Growth Of Hemopoietic Progenitor Cells Research Articles

Minor histocompatibility antigens on canine hemopoietic progenitor cells.

Adoptive immunotherapy with CTL against minor histocompatibility Ags (mHA) provides a promising way to treat leukemia relapse in allogeneic chimeras. Here we describe the in vitro generation of CTL against mHA in the dog. We tested their inhibitory effect on the growth of hemopoietic progenitor cells stimulated by hemopoietic growth factors in a 4-day suspension culture. CTL were produced by coculture of donor PBMC with bone marrow-derived dendritic cells (DCs). These DCs were characterized by morphology, high expression of MHC class II and CD1a, and the absence of the monocyte-specific marker CD14. Characteristically these cells stimulated allogeneic lymphocytes (MLR) and, after pulsing with a foreign Ag (keyhole limpet hemocyanin), autologous T cells. CTL were generated either ex vivo by coculture with DCs of DLA-identical littermates or in vivo by immunization of the responder with DCs obtained from a DLA-identical littermate. In suspension culture assays the growth of hemopoietic progenitor cells was inhibited in 53% of DLA-identical littermate combinations. In canine families mHA segregated with DLA as restriction elements. One-way reactivity against mHA was found in five littermate combinations. In two cases mHA might be Y chromosome associated, in three cases autosomally inherited alleles were detected. We conclude that CTL can be produced in vitro and in vivo against mHA on canine hemopoietic progenitor cells using bone marrow-derived DCs.

Thrombopoietin-independent effect of interferon-gamma on the proliferation of human megakaryocyte progenitors.

Flow cytometric study revealed that almost all CD34+ cells in human umbilical cord blood expressed interferon-gamma receptor (IFN-gammaR). To clarify the precise functional roles of IFN-gammaR in human CD34+ cells, we examined the effect of IFN-gamma alone and in combination with various cytokines on the growth of haemopoietic progenitor cells in CD34+ cells using a serum-free clonal culture. Surprisingly, IFN-gamma alone supported only megakaryocyte (MK) colonies in a dose-dependent manner with a plateau level at 1000 U/ml of IFN-gamma. IFN-gamma at 1000 U/ml induced 10 +/- 1.2 MK colonies from 1 x 10(3) CD34+ cells, whereas thrombopoietin (TPO), interleukin (IL)-3, stem cell factor (SCF) or IL-6 alone induced 22 +/- 4.0, 22 +/- 4.2, 4 +/- 0.6 and 0 MK colonies, respectively. The addition of anti-IFN-gamma monoclonal antibody (mAb) to the IFN-gamma culture completely abrogated MK colony formation, whereas the mAb had no effect on TPO-dependent production of MK colonies. In contrast, although anti-TPO polyclonal Ab almost completely blocked TPO-dependent MK colony formation, it failed to inhibit the generation of MK colonies induced by IFN-gamma, suggesting that the observed effect of IFN-gamma on the proliferation of human MK progenitor cells is independent of TPO. The addition of IFN-gamma to culture with TPO or SCF significantly augmented the development of MK colonies, whereas it did not affect IL-3-dependent MK colony formation. Additionally, IFN-gamma induced the increase of DNA content of cultured glycoprotein IIb/IIIa-positive megakaryocytes. These results suggest that IFN-gamma may have regulatory roles in human megakaryocytopoiesis.

Mechanism of action of antithymocyte globulin in the treatment of aplastic anaemia: in vitro evidence for the presence of immunosuppressive mechanism.

Antithymocyte globulin (ATG) is one of the effective drugs used in the treatment of aplastic anaemia (AA). Although it has been speculated that the mechanism of action of ATG is mediated by its immunosuppressive effect on lymphocytes which might have an inhibitory effect on haemopoietic stem and progenitor cells, no definite evidence of the presence of such a mechanism has been demonstrated. In this study we investigated whether such a mechanism is truly operating in ATG therapy for AA. In five patients who responded to ATG, bone marrow cells were obtained after haematological recovery and CD34-positive cells were separated by immunobeads. Autologous CD34-positive cells were mixed with autologous peripheral CD4- or CD8-positive cells obtained before ATG therapy and after haematological recovery, liquid-cultured for 12h, and then cultured in methylcellulose for 14d in the presence of haemopoietic growth factors. In all five cases studied, only the CD8 cells obtained before ATG therapy suppressed the colony forming unit-granulocyte-macrophage (CFU-GM)- and burst forming unit-erythroid (BFU-E)-derived colony formation. This result is definite evidence that one of the mechanisms of action of ATG in AA is an inhibitory effect on CD8-positive cells which have suppressive activity for the growth of haemopoietic progenitor cells.

