Role of stromal microenvironment in the regulation of bone marrow hemopoiesis after curantyl administration

Role of the stromal microenvironment in regulation of bone marrow hemopoiesis at the administration of the thrombocyte disaggregant curantyl was studied by the method of heterotopic transplantation of the mice bone marrow. It is shown that the action of curantyl on hemopoiesis is realised through the stem stromal cells of the bone marrow. It is noted that the inhibitory action of the preparation on proliferation of osteogenic precursor-cells is followed by activation of bone resorption processes in regenerating ectopic hemopoietic organ. Under the action of curantyl at low bone marrow cellularity in the focus of heterotopic hemopoiesis and femur an increase of mitotic activity in hemopoietic elements is noted. It is revealed that a phenomenon of ineffective megakaryocytopoiesis with intramedullary destruction of megakaryocytes leads to the local excretion of the thrombocyte released growth factor (TRGF) which has a compensatory character.     

2 populations of bone marrow cells transfering the hematopoietic environment]

A bone marrow fragment transplanted under the kidney capsule created a focus of ectopic hemopoiesis, whose isze, measured by the number of hemopoietic cells, was proportional to the implant size. Dimensions of the focus proved to be 11/2--21/2 greater in the irradiated than in the intact recipients. Cells building up the focus of heterotopic hemopoiesis had a different radiosensitivity in the intact and irradiated recipients--their Do constituted about 160 and 350 rad, respectively. In this connection it is supposed that two cell populations of precursors took part in the creation of the focus. Their possible relations with the determined and inducible osteogenic precursor cells are discussed.     

Effect of estrone on formation of the focus of heterotopic hematopoiesis

The effect of estrone injections on the heterotopic bone marrow organ formation was studied in (C57Bl X CBA)F1 mice by bone marrow shaft transplantation under the kidney capsule. Estrone injections resulted in femur marrow fibrosis, the increase in the weight of femur and heterotopic organ ossicle, drastic reduction in heterotopic organ cellularity. Depression of hemopoiesis in ectopic organ cannot be attributed to mechanical displacement of hemopoietic cells because a newly formed bone cavity under the kidney capsule was not closed. Thus, estrone had a detrimental effect on the creation of microenvironment in the heterotopic organ.     

Studies on bone marrow histogenesis: Morphometric and autoradiographic studies of regenerating marrow stroma in extramedullary autoimplants and after evacuation of marrow cavity

Summary The marrow cavity of the rat tibia was mechanically evacuated and autoimplanted to the subcutaneous tissue. The regenerative process which restored the integrity of marrow stroma and hemopoiesis, was morphometrically evaluated in whole mount of tibia. Following evacuation, the clot filled the cavity. The granulation tissue then appeared and expanded, penetrating and replacing the clot. The fibroblasts of the granulation tissue differentiated into osteoblasts forming osteoid bone. Within its interstices, the primordial marrow consisting of loose connective tissue and vascular sinuses appeared and hemopoiesis resumed. Expansion of hemopoiesis resulted in the resorption of bone and within three weeks the tibial cavity was restored to the pre-evacuation state.Autoradiography indicated that the labeling index was initially high in fibroblasts and osteoblasts but was subsequently reduced while it increased in osteocytes, cells of Haversian canals, stromal and hemopoietic cells of marrow. The finding is in disagreement with the view that the regenerative process originates from the Haversian canal. When the label was introduced on day 4 post-operatively, it subsequently appeared in osteocytes, cells of Haversian canal, stromal elements of the marrow, but not in the hemopoietic cells. This indicates complete dissociation of marrow stroma and hemopoietic stem cell.Supported by NASA Contract NSG 9061. Mehdi Tavassoli is the recipient of a CRD Award AM-70551     

Population Dynamics of Clonogenic Stromal Precursor Cells in Hemopoietic and Lymphoid Organs during Bone Marrow Regeneration

This paper describes our study on the regeneration of hemopoietic and stromal components of bone marrow after mechanically emptying the medullar cavity of the guinea pig tibia. The intensity of hemopoiesis was determined from the number of hemopoietic cells, while the concentration and total number of stromal precursor cells were used to estimate the ability of the bone marrow to produce stromal structures, including its ability to restore a specific microenvironment. We found that there was no direct correlation between the recovery characteristics of hemopoietic and stromal cells. An increase in the population size of stromal precursor cells takes place early after curettage, and stromal fibroblasts become phosphatase-positive according to Gomori, which is characteristic of osteogenic tissue. We have also demonstrated that curettage of 3–5 tubular bones results in the growth of this cell population in the bone marrow of nonoperated bones and even in the spleen, which in guinea pigs participates only in lymphopoiesis.     

