Neonatal tolerance induction in the thymus to MHC-class II-associated antigens. III. Significance of hemopoietic stem cells for induction and maintenance of Mls tolerance by continuous supply of tolerance-inducing nonlymphocytes

The role of hemopoietic stem cells and other cell types in the induction and maintenance of immunologic tolerance in the thymus was investigated by intravenous injection of Mls-semi-allogeneic cells into newborn mice less than 24 hr after birth. Mls-specific tolerance was induced by inoculation of peritoneal cells and thymus cells, and the tolerant state was compared with that induced by bone marrow cells which had hemopoietic stem cell activity and were able to create a stable chimera in both central and peripheral lymphoid organs. When peritoneal or thymus cells were injected, the level of tolerance attained was proportional to the number of cells injected, though peritoneal cells were 20 times as effective as thymus cells. In vivo functions of tolerance-inducing cells and their immediate precursors were radiosensitive and belonged to a Thy-1-, nylon-wool-nonadherent (probably non-B), weakly Sephadex G-10-adherent cell population. Tolerance induced by peritoneal cell injections was transient, starting to terminate within the first 2 weeks of life, while tolerance caused by bone marrow cell injections persisted through more than 6 weeks. Such transient tolerance induced by the former became long-lasting when followed by an additional injection of bone marrow cells, which did not cause thymic lymphocyte chimerism. All data indicated that bone marrow stem cells were engaged in tolerance induction and maintenance by continuously supplying tolerance-inducing nonlymphocytes.     

The origin of hematopoietic stem cells in the ontogenesis of vertebrates

A review of the origin of stem blood cells in ontogeny of vertebrates is presented. The comparative analysis of the data on laying, determination and migration of the hemopoietic precursor cells during embryogenesis in various taxonomic groups (teleosteans, urodeleans, anurans, avians and mammals) is performed. The change of the hemopoietic site and erythroid cells populations has been described. The data on sources of blood cell precursors and the origin of hemopoietic cells in the primordiums of hemopoietic organs were classified. A conclusion has been reached that in the course of evolution the hemopoietic anlage is gradually divided into two parts: one part migrates to the extraembryonic (ventral) mesoderm and another one remains intraembryonically and gives rice to the predecessors of definitive hemopoietic stem cells.     

Embryonic Sources of Definitive Hemopoietic Stem Cells

A review of one of the key problems of experimental hematology: the origin of hemopoietic stem cells in the development of vertebrates (amphibians, birds, and mammals). The appearance and functioning of two independent sources of hemopoietic stem cells (extra- and intraembryonic) were considered in amphibians, birds, and mammals. The contribution of each source to the formation of definitive hemopoietic tissue was analyzed. It was shown for amphibians and birds that intraembryonic organs such as the dorsolateral plate and the mesenchyme of dorsal aorta are involved in the formation of adult hemopoietic tissue, while the extraembryonic organs such as ventral islets and the yolk sac are devoid of true stem cells and provide only for the primary, transient hemopoiesis. New data have been considered concerning the previously unknown intraembryonic hemopoietic organ in mammals, a region of aorta–gonad–mesonephros arising in embryogenesis simultaneously with the yolk sac. Two extreme views on the involvement of stem cells of all these organs in the formation of definitive hemopoiesis have been considered. The data are provided on the interaction of the embryonic hemopoietic stem cells and the hemopoietic microenvironment of adult recipients.     

Reaction between the mechanocytes of hematopoietic organs in collagen gel

Interactions between stromal mechanocytes of hemopoietic organs were studied at their cultivation in three-dimentional collagen gel. It was demonstrated that cellular cords appearing between fibroblast colonies and between fragments of hemopoietic organs are of fibroblastic nature. They are not resulted from organic or specific peculiarities, or from discharge of substances attracting fibroblasts. A linear dependence between the amount of fibrorow or spleen was noted. Fibroblast colonies formed by the hamster bone marrow and splenic cells, as well as by passaged fibroblasts of the guinea pig bone marrow were obtained. In order to form colonies by the passaged fibroblasts it is necessary to add of irradiated cells. Its effect, besides the medium conditioning, is evidently, in restriction of fibroblast mobility in collagen gel.     

