Paul Gibbons Roofe,Ph.D. Professor Emeritus of Anatomy

Light microscopic, scanning electron microscopic, and transmission electron microscopic studies of the early developmental stages of chick embryonic bone marrow disclose characteristic associations of the first hematopoietic cells with stromal cells. The first hematopoietic cells, large basophilic cells that we have termed presumptive stem cells, segregate into erythropoietic and granulopoietic regions. Intravascular erythropoietic cells associate with sinusoidal endothelial cells, while granulopoietic cells associate with extravascular reticular cells. Extensive, intimate contacts between erythroid and endothelial cells are maintained, in part, by marginal arrays of microtubules, which promote a flattening of the adherent erythroid cell surface. In addition, cell surface components of opposing cells, visualized by ruthenium red staining, appear to merge and possibly to interact. Granulopoietic cells establish intimate but less extensive associations with reticular cells through cell-surface interactions. Stationary granuloid cells appear to be held in place by small, thin processes emanating from the sheet-like reticular cells. Granuloid cells are capable of moving within the extravascular region, using reticular cell surfaces as a substrate. Intimate associations also occur among granulopoietic cells, the significance of which is unclear. Thus, sinusoidal endothelial cells and reticular cells comprise the critical non-hematopoietic or stromal elements of avian bone marrow, where they have a putative role in segregating presumptive stem cells into erythrocytic and granulocytic compartments. They serve as an architectual, and possibly regulatory, framework on which hematopoiesis occurs.     

Intercellular contacts between migrating blood cells and cells of the sinusoidal wall of bone marrow. An ultrastructural study using tannic acid

The migration of blood cells across the sinusoidal wall of murine bone marrow was studied following fixation with tannic acid-glutaraldehyde. Electron microscopic examination showed regions of close membrane apposition (referred to in this study as “intercellular contacts”) between migrating blood cells and cells of the sinusoidal wall (adventitial and endothelial cells). Ultrastructurally the intercellular contacts are pentalaminar structures resembling gap junctions of other organs after tannic-acid fixation. The possibility that these contacts are regions of intercellular communication and/or sites of membrane attachment utilized for locomotion of the migrating blood cells is discussed.     

Ultrastructural localization of heparan sulfate and chondroitin sulfates associated with granulopoiesis in embryonic chick bone marrow

Sulfated glycoconjugates were ultra-structurally localized within embryonic chick marrow by using the high iron diamine–silver proteinate stain. Stain was concentrated in the extravascular, granulopoietic compartment, indicating that granulopoiesis, but not erythropoiesis, proceeded in a highly sulfated environment. It was likely that most of the stainable material represented sulfated proteoglycans since staining was abrogated by predigesting tissue with enzymes and other treatments known to degrade specific glycosaminoglycan chains. Chondroitinase/hyaluronidase digestion resulted in the removal of most of the stainable material associated with the extracellular matrix and a portion of the stainable material associated with fibroblastic cell surfaces. Unaffected material lay in close proximity to fibroblastic cell membranes. Heparitinase/heparinase digestion had essentially the opposite effect. Sulfated material associated with matrix components was largely unaffected, but the fibroblastic plasmalemmal material was now absent. These results suggest that there are at least two categories of sulfated proteoglycans in the granulopoietic compartment, each differentially distributed. The plasmalemmal material likely represented heparan sulfate which in this tissue appeared to be associated in a uniform layer with fibroblastic stromal cell membranes and not with blood or endothelial cell membranes. Material identified as chondroitin sulfates was found within patches of amorphous matrix that was located on fibroblastic stromal cell surfaces and that was interspersed with fibrils in the extracellular matrix. Chondroitin sulfates were sparsely distributed on granulocytic cell surfaces.     

Gap junctions between cells of bone marrow: An ultrastructural study using tannic acid

In bone marrow of the mouse perfused with fixative containing tannic acid and glutaraldehyde, gap junctions were observed between certain cell types. Gap junctions were seen between adjacent reticular cells, between adjacent macrophages, and between macrophages and reticular cells. Macrophages formed gap junctions with immature neutrophils, eosinophils, monocytes, and erythroblasts. Often a single macrophage had gap junctions with neutrophilic, eosinophilic, and monocytic cells; these blood cells varied from immature to nearly mature forms. In contrast, the macrophages associated with erythroblasts had gap junctions only with erythroblasts and all the erythroblasts were in the same developmental stage. The possible role of the gap junctions in differentiation and mobilization of marrow cells is discussed.     