Hybrid resistance to precursor cells of bone marrow stroma]

A focusl of hemopoiesis appearing after the transplantation of a bone marrow fragment of C57BL mice to syngeneic mice (under the kidney capsule) contained more hemopoietic cells than in transplantation to the semisyngeneic (CBA X C57BL) FI recipient. Experiments were conducted with a secondary seeding by intravenous injection of hemopoietic cells of the C57BL transplant genotype into the transplant depopulated by irradiation; it was shown that these differences were caused by lesser dimensions of the hemopoietic microenvironment in the focus in the hybrid organism in comparison with such in the syngeneic system. Thus, the hybrid resistance was expressed not only to the hemopoietic cells, but also to the stromal precursors transferring the hemopoietic microenvironment.     

Morphologic analysis of guinea pig bone regeneration following curettage]

Interrelation of stromal and hemopoietic elements in the regenerative process of the guinea-pig bone marrow at different time after curettage has been morphologically studied. The bone marrow regenerative process in the depleted medullary cavity of the guinea-pig tibial bone passes through several phases following each other: blood clot formation, rough-fibrillary osseous tissue formation, its resorption, development of sinusoids and formation of reticular stroma with subsequent repopulation of hemopoietic elements in it. The leading role in the process of the bone marrow restoration belongs to the reticular stroma creating specific microconditions for hemopoiesis.     

Long-term maintenance of multilineal hemopoiesis in collagen gel culture.

We examined the long-term maintenance of multilineal hemopoiesis in a collagen gel culture of mouse bone marrow cells. When cells were inoculated into the gel, stromal cells formed foci that were composed of sinusoidlike capillary structures, fibroblastic cells, adipocytes and macrophages. Many small hemopoietic foci similar to granulocyte-macrophage colonies (CFU-GM) appeared within a week and disappeared after two weeks. Several large hemopoietic foci appeared after two to three weeks of culture, without a second challenge of marrow cells. These large hemopoietic foci were composed mainly of myeloid cells. Megakaryocytes and mast cells were also observed. When erythropoietin (EPO) was added to the culture at the beginning, the erythroid focus appeared after 3 weeks and the number of megakaryocytes was greater than that in the culture without EPO. However, when EPO was added to the cultures after 6 or 12 weeks, erythroid cells appeared after 1 week and the number of megakaryocytes increased. This hemopoiesis lasted more than 6 months.     

Wnt signaling regulates hemopoiesis through stromal cells.

Hemopoietic cells develop in a complex milieu that is made up of diverse components, including stromal cells. Wnt genes, which are known to regulate the fate of the cells in a variety of tissues, are expressed in hemopoietic organs. However, their roles in hemopoiesis are not well characterized. In this study, we examined the roles of Wnt proteins in hemopoiesis using conditioned medium containing Wnt-3a. This conditioned medium dramatically reduced the production of B lineage cells and myeloid lineage cells, except for macrophages in the long-term bone marrow cultures grown on stromal cells, although the sensitivity to the conditioned medium differed, depending on the hemopoietic lineage. In contrast, the same conditioned medium did not affect the generation of B lineage or myeloid lineage cells in stromal cell-free conditions. These results suggested that Wnt proteins exert their effects through stromal cells. Indeed, these effects were mimicked by the expression of a stabilized form of beta-catenin in stromal cells. In this study, we demonstrated that Wnt signaling regulates hemopoiesis through stromal cells with selectivity and different degrees of the effect, depending on the hemopoietic lineage in the hemopoietic microenvironment.     

Clonogenic fibroblasts from the hematopoietic organs of the human embryo and fetus at different gestational ages

Fibroblast precursors of hemopoietic organs of 72 embryos and fetuses 5-27 weeks of age have been studied. The study has shown that the increase in the number of clonogenic fibroblasts took place in the bone marrow and spleen 2-3 weeks before the beginning of hemopoiesis, that is during the period of the highest hemopoietic stem cell concentration. These data suggest possible participation of stromal fibroblasts of hemopoietic organs in the formation of microenvironment for hemopoietic stem cell functioning.     