Response of myelodysplastic syndrome marrow progenitor cells to stimualtion with cytokine combinations in a stroma-free long-term culture system.

The effect of an ex vivo expansion culture system using multiple cytokine combinations was evaluated in 38 cases of myelodysplastic syndrome (MDS) with the aim of overcoming the defective in vitro growth of haemopoietic progenitor cells. A combination of four growth factors (GF) including SCF, IL-3, IL-6 and GM-CSF was identified as the optimal combination for expanding clonogenic progenitor cells in MDS bone marrow liquid cultures. The cultures of 50% of the patients (19/38) responded to GF stimulation (mean CFU-GM fold increase 15.65+/-48 at week 4) and showed morphological features of normal and/or dysplastic myeloid differentiation. In 12/38 cases (31%), complete unresponsiveness to multiple cytokine stimulation was observed; a small number of patients (7/38) showed progressive leukaemic growth along the cultures with the presence of 100% immature blasts at week 4. GM-CSF and c-kit receptors, analysed by immuno-histochemistry in 10 patients, were over-expressed in responding patients and either lacking or down-regulated in non-responders. Fluorescence in situ hybridization (FISH) analysis of cultured interphase cells of nine patients (trisomy 8 in eight patients) showed a clear-cut increase in the percentage of cells with three signals in the two responding patients, thus indicating the expansion of a MDS clone. Multiple cytokine liquid cultures seem to be able to override the refractoriness of MDS progenitor cells to GF stimulation in many cases, revealing a heterogeneity which may have prognostic implications and should be considered in ex-vivo and in vivo clinical trials with cytokine combinations.

Expression and function of c-Kit in fetal hemopoietic progenitor cells: transition from the early c-Kit-independent to the late c-Kit-dependent wave of hemopoiesis in the murine embryo

The protooncogene c-kit encodes a receptor type tyrosine kinase and is allelic with the W locus of mice. SLF, the c-Kit ligand which is encoded by the Sl locus, has growth promoting activity for hemopoietic stem cells. Previous studies demonstrated that c-Kit is functionally required for the proliferation of hemopoietic progenitor cells at various differentiation stages in adult bone marrow. However, the absence of functional SLF and c-Kit in fetuses with mutant alleles of Sl and W loci produces only minor effects on the myeloid and early erythroid progenitor cells in the fetal liver, although the level of the late erythroid progenitor cells is significantly affected. We used an anti-c-Kit monoclonal antibody to investigate the expression and function of c-Kit in murine fetal hemopoietic progenitor cells. Flow-cytometric analysis showed that hemopoiesis in the yolk sac and fetal liver started from cells that express c-Kit. The c-Kit expression decreased upon maturation into erythrocytes in each organ. By fluorescence activated cell sorting, the c-Kit+ cell population was enriched with the hemopoietic progenitor cells clonable in vitro (CFU-E, BFU-E and GM-CFC). To elucidate whether c-Kit functions in these progenitor cells in vivo, we took advantage of the antagonistic anti-c-Kit monoclonal antibody, ACK2, which can block the function of c-Kit. Administration of ACK2 after 12.5 days of gestation rapidly eliminated BFU-E and GM-CFC as well as CFU-E from the fetal liver. However, the number of these progenitor cells in the yolk sac and fetal liver was less affected when the fetuses were given ACK2 before 12.5 days of gestation. Our results provide evidence that there are two waves of hemopoiesis in murine embryos relative to c-Kit dependency. The c-Kit has an essential role on the growth of hemopoietic progenitor cells in the fetal liver after 12.5 days of gestation, whereas the progenitor cells in the liver and yolk sac of the earlier embryo do not depend on c-Kit and its ligand SLF.

