Hemopoietic Foci Research Articles

Peculiarities of Gene Transfer into Mesenchymal Stem Cells.

Murine mesenchymal stem cells in long-term bone marrow culture were genetically labeled using lentiviral vector carrying enhanced green fluorescent protein (eGFP) reporter gene under SFFV promoter or without it. We studied the developmental fate of labeled mesenchymal stem cells in stromal cell layers of long-term bone marrow culture and in ectopic hemopoietic foci formed by these stromal layers under the renal capsule of syngeneic mice. The frequency of labeled polypotent stromal precursors (fibroblast CFU) was analyzed in adherent cell layers of long-term culture and ectopic foci formed from them. The proportion of labeled fibroblast CFU in ectopic foci increased by 10 times in case of implantation of adherent cell layers infected with lentivirus containing eGFP reporter gene without promoter. eGFP expression leads to rejection of labeled stromal cells. Labeling with eGFP-carrying vector without promoter makes possible long-term tracking of mesenchymal stromal cells.

Characteristics of Mesenchymal Stromal Precursor Cells Labeled with Lentiviral Vector in Long-Term Bone Marrow Culture

Mouse mesenchymal stromal precursor cells were labeled with lentiviral vector in long-term bone marrow culture. We studied the fate of labeled cells in the stromal sublayer of the long-term bone marrow culture and in ectopic hemopoiesis foci formed from the labeled cultures. The incidence of labeled polypotent fibroblast CFU in sublayers of long-term bone marrow culture and in ectopic hemopoiesis foci formed from these sublayers under the renal capsule of syngeneic mice was also analyzed. It was shown that the marker gene was present in about 40% cells of the stromal sublayer and 30% fibroblast CFU and that effective gene transfer did not affect the total production of hemopoietic cells. The size of ectopic hemopoietic foci formed after implantation of labeled sublayers of the long-term bone marrow culture under the renal capsule did not differ from the control. Differentiated cells of the osseous shell in these foci carried the marker gene in 40% cases. Analysis of fibroblast CFU in these foci showed that despite the total concentration of fibroblast CFU was comparable to that in the bone marrow, the concentration of labeled fibroblast CFU was about 6%, which suggests that one more class of precursors probably exists in the hierarchy of stromal cells presumably between mesenchymal stem cells and fibroblast CFU. Our findings demonstrate the capacities of mesenchymal stem cells to self-maintenance and differentiation without losing the marker gene integrated into the genome.

Early Embryonic Development of the Liver of the One-Humped Camel (Camelus dromedarius)

The present study was carried out to highlight the histomorphological structure of the liver of the one-humped ca-mel during early embryonic life. Samples were collected from pregnant she- camles after slaughtering and evisceration. Different embryonic stages were taken and the crown to rump (CVR) length was measured to the nearest millimeter (ranging from 7 to 185 mm). Morphological study revealed that the liver primordium appeared at 7mm CVR length fetuses. At 12mm CVR length the liver was related cranially to the septum transversum and heart, caudally to the primitive spleen, mesogastrium, and primitive stomach and to the lesser omentum while dorsally it was related to the mesonephrous and ventrally to the floor of the abdominal cavity. Lobation of the liver appeared at fetal lengths of 50 mm CVR length. Histological study revealed that the liver parenchyma consisted of a mass of hepatic foci, haemopoietic cells and irregular wide blood spaces. The primordium of Glisson's capsule appeared in fetuses of 75 mm CVR length. Megakaryocytes were noticed among hepatocytes and heamopoietic foci. Sections stained with theGiemssa stain revealed group of mast cells among hepatocytes. Hepatic tis- sue stained by Methyl green pyroninn stained revealed that hepatocytes showed methyl green positive materials while haemopoietic cells showed pyrinophilic positive granules at 12 mmCVR length fetuses. Small sized mitochondrial granules appeared in the cytoplasm of the hepatocytes stained with Iron haematoxylin in fetuses of 12 mm CVR length. Hepatocytes were faintly stained with PAS. Electron microscopicstudy revealed that the hepatocytes of 185 mm CVR length camel fetuses con- tained large glycogen globlules in addi- tion to abundant mitochondria. Histo- morphometric studies were applied to compare the mean number of hepatic foci to that of haemopoietic foci and also to measure the number and size of megakaryocytes.

