Functional disturbances in hemopoietic microenvironment in various forms of myelodysplastic syndrome

We studied the ability of stromal sublayer of long-term bone marrow cultures and peripheral blood macrophages from patients with various forms of myelodysplastic syndrome to maintain the growth of normal granulocyte-macrophage colony-forming units in mixed cultures. There were changes in the hemopoietic microenvironment in these patients: decreased cellularity of the bone marrow and impaired formation of sublayers in long-term bone marrow cultures, production of growth factors, maintaining the growth of normal granulocyte-macrophage precursors by stromal cells. Dysfunction of macrophages in the stromal microenvironment was probably related to the presence of pathological macrophages. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 130, No. 9, pp. 255–258, September, 2000     

Functional activity of hemopoietic and stromal cells in various types of myelodysplastic syndrome

Using clonal methods for assessment of hemopoietic and stromal cells and long-term bone marrow cell cultures, we have demonstrated heterogeneity of myelodysplastic syndrome. Low content of stromal precursor cells in native bone marrow, peculiarities in the formation of the stromal layer and its hemopoiesis-stimulating capacity in long-term cultures, and altered properties of stromal precursor cells in long-term cultures indicate defect in the stromal microenvironment in myelodysplastic syndrome. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 1, pp. 14–18, January, 1999     

Effect of cytokines (G-CSF and SCF) on stromal precursor cells

Repeated injections of hemopoietic cytokines (granulocyte colony-stimulating factor and stem-cell factor) to normal mice increase the content of bone marrow stromal precursors, which are capable of transferring hemopoietic microenvironment; cytokines have no effect on osteogenic potencies of stromal precursors. In contrast, injection of granulocyte colonystimulating factor during organization of a hemopoietic microenvironment considerably decreases the number of stromal precursors in the ectopic focus. The role of these stromal effects of cytokines in cytokine-induced mobilization of stem cells from the bone marrow into circulation remains unclear. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 125, No. 2, pp. 204–206, February, 1998     

Hemopoietic microenvironment-transferring units and inducible hemopoietic stromal precursors in the bone marrow of thymectomized mice

The method of heterotopic transplantation of the bone marrow was used to study the effect of thymectomy on clonogenic and inducible hemopoietic stromal precursors in adult rats. The self-maintenance or clonogenic capacity of stromal precursors was evaluated by retransplantation of primary hemopoietic foci. The kinetics of the formation of ectopic foci from thymectomized rats is similar to that of normal bone marrow. The presence of inducible stromal hemopoietic precursors was evaluated by the stimulation index (the ratio of the size of hemopoietic focus formed in irradiated to that in nonirradiated recipient). It is found that the growth of ectopic focus in chimeras is stimulated by a nonthymic factor, which suggests thymus-independent regulation of hemopoietic microenvironment precursor cells. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 2, pp. 186–189, February, 1999     

Formation of colonies of clonogenic stromal precursors in bone marrow and splenic cell cultures in the presence of streptococcal antigens in the culture medium

The presence of streptococcal M protein and A polysaccharide in culture medium is shown to have an inhibitory effect on the growth of clonogenic stromal precursors in cultures of healthy murine bone marrow and of healthy guinea pig bone marrow and spleen. The efficacy of colony formation dropped 1.5- to 2-fold in the presence of antigens in a concentration of 25 μg/ml in the medium. The inhibitory effect was absent if antigens were added to adhesive cell cultures. The addition of antigens to cultures originating from animals immunized with streptococcus resulted in inhibition of the efficacy of colony formation in complete cultures and in cultures of adhesive cells. The presence of streptococcal antigens in guinea pig stromal fibroblast cultures of different strains did not affect their growth or colony formation. These data indicate that the effects of streptococcal antigens appear to be aimed at the stromal cells not directly, but rather via another cellular category in the bone marrow and splenic cell cultures, probably lymphocytes. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o 11, pp. 489–492, November, 1994     

Stromal components in rat bone marrow frozen sections compared to long-term rat bone marrow cultures