ELECTRON MICROSCOPIC STUDIES OF MACROPHAGES IN EARLY HUMAN YOLK SACS

Distribution and fine structure of macrophages were studied in 10 human embryos in the 6th and 7th week of gestation, 5.5 to 12 mm in crown-rump length. The yolk sac macrophages were found in the extravascular mesenchymal tissues and intravascular spaces long before the first appearance of bone marrow and lymphatic tissues in the embryos. In addition to the macrophages, the fibroblastic cells and the cells of erythropoietic series were also present in the extravascular space. The macrophages showed a variety of cellular structures suggesting transition from immature cell type with no heterophagolysosomes to mature cell type in phagocytosis. The mature macrophages avidly phagocytized the primitive erythroblasts and occasionally platelets. They were positively stained for lysosomal enzymes and were characterized by numerous pleomorphic heterophagolysosomes which exhibited various stages of digestion of phagocytized blood cells. The origin of intravascular macrophages may be in either migrated extravascular macrophages or phagocytic endothelial cells. The phagocytosis and degradation of erythroblasts appear to be one of the main functions of yolk sac macrophages. The presence of the macrophages in mitosis indicates their proliferation in situ.     

Electron microscopic studies of macrophages in early human yolk sacs

Distribution and fine structure of macrophages were studied in 10 human embryos in the 6th and 7th week of gestation, 5.5 to 12 mm in crown-rump length. The yolk sac macrophages were found in the extravascular mesenchymal tissues and intravascular spaces long before the first appearance of bone marrow and lymphatic tissues in the embryos. In addition to the macrophages, the fibroblastic cells and the cells of erythropoietic series were also present in the extravascular space. The macrophages showed a variety of cellular structures suggesting transition from immature cell type with no heterophagolysosomes to mature cell type in phagocytosis. The mature macrophages avidly phagocytized the primitive erythroblasts and occasionally platelets. They were positively stained for lysosomal enzymes and were characterized by numerous pleomorphic heterophagolysosomes which exhibited various stages of digestion of phagocytized blood cells. The origin of intravascular macrophages may be in either migrated extravascular macrophages or phagocytic endothelial cells. The phagocytosis and degradation of erythroblasts appear to be one of the main functions of yolk sac macrophages. The presence of the macrophages in mitosis indicates their proliferation in situ.     

Murine bone marrow stromal cells sustain in vivo the survival of hematopoietic stem cells and the granulopoietic differentiation of more mature progenitors

The study of the human hematopoietic system would be facilitated by availability of a relevant animal model. Because the medullar microenvironment is made of different types of cells, interactions between hematopoietic cells and stromal cells are difficult to analyze in detail. As an approach for establishing an in vivo model to dissect these interactions, we grafted murine bone marrow fibroblastic cells (MS-5 cell line) with hematopoietic cells into the kidney capsule of syngenic mice. To identify the origin of cells present in the graft, we used green fluorescent protein-stable transfected MS-5 cells for the transplantation. To analyze the evolution of stromal cells and identify hematopoietic cells able to develop in these conditions, we performed morphology, histochemistry, and immunohistology on tissue sections at different times after transplantation. When injected alone, MS-5 cells differentiate into adipocytes. When injected with a bone marrow suspension or with isolated CD45+ cells (leukocytes), the stromal cells keep their fibroblastic morphology and their alkaline phosphatase expression and sustain granulopoiesis. When injected with hematopoietic stem cells called c-kit+ Sca-1+ Lin- suspension, clusters of hematopoietic cells are also observed: They do not present any granulopoietic activity and do not belong to B or T population nor to erythroid lineage. They are quiescent, induce bone marrow recovery and survival of lethally irradiated recipients, are able to form macroscopic colonies in the spleen, and are able to form very few colonies in vitro, suggesting that they are hematopoietic stem cells. In conclusion, our results show that reticular fibroblastic stromal cells MS-5 sustain the survival of stem cells and are not able to induce their differentiation. However, they can control differentiation, proliferation, and/or survival of hematopoietic cells engaged in myeloid lineage.     