A liquid culture method for the in vitro growth of hemopoietic progenitor cells from normal human adult peripheral blood allowing for analysis by multiparameter flow‐cytometry

A liquid culture method has been developed allowing for the in vitro growth of peripheral blood-derived hemopoietic progenitor cells of myeloid, erythroid, monocytic and megakaryocytic lineages. Adherent cell- and CD21-positive cell-depleted PBMC from normal subjects have been cultured in the presence of rhEPO, rhGM-CSF or rhIl-3. Culturing cells in liquid cultures and in plasma clots, a similar dose-response was observed for granulocytic cells/liquid culture and granulocytic colonies/plasma clot with rhGM-CSF, and also for erythroid cells/liquid culture and erythroid colonies/plasma clot with rhEPO. Comparing serum- liquid cultures to serum+ liquid cultures, the ratio of CD13+ cells to CD15+ cells was higher in serum- cultures, indicating a maturation arrest of myecloid cells with serum deprivation. Using dual-colour flow-cytometry, cell-cycle analysis of CD13+ cells, comparing the effects of rhGM-CSF to those of rhIl-3, have been performed. The liquid culture method promises to be a useful tool for the study of in vitro differentiation and proliferation of hemopoietic progenitor cells.

Hemopoietic Colony-stimulating Factors: Biologic Functions and Clinical Potentials

Through use of semisolid tissue culture techniques that sup­ port the clonal growth of hemopoietic progenitor cells, it has become clear during recent years that blood cell production within the bone marrow is regulated by a family of glycopro­ teins referred to as hemopoietic colony-stimulating factors (CSFs) (1). A large body of evidence indicates that these cytokines constitute the endocrine hormones responsible for regulating the production of most if not all of the cells that enter the bloodstream. As illustrated by the data of Nakamura and colleagues, which are reported in the present edition of this journal (2), CSFs are capable of operating both individually and synergistically at various ontogenetic stages of hemopoiesis to stimulate the production of the vari­ ous classes of cells arising from the bone marrow. Many of the genes that encode these regulatory glycoproteins have been cloned, and, as a result, a large number of synthetic recombinant CSFs have been made widely available for in­ vestigation as well as for treatment of various disorders as­ sociated with bone marrow failure (3). To date, at least 14 cytokines in this family which affect the proliferation of phagocytes ' have been identified. Most prominent among these are GM-CSF, which stimulates the production of neutrophils, eosinophils, and macrophages in vitro; IL-3, which supports the growth of all classes of phagocytes; G-CSF, which primarily stimulates the prolifer­ ation of neutrophils; and M-CSF, the effects of which are limited to cells committed to macrophage development. While the effects of G-CSF and M-CSF appear to be re­ stricted to single lineages, both GM-CSF and IL-3 are promiscuous in early hemopoiesis, stimulating the prolifera­ tion of virtually every class of early progenitor cell in the 1 Granulocyte/macrophage colony-stimulating factor (GM-CSF), granu­ locyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), interleukins I through 9 (IL-I through IL-9) , tumor necro­ sis factor-alpha, and interferon-gamma.

Studies on the mechanism of platelet-activating factor production in GM-CSF primed neutrophils: Involvement of protein synthesis and phospholipase A 2 activation

GM-CSF regulates the growth of hemopoietic progenitor cells, enhances the responsiveness of mature PMN and primes these cells for synthesis of leukotrienes and PAF in response to secondary stimuli. The biochemical requirements for PAF production in GM-CSF primed PMN was examined using different metabolic inhibitors. GM-CSF stimulates uridine incorporation into RNA and inhibitors for RNA and protein synthesis decrease PAF synthesis in our model. This suggests a role for gene expression and de novo synthesis of proteins in the action of GM-CSF. Different PLA 2 inhibitors, including a 9 amino-acid peptide derived from a conserved region of hte calpactin superfamily, decrease PAF production, indicating that in GM-CSF primed PMN the chemotactic peptide fMLP triggers lipid mediator synthesis by activating PLA 2.

Clonal growth of haemopoietic progenitor cells from myelodysplastic marrow in response to recombinant haemopoietins

