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Storage of noncryopreserved periphered blood stem cells for transplantation.

Mobilized peripheral blood stem cells (PBSC) were collected in autologous plasma and acid-citrate-dextrose formula A (ACD-A) by leukaphereses using the CS3000 cell separator (Baxter) and stored at 4 degrees C in a refrigerator for 8 days. We have looked at the viability of the nucleated cells with the trypan blue test and the proliferation and differentiation capacity using a standardized progenitor cell cloning assay. The changes in viability, granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-E), and mixed-lineage colony-forming units (CFU-GEMM) were determined daily during the storage period. Viability was 90.8% (SD 8%) at day 0 and declined to a mean of 69.5% (SD 15.5%) at day 8. CFU-GM decreased to 47% (SD 28.7%), CFU-GEMM to 48% (SD 42.2%), and BFU-E to 40.1% (SD 18.4%) after 6 days. After 5 days of storage the mean viability was 79.7% (SD 17.8%), whereas the mean CFU-GM were 65.3% (SD 28.4%) the mean CFU-GEMM were 61.8% (SD 30.4%) and the mean BFU-E were 55.1% (SD 18.2%). At day 4 viability was still 82.5% (SD 17.0%), recovery of CFU-GM was 78.5% (SD 28.8%), recovery of CFU-GEMM was 70.7% (SD 40.4%) and recovery of BFU-E was 65.0% (SD 17.5%). These data show, that PBSC can be stored safely over at least 5 days at 4 degrees C while the patient receives high-dose chemotherapy.

Blood Component Removal↗

[Effect of panaxadiol saponin and panaxtrol saponin on proliferation of human bone marrow hemopoietic progenitor cells].

OBJECTIVE: To observe the effect of panaxadiol saponin (PDS) and panaxtrol saponin (PTS) on proliferation of human bone marrow hemopoietic progenitor cells (HPC). METHODS: PDS and PTS were separated and purified from ginsenosides, and the effects on HPC were studied using in vitro hemopoietic progenitor cell colony-forming technique, by observing the proliferation of human burst forming unit-erythroid progenitor (BFU-E), colony-forming unit-erythroid (CFU-E), colony-forming unit-granulocyte/macrophage (CFU-GM) and colony-forming unit-pluripotent hemopoietic progenitor (CFU-Mix) in mice after PDS and PTS stimulation. RESULTS: Different concentration of PDS (2.5-200 micrograms/ml) could stimulate the proliferation of HPC obviously, showing increase of CFU-E, BFU-E, CFU-GM and CFU-Mix by 54.9 +/- 6.3%, 48.8 +/- 5.1%, 27.6 +/- 4.2% and 48.9 +/- 3.9% respectively, which was higher than that of the control group. While stimulated by PTS of the same concentration, the CFU-E and BFU-E was lower than that of control significantly (P < 0.05); when the terminal concentration of PTS was 200 micrograms/ml, CFU-E and BFU-E was zero respectively. In the CFU-GM culture, PTS in concentration of 12.5 micrograms/ml could cause the proliferation increased by 29.7 +/- 2.2% (P < 0.05), but in concentration of 100 micrograms/ml and 200 micrograms/ml, it showed inhibitory effect on CFU-GM, the inhibition rate being 48.6 +/- 3.9% and 100% respectively. CONCLUSION: PDS is the effective component of ginsenosides in stimulating proliferation of human bone marrow HPC. PTS is an component with inhibitory action on proliferation of CFU-E and BFU-E and its effect on CFU-GM was depending on its concentration.

Bone Marrow Cells↗

Effects of interleukin-11 on the hematopoietic action of granulocyte colony-stimulating factor.