The haematopoietic microenvironment is believed to play an important role in controlling the haematopoietic process. Ultrastructural studies have shown that the haematopoietic stroma is composed of cellular as well as extracellular components. Relatively little is known about the distribution of the different stromal components in the bone marrow. The knowledge on the bone marrow microenvironment is mainly based on studies in which in vitro long-term bone marrow cultures have been used. Although this culture system offers a unique possibility to study haematopoietic in vitro, it does not fully represent the complexity of intact bone marrow.In the present study we describe the immunohistochemical distribution of different cellular and extracellular stromal components in frozen sections of rat bone marrow as well as in long-term bone marrow cultures, in order to compare the haematopoietic microenvironment used in in vitro studies in the in situ situation. We found that in situ a specific compartmentalization of stromal components exists in the bone marrow. Under culture conditions however, most stromal components are indeed present but the architecture present in the in situ situation had almost completely disappeared. The interaction between the different stromal elements was studied in the long-term bone marrow cultures. It appeared that under the chosen conditions, nodules were formed with a core of reticular cells and extracellular matrix. In close contact with this core immature macrophages appeared to proliferate and differentiate into mature, non-dividing macrophages.     

Production of granulocytic colony-stimulating factor in patients with chronic myeloleukemia

After several passages, bone marrow fibroblasts and stromal adherent layers of a long-living bone marrow culture derived from patients with chronic myeloleukemia cannot maintain hemopoiesis in a culture for a long time. Immunofluorescent microscopy and flow cytometry showed that fibroblasts after passing differ from stromal cells of a normal long-living bone marrow culture: they do not produce granulocytic colony-stimulating factor. Adherent layers of bone marrow culture derived from patients with chronic myeloleukemia contain a far lesser number of cells producing granulocytic colonystimulating factor than normal bone marrow cells. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 126, No. 7, pp. 94–97, July, 1998     

Role of Thy 1.2+ cells in hemopoietic regulation in cytostatic-induced hemosuppression

Effect of Thy 1.2⁺ cells (direct and mediated by stromal elements) on the growth of granulomonocyte and erythroid colonies in the bone marrow is studied on CBA mice with cytostatic disease induced by single injection of adriamycin, cyclophosphamide, and 5-fluorouracil in maximum permissible doses. It is shown that Thy 1.2⁺ cells stimulate colony formation in regenerating bone marrow, the effect depending on functional activity of hemopoiesis-inducing microenvironment. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol 125, No. 5, pp. 509–513, May, 1998     

Role of Thy 1,2<Superscript>+</Superscript> Cells in the Regulation of Hemopoiesis during Experimental Neuroses

We studied the direct and stromal cell-mediated effects of bone marrow Thy 1,2⁺ cells on the growth of granulocyte-macrophage and erythroid colonies from the bone marrow of CBA/CaLac mice with experimental neuroses (conflict situation and paradoxical sleep deprivation). Proliferation and differentiation of hemopoietic precursors during neuroses are controlled by regulatory T cells. In conflict situation Thy 1,2⁺ cells stimulate the growth of hemopoietic precursors, which is associated with their direct effect and interaction with adherent cells of the hemopoiesis-inducing microenvironment. The interaction of Thy 1,2⁺ cells with adherent bone marrow cells during paradoxical sleep deprivation stimulates the formation of only granulocyte-macrophage colonies.__________Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 139, No. 6, pp. 608–612, June, 2005     

Effects of bone marrow conditioned media on proliferation of stromal clonogenic cellsin vitro

Fibroblast growth factors aFGF, bFGF, IGF-I, IGF-II, TGF-β, EGF, and PDGF did not stimulate the formation of stromal fibroblast colonies (CFC-F-colonies) in cultured murine adhesive bone marrow cells. It means that colony-stimulating activity of the bone marrow feeder cells with respect to the formation of stromal fibroblast colonies does not depend on the known growth factors. A scheme and conditions of culturing are developed for preparing conditioned media from bone marrow cell cultures which can replace the CFC-F colonystimulating activity of the bone marrow feeder cellsin vitro. Primary separation of conditioned media by ultrafiltration reveals that only the fraction with molecular weight of more than 65 kD exhibits CFC-F colony-stimulating activity. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 2, pp. 218–220, February, 1999     

Growth-inhibitory activity of bone marrow cells during CCl4-induced liver cirrhosis

During CCl₄-induced liver cirrhosis, cells of the hemopoiesis-inducing microenvironment in the bone marrow of BALB/c mice produced activity inhibiting the growth of erythropoiesis and granulomonocytopoiesis precursors. Stimulation with yeast polysaccharide zymosan increased the inhibitory activity (especially in relation to granulomonocytic precursors). The highest growth-inhibitory activity was produced by the bone marrow adherent fraction (residual bone marrow macrophages). Tumor necrosis factor-α is probably responsible for the inhibition of the growth of myeloid precursors in mice with CCl₄-induced liver cirrhosis. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 6, pp. 645–647, June, 1999     

