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.     

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.     

Osteogenic properties of adhesive cells in Dexter culture of the mouse bone marrow

Disaggregated cell suspensions obtained by mouse bone marrow fermentative digestion as well as stromal tissue obtained by marrow mild mechanical destruction were explanted. Both methods yield the cultures in which the hematopoiesis duration is comparable with dexter cultures. Adhesive cells from all of these three culture types were resuspended and in the porous gelatin sponges heterotopically transplanted under the kidney capsule of syngenic recipients. In the transplantation site there develops the hemopoietic organ containing reticular stroma, hemopoietic cells, and in most cases the well developed bone tissue. Thus, the adherent layers of mouse bone marrow dexter and similar cultures contain for a long period (not less than 2-3.5 months) the stromal fibroblast population which maintains its osteogenic and hemopoietic microenvironment transfer capacities.     

Effect of dipyridamole on the interrelations of the stromal and hematopoietic tissues of the bone marrow in experimental studies]

By means of heterotopic transplantation of the bone marrow interrelations of the stromal and hemopoietic tissues of the mice bone marrow have been studied at administration of dipiridamol. Effect of the drug to the hemopoiesis is realized via stem stromal cells of the bone marrow. Under the influence of dipiridamol a focus of heterotopic hemopoiesis the osteogenic component in it is present only in 30% of cases in comparison with the control. Inhibition of the stromal component proliferation is accompanied with increasing mitotic activity of the hemopoietic elements against the background of the bone marrow cellularity decrease both in the femoral bone and in the focus of heterotopic hemopoiesis. At administration of dipiridamol a phenomenon of noneffective megakaryocytopoiesis with the intrabone marrow destruction of megakaryocytes, resulting in local release of thrombocyte growth factor, which has a compensatory character.     

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.     

Molecular-genetic and immunophenotypic analysis of antigen profile and osteogenic and adipogenic potentials of mesenchymal stromal cells from fetal liver and adult bone marrow in rats

Comparative characteristics of mesenchymal stromal cells (MSCs) from adult bone marrow and fetal liver are of great interest due to the similar functions performed by these organs on the organization of a hemopoietic microenvironment at various developmental periods. It is known that MSCs play a pivotal role in the formation of niches for hemopoietic stem cells. The histogenetic relation of MSCs from these two hemopoietic organs cannot be ruled out. An analysis of antigen profile using immunocytochemistry and RT-PCR has confirmed that the studied cell populations fit the MSC criteria and have no contaminations of hemopoietic, lymphoid, and endothelial cells beginning at the second passage. Comparative analysis of osteogenic and adipogenic marker expression revealed MSC from fetal liver to have a weaker potential for adipogenesis and the extremely low capability for terminal osteogenic differentiation, in contrast to pronounced osteo- and adipogenic potentials of adult bone marrow MSC. The similar cell phenotype but different differentiation potentials under identical conditions of cultivation in vitro seem to be due to different developmental programs of the pre- and postnatal histogenesis of these MSC.     

Differentiation of hematopoietic and stromal elements of extramedullary bone marrow transplants in irradiated recipients

The growth of bone marrow transplants under the renal capsule is stimulated in the mice irradiated at a dose of 700 r. The size of transplants increases, primarily, at the expense of hemopoietic tissue. The osteogenic potencies of stromal elements remain at a high enough level as well. Some features which characterize the decrease of viability of hemopoietic cells may be due to incomplete restoration of the system of sinusoidal capillaries.     

Generation of osteoclasts from hemopoietic cells and a multipotential cell line in vitro

Osteoclasts are the cells that resorb bone. It is generally presumed, on the basis of indirect experiments, that they are derived from the hemopoietic stem cell. However, this origin has never been established. We have developed an assay for osteoclastic differentiation in which bone marrow cells are incubated in liquid culture on slices of cortical bone. The bone slices are inspected in the scanning electron microscope after incubation for the presence of excavations, which are characteristic of osteoclastic activity. We have now incubated bone marrow cells at low density, or a factor-dependent mouse hemopoietic cell line (FDCP-mix A4) with 1,25 dihydroxyvitamin D3 (a hormone which we have previously found induces osteoclastic differentiation) with and without murine bone marrow stromal cells, or with and without 3T3 cells, on bone slices. Neither the bone marrow cells nor the bone marrow stromal cells alone developed osteoclastic function even in the presence of 1,25 dihydroxyvitamin D3. However, extensive excavation of the bone surface was observed, only in the presence of 1,25 dihydroxyvitamin D3, on bone slices on which bone marrow stromal cells were cocultured with low-density bone marrow cells or the hemopoietic cell line. Similar results were obtained when the bone marrow stromal cells were killed by glutaraldehyde fixation; 3T3 cells were unable to substitute for stromal cells. These results are strong evidence that osteoclasts derive from the hemopoietic stem cell and suggest that although mature osteoclasts possess neither receptors for nor responsiveness to 1,25 dihydroxyvitamin D3, the hormone induces osteoclastic function through a direct effect on hemopoietic cells rather than through some accessory cell in the bone marrow stroma. The failure of 3T3 cells, which enable differentiation of other hemopoietic progeny from this cell line, to induce osteoclastic differentiation suggests that bone marrow stroma possesses additional characteristics distinct from those that induce differentiation of other hemopoietic cells that are specifically required for osteoclastic differentiation.     

