyrosine phosphorylation of proteins is important for biological activities induced by chemokines, we examined the role of SHP-1 in functions of chemokines using viable motheaten (me(v)/me(v)) mice that were deficient in SHP-1. Chemotactic responses to stromal call-derived factor 1 (SDF-1), a CXC chemokine, were enhanced with bone marrow myeloid progenitor cells as well as macrophages, T cells, and B cells from me(v)/me(v) versus wild-type (+/+) mice. SDF-1-dependent actin polymerization and activation of mitogen-activated protein kinases were also greater in me(v)/me(v) versus +/+ cells. In contrast, immature subsets of me(v)/me(v) bone marrow myeloid progenitors were resistant to effects of a number of chemokines that suppressed proliferation of +/+ progenitors. These altered chemokine responses did not appear to be due to enhanced expression of CXCR4 or lack of chemokine receptor expression. However, expression of some chemokine receptors (CCR1, CCR2, CCR3, and CXCR2) was significantly enhanced in me(v)/me(v) T cells. Our results implicate SHP-1 involvement in a number of different chemokine-induced biological activities.

CD45 cell surface antigens are linked to stimulation of early human myeloid progenitor cells by interleukin 3 (IL-3), granulocyte/macrophage colony-stimulating factor (GM-CSF), a GM-CSF/IL-3 fusion protein, and mast cell growth factor (a c-kit ligand).

CD45 antigens are protein tyrosine phosphatases. A possible link was evaluated between expression of CD45 antigens on human myeloid progenitor cells (MPC) (colony-forming unit-granulocyte/macrophage [CFU-GM], burst-forming unit-erythroid [BFU-E], and colony-forming unit-granulocyte/erythroid/macrophage/megakaryocyte [CFU-GEMM]) and regulation of MPC by colony-stimulating factors (CSF) (interleukin 3 [IL-3], GM-CSF, G-CSF, M-CSF, and erythropoietin [Epo]), a GM-CSF/IL-3 fusion protein, and mast cell growth factor (MGF; a c-kit ligand). Treatment of cells with antisense oligodeoxynucleotides (oligos) to exons 1 and 2, but not 4, 5, or 6, of the CD45 gene, or with monoclonal anti-CD45, significantly decreased CFU-GM colony formation stimulated with GM-CSF, IL-3, fusion protein, and GM-CSF + MGF, but not with G-CSF or M-CSF. It also decreased GM-CSF, IL-3, fusion protein, and MGF-enhanced Epo-dependent BFU-E and CFU-GEMM colony formation, but had little or no effect on BFU-E or CFU-GEMM colony formation stimulated by Epo alone. Similar results were obtained with unseparated or purified (greater than or equal to one of two cells being a MPC) bone marrow cells. Sorted populations of CD343+ HLA-DR+ marrow cells composed of 90% MPC were used to demonstrate capping of CD45 after crosslinking protocols. Also, a decreased percent of CD45+ cells and CD45 antigen density was noted after treatment of column-separated CD34+ cells with antisense oligos to exon 1 of the CD45 gene. These results demonstrate that CD45 cell surface antigens are linked to stimulation of early human MPC by IL-3, GM-CSF, a GM-CSF/IL-3 fusion protein, and MGF.

Increased osteoclast development after estrogen loss: mediation by interleukin-6.

Osteoclasts, the cells that resorb bone, develop from hematopoietic precursors of the bone marrow under the control of factors produced in their microenvironment. The cytokine interleukin-6 can promote hematopoiesis and osteoclastogenesis. Interleukin-6 production by bone and marrow stromal cells is suppressed by 17 beta-estradiol in vitro. In mice, estrogen loss (ovariectomy) increased the number of colony-forming units for granulocytes and macrophages, enhanced osteoclast development in ex vivo cultures of marrow, and increased the number of osteoclasts in trabecular bone. These changes were prevented by 17 beta-estradiol or an antibody to interleukin-6. Thus, estrogen loss results in an interleukin-6-mediated stimulation of osteoclastogenesis, which suggests a mechanism for the increased bone resorption in postmenopausal osteoporosis.

Altered responsiveness to chemokines due to targeted disruption of SHIP.