The transmural migration and release of blood cells in acute myelogenous leukemia

The transmural passage of malignant blood cells from the extravascular parenchyma into the sinusoidal lumen has been studied in the bone marrow of rats with myelogenous leukemia. The Shay myelogenous leukemia was chosen as a model system because an increased bone marrow cellularity is, in this leukemia, usually accompanied by an increase in circulating myeloid cells. By means of light microscopy, transmission electron microscopy (TEM) and scanning electron micrroscopy (SEM) it was found that the sinusoidal endothelial lining of the bone marrow remains intact and continuous even in advanced stages of the disease. SEM shows that the malignant myeloblast-like cell enters the sinusoidal lumen by means of a temporary migration pore, which appears only during the transmural passage of the cell. Certain nondegenerative changes in the sinusoidal blood vessels are associated with the myelogenous leukemia. The normal radial alignment of sinusoids about the central sinusoid is changed into a tortuous pattern, and intraluminal cytoplasmic bridges which impede the blood flow are formed by the endothelial cells.     

Histomorphological study of cellular interactions between stromal and haemopoietic stem cells in normal and leukaemic bone marrow

The interactions between haemopoietic and stromal elements are crucial for stem cell proliferation and differentiation. The bulk of this evidence is derived from experiments in rodents and in-vitro culture studies. We have studied the spatial relationships between the stromal and haemopoietic components and their cellular composition in histological sections of bone marrow (BM) from seven healthy fetuses, 10 normal adults and over 60 patients with acute myeloid leukaemia (AML) and chronic granulocytic leukaemia (CGL) at different stages of the disease. During the early developmental stage (16–18 weeks) fetal BM showed focal haemopoiesis with a characteristic spatial localization of haemopoiesis near bony trabeculae and around small blood vessels. In AML following the treatment-induced hypoplasia, large uniform unilocular fat cells arranged in groups designated ‘structured fat’ developed from scattered multilocular precursor fat cells. Early foci of haemopoietic regeneration were present almost exclusively in areas of structured fat. In the marrow of patients with CGL in blast transformation (BT) treated by intensive therapy and autografting with cryopreserved haemopoietic stem cells, the haemopoiesis was focal. Clusters of regenerating erythroid precursors or of megakaryocytes were seen in intimate contact with marrow sinusoids and granulopoietic precursors in intimate association with small blood vessels and also in close contact with the endosteal surface of bony trabeculae. We conclude that the endosteal cells, fat cells and the vascular endothedial cells comprise the critical non-haemopoietic stromal elements of human BM. The close associations observed between the regenerating haemopoietic cells and the stromal cells provide strong evidence in support of the existence of a permissive haemopoietic micro-environment in man and emphasize the structural and functional interrelationships that exists between bone, fat, the microvascular system and haemopoiesis in human bone marrow.     

Reactions of the blood system and stem cells in bleomycin-induced model of lung fibrosis

On the model of toxic diffuse pulmonary fibrosis induced by intratracheal administration of bleomycin, we studied reactions of the blood system, content of stem cells, committed hemopoietic and stromal progenitor cells in the bone marrow, spleen and peripheral blood of C57Bl/6 mice. It was shown that the development of diffuse pulmonary fibrosis was accompanied by hyperplasia of bone marrow hemopoiesis and leukocytosis in the peripheral blood. Activation of the erythroid and granulocytic hemopoietic stems was related to stimulation of hemopoietic stem cells (polypotent cells, granulocyte/erythroid/macrophage/megakaryocyte precursor cells) and committed erythroid and myeloid progenitor cells in the bone marrow. At the same time, the number of stromal precursors increased. Bleomycin increased the count of hemopoietic stem cells the peripheral blood and spleen and reduced the content of mesenchymal stem cells in the spleen and bone marrow.     