In Vivo Production of Cytokines by Bone Marrow Stromal Cells and Macrophages from Patients with Myelodysplastic Syndrome

We studied functional disturbances in hemopoietic microenvironment and cytokine production by stromal sublayer in long-term bone marrow cultures and peripheral blood macrophages from patients with various forms of myelodysplastic syndrome. Production of factors stimulating the growth of normal erythroid and granulocytic precursors by cells of the stromal sublayer from patients with refractory sideroblast anemia and refractory anemia with excess blasts is impaired compared to cells from healthy donors. The medium conditioned by macrophages from patients with chronic myelomonocytic leukemia displayed a higher ability to stimulate the growth of granulocytes and macrophages compared to media conditioned by cells from donors and patients with refractory sideroblast anemia and refractory anemia with excess blasts. Cultured stromal cells and macrophages produced tumor necrosis factor- and interleukin-6. Their content in media conditioned by cells from patients with myelodysplastic syndrome surpassed that in healthy donors. Our results suggest that production of cytokines by stromal microenvironmental cells is impaired in patients with various forms of myelodysplastic syndrome.     

Age-specific features of formation of hemopoietic microenvironment by stromal precursors from bone marrow of thymectomized mice

Heterotopic transplantation of bone marrow demonstrated that the content of stromal precursor cells capable of hemopoietic microenvironment transfer does not depend on thymus function. Thymectomy of bone marrow donors involves a decrease in the size of foci formed in young donors and an increase in old recipients. The results indicate a thymus-dependent regulation of proliferation of stromal precursors and/or their factor-sensitive category, determining the proliferation of recirculating stem hemopoietic cells. The size of ectopic hemopoiesis focus depends on the age of recipient. Transplantation of syngeneic thymus under renal capsule of thymectomized mice abolished the effect of thymectomy. Osteogenic activity of stromal precursors correlates with the age of bone marrow donors. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 124, No. 4, pp. 457–459, April, 1998     

Changes in the hemopoietic system of mice deficient for tumor necrosis factor or lymphotoxin-alpha

Hemopoietic and stromal precursor cells were studied in mice deficient for tumor necrosis factor or lymphotoxin-α. In normal hemopoiesis the main characteristics of hemopoiesis in knockout mice did not differ from those in wild-type mice. Implantation of bone marrow cells from mice deficient for tumor necrosis factor onto irradiated sublayer of allong-living bone marrow culture led to a notable increase in the number of mature cells and granulocytic-macrophage precursor cells. This can be due to the fact that tumor necrosis factor inhibits proliferation of hemopoietic precursor cells, while in the absence of this factor precursor cells actively proliferate. On the other hand, cell composition and number of colony-forming units of granulocytes-macrophages are significantly decreased in cultures onto which bone marrow cells from lymphotoxin-α-deficient mice were implanted. This can be explained by impaired expression of adhesion molecules in these animals. In addition, the number of stromal precursor cells was changed in mice deficient by genes of the tumor necrosis factor cluster. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 130, No. 7, pp. 76–79, July, 2000     

Changes in the hemopoietic system of mice deficient for tumor necrosis factor or lymphotoxin-α

Hemopoietic and stromal precursor cells were studied in mice deficient for tumor necrosis factor or lymphotoxin-α. In normal hemopoiesis the main characteristics of hemopoiesis in knockout mice did not differ from those in wild-type mice. Implantation of bone marrow cells from mice deficient for tumor necrosis factor onto irradiated sublayer of allong-living bone marrow culture led to a notable increase in the number of mature cells and granulocytic-macrophage precursor cells. This can be due to the fact that tumor necrosis factor inhibits proliferation of hemopoietic precursor cells, while in the absence of this factor precursor cells actively proliferate. On the other hand, cell composition and number of colony-forming units of granulocytes-macrophages are significantly decreased in cultures onto which bone marrow cells from lymphotoxin-α-deficient mice were implanted. This can be explained by impaired expression of adhesion molecules in these animals. In addition, the number of stromal precursor cells was changed in mice deficient by genes of the tumor necrosis factor cluster. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 130, No. 7, pp. 76–79, July, 2000     