Interleukin-11 (IL-11, CAS 145941-26-0) is a cytokine that ameliorates thrombocytopenia induced by chemotherapy. Granulocyte colony-stimulating factor (G-CSF) is frequently used clinically as an adjuvant therapy to ameliorate neutropenia. In this study, it has been investigated whether IL-11 influences the hematopoietic action of G-CSF in vitro. IL-11 alone did not stimulate CFU-Meg colony formation in the absence of interleukin-3 (IL-3), but did in the presence of IL-3. However, IL-11 alone stimulated the formation of CFU-E, BFU-E and CFU-GM colonies, which were further enhanced by IL-3. G-CSF alone stimulated in a concentration dependent manner colony formation of CFU-GM and CFU-E, but not BFU-E or CFU-Meg. The addition of IL-11 enhanced the stimulatory effect of G-CSF on CFU-GM colony formation, and enabled G-CSF to stimulate colony formation of BFU-E, but not CFU-E. The addition of IL-3 further enhanced the stimulatory effect of G-CSF on CFU-E, BFU-E, and CFU-GM colony formation. However, G-CSF was unable to stimulate CFU-Meg colony formation for any of the cytokine combinations tested. These studies demonstrate that IL-11 has significant stimulatory effects on G-CSF-induced CFU-GM colony formation, suggesting that the therapeutic combination of the two growth factors could be beneficial for the treatment of myelosuppression induced by chemotherapy.

Animals↗

Haemopoietic growth factors--from discovery to clinical application.

The Colony Stimulating Factors (CSFs) are a family of haemopoietic hormones that likely share a common ancestral origin and stimulate white blood cell development. They display unique but overlapping biological functions and stimulate the survival, proliferation, differentiation and functional activation of granulocytes and monocytes/macrophages and their precursor cells in vitro and in vivo. Each hormone has been purified and produced in active recombinant form. Recombinant G-CSF and GM-CSF are now being used around the world in a variety of clinical situations (e.g., in conjunction with chemotherapy and bone marrow transplantation) to promote the formation and function of these leukocytes. These molecules are among the first of a new generation of biological agents that will impact enormously on clinical medicine.

Bone Marrow Diseases↗

Inflammatory reactions and severe neutropenia in mice lacking the transcriptional repressor Gfi1.

The transcriptional repressor Gfi1 is a nuclear zinc-finger protein expressed in T-cell precursors in the thymus and in activated mature T lymphocytes. Previous experiments have shown that Gfi1 is involved in T-cell lymphomagenesis and in the development of T-cell progenitors. Here we show that Gfi1 is also expressed outside the lymphoid system in granulocytes and activated macrophages, cells that mediate innate immunity (that is, non-specific immunity). We have generated Gfi1-deficient mice (Gfi1-/-) and show that these animals are severely neutropenic and accumulate immature monocytic cells in blood and bone marrow. Their myeloid precursor cells are unable to differentiate into granulocytes upon stimulation with granulocyte colony-stimulating factor (G-CSF) but can develop into mature macrophages. We found that Gfi1-/- macrophages produce enhanced levels of inflammatory cytokines, such as tumor necrosis factor (TNF), interleukin-10 (IL-10) and IL-1beta, when stimulated with bacterial lipopolysaccharide (LPS) and that Gfi1-/- mice succumb to low doses of this endotoxin that are tolerated by wildtype mice. We conclude that Gfi1 influences the differentiation of myeloid precursors into granulocytes or monocytes and acts in limiting the inflammatory immune response.

Animals↗

Synergistic effect of heme and IL-1 on hematopoietic stromal regeneration after radiation.

