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H Mayani

Publications and source records attributed to H Mayani.

At least 37 records · Page 2Linked to original sources
PubMed1998

Biology of human umbilical cord blood-derived hematopoietic stem/progenitor cells.

Reported in 1989, studies by Broxmeyer, Gluckman, and colleagues demonstrated that umbilical cord blood (UCB) is a rich source of hematopoietic stem/progenitor cells (HSPC) and that UCB could be used in clinical settings for hematopoietic cell transplantation. Since then, a great interest has been generated on the biological characterization of these cells. Over the last nine years, several groups have focused on the study of UCB HSPC, addressing different aspects, such as the frequency of these cells in UCB, the identification of different HSPC subsets based on their immunophenotype, their ability to respond to hematopoietic cytokines, the factors that control their proliferation and expansion potentials, and their capacity to reconstitute hematopoiesis in animal models. Most of these studies have shown that significant functional differences exist between HSPC from UCB and adult bone marrow (i.e., the former possess higher proliferation and expansion potential than the latter). It is also noteworthy that genetic manipulation of UCB HSPC has been achieved by several groups and that genetically modified UCB cells have already been used in the clinic. In spite of the significant advances in the characterization of these cells, we are still in the process of trying to fully understand their biology, both at the cellular and the molecular levels. In the present article, we describe and discuss what is currently known about the biology of UCB HSPC.

Adult↗
PubMed1996

Production of interleukin 1beta by human hematopoietic progenitor cells.

The production of interleukin 1beta (IL-1beta) by human hematopoietic stem/progenitor cells was studied to explore the concept that these cells are not merely responders to stimuli from their microenvironment, but can themselves produce a powerful biomodulator. Cells with a CD34+ CD45RA(lo) CD71(lo) phenotype were purified from human umbilical cord blood and cultured one per well in serum-free medium with a mixture of cytokines. Cells that had divided over 2-5 d to form doublets were identified and the daughter cells were studied individually. 91% (460/506) of daughter cells had clonogenic potential. Analysis of these individual daughter cells by reverse transcription-polymerase chain reaction showed that 29% of them (14/48) were positive for IL-1beta mRNA. One of the cells that was strongly positive for IL-1beta mRNA had a sibling that generated 366,000 cells of multiple lineages after 14 d. IL-1beta converting enzyme mRNA, which is necessary to produce IL-1beta, was also detected by reverse transcription-polymerase chain reaction at the single-cell level. Moreover, enzyme immunoassay for mature secreted IL-1beta in culture supernatants demonstrated the production of IL-1beta protein by these cells. This was confirmed by fluorescent immunostaining of the cells for human IL-1beta which showed a significant portion of positive cells. Taken together, the results demonstrate the capacity of early hematopoietic cells to synthesize IL-1beta. The capacity of human hematopoietic stem/progenitor cells to produce IL-1beta may be involved in regulation of their proliferation and differentiation under certain circumstances and dysregulation of this process may be modified in leukemogenesis.

Actins↗
PubMed1996

Composition and function of the hemopoietic microenvironment in human myeloid leukemia.

In normal adult mammals, blood cell production, hemopoiesis, takes place within the medullary cavity. There, hemopoietic cell proliferation and differentiation are regulated by a network of stromal/accessory cells and their products (ie cytokines and extracellular matrix molecules), known as the hemopoietic microenvironment. Recent in vitro studies indicate that both cell composition and functional abnormalities of the hemopoletic microenvironment are present in a proportion of patients with myeloid leukemia, both chronic (CML) and acute (AML). Cell composition abnormalities have been primarily observed in a subset of patients with AML; these abnormalities include reduced numbers of fibroblast progenitors and, in some cases, reduced numbers of macrophages and adipocytes. In terms of function, it has been shown that the marrow stromal cells from a significant number of both CML and AML patients, possess a deficient hemopoletic supportive capacity in vitro. This seems to be related to the presence of functionally abnormal, malignant macrophages. The mechanisms by which these macrophages alter the hemopoietic function of the marrow stroma, as a whole, are still not fully understood. Whereas in AML, a macrophage-derived soluble inhibitory activity (containing tumor necrosis factor alpha) has been described; in CML, a direct, macrophage-mediated cell-to-cell contact mechanism for hemopoietic inhibition seems to be involved. To date, however, it is not clear whether the abnormalities in the hemopoietic microenvironment are secondary to myeloid leukemia or if they precede clinical CML/AML. Furthermore, it is not known to what extent the functional abnormalities observed in vitro contribute to the hematologic dysfunction that characterizes myeloid leukemia and to the in vivo progression of the disease.