SHIP has been implicated in negative signaling in a number of hematopoietic cell types and is postulated to downregulate phosphatidylinositol-3-kinase- (PI-3K-) initiated events in diverse receptor signaling pathways. Because PI-3K is implicated in chemokine signaling, we investigated whether SHIP plays any role in cellular responses to chemokines. We found that a number of immature and mature hematopoietic cells from SHIP-deficient mice manifested enhanced directional migration (chemotaxis) in response to the chemokines stromal cell-derived factor-1 (SDF-1) and B-lymphocyte chemoattractant (BLC). SHIP(-/-) cells were also more active in calcium influx and actin polymerization in response to SDF-1. However, colony formation by SHIP-deficient hematopoietic progenitor cell (HPCs) was not inhibited by 13 myelosuppressive chemokines that normally inhibit proliferation of HPCs. These altered biologic activities of chemokines on SHIP-deficient cells are not caused by simple modulation of chemokine receptor expression in SHIP-deficient mice, implicating SHIP in the modulation of chemokine-induced signaling and downstream effects.

Effects of recombinant human tumor necrosis factor alpha, recombinant human gamma-interferon, and prostaglandin E on colony formation of human hematopoietic progenitor cells stimulated by natural human pluripotent colony-stimulating factor, pluripoietin alpha, and recombinant erythropoietin in serum-free cultures.

The influences of pure human pluripotent colony-stimulating factor, highly purified pluripoietin alpha, pure recombinant human tumor necrosis factor alpha, pure recombinant human gamma-interferon, and natural prostaglandin E1 (PGE1) were evaluated on colony formation of multipotential and erythroid progenitor cells in the presence of recombinant erythropoietin and hemin and on colony formation of granulocyte-macrophage progenitors in normal human marrow cultured in the presence or absence of serum. Serum was replaced by bovine serum albumin, iron-saturated transferrin, cholesterol, and calcium chloride. Increasing concentrations of pluripotent colony-stimulating factor and pluripoietin alpha stimulated increasing numbers of colonies from nonadherent low-density T-lymphocyte-depleted cells in the absence and presence of serum. Growth was usually greater in the presence of serum and on a unit basis pluripoietin alpha was more active than pluripotent colony-stimulating factor. Recombinant human tumor necrosis factor alpha and recombinant human gamma-interferon suppressed colony formation colony forming unit-granulocyte-macrophage, burst forming unit-erythroid, and colony forming unit-granulocyte-erythroid-macrophage-megakaryocyte; PGE1 suppressed colony formation by colony-forming unit-granulocyte-macrophage, stimulated colony formation by burst forming unit-erythroid, and had no effects on colony formation by colony forming unit-granulocyte-erythroid-macrophage-megakaryocyte in both serum-containing and serum-free medium. The PGE1 enhancing effects on erythroid colony formation required T-lymphocytes. Thus, results are similar using serum-containing and serum-free cultures of human bone marrow cells and serum-free defined culture medium can be used to study the mechanism of action of purified natural and recombinant growth and suppressor molecules in vitro.

Protective influence of lactoferrin on mice infected with the polycythemia-inducing strain of Friend virus complex.

Purified iron-saturated human lactoferrin (LF) was assessed in vivo for effects on the survival rates of C57BL X DBA/2 f1 (hereafter called BD2F1) (Fv-2sr) mice and titers of spleen focus-forming viruses (SFFV) in BD2F1 and DBA/2 (Fv-2ss) mice inoculated with the polycythemia-inducing strain of the Friend virus complex (FVC-P). LF prolonged the survival rates and decreased the titers of SFFV in mice given FVC-P. Titers of SFFV, assayed 14 days after administration of FVC-P, were measured by the spleen focus-forming unit assay in secondary mouse recipients. Decreases in titers of SFFV were apparent when LF was given in vivo as a single bolus dose of 200 micrograms within 2 h of the Friend virus complex (FVC), or as a total dosage of 200 micrograms given on days 1, 2, 4, 7, 9, and 11 after FVC-P, and to a lesser degree when LF was given as a total dosage of 200 micrograms on days 3, 4, 7, 9, and 11 after FVC-P. No decreases in titers of SFFV were detected when LF was given up to 3 days before or more than 3 days after FVC-P. LF did not appear to be directly inactivating the viruses as it did not inactivate the SFFV or the Friend murine leukemia helper virus in vitro. The results suggest that the protective effect of LF in vivo is probably due to an action on cells responding to the FVC or to an action on cells which influence the cells responding to the FVC or which influence the virus. It has been shown elsewhere that LF decreases the percentage of marrow and spleen hematopoietic progenitor cells that are in DNA synthesis in vivo and this may be the means by which the protective effect of LF is mediated in mice given the FVC.