Extrachromosomal circular DNAs from murine hemopoietic tissue cells

Extrachromosomal circular DNA complexes from cells of murine hemopoietic organs, bone marrow, thymus, spleen, and lymph nodes were examined by mica-press-adsorption method (H. Yamagishi, T. Kunisada, and T. Tsuda, 1982, Plasmid 8, 299-306). They showed wide size distribution, from 0.3 to 10 micron. The large-size DNAs of more than 1 micron (3.1 kb) in contour length were more abundant in bone marrow and thymus than they were in spleen and lymph nodes. The appearance of the large size DNAs was examined on splenocytes of athymic nude mice during ontogeny. The large-size DNAs first became detectable after 2 weeks of age and the amount increased thereafter until 9 weeks of age. It appears that large-size circular DNAs appear during differentiation from the hemopoietic stem cells into several descendent cells. Possible immunological implications for the appearance of extrachromosomal circular DNAs are discussed.     

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     

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.     

Effects of226Ra and X-irradiation on the proliferative and differentiative ability of mouse hemopoietic stem cells

Summary The ability of hemopoietic stem cells to repopulate spleens of heavily irradiated syngeneic hosts in form of macroscopically visible clonal colonies of differentiating cells was studied in mice exposed for 32 and 4 weeks to internally deposited²²⁶Ra (0.56 and 0.46 µCi per mouse respectively) or to 100 rad X-irradiation. Exocolonizing test and cytological techniques were used for quantitative evaluation. The size of stem cell compartment was reduced and the function of the surviving stem cells was altered by radium and X-ray irradiation. The proliferation and maintenance of hemopoietic cell populations were found to depend not only on the numbers of stem cells but also on their multiplicative and differentiative capability.     

Changes in the histostructure of functional activity of the immunocompetent organs in damaged portal blood supply of the liver

In CBA mice calibrated stenosis of the portal vein was produced. Liver and immunocompetent organs were morphologically analyzed. The total number of hemopoietic stem cells in the bone marrow was estimated by the colony-forming cells and in the spleen after immunization with sheep red cells by the plaque forming method. It is established that stenosis of the portal vein (on the average by 45% and 58%) produced the histostructural changes in the liver and in the immunocompetent organs. Expression of morphological changes depended on the time elapsed after operation and the degree of the portal vein stenosis. These changes were the most pronounced on the 16-17th day when stenosis of the portal vein was 58%. The character of the changes in the number of the hemopoietic stem cells in the bone marrow and in that of antibody-forming cells in the spleen depended on the degree of the liver damage. These changes increased with the degree of the liver histostructure damage. The maximal liver damage was accompanied by a decrease of these indices.     

Compensation mechanisms in hemopoietic stem cell pool (CFUs) under the conditions of experimental chronic gamma-irradiation

The kinetics, proliferation and differentiation potentials of hemopoietic stem cells (CFUs) of bone marrow and spleen were investigated in CBA-line mice in the early period (1-30 days) of chronic gamma-irradiation at a dose rate of 0.16 Gy/day to attain a cumulative dose of 4.8 Gy. The results of the experimental study showed the prevalent maintenance of productivity of granulocytic and erythrocytic hemopoietic cell series within the range of reference values, persistent inhibition of the megakaryocytic series (in terms of all hemopoiesis parameters of interest), more marked suppression of the population of polypotential CFUs in the bone marrow as compared with that in the spleen. The obtained results indicated that the mechanisms of hemopoiesis compensation at stem cell pool level were as follows: the increase in proliferation potency of erythrocytic and in polypotential precursors, the rise in the proportion of granulocytic precursors in the real differentiation potential of CFUs, and the processes of repopulation manifested with different intensity in all stem cell populations under study. For maintenance of the necessary productivity of CFUs in each of hemopoietic cell series, consecutively or simultaneously, several compensatory-adaptive mechanisms are started, which allows the avoidance of a sharp competition between hemopoietic cell series under the conditions of stem cell pool depopulation, and preservation of the hemopoiesis as a whole.     

Cells that maintain long-term hematopoiesis in culture do not possess the capacity for regeneration following irradiation

It has been revealed by competitive repopulation assay that hemopoietic stem cells capable of supporting long-term hemopoiesis in the culture failed to regenerate after irradiation. 19 weeks after irradiation with 4 Gy the content of hemopoietic stem cells was 0.5% normal, while regeneration of CFUs was achieved up to subnormal level.     

Compensatory adjustment of hemopoietic stem cell pool of CBA mice after acute intake of 90Sr

It was investigated the functional status of stem cell pool (CFUs) of bone marrow, spleen and peripheral blood in mice (CBA) in early (1-30 days) and late (180-360 days) period after acute intake of 90Sr (29.6 kBq/g). Cumulative dose in red bone marrow due to incorporated 90Sr was 0.98-87.7 Gy. The kinetics, proliferative and differentiative potential of stem hemopoietic cells (CFUs) and productivity of hemopoietic tissues were significantly influenced by dose rate, absorbed dose and degree of suppresssion of bone marrow functions.The obtained results indicated that the sarcomogenous doses of 90Sr (29.6 kBq/g) resulted in realization of compensatory reactions in hemopoietic stem cell pool to support the life ability of irradiated animals: higher proliferative potential of CFUs and its repopulation, redistribution of cell subpopulations during differentiation and activation of spleens hemopoiesis.     

