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A mixture of stromal cells from murine bone marrow placed upon cellulose ester membranes (CEM) and then implanted intraperitoneally (i.p.) in mice results in a regenerated hematopoietic microenvironment which supports trilineal hematopoiesis. We used this model to study the capacity of 5 cloned murine stromal cell lines of marrow origin to support hematopoiesis in vivo: MBA-1 (fibroblast); MBA-2 (endothelial); MBA-13 (fibroendothelial); 14F1.1 (endothelial-adipose); and 14M1.4 (macrophage).10(7) stromal cells of a single cell line were applied to 1.5 cm2 CEM, which were folded into tubes and implanted i.p. into mice. Similarly, combinations of 4, 3 and 2 stromal cell lines were applied to CEM and implanted i.p. Single lines were implanted into syngeneic hosts of the same murine strain from which the clone was derived and into nude mice. Combinations of stromal cells were implanted only in nude mice to avoid allogeneic incompatibility. CEM implants were removed after intervals of 5 to 36 weeks and examined histologically. 1) Stromal cells of a single phenotype did not develop hematopoiesis. 2) A combination of 4 stromal phenotypes (MBA-1, MBA-2, MBA-13 and 14F1.1) formed a hematopoietic microenvironment supportive of trilineal hematopoiesis and bone. 3) The combination of 14F1.1 (endothelial adipose) + a second stromal phenotype--MBA-1 (fibroblast) or MBA-2 (endothelial) or MBA-13 (fibroendothelial) also supported trilineal hematopoiesis and bone. 4) CEM coated with MBA-13 or MBA-1 developed bone but no hematopoiesis. The endothelial-adipose phenotype appears to be essential to support hematopoiesis but requires other types of stromal cells--fibroblast, fibroendothelial or endothelial phenotype.
Although IL-12 has been reported to synergize with c-kit ligand (KL) in promoting hematopoietic stem cell proliferation in vitro, administration of recombinant mouse IL-12 (rIL-12) to normal mice caused a dose- and time-dependent anemia, leukopenia, and thrombocytopenia in vivo. Decreased numbers of bone marrow cells were recovered from the tibiae of IL-12-treated mice, and histologic examination of the marrow revealed a loss of mature neutrophils and red blood cell precursors. However, simultaneously with the suppression of hematopoiesis in the bone marrow, the IL-12-treated mice developed splenomegaly, which was largely caused by a marked enhancement of splenic extramedullary hematopoiesis of the erythroid, myeloid, and megakaryocytic lineages. These histologic observations were confirmed by colony-forming cell assays in which administration of IL-12 was shown to cause a time-dependent decrease in bone marrow CFU-GM, CFU-E, and BFU-E hematopoietic colony-forming cells while causing an increase in splenic CFU-GM and BFU-E colony-forming cells. All these effects were reversible upon cessation of IL-12 treatment. The observation that in IL-12-treated mice hematopoiesis was suppressed in the marrow but enhanced in the spleen suggests that myelosuppression was not caused by a direct effect of IL-12 on hematopoietic progenitors. It seems likely that myelosuppression was caused instead by an IL-12-induced alteration in the local environment of the marrow.
A cellular model of hematopoiesis which would be more convenient than bone marrow (BM) progenitors and directly relevant to human pathology is needed in order to investigate xenobiotic toxicity. Human umbilical cord blood (HCB), previously shown to be able to repopulate BM, provides a powerful in vitro model of normal human hematopoiesis. In order to validate the use of normal HCB progenitors as targets for dose-related myelosuppression, we used clonogenic assays and expansion in a liquid culture of progenitor-enriched cell suspensions from HCB. A series of 8 reference molecules, doxorubicin, cytosine-arabinoside, 5-fluorouracil, 3'-azido-3'-deoxythymidine, acetylsalicylic acid, sodium valproate and two cephalosporin antibiotics, were tested. In vitro 50% inhibition concentrations (IC50) were compared to those observed or reported with BM progenitors, and to the values of plasma concentrations from treated patients. HCB progenitors as in vitro targets for cytotoxic molecules were easy to access and handle, and their use was sensitive, specific and reproducible. They gave results similar to BM progenitors and allowed a qualitative approach to cellular metabolism and toxicity using morphological, flow cytometric and chromatographic methods.