Results from this study show that a combination of heme and interleukin-1 (IL-1) treatment resulted in the most improved recovery of hematopoietic-stromal regeneration after sublethal irradiation. Less pronounced effects were obtained when heme or IL-1 were given singly. Sublethal irradiation of mice produced an initial (as early as day 1) intense depression of the hematopoietic system as evidenced by leukopenia. In vivo treatment of animals with heme in combination with IL-1, accelerated hematopoietic and stromal regeneration as determined by hematopoietic spleen colony forming unit assay (CFU-S), erythroid (BFU-E), myeloid (CFU-GM) clonal cultures, long-term bone marrow cultures (LTBMC), and the ability to regenerate hematopoiesis by ectopic (renal) stromal hemopoietic transplantation. Sixteen days after irradiation, leukocyte levels in heme and IL-1 treatment groups were higher than non-treated animals and were near normal values by 27 days. One day after irradiation, the capacity of stromal progenitors to form new bone and hematopoietic cells (ectopic foci) was severely impaired, but recovered after 2-4 weeks. This recovery process was accelerated in heme and IL-1-treated animals. BFU-E, CFU-GM, and CFU-S capacity was also severely impaired in all animals 1-27 days after irradiation. CFU-S was only 0.15% of control by day 1 and 5% of control by day 16. Treatment with heme or IL-1 improved recovery by as much as 70% after 27 days of irradiation. A similar but enhanced recovery was seen for BFU-E and CFU-GM, with erythroid recovery the best. Total cellularity, adherent cell layer (ACL) formation, and clonogenic capacity by LTBMCs (10 weeks) derived from irradiated animals was severely reduced, whereas the hematopoietic capacity by LTBMCs derived from heme- and IL-1-treated animals had recovery values similar to non-irradiated controls. These results suggest therapeutic use of heme and IL-1 after chemotherapy or bone marrow depression may be beneficial.

Animals↗

Development of the liver in the chicken embryo. II. Erythropoietic and granulopoietic cells.

Hepatic hemopoiesis is apparent in the chicken embryo on day 7 of incubation (Hamburger and Hamilton Stage 30), and a peak in hemopoietic activity occurs on day 14 (Stage 40). During this period, the differentiation of hemopoietic cells was examined by light microscopy and by transmission and scanning electron microscopy. Glycol methacrylate sections were used in lieu of smears to study hemopoietic cells, thus minimizing the problems of cell shrinkage and rupture. The sections were superior to smears for close examination of nuclear and cytoplasmic morphologies and for precise localization of hemopoietic cells to intravascular and extravascular sites. The avian liver is involved directly with erythropoiesis and granulopoiesis only. Erythropoietic cells, occurring in intravascular and extravascular locations, appear throughout the time frame examined. Blood islands with granulopoietic cells were not observed until days 8-9 (Stage 35). Granulopoiesis in the liver produces only eosinophilic leukocytes. Individual granulopoietic cells appear first in the connective tissue sheaths of hepatic vessels, and these cells subsequently congregate into blood islands. Endothelial cells of the sinusoidal linings, through asymmetric divisions, frequently release daughter cells into the circulation, and Kupffer cells are actively engaged in phagocytosis of erythrocytes. From a comparative standpoint, the elements deemed critical to hemopoiesis in the mammalian liver--prehepatocyte population, hepatic vasculature, and compartments for stem cell differentiation--may not hold the same importance in the bird, owing to an inordinate reliance on intravascular hemopoiesis in this vertebrate class.

Animals↗

Synergy among erythropoietin, interleukin 3, stem cell factor (c-kit ligand) and interferon-gamma on early human hematopoiesis.

The stimulatory effect of interferon-gamma (IFN-gamma) on human early hematopoiesis was investigated using CD34+ cells that were purified from peripheral blood as a target. In the presence of human interleukin 3 (IL-3) and stem cell factor (SCF), the growth of colony forming units for granulocyte/macrophage (CFU-GM) in serum-free methylcellulose culture was enhanced up to 5.5-fold over baseline colony formation (n = 10, mean +/- SE, 3.18 +/- 1.00) by coculture with 2.5 x 10(3) U/ml IFN-gamma, whereas burst forming units-erythroid (BFU-E) growth induced by additional erythropoietin (Epo) was reduced dose-dependently. Liquid-suspension culture of the target cells for 24 h with IFN-gamma before secondary plating in serum-free methylcellulose resulted in a marked increase in the colony numbers; 3.62 +/- 1.04-fold for CFU-GM and 5.72 +/- 2.32-fold for BFU-E (n = 5). The effect was most striking in the growth of erythroid lineage supported by Epo alone in serum-free culture. Delayed addition of IFN-gamma between days 4 and 7 of methylcellulose culture suppressed CFU-GM growth, but not that of BFU-E. These results suggest that IFN-gamma is an essential cytokine that affects human hematopoiesis and exerts a bimodal effect on stem cell maturation; potentiating or replacing the effect of early-acting cytokines such as SCF or IL-3, and suppressing the growth of matured progenitor cells.