Effect of leukemia inhibitory factor on hemopoietic and stromal precursor cells in prolonged culture of mouse bone marrow

Treatment of prolonged bone marrow cultures with leukemia inhibitory factor during the first 2 weeks after explantation has no appreciable effect on the production of precursors and mature hemopoietic cells during 4 weeks of culturing. However, the proliferative potential of polypotent hemopoietic precursors in these cultures increases substantially. The addition of exogenous cytokine has a pronounced effect on the hemopoietic stroma, specifically, on the content of osteogenic precursors and cells transporting the hemopoietic microenvironment to prolonged bone marrow cultures treated by leukemia inhibitory factor. This effect is confirmed by formation of ectopic hemopoietic fociin vivo, being 2–3 times higher than in the control. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 122, No. 9, pp. 325–328, September, 1996     

Long-term self-maintenance of hemopoietic precursors in bone marrow culture derived from tumor necrosis factor-deficient mice is not a result of neoplastic transformation

In long-term bone marrow cultures derived from tumor necrosis factor-deficient mice the total cell production and the total duration of hemopoiesis are increased (the latter is comparable with mouse life span). Telomerase activity in cells of nonadherent fraction of long-term bone marrow cultures from tumor necrosis factor-deficient mice increases with time and peaks after 1-year culturing. Karyotyping of nonadherent and adherent cells of long-term bone marrow cultures revealed instability of nonadherent cells and hyperploidy of the stromal sublayer cells, which attested to the presence of a neoplastic transformation. However, cell differentiation is not blocked in long-term bone marrow cultures. The nonadherent fraction of long-term bone marrow cultures from tumor necrosis factor-deficient mice cannot be cultured without exogenous growth factors; in the presence of growth factors the cells proliferate, but cannot be passaged; stromal sublayer cells cannot be passaged as well. Intraperitoneal and intravenous injections of nonadherent cells to recipients with normal and radiation-attenuated immunity induced no tumor growth. Hence, peculiar dynamics of long-term bone marrow cultures from tumor necrosis factor-deficient mice cannot be explained by neoplastic transformation.     

Structural and functional organization of bone marrow of AKR/J mice during aging

Expression of sialoadhesin and erythroblast receptors on macrophages and structural and functional organization of the bone marrow during aging were studied on AKR/J mice. It is shown that progressive accumulation of granulocyte hemopoietic islets can be a compensatory reaction to a decreased capacity of their central stromal elements to bind young granulocytopoietic cells. Expression of erythroblast receptors on macrophages from 4-month-old AKR/J mice is considerably higher than in young and old mice and than in 4-month-old (CBA×AKR)F₁ mice. The high concentration of erythroid precursors in the bone marrow of AKR mice is not accompanied by enhanced erythropoiesis, probably due to a decreased yield of erythroid hemopoietic islets. Thus, a marked imbalance in structural and functional organization of the bone marrow during aging is noted in highly leukemic AKR/J mice, which provides a basis for the development of reliable diagnostic and prognostic criteria of leukemic progression. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 125, No. 3, pp. 266–268, March, 1998     

Observations on the haemopoietic response to critical illness.

Peripheral blood cytopenias are common in patients receiving intensive care, particularly in those with multiple organ failure. To assess the contribution of bone marrow hypoplasia in such patients 44 bone marrow samples from 24 patients under intensive care were studied by standard morphological techniques and by the granulocyte-macrophage colony forming cell (GM-CFC) assay. Frequently observed morphological abnormalities in the bone marrow included the following: (i) a reduction in overall cellularity in seven patients, with a progressive decrease in most patients studied sequentially; (ii) an increase in the number of actively phagocytic macrophages; and (iii) a disruption of normal bone marrow architecture with the accumulation of intercellular hyaluronic acid glycosaminoglycan. Mean GM-CFC growth was significantly reduced when compared with that in a group of normal controls. In four of five patients studied sequentially GM-CFC growth became subnormal in association with a reduction in bone marrow cellularity. Inhibitory serum factors were not identified. These morphological abnormalities are similar to the changes observed in gelatinous degeneration of the bone marrow. In both situations disruption of the haemopoietic microenvironment, with the accumulation of hyaluronic acid proteoglycan, may be an important factor in the inhibition of haemopoietic progenitor cell growth. The proliferation of macrophages, by the release of a variety of cytokines or reactive oxygen intermediates, may also be implicated in impaired haemopoiesis and the development of disordered erythropoiesis.