The aim of the present study was to analyze whether or not leukemic clonogenic cells are restricted to the CD34+ cell fraction and to investigate the effect of IL-3 and G-CSF on blast cell populations dissected according to their CD34 reactivity. For this purpose 34 patients were studied. Patients were classified into three groups according to CD34 antigen expression: (1) cases in which all blast cells (100%) were positive for the CD34 Ag (n = 9); (2) cases in which all blast cells lacked the expression of this antigen (n = 10); and (3) patients in whom both, CD34 positive and negative blast cell subsets coexisted (n = 15). In 15 cases immunomagnetic cell selection was performed and two subpopulations were separated: one, phenotypically more immature (CD34+), and another, theoretically more differentiated (CD34-/33+). In addition, in three cases both CD34+ and CD34- blast cell subpopulations were sorted using a FACStar flow cytometer. Blast colony assays were performed using 0.9% methylcellulose and two different recombinant human hematopoietic growth factors (HGFs), IL-3 and G-CSF, were used as growth stimulants. Either, a single or a combination of the growth factors was added to cultures. Colony formation was observed in both 100% positive or 100% negative cases for the CD34 antigen as well as in the CD34+ and CD34- cell fractions separated by immunomagnetic selection or flow cytometry. The effect of G-CSF and IL-3 on both cell fractions was as follows: cases with a uniform population according to CD34 expression (100% positive or negative) showed a better growth response with IL-3 especially for the CD34+ cases (87% vs 40% of CD34+ and CD34- cases, respectively). Within the CD34-/33+ selected fractions, IL-3 tended to induce a higher proliferative response than G-CSF while the opposite was found within the CD34+ cell selected fractions. In contrast it was observed that both IL-3 and G-CSF induced a higher PE on the CD34- blast cells (both selected and 100% negative), although the difference was not statistically significant. The existence of a possible synergistic effect (SE) between HGFs was also explored. Overall, a synergistic growth was observed in nine out of the 13 selected cases studied and this effect could be seen in both CD34- or CD34+ blast cell fractions. The analysis of the complete phenotypic characteristics of these cells revealed that cell fractions showing SE were more immature according to the expression of CD15 and HLA-DR antigens. We can conclude that in leukemic hematopoiesis, CD34 antigen expression does not have the same significance as it does in normal hematopoiesis since clonogenic cells are not restricted to the CD34+ acute myeloid leukemia (AML) blast cell fraction. Moreover, our study shows that the heterogeneous response to HGFs observed in AML patients may be associated with the existence of immunophenotypically different blast cell subsets.
The enhancement of in vitro human hematopoiesis by the addition of a noncytotoxic monoclonal antibody, 9.1C3, is described. Enhancement of all aspects of in vitro hematopoiesis was observed on addition of 9.1C3 antibody to cultures of mononuclear cells from normal bone marrow, cord blood, and peripheral blood from beta-thalassemia major patients. In cultures with no exogenous colony-stimulating factor (CSF), the addition of 9.1C3 resulted in a two- to eightfold increase in nonerythroid colony formation. Similarly, for cultures maximally stimulated with CSF, the addition of 9.1C3 antibody resulted in a one- to fourfold increase in colony formation. These effects were abrogated by the removal of either adherent, Leu-M3+ or Leu-7+ cells. Colony-forming cells were shown to be present among the 9.1C3-negative cells when mononuclear cells were sorted by flow cytometry. Media conditioned in the presence of 9.1C3 and mononuclear cells were able to enhance colony formation in vitro for normal nonadherent bone marrow cells beyond that achieved with supramaximal amounts of human placental-conditioned medium and erythropoietin. The data suggest that natural killer cells interact with monocytes to exert a negative regulatory control on in vitro granulopoiesis and erythropoiesis. Consequently, the number of progenitor and multipotential cells in cultures of unfractionated cell populations may be greatly underestimated.
CAMAL (common antigen of myelogenous acute leukemia) is an antigenic preparation isolated in this laboratory from the bone marrow or peripheral blood leucocytes of persons with myeloid leukemias and shown using an immunoperoxidase slide test to be diagnostic of these leukemias. Material further purified from CAMAL preparations, which migrates in the range of 30-35 kilodaltons (kDa) by sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE), which is referred to as P30-35 CAMAL, and was previously shown to be inhibitory to colony formation by progenitor cells from normal healthy human donors in vitro. This inhibitory activity was directed toward neutrophilic granulocyte colonies (CFU-G) in particular. We now report that P30-35 CAMAL is inhibitory to colony formation by murine progenitor cells in vitro. Colonies from P30-35 CAMAL-treated cultures of murine bone marrow cells were reduced in number and in size, an effect similar to that seen in cultures of human cells. As in assays using human cells, murine CFU-G appeared to be preferentially targeted by the inhibitory activity of P30-35 CAMAL. In addition, day 10 spleen colony formation was inhibited by P30-35 CAMAL in an ex vivo assay. Hence, the effects of P30-35 CAMAL on murine progenitor cells appear to parallel the effects observed using human cells. These observations support the possibility that CAMAL might be a regulatory protein in hematopoiesis which is conserved between species.