Effect in vivo of multiple injections of purified murine and recombinant human macrophage colony-stimulating factor to mice.

Hematopoietic efficacy in vivo of multiple injections of purified murine L-cell and recombinant human macrophage colony-stimulating factors (M-CSF; specific activity, greater than 2 x 10(7) units/mg) was assessed in mice. Injections i.v. of sterile saline or 20,000 units of M-CSF were administered once (at 0 h), twice (at 0 and 12 h), or three times (at 0, 12, and 24 h) to C57BL/6 x DBA/2 F1 mice. Numbers and cycling rates of marrow and spleen granulocyte-macrophage, erythroid, and multipotential progenitor cells were assessed 32-36 h after the first injection. Marrow, spleen, and peripheral blood cellularity was assessed at intervals of up to 105 h. Progenitor cell cycling rates were significantly increased after one and two injections of M-CSF but were reduced to a slow or noncycling state after three injections. For marrow cells, the third injection resulted in a significant suppression of hematopoietic progenitor cell cycling compared to the control group. No significant changes were noted for number of progenitors per femur or spleen, for marrow, spleen, or peripheral blood cellularity, or for differential cell counts in these organs after any of the M-CSF treatment schedules. Suppression of progenitor cell proliferation noted after three injections of M-CSF may at least partially explain why repeated injections of 20,000 units of M-CSF fails to increase bone marrow, spleen, or blood cellularity even though one injection of M-CSF increases cycling rates of the hematopoietic progenitors.

Activation of OCT4 enhances ex vivo expansion of human cord blood hematopoietic stem and progenitor cells by regulating HOXB4 expression.

Although hematopoietic stem cells (HSC) are the best characterized and the most clinically used adult stem cells, efforts are still needed to understand how to best ex vivo expand these cells. Here we present our unexpected finding that OCT4 is involved in the enhancement of cytokine-induced expansion capabilities of human cord blood (CB) HSC. Activation of OCT4 by Oct4-activating compound 1 (OAC1) in CB CD34(+) cells enhanced ex vivo expansion of HSC, as determined by a rigorously defined set of markers for human HSC, and in vivo short-term and long-term repopulating ability in NSG mice. Limiting dilution analysis revealed that OAC1 treatment resulted in 3.5-fold increase in the number of SCID repopulating cells (SRCs) compared with that in day 0 uncultured CD34(+) cells and 6.3-fold increase compared with that in cells treated with control vehicle. Hematopoietic progenitor cells, as assessed by in vitro colony formation, were also enhanced. Furthermore, we showed that OAC1 treatment led to OCT4-mediated upregulation of HOXB4. Consistently, siRNA-mediated knockdown of HOXB4 expression suppressed effects of OAC1 on ex vivo expansion of HSC. Our study has identified the OCT4-HOXB4 axis in ex vivo expansion of human CB HSC.

Small-molecule inhibitors targeting the DNA-binding domain of STAT3 suppress tumor growth, metastasis and STAT3 target gene expression in vivo.

Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in malignant tumors and has important roles in multiple aspects of cancer aggressiveness. Thus targeting STAT3 promises to be an attractive strategy for treatment of advanced metastatic tumors. Although many STAT3 inhibitors targeting the SH2 domain have been reported, few have moved into clinical trials. Targeting the DNA-binding domain (DBD) of STAT3, however, has been avoided due to its 'undruggable' nature and potentially limited selectivity. In a previous study, we reported an improved in silico approach targeting the DBD of STAT3 that resulted in a small-molecule STAT3 inhibitor (inS3-54). Further studies, however, showed that inS3-54 has off-target effect although it is selective to STAT3 over STAT1. In this study, we describe an extensive structure and activity-guided hit optimization and mechanistic characterization effort, which led to identification of an improved lead compound (inS3-54A18) with increased specificity and pharmacological properties. InS3-54A18 not only binds directly to the DBD and inhibits the DNA-binding activity of STAT3 both in vitro and in situ but also effectively inhibits the constitutive and interleukin-6-stimulated expression of STAT3 downstream target genes. InS3-54A18 is completely soluble in an oral formulation and effectively inhibits lung xenograft tumor growth and metastasis with little adverse effect on animals. Thus inS3-54A18 may serve as a potential candidate for further development as anticancer therapeutics targeting the DBD of human STAT3 and DBD of transcription factors may not be 'undruggable' as previously thought.