Hemothorax due to extramedullary erythropoietic masses

We describe a 27-year-old male patient suffering from β-thalassemia intermedia who presented with a nontraumatic spontaneous hemothorax due to extramedullary hemopoietic foci. In reviewing the literature, four similar reports were found. The details of this unusual entity are discussed.

Hemopoiesis on Acetate Cellulose Membranes in the Presence of Lipopolysaccharide

The formation of hemopoietic colonies on acetate cellulose membranes in the peritoneal cavity of mice was markedly enhanced after the injection of bacterial lipopolysaccharide. In addition to granulocytic-macrophagal differentiation, the foci of erythropoiesis appeared. The stimulating effect of lipopolysaccharide was not expressed in nonirradiated mice and during the formation of hemopoietic foci on acetate cellulose membranes in the subcutaneous connective tissue.

Expression of alphaVbeta3 integrin in the chick embryo aortic endothelium.

The integrin chain alphaV, expressed in association with beta3, by cells of the megakaryocytic/thrombocytic and endothelial lineages is thought to play an important role in angiogenesis. alphaVbeta3 expression by endothelial cells is not constitutive but induced by various stimuli in avian and human models. Here the developmental pattern of alphaVbeta3 expression was analysed in the chick embryo by immunocytochemistry, using a specific monoclonal antibody. On day 2 of development alphaVbeta3 expression was restricted to rare cells in the blood stream, in the embryo proper and in the yolk sac blood islands. AlphaVbeta3 expression by endothelial cells became detectable on day 3 and was restricted to the dorsal aorta. Interestingly it was absent from the intra-aortic hemopoietic clusters (E3.5) which, as we have showed previously, express the alphaIIbbeta3 integrin and display progenitor potentialities. However the endothelium underlying intra-embryonic hemopoietic clusters expressed this integrin. In contrast E6-7 para-aortic hemopoietic foci contained numerous alphaVbeta3 positive cells. Both alphaVbeta3 and alphaIIbbeta3 were expressed in these latter hemopoietic sites, while alphaVbeta3 was still selectively expressed by the aortic endothelium until E6. Thereafter, at E7 the pulmonary artery also expressed it. Since alphaIIbbeta3 is expressed by avian and murine multilineage hemopoietic progenitors, we then studied the hemopoietic potentialities of alphaVbeta3/alphaIIbeta3 double positive cells from embryonic bone marrow differentiating in vitro in erythro-myeloid conditions. Thrombocytic, erythroid and myeloid progenitor potentialities were found within the cell population expressing both beta3 integrins.

Crucial role of donor-derived stromal cells in successful treatment for intractable autoimmune diseases in mrl/lpr mice by bmt via portal vein.

We have recently established a new bone marrow transplantation (BMT) method for the treatment of intractable autoimmune diseases in MRL/lpr mice; the method consists of fractionated irradiation (5.5 Gy x 2), followed by BMT of whole bone marrow cells (BMCs) from allogeneic C57BL/6 mice via the portal vein (abbreviated as 5.5 Gy x 2 + PV). In the present study, we investigate the mechanisms underlying the early engraftment of donor-derived cells in MRL/lpr mice by this method. In the mice treated with this method, the number of donor-derived cells possessing the mature lineage (Lin) markers rapidly increased in the BM, spleen, and liver; almost 100% were donor-derived cells by 14 days after the treatment. The number of donor-derived hemopoietic progenitor cells (defined as c-kit(+)/Lin(-) cells) increased in the BMCs, hepatic mononuclear cells, and especially spleen cells by 14 days after the treatment. Simultaneously, hemopoietic foci adjoining donor-derived stromal cells were observed in the liver when injected via the PV, but not via the peripheral vein (i.v.). When adherent cell-depleted BMCs were injected via the PV, recipients showed a marked reduction in the survival rate. However, when mice were transplanted with adherent cell-depleted BMCs with cultured stromal cells, all the recipients survived. These findings suggest that not only donor hematopoietic stem cells (HSCs) but also donor stromal cells administered via the PV were trapped in the liver, resulting in the early engraftment of donor HSCs in cooperation with donor-derived stromal cells. This new strategy to facilitate the early recovery of hemopoiesis would therefore be of great advantage in human application.