A study of intercellular relationships between trabecular bone and marrow stromal cells in the murine femoral metaphysis

The cellular relationship between the substantia spongiosa of bone (cancellous or trabecular bone) and the haematopoietic bone marrow in the femoral metaphysis of C57BL/6NJCL mice was studied by transmission electron microscopy (TEM). Special attention was directed to intercellular junctions between osteocytes, osteoblasts, and bone marrow reticular cells. These were gap junctions and adhesive devices of simple architecture referred to as primitive junctions or zonula adherens-like junctions. Gap junctions were observed between osteocytes (within the trabeculae) and osteoblasts (at the trabecular surface) and between osteoblasts and marrow reticular cells. Gap junctions were also observed between the same cell type within each of these categories. These junctions involved the plasmalemmal membranes of adjacent cell bodies and of processes. Primitive cell junctions had a similar cellular distribution. Quantitative analysis of the cell types covering or positioned around the trabecular bones and of gap junctions between these and other cells was carried out by TEM. It was found that osteoblasts were the most numerous cell type, occupying 31% of the total of each cell type positively identified around the trabeculae (31%), while pre-osteoblasts, (flattened bone marrow reticular cells) took up 26%. These data emphasise the intimate relationship of the various mesenchymal cells based on processes and intercellular junctions, and point to an anatomical and probably functional integration of trabeculae and marrow. The functional significance and putative regulatory activity of this unit are discussed.     

A histological study and three-dimensional reconstruction of F4/80-positive reticular cells and macrophages at the onset of murine bone marrow hematopoiesis

Adult bone marrow consists of two different compartments, a vascular compartment of sinusoid and a hematopoietic compartment consisting of stromal cells and hematopoietic cells. In the hematopoietic compartment, stromal cells play an important role in the formation of the microenvironment for hematopoiesis. To clarify the relationship between hematopoietic cells and stromal cells, particularly reticular cells and macrophages, we examined the femur bone marrow of ICR mouse fetuses and neonates using F4/80 immunostaining and three-dimensional reconstruction under light and electron microscopy. In the fetal femurs, the marrow cavity formed early from 15 days of gestation, and it showed a marked increase in volume thereafter. On the basis of the appearance of hematopoietic cells, marrow development could be classified into two stages, a pre-hematopoietic stage from 15 days of gestation to two days of age, and a beginning stage of hematopoiesis thereafter. The pre-hematopoietic bone marrow contains not only stromal reticular cells but also macrophages, and both types of stromal cells were strongly positive to F4/80 monoclonal antibody. These F4/80-positive reticular cells had a triangular cell profile with long and slender cytoplasmic processes. Reticular cells often contained large lysosomes of not only dying neutrophils but also erythroblast nuclei. A few erythroblasts accumulated around the processes, and the number of erythroblasts around reticular cells increased with bone marrow development. On the other hand, macrophages were located either close to sinusoids or in sinusoid lumen, and a close relationship to hematopoietic cells was hardly noticeable. At the beginning stage of hematopoiesis, F4/80-positive reticular cells extended their long and slender cytoplasmic processes, and the number and length of the processes appeared markedly increased. The three-dimensional cell surface of the F4/80-positive reticular cells became very complex. Numerous erythroblasts accumulated around the processes, and erythroblastic islands could gradually be recognized after four days of age. In the erythroblastic islands, central reticular cells were F4/80-positive and contained numerous large phagosomes originating from the expelled nuclei of erythroblasts. Although macrophages contained large phagosomes, the relationship between macrophages and hematopoietic cells could not clearly be elucidated even at the beginning stage of hematopoiesis. At the onset of bone marrow hematopoiesis, the hematopoietic compartment contained two kinds of F4/80-positive phagocytes, i.e., reticular cells and macrophages. In marrow erythroblastic islands, not macrophages but F4/80-positive reticular cells were located at the center of each island.     