Proliferation activity of stromal stem cells (CFU-f) from hemopoietic organs of pre- and postnatal mice

Stromal stem cells (CFU-f assay) from hemopoietic organs of fetuses, in contrast to adult animals, exhibit a high proliferation activity. This implies that these CFU-f are radiosensitive and potential target cells after radioactive contamination of fetuses. Furthermore, the percentage of CFU-f in DNA synthesis is correlated with the hemopoietic activity in liver, spleen, and bone marrow. As hemopoiesis starts, high numbers of CFU-f are in S phase. In fetal liver, spleen, and bone marrow, values of 70, 43, and 58%, respectively, are reached. As hemopoietic activity decreases in liver and stabilizes in spleen and bone marrow, mitotic activity of these stromal stem cells becomes undetectable.     

Developments in modern hematology.

In the past 40 years our concepts about hemopoiesis have been changed dramatically. The results of bone marrow transplantation into lethally irradiated mice since the mid-fifties suggested the existence of a hemopoietic stem cell, which was initially identified as a spleen colony forming cell (CFU-S). Later experiments showed that the stem cell compartment is rather heterogeneous and that the most primitive stem cell, unlike the CFU-S, has the ability for long-term engraftment of an irradiated recipient. Daughter cells of such primitive quiescent stem cells lose their capacity for self-generation gradually with each mitosis and become more and more committed to a specific differentiation lineage. In vitro culture techniques in a serum-free semi-solid medium enabled the establishment and analysis of specific hemopoietic growth factors. Such factors, which are essential for the maintenance, proliferation and differentiation of progenitor cells and the functional activity of mature cells can now be produced with recombinant DNA techniques in pure form and large quantities. Hemopoiesis requires an appropriate microenvironment, consisting of various stromal cell types and an extracellular matrix. Intercellular contacts, adhesion of cells and growth factors to the matrix molecules seem essential in the regulating action of this hemopoietic microenvironment. In long-term bone marrow cultures the development of a stromal hemopoietic microenvironment can facilitate long-term maintenance of stem cells and hemopoietic differentiation. For bone marrow transplantation and infusion of hemopoietic growth factors many clinical indications are well established and our possibilities to interfere in the regulation of hemopoiesis are still growing.     

Interrelation of the osteogenic and hemopoietic tissues of the bone marrow during the development of estrogenic myelofibrosis

By means of estrogenic myelofibrosis, using the method of heterotopic transplantation of the bone marrow, interrelations of osteogenic and hemopoietic tissues have been studied. Under estrone effect disorders in stromal microenvironment take place at the level of stem osteogenic cells of the bone marrow. Deficiency in cells of monocytic-macrophagal line, inhibiting proliferation of the bone marrow connective tissue cells, contributes to development of myelofibrosis. The hormone acts mainly in committed hemopoietic cells, singly launching them from G0- into S-period of the cycle, and then--into differentiation, accompanied with an enhanced discharge of cells from the bone marrow. There is neither direct, nor indirect dependence between the osteogenic mass and the cellularity of the hemopoietic tissue of the bone marrow. The changes, that take place in the bone marrow under estrone effect, are reversible.     

Quantitative characteristics of the transfer of the hematopoietic microenvironment

Heterotopic transplantation of marrow fragments results in the formation of new bone marrow organs. The amount of hemopoietic cells populating these organs is connected nonlinearly with the volume of the grafted tissue or with the number of transplanted marrow cells. Statistical analysis points out that the number of the populating cells depends on the radius of the initial bone marrow transplant. Thus, the previous data on the radiosensitivity of the microenvironmental transfering stromal cells and on their concentration obtained by measuring the size of the heterotopic bone marrow organs have turned out incorrect.     

The role of T-lymphocytes, macrophages and stromal mechanocytes in regulating bone marrow hematopoiesis during stress

The role of T-lymphocytes, macrophages and stromal mechanocytes in regulation of bone marrow hemopoiesis has been studied at stress. A reliable increasing of absolute number of erythroid, granulocyte and mixed hemopoietic islets [correction of elots] in animals after 10-hours immobilisation has been found. The antithymocyte sera blockade of T-cells inhibits the appearance of new hemopoietic islets [correction of elots] in hemopoietic tissue at stress.     