Adipocytes↗
PubMed1995

Differential effects of the hematopoietic inhibitors MIP-1 alpha, TGF-beta, and TNF-alpha on cytokine-induced proliferation of subpopulations of CD34+ cells purified from cord blood and fetal liver.

We have previously characterized the proliferative response of primitive CD34+ cells, purified from adult bone marrow, umbilical cord blood, and fetal liver, to a mixture of hematopoietic stimulators (steel factor [SF], interleukin-3 [IL-3], IL-6, and erythropoietin [Epo]) in serum-free liquid cultures. In the present study, we assessed the effects of the hematopoietic inhibitors, macrophage inflammatory protein-1 alpha (MIP-1 alpha), transforming growth factor-beta (TGF-beta), and tumor necrosis factor-alpha (TNF-alpha), on the cytokine-induced proliferation of three different CD34+ cell subpopulations derived from cord blood and on total CD34+ cells derived from fetal liver. In cultures of cord blood cells, addition of MIP-1 alpha inhibited the numerical expansion of primitive CD34+ cells (CD34+ CD45RAlow CD71low cells) without inhibiting the proliferation of more mature subpopulations enriched for myeloid (CD34+ CD45RA+ CD71low cells) or erythroid (CD34+ CD45RAlow CD71+ cells) progenitors. TGF-beta significantly reduced the proliferation of all three subpopulations, although its effects were more pronounced on cells of the erythroid lineage, particularly immature erythroid progenitors. Similarly, TNF-alpha preferentially inhibited total nucleated and CD34+ cell production in the subpopulation enriched for erythroid cells. However, in contrast to TGF-beta, TNF-alpha preferentially inhibited the proliferation of more mature erythroid progenitors. In a separate set of experiments, MIP-1 alpha, TGF-beta, and TNF-alpha were added to cultures of total CD34+ cells purified from fetal liver. In keeping with the fact that the majority of the progenitors contained in these cells were erythroid progenitors, the inhibitory effects of the three cytokines were similar to those observed in cultures of CD34+ CD45RAlow CD71+ cord blood cells. The results of the present study demonstrate that MIP-1 alpha, TGF-beta, and TNF-alpha have the capacity to modulate cytokine-induced proliferation of cord blood and fetal liver progenitors. The differential effects of these three cytokines confirm their pleiotropic nature as regulators of hematopoiesis.

Antigens, CD↗
PubMed1995

Proliferation of individual hematopoietic progenitors purified from umbilical cord blood.

In previous studies, we described long-term cultures of subpopulations of CD34+ cord blood cells that were fractionated using antibodies specific for CD45RA and CD71. In the present study, we plated the most primitive CD34+CD45RAlowCD71low cells individually and analyzed the proliferation and expansion kinetics of the various hematopoietic progenitors included in this subpopulation. The proliferation capacity of single progenitors was assessed by the total number of cells produced, and their expansion capacity was assessed by the production of CD34+ and colony-forming cells. Monolineage progenitors (erythroid, granulocyte, and macrophage progenitors) showed the lowest expansion capacity, which correlated with a relatively short life span (< 37 days) of the generated colonies. Among these cells, erythroid progenitors showed the highest proliferative potential. Based on their relative cell content and proliferation/expansion capacities, two types of both granulomacrophagic (GM) and multipotent (Mix) progenitors were identified. GM progenitors producing colonies with > 70% granulocytes had a low expansion capacity and gave rise to colonies that were sustained for < 42 days. A second type of GM progenitors, giving rise to colonies with > 90% macrophages, had a higher expansion capacity and the colonies they produced were sustained for > 72 days. On the other hand, Mix progenitors producing colonies with > 70% erythroid cells had a lower expansion capacity and life span (< 46 days in culture), than Mix progenitors producing colonies with > 90% myeloid cells (life span of > 72 days). These results demonstrate, at the single-cell level, a correlation between the type of progenitor cell and its expansion capacity. In turn, the expansion capacity of the progenitors was correlated with the life span of the colonies produced. Furthermore, our results suggest that in cord blood, at least two types of bipotential myeloid (GM) progenitors and two types of multipotent (Mix) progenitors can be distinguished, that have respectively low and high proliferative and expansion capacity.