Mechanisms of regulation of hemopoiesis during experimental cytostatic myelosuppression induced by carboplatin

The mechanisms of suppression and recovery of the bone marrow erythroid stem were studied on the model of myelosuppression induced by administration of carboplatin in the maximum tolerated dose. Single administration of the cytostatic led to the development of long-term hypoplasia of hemopoiesis. Despite enhanced proliferation of erythroid precursor caused by increased erythropoietic activity of blood plasma and bone marrow cells, inhibition of cell maturation prevented recovery of the content of morphologically recognizable erythrokaryocytes in the bone marrow. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 5, pp. 515–518, May, 2007     

Hemopoiesis recovery of irradiated rats conjugated with normo- and polycythemic animal by aortic anastomoses

The experiment was designed to observe the possible relation between myelopoietic and erythropoietic activities of circulating nucleated cells. Wistar rats were lethally irradiated with 60Co, 100 r once. Two days after irradiation the bone marrow cells had faded completely. At this stage animals were conjugated with normocythemic or polycythemic rats by aortic anastomoses. After conjugation the aplastic bone marrow of the irradiated animal rapidly regained its hemopoietic activity in cases having normocythemic and polycythemic partners. Active erythropoiesis and myelopoiesis were found 96 h after parabiosis in those having normocythemic partners. In animals having polycythemic partners, however, erythropoiesis was successfully suppressed. An increase in lymphoid cell numbers was found in place of decreased erythroid cells, but there was no change in the myeloid cell proliferation rate. No hemopoietic precursor cells or immature cells were found in circulating blood all through the experimental period before and after parabiosis. The data suggest that circulating nucleated cells have marked erythropoietic activity. Erythropoietic cells may be somehow related to lymphoid cells independent of myelopoietic activity.     

Development of the liver in the chicken embryo. II. Erythropoietic and granulopoietic cells

Hepatic hemopoiesis is apparent in the chicken embryo on day 7 of incubation (Hamburger and Hamilton Stage 30), and a peak in hemopoietic activity occurs on day 14 (Stage 40). During this period, the differentiation of hemopoietic cells was examined by light microscopy and by transmission and scanning electron microscopy. Glycol methacrylate sections were used in lieu of smears to study hemopoietic cells, thus minimizing the problems of cell shrinkage and rupture. The sections were superior to smears for close examination of nuclear and cytoplasmic morphologies and for precise localization of hemopoietic cells to intravascular and extravascular sites. The avian liver is involved directly with erythropoiesis and granulopoiesis only. Erythropoietic cells, occurring in intravascular and extravascular locations, appear throughout the time frame examined. Blood islands with granulopoietic cells were not observed until days 8–9 (Stage 35). Granulopoiesis in the liver produces only eosinophilic leukocytes. Individual granulopoietic cells appear first in the connective tissue sheaths of hepatic vessels, and these cells subsequently congregate into blood islands. Endothelial cells of the sinusoidal linings, through asymmetric divisions, frequently release daughter cells into the circulation, and Kupffer cells are actively engaged in phagocytosis of erythrocytes. From a comparative standpoint the elements deemed critical to hemopoiesis in the mammalian liver–prehepatocyte population, hepatic vasculature, and compartments for stem cell differentiation–may not hold the same importance in the bird, owing to an inordinate reliance on intravascular hemopoiesis in this vertebrate class. © 1993 Wiley-Liss, Inc.     

Bone marrow stromal cells as targets for gene therapy

The bone marrow (BM) is composed of the non-adherent hematopoietic and adherent stromal cell compartment. This adherent BM stromal cell fraction contains pluripotent mesenchymal stem cells (MSCs) and differentiated mesenchymal BM stromal cells. The MSCs self-renew by proliferation while maintaining their stem-cell phenotype and give rise to the differentiated stromal cells which belong to the osteogenic, chondrogenic, adipogenic, myogenic and fibroblastic lineages. A more primitive adherent stem cell was recently identified, the multipotent adult progenitor cell (MAPC) or mesodermal progenitor cell, which co-purifies with MSCs. These MAPCs differentiate into MSCs, endothelial, epithelial and even hematopoietic cells. BM stroma cells, including the primitive pluripotent MSCs and MAPCs, are attractive targets for cell and gene therapy. The BM stromal cell population and its multipotent stem cells can be engineered to secrete a series of different proteins in vitro and in vivo that could potentially treat a variety of serum protein deficiencies and other genetic or acquired diseases, including bone, cartilage and BM stromal disorders or even cancer.     