Regulation by cytokines of leptin expression in human bone marrow adipocytes.

Leptin is a hormone secreted by adipocytes. Besides controlling appetite and body weight, it has been suggested that leptin plays a role in inflammation and hemopoiesis. In this study we demonstrate that the pro-inflammatory/hemopoietic cytokines, IL-1beta, IL-6, TNF-alpha, and interferon-gamma, significantly inhibit gene expression and secretion of leptin by bone marrow adipocytes. These findings are in agreement with the data recently obtained from non-medullary adipose tissues. Within the bone marrow environment, leptin regulation by these pleiotropic cytokines could contribute to controlling the proliferation and differentiation of hemopoietic precursors as well as the maturation of stromal cells.     

Stromal cells of hemopoietic origin

Hemopoiesis is a multistep process involving stem cell renewal, commitment, differentiation, maturation and consequent positioning of the cells within the tissue. Stromal cells are a major component of the hemopoietic microenvironment. The in vitro culture of cloned stromal cells has enabled detailed analysis of their functions and has provided answers relating to the contribution of stromal cells to the control of hemopoiesis. Cultured stromal cells were found to support the renewal of stem cells through a mechanism that did not seem to involve already known cytokines. Cloned stromal cells from both marrow and thymus supported the in vitro accumulation of myeloid as well as T and B lymphoid cells. Thus, cloned stromal cells had the ability to induce multilineage hemopoiesis, irrespective of the organ from which they were derived. Invariably, stromal cells tended to select in culture for hemopoietic cells at early differentiation stages and restricted the accumulation of mature cells. These functions may be part of the mechanism that protects the stem cell pool from excess differentiation.     

Stromal cells of the regenerating bone marrow in rats (an immunocytochemical electron microscopy study)

Process of the bone marrow regeneration has been studied after its removal out of the rat femoral bone cavity. The stage of stroma formation precedes hemopoiesis. The stromal cells during its reconstruction (the 4th-5th day after removal of the bone marrow) are analyzed by means of the indirect immune-peroxidase electron microscopical method with antiserum applied against insoluble antigens of the rat bone marrow cells. Most of the stromal cells do not fix the antiserum used, as do the hemopoietic cells, macrophages and preosteoclasts. Some part of the stromal cells (not more than 30%) demonstrate the immune-peroxidase label. The labelled stromal cells have some ultrastructural signs of poorly differentiated elements of fibroblastic and osteoblastic raws. In the regeneration area, there are non-labelled poorly differentiated cells, which do not differ, at the ultrastructural level, from labelled poorly differentiated stromal elements. Possible causes of the difference revealed among the poorly differentiated stromal cells concerning their fixing of the anti-bone-marrow antiserum are discussed.     

Polypeptide factors regulating osteogenesis and bone marrow repair

Osteogenic growth polypeptides regulate bone cell function in vitro and may act in vivo in an autocrine, paracrine, or endocrine manner. Several of these polypeptides are present in the blood in an inactive form. During postablation bone marrow regeneration these factors may be activated, released from the blood clot, and together with locally produced polypeptides mediate the initial intramedullary/systemic osteogenic phase of this process. Then, the same and/or other polypeptides expressed by stromal cells have the potential to promote the second phase of regeneration that consists of osteoclastogenesis, resorption of the transient intramedullary bone, and hemopoiesis. This may be an indirect influence since these polypeptides can regulate the stromal cell expression of some of the hemopoietic factors. Clinically, the osteogenic growth polypeptides that regulate osteogenesis and hemopoiesis have a potential role in osteoporosis therapy, implant bone surgery, and bone marrow transplantation. © 1994 Wiley-Liss, Inc.     

The role of the hemopoiesis-inducing microenvironment in the mechanisms of the regeneration of hemopoiesis after cytostatic exposure]

On the model of adriamycine-induced hemopoiesis hypoplasia it has been shown that the accelerated differentiation of hemopoietic precursors regeneration is of great importance in regeneration of hemopoiesis. This differentiation is provided by preceded recovery of bone marrow structural-functional organization. The important role in regeneration of hemopoiesis belongs to hemopoietic microenvironmental elements which express its induced influence by the increase of colony-stimulating and erythropoietic activities, interleukin-1 and interleukin-3 production.