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The ultrastructure of developing osseous and hematopoietic tissue and supporting stroma was examined within intraperitoneally implanted, marrow-coated cellulose ester membranes (CEMs). During initial periods of implantation (two weeks), coated CEMs were shallowly infiltrated and surface-lined with mainly two stromal cell types--primitive mesenchymal cells and large, pleomorphic, multinucleated monocytoid-like stromal cells--and, in addition, endothelial cells in early stages of vessel development. Selective proliferation, maturation, and orientation of these cell types along the CEM's surface (one month), resulted in the formation of primitive osseous and hematopoietic tissue sites. Osseous sites developed as undifferentiated mesenchymal cells transformed into well-differentiated secretory cells residing in an electron-dense, pre-mineralized extracellular matrix, that upon extended implantation (3-6 months) formed bone. Hematopoietic sites developed as mesenchymal cells, in intimate contact with the monocytoid-like stromal cells, extended elongated branches into the medullary cavity, and enveloped newly formed surface-associated vascular structures. With subsequent maturation of vascular sinuses and supporting adventitial stroma (3-6 months), the sites were colonized with either uni-, bi-, or trilineal hematopoietic elements. These observations provide evidence that common, marrow-derived precursor stromal cells, i.e., mesenchymal and multinucleated monocytoid-like cells, cooperate in the development of both osseous and hemic tissue sites.
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The paper discusses the mechanisms of hemopoiesis regulation on the models of bone marrow hypoplasia induced by cyclophosphane, adriamycin, sublethal and lethal X-ray radiation followed by bone marrow transplantation. The findings suggest that there is the unique complexly organized system of local hemopoiesis regulation in the body (cell elements of the hemopoiesis-inducing microenvironment and their produced humoral factors) whose functional activity increases naturally under the action of different myelotoxic agents. The specific features of hemopoiesis-inducing microenvironmental functioning are largely determined by the extent and specificity of the damaging action of an agent on some individual components of the local regulation regulatory system.
Essential thrombocythemia (ET) is a myeloproliferative disorder, characterized by sustained thrombocytosis. Diagnosis requires the elimination of all known causes of thrombocytosis. ET is believed to be a clonal disorder, and we investigated the frequency of a clonal hematopoiesis in this disease with the aim of using this as a positive diagnostic criterion. However, a non-random inactivation pattern can be encountered in normal females which mimics clonal hematopoiesis. In addition, the percentage of normal females with skewed lyonization seems higher using techniques based on the difference in DNA methylation, compared to G6PD enzyme polymorphism. Recently, new techniques based on transcript analysis have been developed. We report here the results of clonality studies of hematopoiesis in 53 ET patients using two different techniques based on DNA and RNA polymorphisms, and T-lymphocytes as a control tissue of lyonization. The majority of ET patients showed monoclonal hematopoiesis in the presence of polyclonality of T-lymphocytes. Because all ET patients did not show the same clonal pattern of hematopoiesis, we searched for inappropriate secretion of thrombopoietin (TPO) in patients with polyclonal disease. This assay was performed in 48 patients, of whom 9 showed polyclonal hematopoiesis and 27 monoclonal hematopoiesis. We found no difference in TPO levels between ET patients and normal controls, nor between patients with polyclonal hematopoiesis and those with monoclonal hematopoiesis. Our results confirm the high frequency of monoclonal hematopoiesis in ET, the usefulness of RNA markers, and the possibility of using T-lymphocytes as a control tissue for X-chromosome inactivation patterns. On the other hand, TPO levels are not decreased even in ET patients with high platelet counts, suggesting an increased production or decreased clearance of TPO in this disease.