Modulation of Friend virus infectivity in vivo by administration of purified preparations of human lactoferrin and recombinant murine interleukin-3 to mice.

Purified iron-saturated human milk lactoferrin (LF) and purified recombinant murine interleukin-3 (IL-3) were assessed in vivo for their effects on replication of spleen focus forming viruses (SFFV) in spleens of DBA/2 mice injected with the polycythemia-inducing strain of the Friend virus complex. LF and IL-3, inoculated 2 hr prior to the administration of the polycythemia-inducing strain of the Friend virus complex, respectively decreased and increased the replication of SFFV in mice as assessed by the spleen focus forming unit assay in primary and secondary DBA/2 mice. Since virus infectivity is associated with the DNA synthetic phase of the cell cycle and it has been shown elsewhere that LF decreases and IL-3 increases the percent of hematopoietic progenitor cells in S-phase in vivo, the results suggest that the opposing actions of LF and IL-3 on replication of SFFV may reflect the actions of these molecules on cycling of the target cells for SFFV.

Influence of T-lymphocytes and lactoferrin on the survival-promoting effects of IL-1 and IL-6 on human bone marrow granulocyte-macrophage and erythroid progenitor cells.

Purified recombinant human (rhu) interleukin (IL)-1 alpha, rhuIL-6, iron saturated lactoferrin (LF), and T-lymphocytes were assessed for their effects on the survival of granulocyte-macrophage (granulocyte-macrophage colony-forming units, CFU-GM) and erythroid (erythroid burst-forming units, BFU-E) progenitor cells from human low-density (LD) and nonadherent LD T-lymphocyte-depleted (NALT-) bone marrow (BM) cells. Colony-stimulating factor (CSF) deprivation studies showed that 10 ng/ml IL-1 alpha could promote the survival of CFU-GM and BFU-E from NALT- BM cells. Concentrations of 1 ng/ml IL-1 alpha and 1-100 ng/ml IL-6 alone could not promote progenitor cell survival from NALT- BM cells; however, concentrations of 1 ng/ml each of IL-1 alpha and IL-6 could synergize to promote the survival of CFU-GM but not of BFU-E. The combination of these low concentrations of IL-1 alpha and IL-6 could, however, support the survival of BFU-E in the presence of purified T-lymphocytes. LF could decrease the survival of CFU-GM and BFU-E from LD but not from NALT- BM cells, apparently due to the inhibition of IL-1 release from monocytes in this cell population. The suppressive effect of LF on the survival of those progenitor cells was abolished by concentrations of 10 ng/ml IL-1 alpha or 1 ng/ml each of IL-1 alpha and IL-6. These results demonstrate that the survival of human marrow CFU-GM and BFU-E can be influenced by IL-1, IL-6, LF, and T-lymphocytes.

Regulation of myelopoiesis by murine fibroblastic and adipogenic cell lines.

The regulation of hematopoiesis has been suggested to take place in close association with various cell types found in the bone marrow (BM) microenvironment. In the present study the role of fibroblasts, adipocytes and cell surface heparan sulfate in regulating hematopoiesis in an in vitro mouse system was examined. Mouse BM cells were allowed to adhere to a mouse embryo fibroblast cell line (C3H 10T1/2) or a clonally derived adipogenically determined derivative (Clone D) of the 10T1/2 cell line. Nonadherent cells were removed, cultures were overlaid with semisolid media supplemented with growth factors and colony formation by granulocyte-macrophage (CFU-GM), erythroid (BFU-E) and multipotential (CFU-GEMM) progenitor cells was quantitated. Adherence and co-culture of BM cells with the fibroblast cell line resulted in increased numbers of total CFU-GM and CFU-GEMM colonies. In contrast, adherence and co-culture of BM cells with the adipocytic cell line resulted in an increase only in CFU-GEMM colonies. Morphological analysis revealed a preferential adherence/growth of granulocyte and macrophage progenitors at the expense of bipotent granulocyte-macrophage progenitors to the fibroblastic cell line and an increase in the adherence/growth of granulocyte progenitors to the adipogenic cell line. Progenitor cell adherence was abolished when the fibroblastic or adipocytic cell lines were pretreated with heparitinase. These results demonstrate enhanced proliferation/differentiation of hematopoietic progenitor cells when there is direct contact between hematopoietic progenitors and cell types characteristic of those found in the microenvironment and that heparan sulfate and different types of stromal cells appear to play different roles in this interaction.