Antigens, CD↗
PubMed1994

Expression of interleukin-1 beta gene in candidate human hematopoietic stem cells.

Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect interleukin-1 beta (IL-1 beta) mRNA in candidate human hematopoietic stem cells. The cells, obtained from adult bone marrow (BM) or umbilical cord blood, had a CD34+ CD45RAlo CD71lo phenotype and were further fractionated into CD38+ and CD38- or Thy-1+ and Thy-1- subpopulations. The purity of these fractions was always more than 99%. IL-1 beta and CD34 mRNA were detected in pools of 30 BM-derived CD34+ CD45RAlo CD71lo cells. To further exclude any contribution by contaminating cells, individual cells were analyzed for CD34 and IL-1 beta mRNA. Positive results were obtained with 2 of 5 individual BM-derived CD34+ CD45RAlo CD71lo CD38+ cells isolated by micromanipulation after overnight culture in serum-free medium without any exogenous cytokines, and 1 of 10 individual CD34+ CD45RAlo CD71lo CD38- cells isolated immediately after sorting. Moreover, of 10 pools of three BM-derived CD34+ CD45RAlo CD71lo cells cultured overnight in the presence of a mixture of various cytokines (Steel factor, IL-3, IL-6, macrophage colony-stimulating factor [M-CSF], erythropoietin, and IL-3/granulocyte-macrophage colony-stimulating factor [GM-CSF] fusion protein), 5 were positive for IL-1 beta mRNA. This result was compatible with more than 20% (95% confidence limit 0.06-0.61) of the BM cells with the CD34+ CD45RAlo CD71lo phenotype expressing IL-1 beta mRNA. IL-1 beta expression was also consistently observed from day 0 to day 9 in liquid cultures of cord-blood-derived CD34+ CD45RAlo CD71lo Thy-1+ or Thy-1- cells. The cultures contained the same combination of cytokines and resulted in an expansion of cell numbers of up to 400-fold. GM-CSF mRNA was not detected in the equivalent of 75 cells at any day, even though it could be detected with high sensitivity in control stromal cells. Because IL-1 beta is a powerful and pleiotropic biomodulator of cytokines and adhesion molecules, our observations suggest that at least some primitive hematopoietic cells do not merely respond passively to signals from their environment, but may themselves regulate the paracrine production of cytokines from neighboring stromal cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Antigens, CD↗
PubMed1994

Thy-1 expression is linked to functional properties of primitive hematopoietic progenitor cells from human umbilical cord blood.

We have previously shown that the most primitive human hematopoietic cells are included within a cell subpopulation expressing high levels of CD34 and low or undetectable levels of CD45RA and CD71. In this study, cord blood cells with this phenotype were sorted and further separated based on their expression on the Thy-1 antigen. The proliferation and differentiation of the purified cell fractions in response to a mixture of hematopoietic cytokines was analyzed in serum- and stroma-free liquid cultures. Thy-1+ cells (25% of CD34+ CD45RAlo CD71lo cells) were particularly enriched for high proliferative potential colony-forming cells (HPP-CFC; up to 45% of the clonogenic cells), whereas Thy-1- cells were enriched for multipotential colony-forming cells (CFU-MIX; up to 46% of the clonogenic cells). When both subpopulations were cultured in serum-free liquid cultures supplemented with a cytokine mixture that included steel factor, interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF)/IL-3 fusion protein, M-CSF, G-CSF, and erythropoietin, Thy-1+ cells showed a much higher numerical expansion of CD34+ cells (30,000-fold) and colony-forming cells (4,700-fold) than was observed in cultures initiated with Thy-1- cells (900-fold increase in CD34+ cell numbers and 241-fold increase in CFC numbers). Cells coexpressing CD34 and Thy-1 were only transiently expanded (up to 29-fold) and were not detected after day 22 of culture. When CD34+ CD45RAlo CD71lo Thy-1+ cells were cultured, either in semi-solid or liquid cultures, in the presence of anti-Thy-1 antibody, a significant reduction in progenitor cell numbers (particularly HPP-CFC) was observed. In contrast, CD34+ CD45RAlo CD71lo Thy-1- cells were not affected by anti-Thy-1. The results of this study indicate that Thy-1 is expressed on primitive cord blood progenitors with the highest in vitro proliferative potential, and further suggest that Thy-1 is involved in hematopoietic cell development, possibly by mediating a negative signal that results in inhibition of primitive cell proliferation.