骨髓基质细胞的辐射效应及其临床意义

小鼠骨髓基质细胞团在γ线照射后的Do值为2.40Gy,但其成灶能力损伤后持续时间较久。正常骨髓基质细胞能促进骨髓GM-CFU-C的生长;照射10-80Gy后的骨髓基质细胞失去这种促进作用。文中讨论了骨髓基质细胞的辐射效应及其临床意义,提出了谨慎选择放射治疗剂量的必要性。     

Ultrastructural characteristics of stromal mechanocytes and their interaction with hematopoietic cells in regenerating bone marrow transplants

Extramedullar grafts of the bone marrow have been studied electron microscopically 2-8 days after transplantation. The data on ultrastructural peculiarities of the stromal mechanocytes have been obtained at various stages of their differentiation, as well as topographic interrelationships between the mechanocytes and the hemopoietic cells. Digital junctions are described between the stromal mechanocytes (primitive) and the hemopoietic cells (in 3-day-old grafts) which are, probably, the first morphological signs demonstrating restoration of the hemopoietic microenvironment in the bone marrow grafts.     

Stromal cells from murine developing hemopoietic organs: comparison of colony-forming unit of fibroblasts and long-term cultures.

The adherent stromal layer in long-term marrow cultures is essential to the proliferation and differentiation of hemopoietic cells. Adhering cells are heterogeneous and morphologically not adequately characterized. Comparative morphological studies were conducted on adherent cells in short-term clonal assays and long-term cultures derived from liver and bone marrow. Liver and bone marrow at different developmental ages have different hemopoietic activities in vivo and in vitro, as tested via CFU-GM recovery in long-term cultures. Adherent cells from each organ were recovered at an age with high hemopoietic activity (fetal liver and adult bone marrow) and at an age with low hemopoietic activity (neonatal liver and bone marrow). The presence of macrophages, alkaline phosphatase, acid phosphatase, myeloperoxidase, sulfated and non-sulfated glycosaminoglycans (GAGs) and fibronectin was compared. For a given organ, CFU-f colonies showed characteristics similar to those of the confluent adherent stromal layer in long-term cultures. The presence of macrophages and GAGs (sulfated and non-sulfated) in the adherent layer were directly related to the hemopoietic activity. The amount of alkaline phosphatase-positive cells and the amount of fibronectin showed no correlation with the hemopoietic activity of the cultures.     

The development relationship between osteocytes and osteoclasts: a study using the quail-chick nuclear marker in endochondral ossification.

Interspecific grafts of limb buds and femurs on the chorioallantoic membrane of 5-day-old hosts and into the somatopleure of 3-day-old hosts were carried out between quail and chick embryos. Due to their different nuclear features, the cells of the two species can be identified in the chimeric bones resulting from the endochondral ossification which occurs in the explanted tissues. By following the cell lineage in the bone and marrow we were able to show that the hemopoietic and the osteogenic (comprising osteoblasts, osteocytes, and chondrocytes) cell lines have different embryological origins. The osteogenic line is derived from the limb bud mesenchyme, while the hemopoietic cells are brought into the bone marrow via the circulation. In the fixed cells of the marrow two categories have to be distinguished: the reticular cells originating from the bone rudiment and the endothelial cells which invade the cartilage and are of hematogenous origin. The osteoclasts belong to the hemopoietic cell line and are not derived from any cell type of the osteogenic line.     

A possible role for CXCR4 and its ligand, the CXC chemokine stromal cell-derived factor-1, in the development of bone marrow metastases in neuroblastoma

The homing of hemopoietic stem cells to the bone marrow is mediated by specific interactions occurring between CXCR4, which is expressed on hemopoietic stem cells, and its ligand, stromal cell-derived factor-1 (SDF-1), a CXC chemokine secreted by bone marrow stromal cells. In the present study we evaluated the possibility that neuroblastoma cells use a mechanism similar to that used by hemopoietic stem cells to home to the bone marrow and adhere to bone marrow stromal cells. Our study suggests that CXCR4 expression may be a general characteristic of neuroblastoma cells. SH-SY5Y neuroblastoma cells express not only CXCR4, but also its ligand, SDF-1. CXCR4 expression on SH-SY5Y neuroblastoma cells is tightly regulated by tumor cell-derived SDF-1, as demonstrated by the ability of neutralizing Abs against human SDF-1alpha to up-regulate CXCR4 expression on the tumor cells. The reduction in CXCR4 expression following short term exposure to recombinant human SDF-1alpha can be recovered as a result of de novo receptor synthesis. Recombinant human SDF-1alpha induces the migration of CXCR4-expressing SH-SY5Y neuroblastoma cells in CXCR4- and heterotrimeric G protein-dependent manners. Furthermore, SH-SY5Y cells interact at multiple levels with bone marrow components, as evidenced by the fact that bone marrow-derived constituents promote SH-SY5Y cell migration, adhesion to bone marrow stromal cells, and proliferation. These results suggest that SH-SY5Y neuroblastoma cells are equipped with adequate machinery to support their homing to the bone marrow. Therefore, the ability of neuroblastoma tumors to preferentially form metastases in the bone marrow may be influenced by a set of complex CXCR4-SDF-1 interactions.     