Chondrogenesis and Osteogenesis in Ectopic Transplants of the Fetal Liver in Mice

It was shown for the first time that when the livers of 14 day-old mouse fetuses was transplanted under the renal capsule or in the subcutaneous connective tissue of adult recipients, successive de novo formation of hyaline cartilage, bone, and hemopoietic foci took place. We propose that the liver mesenchyme, which preserves wider differentiation potencies in fetuses, is the cellular source of different types of mechanocytes: cartilaginous, bone, and reticular cells.

Study on Localization of Hemopoietic Tissue in Sturgeon

Morphological and autoradiographic studies on various hemopoietic tissues in sturgeon are presented. The classic hemopoietic organs characteristic of lower vertebrates, such as the kidney and spleen, are studied as well as unique hemopoietic structures described only in the evolutionarily most ancient fish species (hemopoietic tissues of cartilaginous skull capsules and epicardium). The intensity of cell divisions in hemopoietic foci has been characterized by autoradiography. The results obtained provide a basis for the revision of traditional views about the phylogeny of hemopoiesis. They provide evidence that the osteogenic gravitation of hemopoietic tissue shows up in evolution alongside the appearance of the inner skeleton.

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.

Splenic cultures from rainbow trout,Oncorhynchus mykiss: establishment and characterisation

This study describes the culture conditions and the phenotypic features of different types of splenic cultures established from explants. Using the same culture technique it was possible to grow splenic explants from which monolayers of reticular origin, long-term haematopoietic cultures, and subcultures were obtained. The cultures were characterised by light and electron microscopy, cytochemical and immuno-cytochemical analyses, phagocytic activity and susceptibility to virus. The cultures comprised multilayers of epithelioid and fibroblastoid cells with haemopoietic foci, melanomacrophages and eosinophilic granular cells. The cytochemical and immuno-cytochemical analyses revealed that the stromal cells were always positive for ANAE activity. The stromal cells in primary cultures were negatively or weakly stained by antibodies directed against cytokeratins and S-100, but in the subcultures they were strongly stained by these antibodies. The stromal cells had very poor phagocytic activity and were susceptible to VHS virus.

Experimental analysis of cell interactions during hemopoiesis.

The principal approach was to study hemopoiesis on different stromal cell underlayers (fibroblasts or fibroblast-like cells covering a foreign body implanted into the peritoneal cavity of mice or other rodents) after intraperitoneal transplantation of syngeneic, allogeneic, and xenogeneic hemopoietic cells. The data obtained are compared with the results of experiments on repopulation of ectopic hemopoietic territories (under the mouse kidney capsule) by syngeneic and xenogeneic hemopoietic cells. Competitive cell interactions are described that occur during repopulation of the hemopoietic stroma or formation of the hemopoietic foci on cellulose acetate membranes (CAMs) in the peritoneal cavity of irradiated mice by genetically different hemopoietic cells transplanted to these animals (multicomponent radiation chimeras). The model of xenogeneic and multicomponent radiation chimeras was reproduced in long-term bone marrow cultures, where hemopoietic cells of different genotypes coexisted, without any competitive cell elimination. The second part of this review deals with hemopoiesis on stromal cell underlayers, formed by cells of different origin, different stages of development, and obtained from other sources. These underlayers were formed on CAMs in vitro and then transferred into the peritoneal cavity of irradiated mice, which subsequently received intraperitoneal injections of donor hemopoietic cells. Specific features of hemopoiesis on stromal underlayers formed by the following cell types are described: (1) fibroblasts from mouse embryos at different developmental stages; (2) fibroblasts from the skin, liver, and bone marrow of 17-day mouse fetuses and newborn mice; (3) fibroblasts from the monolayer cultures of mouse and rat bone marrow; (4) 3T3 cell line; (5) hepatocytes of 17-day mouse fetuses or sexually mature rats; (6) newborn mouse kidney cells; and (7) cells transgenic for the erythropoietin gene. The phenomena observed in these experiments and their probable mechanisms are discussed.

Vascular networks within the stroma of human long-term bone marrow cultures.