Basement membrane of mouse bone marrow sinusoids shows distinctive structure and proteoglycan composition: a high resolution ultrastructural study

Venous sinusoids in bone marrow are the site of a large-scale traffic of cells between the extravascular hemopoietic compartment and the blood stream. The wall of the sinusoids consists solely of a basement membrane interposed between a layer of endothelial cells and an incomplete covering of adventitial cells. To examine its possible structural specialization, the basement membrane of bone marrow sinusoids has now been examined by high resolution electron microscopy of perfusion-fixed mouse bone marrow. The basement membrane layer was discontinuous, consisting of irregular masses of amorphous material within a uniform 60-nm-wide space between apposing endothelial cells and adventitial cell processes. At maximal magnifications, the material was resolved as a random arrangement of components lacking the "cord network" formation seen in basement membranes elsewhere. Individual components exhibited distinctive ultrastructural features whose molecular identity has previously been established. By these morphological criteria, the basement membrane contained unusually abundant chondroitin sulfate proteoglycan (CSPG) revealed by 3-nm-wide "double tracks," and moderate amounts of both laminin as dense irregular coils and type IV collagen as 1-1.5-nm-wide filaments, together with less conspicuous amounts of amyloid P forming pentagonal frames. In contrast, 4.5-5-nm-wide "double tracks" characteristic of heparan sulfate proteoglycan (HSPG) were absent. The findings demonstrate that, in comparison with "typical" basement membranes in other tissues, the bone marrow sinusoidal basement membrane is uniquely specialized in several respects. Its discontinuous nature, lack of network organization, and absence of HSPG, a molecule that normally helps to maintain membrane integrity, may facilitate disassembly and reassembly of basement membrane material in concert with movements of adventitial cell processes as maturing hemopoietic cells pass through the sinusoidal wall: the exceptionally large quantity of CSPG may represent a reservoir of CD44 receptor for use in hemopoiesis.     

Ulrastructure of cytoskeletal element of endothelial and stromal cells of rat marrow

This study reports the distribution of microfilaments, microtubules, and intermediate filaments in endothelial cells, reticular cells, and macrophages of bone marrow of rats following fixation with glutaraldehyde, tannic acid, and saponin. In endothelial cells bundles of microfilaments are seen along the basal surface, where these cells adhere to underlying extracellular materials. The reticular cells, especially those that are closely associated with the endothelium of sinusoids, contain many intermediate filaments and microtubules as well as microfilaments. The reticular cell processes that partially cover the endothelium and extend among the blood cells have numerous microtubules and intermediate filaments arranged longitudinally within them; these cytoskeletal elements appear to provide mechanical support for the processes. Macrophages also have many microtubules and intermediate filaments but these organelles do not extend into the thin processes of these cells as is the case with reticular cells. Bundles of microfilaments are observed in the cytoplasm of adventitial and endothelial cells at sites where migrating blood cells are attached to these cells producing local regions of stress.     

Ultrastructure of cytoskeletal elements of endothelial and stromal cells of rat marrow

This study reports the distribution of microfilaments, microtubules, and intermediate filaments in endothelial cells, reticular cells, and macrophages of bone marrow of rats following fixation with glutaraldehyde, tannic acid, and saponin. In endothelial cells bundles of microfilaments are seen along the basal surface, where these cells adhere to underlying extracellular materials. The reticular cells, especially those that are closely associated with the endothelium of sinusoids, contain many intermediate filaments and microtubules as well as microfilaments. The reticular cell processes that partially cover the endothelium and extend among the blood cells have numerous microtubules and intermediate filaments arranged longitudinally within them; these cytoskeletal elements appear to provide mechanical support for the processes. Macrophages also have many microtubules and intermediate filaments but these organelles do not extend into the thin processes of these cells as is the case with reticular cells. Bundles of microfilaments are observed in the cytoplasm of adventitial and endothelial cells at sites where migrating blood cells are attached to these cells producing local regions of stress.