The growth factor requirements of granulocyte-macrophage (GM) and erythroid marrow progenitor cells from 12 myelodysplastic (MDS) patients have been analysed. GM progenitors from two of six patients who grew normal numbers of colonies in response to conditioned medium + erythropoietin (5637CM + Epo) showed defective responses to either GMCSF and/or IL-3. Of all the recombinant factors tested (IL-3, IL-1, GCSF, GMCSF, MCSF), GMCSF was the strongest stimulator of myeloid clonal growth, inducing normal numbers of GM colonies from marrow of six patients (two of whom were neutropenic). Erythroid colonies were low in 5637CM + Epo-supplemented cultures of marrow from all but one patient and remained poor in the presence of any of the haemopoietins tested. Supraoptimal doses (for normal marrow) of these haemopoietins improved colony growth in only one patient (GM colonies in response to IL-3). Combinations of factors were also largely ineffective at raising myeloid or erythroid colony numbers. These data indicate that the defective response of MDS progenitor cells to growth factors is not amenable to experimental manipulation of recombinant factor levels or combinations. Clonal assays might suggest a role for GMCSF therapy in a subpopulation of neutropenic MDS patients but their potential now needs to be evaluated in association with clinical trials.

Interleukin 4 (B-cell stimulatory factor 1) can enhance or antagonize the factor-dependent growth of hemopoietic progenitor cells.

Our studies show that although interleukin 4 (IL-4) fails to stimulate significant colony formation by bone marrow progenitor cells, it enhances erythroid, granulocyte, macrophage, and mast-cell colony formation when used as a costimulant with erythropoietin, granulocyte colony-stimulating factor, macrophage colony-stimulating factor, and interleukin 3 (IL-3), respectively. In contrast, IL-4 suppresses IL-3-dependent colony formation by granulocyte and macrophage progenitor cells and by multipotential progenitor cells. Furthermore, it appears to inhibit the in vitro generation of colony-forming progenitor cells from immature IL-3-dependent stem cells. We also found that IL-4 inhibits stromal cell-dependent growth of bone marrow-derived pre-B cells. The ability of IL-4 to directly or indirectly regulate both positive and negative aspects of progenitor cell growth is discussed.

Defective in vitro growth of the hemopoietic progenitor cells in the acquired immunodeficiency syndrome.

In addition to immunologic derangement, hematological abnormalities have been reported in the majority of patients with acquired immunodeficiency syndrome (AIDS). In this study 15 patients with AIDS or AIDS-related complex (ARC) were evaluated for the in vitro growth of hemopoietic progenitor cells. In all patients a significant reduction of growth (mean +/- SEM) of colony-forming unit-granulocyte, erythrocyte, macrophage, (megakaryocyte) (CFU-GEM) (1.2 +/- 0.3), burst-forming unit-erythroid (BFU-E) (17 +/- 10), CFU-megakaryocyte (CFU-Mk) (1.7 +/- 0.6), and CFU-granulocyte-macrophage (CFU-GM) (35 +/- 10) was observed in comparison with normal controls. Depletion of T cells from the bone marrow before culture led to a significant increase in colony growth, which indicated an imbalance of the normally modulating T cell subsets. This increase was reversed by readdition of autologous T cells causing a decrease in colony growth to a degree, dependent on the T4 to T8 ratio. A decreased number of hemopoietic progenitor cells and/or a defective modulation of progenitor cell growth, normally carried out by T lymphocyte subsets, might be the cause of the hematological abnormalities in AIDS patients.

In vitro homing of hemopoietic stem cells is mediated by a recognition system with galactosyl and mannosyl specificities.

We synthesized a number of neoglycoprotein probes by covalently linking three biologically relevant sugars (mannose, galactose, and fucose) to a protein molecule so as to retain the pyranose (ring) form of sugars necessary for their interaction with lectins. In the presence of galactosyl and mannosyl but not fucosyl probes, the production of CFU-S [colony-forming unit(s) in spleen] and total cells was halted in murine long-term marrow cultures. Cobblestone areas disappeared in these cultures, indicating the inhibition of binding of hemopoietic cells to the stroma. Electron microscopy revealed no alterations of the stroma, and the probes did not have direct cytotoxic or inhibitory effects on the growth of CFU-S or CFU-C [colony-forming unit(s) in culture]. Stroma grown for 5 weeks in the presence of the probes could subsequently support the growth of hemopoietic progenitor cells when the probes were removed from the medium. Conversely, the proliferative capacity of CFU-S in the supernate, grown in the presence of the probes, was retained upon grafting to control stroma. Galactosyl and mannosyl but not fucosyl probes differentially agglutinated CFU-S in whole-marrow-cell suspensions, suggesting the presence of membrane lectins with specificity for these sugars on the surface of CFU-S. We conclude that the binding of CFU-S to marrow stroma (homing) is mediated by a recognition system with galactosyl and mannosyl specificities.