Experimental studies on hemopoiesis in the pronephros of Rana pipiens.

Embryogenesis of hemopoietic cell populations in the pronephros of Rana pipiens was examined during embryonic and early larval development. Differential cell counts of Wright-Giemsa-stained cell suspensions demonstrated that granulopoiesis is the predominant hemopoietic activity in the pronephros, erythropoiesis accounts for a minor component of the hemopoietic activity (less than 10%), and lymphopoiesis within the organ is negligible. Microdensitometric analysis of Feulgen-DNA stained granulocyte populations in pronephroses from larvae that had received chromosomally labeled pronephric analgen transplants between 84 and 96 h of development demonstrated that hemopoiesis in this organ is dependent on colonization by an extrinsic hemopoietic stem cell. A similar analysis of pronephric hemopoiesis in larvae which had received chromosomally labeled, presumptive ventral blood island transplants between 62 and 67 h of development, indicates that granulopoietic cells are not derived from the embryonic blood islands. It is proposed that the pronephros may be the initial site of granulocyte differentiation during early embryogenesis. Although the embryonic origin of the hemopoietic stem cell is unknown, indirect evidence from this study indicates a dorsal stem cell compartment.     

Experimental Studies on Hemopoiesis in the Pronephros of Rana pipiens

Embryogenesis of hemopoietic cell populations in the pronephros of Rana pipiens was examined during embryonic and early larval development. Differential cell counts of Wright-Giemsa-stained cell suspensions demonstrated that granulopoiesis is the predominant hemopoietic activity in the pronephros, erythropoiesis accounts for a minor component of the hemopoietic activity (> 10%), and lymphopoiesis within the organ is negligible. Microdensitometric analysis of Feulgen-DNA stained granulocyte populations in pronephroses from larvae that had received chromosomally labeled pronephric anlagen transplants between 84 and 96 h of development demonstrated that hemopoiesis in this organ is dependent on colonization by an extrinsic hemopoietic stem cell. A similar analysis of pronephric hemopoiesis in larvae which had received chromosomally labeled, presumptive ventral blood island transplants between 62 and 67 h of development, indicates that granulopoietic cells are not derived from the embryonic blood islands. It is proposed that the pronephros may be the initial site of granulocyte differentiation during early embryogenesis. Although the embryonic origin of the hemopoietic stem cell is unknown, indirect evidence from this study indicates a dorsal stem cell compartment     

Steel factor stimulates the tyrosine phosphorylation of the proto-oncogene product, p95vav, in human hemopoietic cells.

Steel factor (SF) (also called stem cell factor, mast cell growth factor, or c-kit ligand) is a recently cloned hemopoietic growth factor that is produced by bone marrow stromal cells, fibroblasts, and hepatocytes. In both mouse and man it acts synergistically with several colony stimulating factors, including interleukin-3 (IL-3) and granulocyte macrophage-colony stimulating factor (GM-CSF), to induce the proliferation and differentiation of primitive hemopoietic precursor cells. In order to study its mechanism of action and to explore the molecular basis for its synergistic activity we have examined the proteins that become tyrosine phosphorylated in response to SF, IL-3, and GM-CSF. We report herein that SF, but not IL-3 or GM-CSF, dramatically stimulates the tyrosine phosphorylation of the product of the recently discovered proto-oncogene, vav, in two SF-responsive human cell lines, M07E and TF-1. Although phosphorylation is very rapid, reaching maximal levels within 2 min at 37 degrees C, co-immunoprecipitation studies suggest that c-kit may either not associate directly with p95vav or bind to it with very low affinity. Nonetheless, our data suggest that c-kit may utilize p95vav to mediate downstream signaling in hemopoietic cells.     