The formation of branching and anastomosing vascular structures has been demonstrated within the stromal layers of human long-term bone marrow cultures (LTBMCs). Such organized vascular structures have not been described previously and may be of functional importance, as adherent granulocyte-predominant haemopoietic foci were more numerous in their interstices and margins than elsewhere in the stromal layers. Mature granulocytes were seen within vascular lumina in a few instances, possibly indicating an attempt to recapitulate the normal process of egress of neutrophils from bone marrow into the circulation. Immunostaining showed that the vascular structures expressed CD31, CD34, and CD105 (endoglin), which were not expressed by stromal fibroblastic cells. Collagen IV and laminin, expressed throughout the stroma, were present in increased amounts where vascular arrays were seen. In contrast, von Willebrand factor (VWF) and vascular cell adhesion molecule-1 (VCAM-1) were expressed equally by the vascular arrays and by stromal fibroblastic cells. Neither the arrays nor the general stroma expressed intercellular adhesion molecule-1 (ICAM-1), ICAM-2, ICAM-3, or endothelial leukocyte adhesion molecule-1 (ELAM-1).

Immunohistochemical characterization of intact stromal layers in long-term cultures of human bone marrow.

We have performed an immunohistochemical study of intact adherent layers of human long-term bone marrow cultures (hLTBMC) in order to characterize the cell types present. Our panel of antibodies was selected to demonstrate various mesenchymal and haemopoietically derived cell types and to assess the presence of molecules of potential importance as adhesive ligands between haemopoietic cells and stroma. Subpopulations of fibroblasts and macrophages were identified which differed in immunophenotype. We were able to demonstrate modulation of fibroblast and extra-cellular matrix immunophenotypes between 2 and 12 weeks in culture. Stromal cells and matrix expressed a wide variety of antigens of potential importance in haemopoietic cell adhesion, but no ICAM-1, 2 or 3 could be demonstrated to correspond to the strong LFA-1 expression seen in haemopoietic precursor cells. No localization of antigen expression by stromal elements was found to account for the formation of haemopoietic foci at particular sites. However, granulocyte-predominant foci preferentially occupied the interstices and margins of structures which appeared to be vascular arrays.

Promotion by estriol of the development of grafted fetal livers in mice.

Mouse fetal livers of 12 days gestation were transplanted beneath the kidney capsule of syngeneic castrated male hosts. Recipients received a single intraperitoneal injection of 5 mg estriol (E3) immediately after transplantation and were sacrificed 4, 7 and 14 days later. Control mice received injections of solvent only. The fetal liver grafts of the E3 groups showed remarkable growth compared with the control grafts at each corresponding time. In the grafted fetal livers of the E3 groups, many more basophilic hepatocytes appeared for much longer periods, and the early formation of wide sinusoids was noted. Although hemopoietic activity had almost ceased a short time after transplantation in the control grafts, many prominent extrasinusoidal erythroid, granulocytic and megakaryocytic hemopoietic foci developed and persisted to the end of the experiments in the grafted fetal livers of the E3 groups. In the next experiment, the recipients received three injections of 0.5-5 mg E3 at 5-day intervals, and were sacrificed 21 days after transplantation. Control animals received solvent only in the same manner. The fetal liver grafts of the E3 groups also showed remarkable, dose dependent growth. The growth was particularly striking at doses of 3-5 mg E3. In the grafts of these groups, basophilic hepatocytes were predominant, and hepatic cords and sinusoids were well formed. Moreover, sinusoidal erythropoiesis was intense. These results provide evidence for the possible role of E3, secreted from the placenta into the fetal circulation, in the development of the fetal liver.

The ultrastructure of intravascular and coelomic scavenger macrophages of the mouse embryo.

The aim of this study was to establish whether or not mononuclear cells which appear in both the vitelline vessels and embryonic coelom in mice prior to liver hemopoiesis are specialized scavengers. Before the initiation of liver hemopoiesis, the majority of the embryonic blood cells were primitive erythroblasts derived from yolk sac hemopoietic foci. In addition, the peripheral blood contained a few free phagocytes as early as 10 days of gestation. The phagocytes devoured various cell elements such as degenerating erythroblasts and cell fragments. Ultrastructurally, they had long filamentous cytoplasmic projections on their cell surface, clear subsurface vacuoles or vesicles, lipid droplets, a few lysosomal granules, large heterogeneous phagolysosomes and residual bodies. Mononuclear phagocytes with ultrastructural features similar to those of the intravascular phagocytes also could be observed in the intraembryonic peritoneal cavities at 10 days of gestation; they sometimes engulfed possible mesothelial cells undergoing degeneration. Based on fine structural criteria, these intravascular and coelomic phagocytes were considered to be specialized scavenger macrophages with the function of clearing the blood and tissue fluid of whatever has been ingested. In so doing, they serve as the most primitive discriminating filter set in embryonic circulation.