Antibodies↗
PubMed1994

Age-related decline in proliferative potential of purified stem cell candidates.

Recent studies in our laboratory have shown striking differences in the functional properties of candidate hematopoietic stem cells purified from fetal, neonatal, and adult human tissues. These differences include the ability to produce CD34+ cells, the turnover rate, and the fraction of cells that respond to a mixture of cytokines. All these parameters decrease with the age of the cell donor, and some of these observations are summarized here. Extensive qualitative changes in hematopoietic cells from various stages of development should be taken into account in the design of novel therapeutic strategies.

Adult↗
PubMed1993

Characterization of functionally distinct subpopulations of CD34+ cord blood cells in serum-free long-term cultures supplemented with hematopoietic cytokines.

We have previously identified, based on the expression of the CD45RA and CD71 antigens, three major subpopulations of CD34+ cells derived from human umbilical cord blood: CD34+ CD45RAloCD71lo cells (up to 42% multipotent progenitors), CD34+ CD45RA+ CD71lo cells (90% myeloid progenitors), and CD34+ CD45RAloCD71+ cells (70% erythroid progenitors). In the present study, we have investigated the long-term proliferation and differentiation of these subpopulations in response to hematopoietic cytokines. Cells from each subpopulation were cultured for 38 days in serum- and stroma-free liquid cultures supplemented with cytokine combinations that favor either erythropoiesis or myelopoiesis. In keeping with their high content of primitive progenitors, CD34+ CD45RAloCD71lo cells showed the highest CD34+ cell expansion (up to 532-fold) throughout the culture period, followed by CD34+ CD45RA+ CD71lo (130-fold) and CD34+ CD45RAloCD71+ (28-fold) cells. Interestingly, the cytokine combination favoring myelopoiesis was always more efficient in inducing CD34+ cell expansion than the one favoring erythropoiesis. In all but one of the cultures, a predominance of myelopoiesis was observed after 2 weeks, even in those supplemented with the cytokine mixture that favors erythropoiesis. Only when CD34+ CD45RAloCD71+ cells were cultured in the presence of erythroid cytokine mixture, erythropoiesis was evident at all time points. However, such cultures could be sustained for only 29 days. The results of this study demonstrate that the cord blood-derived CD34+ cell compartment consists of functionally distinct cell subpopulations that possess different proliferative capacities in vitro. Our results also show that the cytokine combinations used here were able to modulate proliferation and, to a much lesser extent, differentiation of such subpopulations, probably by favoring the expansion of committed progenitors rather than by acting on uncommitted cells.

Antigens, CD↗
PubMed1993

Ontogeny-related changes in proliferative potential of human hematopoietic cells.

Blood cells originate from hematopoietic stem cells that are located at different sites during ontogeny. Production of human stem cells and their progeny in culture is expected to have important implications for experimental therapeutic strategies involving gene transfer and transplantation. Here we report striking differences between primitive hematopoietic cells purified from adult bone marrow, umbilical cord blood, and fetal liver in cytokine-supplemented, serum-free cultures. In such cultures both the fraction of responding cells and their ability to produce CD34+ progenitor cells decreased markedly with the age of the cell donor. These results document extensive, ontogeny-related functional differences between primitive hematopoietic cells.