Growth characteristics of marrow hematopoietic progenitor/precursor cells from patients on a phase I clinical trial with purified recombinant human granulocyte-macrophage colony-stimulating factor.

Bone marrow cells from patients with leukemia, myelodysplastic syndromes, cancer, and other disorders on a phase I clinical trial with recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) were assessed in vitro for numbers of granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells, and for growth patterns (colony-to-cluster ratio) of CFU-GM, cycling rates of CFU-GM, and responsiveness in vitro to colony-stimulating and colony-inhibiting factors. The colony-to-cluster ratio of CFU-GM and the dose-response curves of CFU-GM to stimulation by rhGM-CSF in vitro did not change during the clinical trial. However, the percentage of CFU-GM in DNA synthesis, which is a measure of the proliferative rates of these cells, determined by the high specific activity tritiated thymidine kill technique in vitro, was markedly enhanced in a reversible fashion after administration in vivo of rhGM-CSF. The increased cycling rates of CFU-GM were consistent with the induced increase in neutrophil counts in these patients that has been reported elsewhere. Additionally, marrow CFU-GM from patients given rhGM-CSF in vivo were increased in sensitivity to inhibition in vitro by recombinant human H-subunit (acidic) ferritin in two of eight cases, and were increased in sensitivity to inhibition by lower dosages of recombinant human tumor necrosis factor alpha in all patients evaluated. The sensitivity of CFU-GM to inhibition in vitro by recombinant human interferon gamma and prostaglandin E1 did not change during the clinical trial. These studies demonstrate that the rhGM-CSF is having an effect on CFU-GM in the patients on the phase I clinical trial. This information may be of significance in planning future clinical studies combining rhGM-CSF with chemotherapy and/or other biotherapy.

Effects in vivo of recombinant human inhibin on myelopoiesis in mice.

Inhibin, a protein dimer, has been implicated in negative regulation of human erythropoiesis in vitro. In this study, purified recombinant human (rhu) inhibin was assessed for its effect in vivo on the proliferation of hematopoietic progenitor cells in C3H/HeJ mice. Administration of single doses of inhibin i.v. to mice resulted 24 hrs later in significant decreases in cell cycling status of marrow and splenic granulocyte-macrophage (CFU-GM), erythroid (BFU-E) and multipotential (CFU-GEMM) progenitors. While no apparent effect was observed on marrow cellularity or on absolute numbers of marrow CFU-GM and BFU-E, inhibin significantly reduced absolute numbers of marrow CFU-GEMM, spleen nucleated cellularity and also absolute numbers of CFU-GM, BFU-E and CFU-GEMM in the spleen. The results demonstrate in vivo myelosuppressive effects for inhibin and demonstrate that effects in vivo are not restricted to erythropoiesis.

Influence of murine mast cell growth factor (c-kit ligand) on colony formation by mouse marrow hematopoietic progenitor cells.

Purified natural and recombinant murine mast cell growth factor (MGF, a c-kit ligand) were evaluated alone and in combination with other cytokines for effects in vitro on colony formation by multipotential (CFU-GEMM), erythroid (BFU-E) and granulocyte-macrophage (CFU-GM) progenitor cells from BDF1 mouse bone marrow. Both preparations stimulated Epo-dependent CFU-GEMM and enhanced Epo-dependent BFU-E colony numbers and size. MGF had some stimulating activity for CFU-GM. When used in combination with plateau concentrations of pokeweed mitogen mouse spleen cell conditioned medium or granulocyte-macrophage colony stimulating factor (CSF), MGF enhanced in greater than additive fashion colony formation by CFU-GM. MGF also enhanced the size of colonies formed, an enhancement greatest for colonies containing granulocytes and macrophages. MGF did not enhance Macrophage-CSF stimulated colony numbers or size. MGF seems to be an early acting cytokine with preferential effects on the growth of more immature hematopoietic progenitor cells.