Antigens, CD↗

The nucleoside diphosphate kinase activity of DRnm23 is not required for inhibition of differentiation and induction of apoptosis in 32Dcl3 myeloid precursor cells.

DRnm23 belongs to a multigene family which includes nm23-H1, the first bona fide metastasis suppressor gene, nm23-H2, nm23-H4, and nm23-H5. Like nm23-H1, nm23-H2, and nm23-H4, DRnm23 possesses nucleoside diphosphate kinase (NDPK) activity. Upon overexpression in myeloid precursor 32Dcl3 cells, DRnm23 inhibits granulocytic differentiation and promotes apoptosis. Two specific mutants of DRnm23 (H134Q and S136P), at residues required for the NDPK activity, inhibit differentiation and promote apoptosis of 32Dcl3 cells. By contrast, substitution of serine 61 with proline (S61P) or deletion of the RGD domain (DeltaRGD) abrogates the effects of wild-type DRnm23. Like wild-type DRnm23, all four mutants show a predominantly mitochondrial subcellular localization. These studies indicate that the enzymatic activity of DRnm23 is not required for the effects observed in 32Dcl3 cells. Moreover, the inability of the S61P and DeltaRGD DRnm23 mutants to inhibit differentiation and promote apoptosis may be due to defective protein-protein interactions at the mitochondria, the predominant site of DRnm23 subcellular localization.

Animals↗

Effects of various recombinant human hemopoietic growth factors (rhEpo, rhG-CSF, rhGM-CSF, rhIl-3) on the growth of peripheral blood progenitor cells (BFU-E, CFU-GM).

The effects of rhEpo 1, rhG-CSF, rhGM-CSF and rhI1-3 on the growth of both CFU-GM and BFU-E from normal human adult peripheral blood have been studied in plasma clot cultures. Using optimal concentrations of all growth factors, alone and in combination with all other factors, rhI1-3 showed the highest activity in regard to growth of both CFU-GM and BFU-E, whereas rhGM-CSF treatment resulted only in half-maximal colony growth compared to rhI1-3. No synergism or additive effect was seen with the combination of rhI1-3 and rhGM-CSF. Treatment with rh-G-CSF had no additional effect with optimal concentrations of rhI1-3 and/or rhGM-CSF. When suboptimal concentrations of rhGM-CSF and rhI1-3 were applied, however, they showed a marked synergism on both BFU-E and CFU-GM. RhG-CSF, added to a suboptimal concentration of rhGM-CSF, resulted in a marked growth increase of CFU-GM but had no effect on BFU-E.

Adult↗

Analysis of platelet recovery after autologous transplantation with G-CSF mobilized CD34+ cells purified from leukapheresis products.