Adult↗
PubMed1993

Cytokine-induced selective expansion and maturation of erythroid versus myeloid progenitors from purified cord blood precursor cells.

To study the role of different cytokine combinations on the proliferation and differentiation of highly purified primitive progenitor cells, a serum-free liquid culture system was used in combination with phenotypic and functional analysis of the cells produced in culture. CD34+ CD45RAlo CD71lo cells, purified from umbilical cord blood by flow cytometry and cell sorting, were selected for this study because of their high content of clonogenic cells (34%), particularly multipotent progenitors (CFU-MIX, 12% of all cells). Four cytokine combinations were tested: (1) mast cell growth factor (MGF; a c-kit ligand) and interleukin-6 (IL-6); (2) MGF, IL-6, IL-3, and erythropoietin (Epo); (3) MGF, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF)/IL-3 fusion protein (FP), macrophage colony-stimulating factor (M-CSF), and granulocyte-CSF (G-CSF); and (4) MGF, IL-6, FP, M-CSF, G-CSF, and Epo. Maximum numbers of erythroid progenitors (BFU-E, up to 55-fold increase) and mature erythroid cells were observed in the presence of MGF, IL-6, IL-3, and Epo, whereas maximum levels of myeloid progenitors (CFU-C, up to 70-fold increase) and mature myeloid cells were found in cultures supplemented with MGF, IL-6, FP, M-CSF, and G-CSF. When MGF, IL-6, FP, M-CSF, G-CSF, and Epo were present, maximum levels of both erythroid and myeloid progenitors and their progeny were observed. These results indicate that specific cytokine combinations can act directly on primitive hematopoietic cells resulting in significant expansion of progenitor cell numbers and influencing their overall patterns of proliferation and differentiation. Furthermore, the observations presented in this study suggest that the cytokine combinations used were unable to bias lineage commitment of multipotent progenitors, but rather had a permissive effect on the development of lineage-restricted clonogenic cells.

Antigens, CD↗
PubMed1993

Lineage commitment in human hemopoiesis involves asymmetric cell division of multipotent progenitors and does not appear to be influenced by cytokines.

Different models have been proposed to explain lineage commitment in hemopoiesis. Some suggest that lineage commitment occurs in a stochastic manner without the direct influence of extracellular factors; others postulate that cytokines determine whether multipotent cells will become erythroid or granulocyte/macrophage progenitors. In the present study, the patterns of proliferation and differentiation of individually sorted human cord blood-derived primitive hemopoietic cells (highly enriched for multipotent progenitors) were analyzed in a serum-free culture system supplemented with different cytokine combinations. In a first set of experiments, the response of individual cells to different cytokine combinations was compared, whereas in a second set of experiments, single cells were allowed to undergo one division after which the two daughter cells were physically separated and cultured in either the same or different cytokine combinations. Proliferation of progenitor cells was absolutely dependent on cytokines, and the combination of mast cell growth factor plus interleukin 6 was sufficient to induce mitosis. When cytokine combinations favoring erythropoiesis and/or myelopoiesis were added to the cultures, a more vigorous proliferative response of the sorted primitive progenitors was observed. Interestingly, the relative proportions of granulocyte/macrophage, erythroid, and multipotent progenitors remained more or less the same regardless of the cytokine combination used, indicating a permissive rather than an instructive role for cytokines in hemopoietic differentiation. Asymmetric cell divisions, defined as a division that yields two daughter cells with distinct functional properties, were observed in 3-17% of the progenitor cells capable of forming colonies under our experimental conditions. In the rest, symmetric divisions involving multipotent and lineage-committed progenitors were observed. The results of this study demonstrate that the asymmetric cell divisions that occur in the early stages of hemopoiesis at the level of multipotent progenitors cannot be skewed by the addition of specific cytokine combinations. These findings support the hypothesis that lineage commitment in hemopoiesis occurs in a stochastic manner by mechanisms that remain to be elucidated.

Antigens, CD↗
PubMed1993

Human preleukemia: cellular, molecular and clinical aspects.