The comparative enhancing effects of prostaglandin E1 on colony formation by erythroid progenitor (BFU-E) cells from bone marrow of mice differing in the Fv-2 locus.

Prostaglandin E1(PGE1) was assessed for its colony enhancing effects on erythroid (BFU-E) progenitor cells and for its colony suppressive effects on granulocyte-macrophage (CFU-GM) progenitor cells from bone marrows of mice differing in the Fv-2 locus. The Fv-2 locus has been reported by others to control the proportion of BFU-E, but not of CFU-GM, in DNA synthesis. PGE1 significantly enhanced erythroid colony formation by marrow cells from DBA/2 (Fv-2ss), C57BL/6 (Fv-2rr) and BDF1 (Fv-2rs) mice, but the DBA/2 cells were more sensitive to the PGE1 enhancing effects than were cells from C57BL/6 or BDF1 mice. The enhanced sensitivity of DBA/2 cells to PGE1 was associated with the higher cycling rate of DBA/2-BFU-E in comparison with C57BL/6- and BDF1-BFU-E, and removal of S-phase DBA/2-BFU-E by pulse exposure of cells to high specific activity tritiated thymidine in vitro eliminated the erythroid colony enhancing effects of PGE1. The differences in sensitivity of BFU-E from mice differing in the Fv-2 locus to the effects of PGE1 were verified using Fv-2 congenic mice. In contrast, no significant differences were noted in sensitivity of CFU-GM from these different mouse strains, including mice congenic for the Fv-2 locus, to the suppressive effects of PGE1. This correlated with the similar cycling characteristics of CFU-GM from these mice, and the non-cycle specific effects of PGE1 on mouse CFU-GM. These studies substantiate further the regulatory effects of the Fv-2 locus on mouse erythroid progenitor cells which are a manifestation of the cycling rates of these cells.

Clinical and biological aspects of human umbilical cord blood as a source of transplantable hematopoietic stem and progenitor cells.

Presented is a short review of the current status fo the clinical and biological aspects of using human umbilical cord blood as a source of transplantable hematopoietic stem and progenitor cells.

Effects of CC, CXC, C, and CX3C chemokines on proliferation of myeloid progenitor cells, and insights into SDF-1-induced chemotaxis of progenitors.

Chemokines have been implicated in the regulation of stem/progenitor cell proliferation and movement. The purpose of the present study was to assess a number of new chemokines for suppressive activity and to delve further into SDF-1-mediated chemotaxis of progenitor cells. This report extends the list of chemokines that have suppressive activity against immature subsets of myeloid progenitors stimulated to proliferate by multiple growth factors to include: MCP-4/CK beta-10, MIP-4/CK beta-7, I-309, TECK, GCP-2, MIG and lymphotactin. The suppressive activity of a number of other chemokines was confirmed. Additionally, pretreatment of the active chemokines with an acetylnitrile solution enhanced specific activity of a number of these chemokines. The new chemokines found to be lacking suppressive activity include: MCP-2, MCP-3, eotaxin-1, MCIF/HCC-1/CK beta-1, TARC, MDC, MPIF-2/eotaxin-2/CK beta-6, SDF-1 and fractalkine/neurotactin. Overall, 19 chemokines, crossing the CC, CXC, and C subgroups, have now been found to be myelosuppressive, and 14 chemokines crossing the CC, CXC and CX3C subgroups have been found to lack myelosuppressive activity under the culture conditions of our assays. Because of the redundancy in chemokine/chemokine receptor interactions, it is not yet clear through which chemokine receptors many of these chemokines signal to elicit suppressive activities. It was also found that SDF-1-induced chemotaxis of progenitors can occur in the presence of fibronectin (FN) and extracellular matrix components and that FN effects involve activation of beta 1-, and possibly alpha 4-, integrins.