RESPONSE OF THE PREOSTEOBLAST AND STEM CELL OF RAT BONE MARROW TO A LETHAL DOSE OF X-IRRADIATION OR CYCLOPHOSPHAMIDE

Following syngeneic or autotransplantation of hemopoietic tissue to a heterotopic location, bone formation has been observed to occur in the implanted tissue. the characteristics of the cell residing in hemopoietic tissue with bone forming potential (preosteoblast) are unknown. to define some properties of this cell, its response to X-irradiation and cyclophosphamide (CTX) was compared to the response of the hemopoietic stem cell. Adult, male rats were exposed to 900 R whole body X-irradiation or 220 mg/kg of intraperitoneal CTX. With either treatment the dose was sufficient to kill the animals by bone marrow failure. At intervals following the X-irradiation or CTX, hemopoietic tissue was examined for the presence of viable hemopoietic stem cells and preosteoblasts. Following X-irradiation, viable hemopoietic stem cells and preosteoblasts could not be detected. Following CTX these cells could be detected. It is suggested that in the rat CTX at 220 mg/kg, although causing death by bone marrow failure, does not reduce the population of the preosteoblast or hemopoietic stem cell as effectively as 900 R X-irradiation.     

Substrate elasticity provides mechanical signals for the expansion of hemopoietic stem and progenitor cells

Surprisingly little is known about the effects of the physical microenvironment on hemopoietic stem and progenitor cells. To explore the physical effects of matrix elasticity on well-characterized primitive hemopoietic cells, we made use of a uniquely elastic biomaterial, tropoelastin. Culturing mouse or human hemopoietic cells on a tropoelastin substrate led to a two- to threefold expansion of undifferentiated cells, including progenitors and mouse stem cells. Treatment with cytokines in the presence of tropoelastin had an additive effect on this expansion. These biological effects required substrate elasticity, as neither truncated nor cross-linked tropoelastin reproduced the phenomenon, and inhibition of mechanotransduction abrogated the effects. Our data suggest that substrate elasticity and tensegrity are important mechanisms influencing hemopoietic stem and progenitor cell subsets and could be exploited to facilitate cell culture.     

Fibronectin-plasma membrane interaction in the adhesion of hemopoietic cells

Many hemopoietic cell lines were examined for their ability to adhere to culture dishes coated with extracellular matrix proteins. Adhesion assay was performed with murine and human leukemic cell lines representative of different stages of differentiation along both erythroid and myeloid lineages. All the hemopoietic cell lines tested adhered to fibronectin but not to laminin, types I, III, and IV collagen, serum-spreading factor, and cartilage proteoglycans. In addition to immortalized cell lines, immature erythroid and myeloid mouse bone marrow cells adhered to fibronectin. To define the fibronectin region involved in hemopoietic cell adhesion, proteolytic fragments, monoclonal antibodies, and synthetic peptides were used. Among different fibronectin fragments tested, only a 110-kD polypeptide, corresponding to the fibroblast attachment domain, was active in promoting adhesion. Moreover, a monoclonal antibody to the cell binding site located within this domain prevented hemopoietic cell adhesion. Finally, the tetrapeptide Arg-Gly-Asp-Ser, which corresponds to the fibronectin sequence recognized by fibroblastic cells, specifically and competitively inhibited attachment of hemopoietic cells to this molecule. The cell surface molecule involved in the interaction of mouse hemopoietic cells with fibronectin was identified as a 145,000-D membrane glycoprotein by adhesion-blocking antibodies. This glycoprotein was found to be antigenically and functionally related to the GP135 membrane glycoprotein involved in the adhesion of fibroblasts to fibronectin (Giancotti, F. G., P. M. Comoglio, and G. Tarone, 1986, Exp. Cell Res., 163:47-62). On the basis of these data, we conclude that interaction of hemopoietic cells with fibronectin involves a specific fibronectin sequence and a 145,000-D cell surface glycoprotein. We speculate that this property might be relevant for the interaction of hemopoietic cells with the bone marrow stroma, which represents the natural site of hemopoiesis.     

Collection of hematopoietic stem cells from canine peripheral blood and their ability to regenerate hematopoiesis]

A procedure is presented for the collection of a large number of hemopoietic stem cells from the peripheral blood of dogs by means of a single leukapheresis using the NCI-IBM Blood Cell Separator. In the course of a leukapheresis of about 285 min duration a mean of 23 x 10-9 leukocytes is collected from the blood. The hemopoietic stem cells among such separated leukocytes initiate repopulation of bone marrow within 10 days after whole body X-irradiation with 1200 R. The cell numbers in a defined histological section of femoral bone marrow are evaluated 9 to 10 days after irradiation and subsequent autologous transfusion of 6.72 x 10-9 separated mononuclear leukocytes. The results indicate that the bone marrow cell numbers of transfused dogs are significantly greater than in dogs given only 1200 R and reach a level of approximately 49% of the normal value. Possible ways of increasing the yield of hemopoietic stem cells from the peripheral blood will be considered.