Cross-linking of major histocompatibility complex (MHC) class II antigens by anti-HLA-DR monoclonal antibody (MoAb; H81.9; IgG2a) results in inhibition of hematopoiesis in canine and human models. Inhibition of hematopoiesis is associated with apoptosis in a proportion of marrow cells. Since in murine macrophages class II cross-linking triggers nitric oxide (NO) production, and NO is thought to affect regulation of hematopoiesis, we investigated whether NO was involved in our models. In murine J774 monocytes/macrophages, MoAb H81.9 did induce NO. NO production was blocked by N(G)-monomethyl-L-arginine (NMMA), an inhibitor of NO synthase (NOS), and by the antioxidant N-acetylcysteine (NAC). In human and canine long-term marrow cultures (LTMCs) and in enriched marrow monocytes, however, no measurable increase in NO production was noted after H81.9 exposure. Nevertheless, NAC protected LTMCs against H81.9 induced inhibition of hematopoiesis. Therefore, we determined the effect of an exogenous NO donator, sin-1 (3-morpholinosydnonimine), on canine and human LTMCs and on apoptosis. Sin-1 at concentrations > or =100 microg/mL inhibited LTMCs and induced apoptosis; at low concentrations (1 microg/mL), however, sin-1 stimulated the generation of colony-forming unit granulocyte-macrophage. Combined treatment with sin-1 at 100 microg/mL and MoAb H81.9 resulted in profound inhibition of hematopoiesis in both canine and human LTMCs, and had an additive effect on apoptosis. At 1 microg/mL sin-1 counteracted the effect of H81.9 on hematopoiesis. The effect of sin-1 on apoptosis and hematopoiesis in LTMC was largely prevented by NAC. These results are consistent with the hypothesis that HLA-DR mediated apoptosis and inhibition of hematopoiesis involve oxidative stress. However, the biphasic response of hematopoiesis to sin-1 suggests a complex regulatory network possibly related to differences in NO sensitivity of distinct subpopulations of cells. Signals in addition to NO appear to be involved in the effect of anti-HLA-DR MoAb on hematopoiesis.
BACKGROUND: Extramedullary hematopoiesis is a response of the organism to a deficient production of blood-forming cells within the bone marrow. It may coincide with some hematologic diseases. Two patients with paravertebral mass lesions representing extramedullary hematopoiesis are discussed. Characteristic findings of extramedullary hematopoiesis are presented along with a review of the literature on this topic. CASE REPORTS: A 76-year-old male with a known myelodysplastic syndrome presented with pneumonia. In addition, he had symptoms of a cauda equina syndrome with complaints of pain and hypesthesia of the lower limbs as well as urinary retention. A 63-year-old female presented with aggravated complaints of the lower thorax, low back pain radiating to the upper left leg, and dysesthesia of both feet. In her past medical history, she had polycythemia vera and a splenectomy. Both patients showed paravertebral and intraspinal lesions located in the thoracic and sacral spine which were regarded as extramedullary hematopoiesis according to the imaging findings in MRI and CT. Radiation therapy showed marked improvement in their neurologic complaints following the initial sessions. CONCLUSION: Clinical presentation, knowledge of the underlying disease and of imaging findings are essential in the diagnosis of extramedullary hematopoiesis. MRI is the imaging modality of choice in the primary diagnosis of extramedullary hematopoiesis. Possible extension of the disease into the intraspinal space can be evaluated with high accuracy and differential diagnosis can be facilitated. In addition, MRI is of use in the accurate planning of radiation fields as well as during follow-up of extramedullary hematopoiesis.
Signaling by vascular endothelial growth factors (VEGFs) through VEGF receptors (VEGFRs) plays important roles in vascular development and hematopoiesis. The authors analyzed the function of VEGF-C signaling through both VEGFR-2 and VEGFR-3 in vasculoangiogenesis and hematopoiesis using a coculture of para-aortic splanchnopleural mesoderm (P-Sp) explants from mouse embryos with stromal cells (OP9). Vasculogenesis and angiogenesis were evaluated by the extent of vascular bed and network formation, respectively. Addition of VEGF-C to the P-Sp culture enhanced vascular bed formation and suppressed definitive hematopoiesis. Both vascular bed and network formations were completely suppressed by addition of soluble VEGFR-1-Fc competitor protein. Formation of vascular beds but not networks could be rescued by VEGF-C in the presence of the competitor, while both were rescued by VEGF-A. VEGFR-3-deficient embryos show the abnormal vasculature and severe anemia. Consistent with these in vivo findings, vascular bed formation in the P-Sp from the VEGFR-3-deficient embryos was enhanced to that in wild-type or heterozygous embryos, and hematopoiesis was severely suppressed. When VEGFR-3-Fc chimeric protein was added to trap endogenous VEGF-C in the P-Sp culture of the VEGFR-3-deficient embryos, vascular bed formation was suppressed and hematopoiesis was partially rescued. These results demonstrate that because VEGF-C signaling through VEGFR-2 works synergistically with VEGF-A, the binding of VEGF-C to VEGFR-3 consequently regulates VEGFR-2 signaling. In VEGFR-3-deficient embryos, an excess of VEGF-C signals through VEGFR-2 induced the disturbance of vasculogenesis and hematopoiesis during embryogenesis. This indicates that elaborated control through VEGFR-3 signaling is critical in vasculoangiogenesis and hematopoiesis. (Blood. 2000;96:3793-3800)