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.     

A stromal cell line from myeloid long-term bone marrow cultures can support myelopoiesis and B lymphopoiesis

The production of B lymphocytes and myeloid cells occurs in the bone marrow in association with a supporting population of stromal cells. To determine whether these processes are dependent upon the same or different populations of stromal cells, stromal cell lines were generated from the adherent layer of a Dexter type long-term bone marrow culture. These cultures support myeloid cells and their precursors, a B cell precursor, and the adherent layer cells with support B cell differentiation under appropriate conditions. Two of the lines examined, S10 and S17, express class I histocompatibility antigens but not other hemopoietic cell surface determinants such as Thy-1, Lyt-1, Ig, Ia, Mac-1, or BP-1. Both lines could support myelopoiesis under Dexter conditions upon seeding with nylon wool-passed bone marrow. The nylon wool passage depletes stromal cells capable of forming adherent layers in vitro but retains hemopoietic precursors. The number of cells and colony-forming units-granulocytes/macrophages in the nonadherent cell population recovered 3 wk post-seeding had increased 19-fold and 10-fold, respectively, in the reseeded cultures of S10 and S17. After 3 wk of growth in Dexter conditions, the reseeded cultures were transferred to conditions optimal for B cell differentiation described by Whitlock and Witte. After 4 wk of growth, hemopoietic cells were consistently recovered from S17 cultures but not those of S10. A proportion of these cells from S17 cultures expressed the 14.8 antigen and were surface IgM positive. Surviving hemopoietic cells present in cultures of S10 were primarily macrophages. These findings indicate that S17 but not S10 can support both myelopoiesis and B lymphopoiesis and suggest that one stromal cell population has the capacity to form a hemopoietic microenvironment for both lineages.     

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.     

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.     

Adipocytes and reticular cells of the bone marrow stroma in the human iliac bone

The results of the histological and electron microscopic investigation of adipose and reticular cells and their interconnections with blood cells are presented in the material of trephine biopsies of the iliac bone. A possibility for development of adipocytes from the adventitial reticular cells is demonstrated. Close contacts are revealed between pre-adipocytes and young hemopoietic cells. Two types of the reticular cells are characterized, they differ in their position, structural organization and interconnection with the young hemopoietic elements. The peculiarities revealed in the morphofunctional state of the microenvironmental structures demonstrate functional variegation of the stromal elements, and also attest an essential importance of intercellular contacts of the hemopoietic predecessors and the stromal cells in maintaining the hemopoietic function of the bone marrow.     

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.     

Role of Nanog in the maintenance of marrow stromal stem cells during post natal bone regeneration

Post natal bone repair elicits a regenerative mechanism that restores the injured tissue to its pre-injury cellular composition and structure and is believed to recapitulate the embryological processes of bone formation. Prior studies showed that Nanog, a central epigenetic regulator associated with the maintenance of embryonic stem cells (ESC) was transiently expressed during fracture healing, Bais et al. In this study, we show that murine bone marrow stromal cells (MSCs) before they are induced to undergo osteogenic differentiation express ～50× the background levels of Nanog seen in murine embryonic fibroblasts (MEFs) and the W20-17 murine marrow stromal cell line stably expresses Nanog at ～80× the MEF levels. Nanog expression in this cell line was inhibited by BMP7 treatment and Nanog lentivrial shRNA knockdown induced the expression of the terminal osteogenic gene osteocalcin. Lentivrial shRNA knockdown or lentiviral overexpression of Nanog in bone MSCs had inverse effects on proliferation, with knockdown decreasing and overexpression increasing MSC cell proliferation. Surgical marrow ablation of mouse tibia by medullary reaming led to a ～3-fold increase in Nanog that preceded osteogenic differentiation during intramembranous bone formation. Lentiviral shRNA knockdown of Nanog after surgical ablation led to an initial overexpression of osteogenic gene expression with no initial effect on bone formation but during subsequent remodeling of the newly formed bone a ～50% decrease was seen in the expression of terminal osteogenic gene expression and a ～50% loss in trabecular bone mass. This loss of bone mass was accompanied by an increased ～2- to 5-fold adipogenic gene expression and observed increase of fat cells in the marrow space. In summary these data show that Nanog is expressed during surgically induced marrow bone formation and is functionally involved in post natal marrow stromal cell maintenance and differentiation.