THE GROWTH OF HEMOPOIETIC STEM CELLS IS INHIBITED BY NATURAL KILLERS ONLY IN THE NONSYNGENEIC MICROENVIRONMENT

The growth of hemopoietic cells of some inbred animal lines is repressed in hybrids of the first generation (F1)--the phenomenon of hybrid resistance. We have investigated the mechanism of hybrid resistance by studying the growth of hemopoietic cells of C57BL/6 mice in syngeneic and semisyngeneic heterotopic hemopoietic foci formed under the kidney capsule in (CBA*C57BL/6)F1 recipient mice. We found that hybrid resistance depended upon the joint involvement of NK cells and semisyngeneic hemopoietic stroma. It is concluded that NK cells recognize antigenic markers appearing on target cells located in a nonsyngeneic microenvironment.

Localisation of granulocyte macrophage colony-stimulating factor in human long-term bone marrow cultures. Biological and immunocytochemical characterisation.

The distribution of granulocyte macrophage colony-stimulating factor (GM-CSF) in human long-term bone marrow cultures (HLTBMC) was examined using two monoclonal antibodies raised using purified recombinant GM-CSF and a third commercially available GM-CSF antibody. The antibodies were able to bind to purified recombinant GM-CSF and showed inhibition of GM-CFC colonies in the presence of both recombinant and native protein. All antibodies displayed similar patterns of distribution in both permeabilised and non-permeabilised stromal cell preparations. Fibroblasts were labelled at their periphery in early cultures and both endothelial cells and fibroblasts showed cytoplasmic labelling with anti-GM-CSF. The fact that GM-CSF appears to be sequestered by cells of the bone marrow stroma raises the possibility that it is synthesized by these cells and may regulate activity of the progenitor cells in the haemopoietic foci. In contrast, early progenitor cells within the foci did not stain with any of the anti-GM-CSF antibodies. Adipocytes, which differentiate from fibroblasts in these cultures, showed a diffuse staining pattern. Two types of macrophage staining were observed in the non-permeabilised cells; those exhibiting only autofluorescence and those that bound the antibody. Intracellular staining was apparent in a small sub-population. Generally, the staining persisted up to eight weeks of culture and thereafter declined, becoming virtually undetectable after 12 weeks. This correlates with the pattern of GM-CFC production in long-term bone marrow cultures.

Immature endodermal teratoma of the ovary: Embryologic correlations and immunohistochemistry

Two grade 2 ovarian immature, predominantly endodermal teratomas are reported. The teratomas were in stage I and occurred in two girls, 9 and 10 years of age, who were treated with triple chemotherapy. These neoplasms differed from the usual immature ovarian teratoma as they contained no neuroectodermal components and had high α-fetoprotein and low human chorionic gonadotropin levels as their serum markers despite the absence of other concomitant germ cell tumors. The epithelia of the teratomas demonstrated exclusively the embryologic development of endoderm, ranging from early endoderm to tissues similar to esophagus, liver, and intestinal structures. All epithelial derivatives were positive for α-fetoprotein and α 1-antitrypsin. Liver and esophagus expressed fibrinogen, while intestine and esophagus were positive not only for carcinoembryonic antigen and chromogranins but also for thyroglobulin, thus reflecting yet another type of endodermal differentiation into thyroid. Focal human chorionic gonadotropin positivity associated with primitive intestinal and esophageal epithelia may reflect the early embryologic relationships between endoderm and trophoblast. These cases demonstrate that simultaneous α-fetoprotein and human chorionic gonadotropin secretion may occur in immature teratoma. The mesenchymal component also showed a wide range of differentiation, from primitive mesoblastic cells to differentiated cells, such as hemopoietic foci, smooth muscle, bone, and cartilage. Both the primitive endoderm and the mesenchyme co-expressed vimentin and keratin, reflecting their intimate developmental relationships and possibly supporting the hypothesis of mesenchyme originating from endoderm, as suggested by previous embryologic studies. Since endodermal and mesenchymal areas similar to those described here are found in association with yolk sac tumors and embryonal carcinoma, it is possible that the present cases may represent an endodermal differentiation accomplished by either of these developmentally related germ cell tumors.

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.