In a significant proportion of patients with acute myeloid leukemia (AML), a series of hematological alterations--including refractory anemia, neutropenia, thrombocytopenia, abnormal iron metabolism, and elevated levels of blast cells both in peripheral blood and bone marrow--are observed before the diagnosis of AML is made. This preleukemic state has called the attention of several investigators around the world, since it represents a way to study the origin and progression of leukemia in man. During the past 5 years, major advances in the molecular and cellular biology of this disease have been achieved. It is now known that preleukemia is a clonal disorder that arises from a malignant transformation at the level of primitive pluripotent hemopoietic stem cells. The hemopoietic progenitors in preleukemic patients have abnormal responses to hemopoietic regulators, thus, they do not seem to follow the controlled proliferation observed in the hemopoietic system under normal conditions. The mechanisms of cell differentiation and maturation are also altered, leading to the production of immature (blast) cells, instead of the development of fully mature erythrocytes, granulocytes, platelets and lymphocytes. Several oncogenes, such as C-FMS and RAS, have been found to be structurally altered in a significant proportion of preleukemic patients, suggesting that they may be involved in the pathogenesis of the disease. In spite of the advances made during the last few years, major questions regarding the biology of this hematological disorder are still unanswered.(ABSTRACT TRUNCATED AT 250 WORDS)

Humans↗
PubMed1993

Functional characterization of fibroblastic cells in long-term marrow cultures from patients with acute myelogenous leukemia.

We have investigated the role of acute myelogenous leukemia (AML) marrow-derived fibroblastic cells in the development of the hemopoietic microenvironment in vitro, and compared their functional integrity with that of their normal marrow-derived counterparts. Our results indicate that the development of stromal adherent layers in AML long-term marrow culture (LTMC) depends to a large extent on the presence of fibroblastic progenitors (fibroblast colony-forming units, CFU-F) in the bone marrow inoculum. Positive correlations were observed between the numbers of CFU-F in fresh AML marrow samples (nine cases studied) and the numbers of adherent nucleated cells (r = 0.91), adherent CFU-F (r = 0.87), adherent hemopoietic progenitors (r = 0.71), and colony-stimulating factor-1 (CSF-1) levels (r = 0.74) in LTMC. All the above parameters were drastically reduced in LTMC derived from AML marrows that contained low numbers of CFU-F (i.e. below the normal range of 76-130 CFU-F per 10(6) bone marrow cells) when compared with LTMC established from normal marrows. In contrast, no difference was observed in such parameters when normal LTMC and LTMC from AML patients with normal levels of marrow CFU-F were compared. Fibroblast adherent layers derived from AML patients with normal marrow CFU-F numbers produced normal levels of CSF-1 and showed a similar hemopoietic supportive capacity to that of fibroblast layers from normal bone marrow. Thus, our results suggest that (i) the presence of CFU-F in adequate levels in the bone marrow inoculum is critical for the adequate development of the hemopoietic microenvironment in LTMC, and (ii) AML marrow-derived fibroblasts seem to be functionally normal, at least in some AML patients.

Bone Marrow↗
PubMed1992

Biology of the hemopoietic microenvironment.

In adult mammals, hemopoiesis takes place primarily in the bone marrow. The steady-state production of blood cells depends to a large extent on the interaction between hemopoietic stem/progenitor cells (HPC) and the different components of the microenvironment present in the medullary cavity. During the last three decades, in vivo and in vitro studies have allowed significant advances in understanding of the biology of such a hemopoietic microenvironment. Although not evident in histological sections, it is well known that the hemopoietic microenvironment is a highly organized structure that regulates the location and physiology of HPC. The hemopoietic microenvironment is composed of stromal cells (fibroblasts, macrophages, endothelial cells, adipocytes), accessory cells (T lymphocytes, monocytes), and their products (extracellular matrix and cytokines). Microenvironmental cells can regulate hemopoiesis by interacting directly (cell-to-cell contact) with HPC and/or by secreting regulatory molecules that influence, in a positive or negative manner, HPC growth. Recent in vitro studies suggest that functional abnormalities of the hemopoietic microenvironment may be implicated in the manifestation of certain hematological disorders such as aplastic anemia, and acute and chronic myelogenous leukemia. Thus, the characterization of the structure and function of the human hemopoietic microenvironment may have relevance in understanding and treating different hematological disorders.