We studied platelet recovery in relation to graft content in CFUs and CD34+ cells in 31 patients with multiple myeloma (21) or non-Hodgkin lymphoma (10) receiving marrow-ablative therapy followed by autologous transplantation with G-CSF mobilized CD34+ cells purified from leukapheresis products. Twelve patients had prolonged post-transplantation thrombopenia (> or = 14 days): their graft contents in CD34+ cells, CFU-GM and BFU-E were significantly inferior to those of patients with rapid platelet recovery. Although numbers of infused CD34+ cells and CFU-GM or BFU-E were well correlated, the graft content in CD34+ cells was the only parameter predictive of platelet recovery (r = -0.38, p = 0.04), with a threshold of 2.5 x 10(6) CD34+ cells/kg. However, because rapid platelet reconstitution was obtained for 4 of 16 patients re-infused with < 2.5 x 10(6) CD34+ cells/kg, we investigated whether the graft CFU-MK content might be a better predictor of platelet reconstitution than the CD34+ cell content. Eighteen CD34 grafts were studied for CFU-MK content: CD34 and CFU-MK contents were weakly correlated (r = 0.52, p = 0.03), but there was no correlation between numbers of infused CFU-MK and time to platelet recovery. We conclude that, for autologous CD34 grafts, CFU-MK assays, like CFU-GM or BFU-E assays, cannot be used to predict platelet recovery. A CD34+ cell content > or = 2.5 x 10(6)/kg remains the only reliable indicator of the platelet reconstitution capacity of a CD34 graft.

Adult↗

Hemopoietic precursors: mechanisms of regulation under conditions of chronic stress.

We studied the content of hemopoietic precursor cells in the bone marrow, production of IL-1, IL-3, and colony-stimulating and erythropoietic activities by bone marrow cells under conditions of chronic stress. It was shown that in mice subjected to 15-h immobilization no hyperplasia of the bone marrow developed against the background of increased content of committed erythro- and granulomonocytopoietic precursors and enhanced production of short-distant humoral regulators of hemopoiesis, which reflects dysregulation of hemopoietic cells proliferation and differentiation processes in stressed animals.

Animals↗

Opposing effects of PI3 kinase pathway activation on human myeloid and erythroid progenitor cell proliferation and differentiation in vitro.

OBJECTIVE: To investigate 1) the effects of lineage-specific cytokines (G-CSF and EPO) combined with ligands for different classes of cytokine receptors (common beta chain, gp130, and tyrosine kinase) on proliferation by human myeloid and erythroid progenitor cells; and 2) the signal transduction pathways associated with combinatorial cytokine actions. PATIENTS AND METHODS: CFU-GM and BFU-E were cloned in vitro. Secondary colony formation by replated CFU-GM and subcolony formation by BFU-E provided measures of progenitor cell proliferation. Studies were performed in the presence of cytokine combinations with and without signal transduction inhibitors. RESULTS: Proliferation by CFU-GM and BFU-E was enhanced synergistically when common beta chain receptor cytokines (IL-3 or GM-CSF) were combined with G-CSF or EPO, but not with gp130 receptor cytokines (LIF or IL-6) or tyrosine kinase receptor cytokines (SCF, HGF, Flt-3 ligand, or PDGF). Delayed addition studies with G-CSF+IL-3 and EPO+IL-3 demonstrated that synergy required the presence of both cytokines from the initiation of the culture. The Jak2-specific inhibitor, AG490, abrogated the effect of combining IL-3 with EPO but had no effect on the enhanced CFU-GM proliferation stimulated by IL-3+G-CSF. The PI3 kinase inhibitors LY294002 and wortmannin substituted for G-CSF in combination with IL-3 since proliferation in the presence of LY294002/wortmannin+IL-3 was enhanced to the same extent as in the presence of G-CSF+IL-3. In contrast, LY294002 and wortmannin inhibited proliferation in the presence of EPO and in the presence of EPO+IL-3. CONCLUSION: 1) IL-3 may activate different signal transduction pathways when combined with G-CSF and when combined with EPO; 2) different signal transducing intermediates regulate erythroid and myeloid progenitor cell proliferation; and 3) inhibition of the PI3 kinase pathway suppresses myeloid progenitor cell differentiation and thereby increases proliferation.

Cell Differentiation↗

Normal human bone marrow CD34(+)CD133(+) cells contain primitive cells able to produce different categories of colony-forming unit megakaryocytes in vitro.