Animals↗
PubMed1992

Production of tumor necrosis factor-alpha in human long-term marrow cultures from normal subjects and patients with acute myelogenous leukemia: effect of recombinant macrophage colony-stimulating factor.

We have recently reported that normal long-term marrow cultures (LTMC) treated with recombinant human macrophage colony-stimulating factor (rhCSF-1), as well as LTMC from patients with acute myelogenous leukemia (AML), produce a soluble activity capable of inhibiting hemopoietic colony formation in semisolid cultures. In the present study, we have found that such an activity is produced, both in normal and AML LTMC, by an adherent, nonfibroblastic cell population (most likely macrophages), and also by blast cells developed in AML LTMC. The presence of the inhibitory activity correlated with increased levels of tumor necrosis factor (TNF) in the culture supernatants. Part of the activity (30%) produced in rhCSF-1-treated normal LTMC was neutralized in colony assays by anti-TNF alpha monoclonal antibody. In contrast, the soluble inhibitory activity from AML LTMC was completely neutralized by anti-TNF alpha. However, addition of anti-TNF alpha (every 72 h, from day 0 to 21, at 125 ng/ml) to AML LTMC resulted in only partial neutralization of the inhibitory activity, indicating that production of TNF alpha is just one of the mechanisms by which normal hemopoiesis is inhibited in AML LTMC, and that other factors are involved in this process. In keeping with this idea, we found very high levels of prostaglandin E, a hemopoietic inhibitor, in the supernatant of cultures that contained the soluble inhibitory activity. Interestingly, rhCSF-1 showed opposite effects on TNF production in normal (up-regulation) and AML (down-regulation) LTMC, which suggests the presence of functionally abnormal, leukemia-derived macrophages in AML LTMC.

Bone Marrow↗
PubMed1992

Composition and functional integrity of the in vitro hemopoietic microenvironment in acute myelogenous leukemia: effect of macrophage colony-stimulating factor.

In the present work, we have investigated the composition and hemopoietic supportive capacity of eleven normal and six acute myelogenous leukemia (AML) marrow-derived stromal adherent layers, established in the absence or in the presence of recombinant human colony-stimulating factor 1 (rhCSF-1, macrophage colony-stimulating factor). Two of six AML adherent layers were deficient in composition (i.e., no confluency, reduced numbers of macrophages and fibroblastic progenitors, and no fat cell formation), resulting in reduced CSF-1 production and a poor hemopoietic supportive capacity (assessed by the ability of an irradiated stroma to sustain the growth of myeloid, erythroid, and multipotential progenitors derived from a second innoculum of normal bone marrow). Four out of six AML adherent layers showed levels of macrophages, fibroblastic progenitors, fat cells, and CSF-1 similar to those observed in adherent layers from normal bone marrow; however, their capacity to sustain normal hemopoiesis was still significantly reduced. The deficient hemopoietic supportive capacity of all AML adherent layers correlated with the presence of a soluble activity in the culture supernatant that inhibited hemopoietic colony formation. Addition of rhCSF-1 during the establishment of AML adherent layers significantly increased their hemopoietic supportive capacity. In contrast, the hemopoietic supportive capacity of normal adherent layers was reduced by rhCSF-1. The opposite effects of rhCSF-1 on the hemopoietic supportive capacity of normal and AML adherent layers correlated with the levels of the soluble inhibitory activity, that is, increased levels in cultures containing rhCSF-1-treated normal adherent layers, and slightly reduced levels in cultures of rhCSF-1-treated AML layers. These results indicate that, despite a morphologically normal composition in most cases (four out of six), the hemopoietic microenvironment developed in long-term marrow culture (LTMC) from all AML marrows analyzed has a deficient hemopoietic supportive capacity, due, at least in part, to the production of hemopoietic inhibitor(s). Such a deficiency can be partially overcome by establishing the stroma layers in the presence of rhCSF-1.

Bone Marrow↗