OBJECTIVE: To evaluate the megakaryocyte potential of normal bone marrow (NBM) CD34(+)CD133(+) cells, a subset offering a possible alternative for clinical CD34 immunoselection, we evaluated their colony-forming unit megakaryocyte (CFU-Mk) content and their ability to produce clonogenic Mk progenitors in comparison with the CD133(-) subset. MATERIALS AND METHODS: Sorted NBM CD34(+)CD133(+) and CD34(+)CD133(-) subsets were evaluated for Mk clonogenic capacity before and after in vitro proliferation in serum-free liquid culture containing kit ligand, Flt3 ligand, thrombopoietin, interleukin-3, and interleukin-6. The segregation of CFU-Mk according to the expression of CD34, CD133, and CD41 was compared between fresh BM cells and expanded cells. RESULTS: Although the fresh NBM CD133(-)CD34(+) subset included two thirds CFU-Mk, only the CD133(+) subset contained primitive cells able to produce all categories of CFU-Mk in vitro. Immunophenotyping confirmed that CD41 antigen is nonspecific for Mk lineage and showed that the usual CD34(+)CD41(+) subset does not specifically define a CFU-Mk population. The segregation of CFU-Mk before and after expansion according to CD34, CD41, or CD133 was modified in relation with down-regulation of CD34 and CD133 antigens and up-regulation of CD41 antigen. CONCLUSIONS: The NBM CD133(+) subset contains primitive cells able to generate CFU-Mk, a subset probably relevant to platelet recovery after infusion. The alteration of antigen expression during in vitro proliferation calls for caution in the identification of the different categories of Mk subsets produced and in the assessment of their predictivity for in vivo platelet production.

AC133 Antigen↗

Characterization and partial purification of a haemopoietic cell growth factor in WEHI-3 cell conditioned medium.

A myelomonocytic leukaemia cell line, WEHI-3, releases into its growth medium factors which stimulate the development of pluripotential cells, granulocyte/macrophage progenitor cells, megakaryocytic and erythroid progenitor cells. Also present is a factor which is essential for the continued proliferation in vitro of a variety of haemopoietic precursor cell lines of a granulocytic nature (FDC-P cells). Characterization of this growth factor has demonstrated that it is a glycoprotein of apparent Mr 25 800, in which the carbohydrate component appears to be important for activity. After several purification steps, there is an increase in specific activity of approx. 4000-fold over the starting material. At each stage of purification, the factor necessary for the proliferation of FDC-P cells 'co-purifies' with activity which stimulates the proliferation and development of normal multipotential haemopoietic cells as well as megakaryocytic, erythroid and granulocytic committed progenitor cells. This 'co-purification' occurs to the extent that the multilineage stimulating factor and the FDC-P growth factor can be eluted from the same region of sodium dodecyl sulphate/polyacrylamide gels. Thus, evidence so far, using different starting methods and purification regimes, suggests that one molecule may have multiple activities on diverse cell types.

Animals↗

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.

Animals↗

Human parathyroid hormone does not influence human erythropoiesis in vitro.

BACKGROUND: Although renal anaemia is associated with secondary hyperparathyroidism, the relationship of both conditions remains obscure. Previously it was reported that high levels of bovine parathyroid hormone (PTH) did not inhibit in vitro human erythropoiesis, but whether human PTH inhibits in vitro human erythropoiesis has not been determined. METHOD: To clarify the direct effects of human biologically active N-terminal (1-34) PTH and intact (1-84) PTH on human haematopoietic progenitor growth, we investigated colony assays of human erythropoiesis and granulomonopoiesis. RESULTS: Neither N-terminal PTH (300 ng/ml) nor intact PTH (5000 pg/ml) inhibited haematopoietic progenitor growth. CONCLUSION: Our findings confirm that human PTH does not directly inhibit human erythropoiesis.

Animals↗