Evaluation of the marrow culture on the macrophage layer covering intraperitoneally implanted membrane as an assay for hemopoietic progenitor cells

When cellulose acetate membranes are implanted into abdominal cavity of mice they turn into a foreign body overgrown with macrophages. Such macrophage layer has been shown by other authors to be able to support the growth of hemopoietic colonies formed by intraperitoneally injected hemopoietic cells. This study confirms and extends this observation by showing that both granulopoietic and erythropoietic colonies may be observed. The number of colonies grown is in linear correlation with that of injected hemopoietic cells. The frequency of erythropoietic colonies was greatly enhanced by blood letting of the host mice. Colony forming cells were most numerous in the bone marrow then in the spleen and peripheral blood and hardly in the thymus. Prior irradiation of the host mice was essential for obtaining colony growth and the optimal dose was determined to be 6.0 Gy. This technique opens the way to studies into hemopoietic progenitor cells for laboratories having no sophisticated tissue culture equipment and where necessary reagents are easily available.     

Preliminary observation of blood cells and hematopoietic organs in Lutjanus herenbergi and Lutjanus flaviflammus

The morphology of differentiated and differentiating cells of the red and white series in Lutjanus herenbergi and in Lutjanus flaviflammus is described. Early stages of red and white blood cells may be found only in smears of hemopoietic organs. Polychromatic erythroblasts, myelocytes and lymphoblasts may also occasionally be found in blood smears. Mature blood cells may be found both in blood smears and in hemopoietic organs. Differential white cell counts seem to demonstrate that the granulocytic series elements are the most common leukocytes in blood smears. Almost all granulocytes may be classified in the first three Arneth classes. An analysis of hemopoietic organs in these species was also performed. It was found that the only organs carrying on a hemopoietic function are the kidney and the spleen. The kidney is essentially a site of granulocytic differentiation while the spleen is a lymphopoietic organ. An erythropoietic activity may generally be observed in the kidney although weak erythropoietic activity may at times be found in the spleen.     

Splenic hemopoiesis of the platypus (Ornithorhynchus anatinus): evidence of primary hemopoiesis in the spleen of a primitive mammal

The generation of blood cells has been observed in the spleen and in the bone marrow of the platypus. Hemopoiesis was found to be far more active in the spleen than in the bone marrow judging by the number of proliferating hemopoietic elements within a unit area of tissue from each organ. Granulocytes, erythroblasts, and megakaryocytes, with the related immature forms for each cell line, were noted in the spleen. In contrast, there were very few examples of immature forms of these cell lines and a complete absence of mature megakaryocytes in the bone marrow. These findings suggest that the spleen is the primary hemopoietic organ in the platypus. Since the platypus is one of two species representing the most primitive existing mammals, it seems likely that the spleen may be the primary hemopoietic organ in mammalian evolution.     

Regulation of blood cell diferentiation.

The hemopoietic (blood forming) system contains pluripotent stem cells able to give rise to a variety of differentiated progeny, including erythrocytes, granulocytes, megakaryocytes, monocytes, macrophages, and possible other cell types. Although a good deal is known about cell lineage relationships in the hemopoietic system, only limited information is available about the mechanisms regulating the proliferation and differentiation of the stem cells and their progeny. An approach to this latter problem has been provided by the develoment of new techniques for the cultivation of hemopoietic cells in short-term cultures. In such cultures, the proliferation and differentiation of hemopoietic cells can be studied under controlled conditions. Two areas of investigation show particular promise: elucidation of the role of the cell surface membrane in regulation; and the possible development, through a detailed investigation of the properties of leukoviruses, of new methods for the genetic analysis of hemopoietic cells.     

Fine Structure of the Hemopoietic Tissues in the Mealworm Beetle, Tenebrio molitor

The fine structure of the hemopoietic tissue and its detailed reticular organization in the mealworm beetle, T. molitor were examined using light and scanning electron microscopes. The major hemopoietic tissues in the abdomen were located on the upper surface of the dorsal diaphragm which continuous over the ventral wall of the heart. Histologic characteristics of this hemopoietic tissues are dense clusters of cells. They are irregular in outline and are not surrounded by any connective tissue sheath. The hemopoietic tissue of this insect is consisted of three cellular components which are the reticular cells, hemocytic stem cells and several kinds of mature hemocytes. The reticular cells had numerous cytoplasmic processes and forming a complex network. The stem cells give rise to differentiating hemocytes of the different cell lineages. Mature hemocytes within this hemopoietic tissue are originated from the stem cells and differentiated into several types of hemocytes including prohemocytes, plasmatocytes, and granulocytes.     

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.     

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.     

Expansion of umbilical cord blood for clinical transplantation

Since the first successful cord blood transplant was performed in 1988 there has been a gradual increase in the use of cord blood for hemopoietic stem cell transplantation. Worldwide, over 8,000 unrelated cord blood transplants have been performed with the majority being for children with hemopoietic malignancies. Transplantation for adults has increased but is limited by the low number of nucleated cells and CD34(+) cells within a single cord blood collection. Cord blood hemopoietic stem cells are more primitive than their adult counterparts and have high proliferative potential. Cord blood ex vivo expansion is designed to improve transplant outcomes by increasing the number of hemopoietic stem cells with long term repopulating potential and their differentiated progeny. However, despite a large amount of research activity during the last decade, this aim has not been realized. Herein we discuss the rationale for this approach; culture methods for ex vivo expansion, ways to assess the functional capacity of ex vivo generated hemopoietic stem cells and clinical outcomes following transplantation with ex vivo expanded cord blood.     

Physical separation of colony stimulating cells from in vitro colony forming cells in hemopoietic tissue

Hemopoietic colony formation in agar occurred spontaneously in mass cultures of marrow cells obtained from a number of species (guinea pig, rat, lamb, rabbit, pig, calf, human and Rhesus monkey). This contrasted with the observation that colony formation by mouse bone marrow exhibited an absolute requirement for an exogenous source of a colony stimulating factor. Analysis of spontaneous colony formation in Rhesus monkey marrow cultures revealed the presence of a cell type in hemopoietic tissue, capable of elaborating colony stimulating factor when used to condition media or as feeder layers. Equilibrium density gradient centrifugation separated colony stimulating cells from in vitro colony forming cells in monkey bone marrow. Separation studies on spleen, blood and marrow characterized the stimulating cells as of intermediate density, depleted or absent in fractions enriched for cells of the granulocytic series and localized in regions containing lymphocytes and monocytes. Adherence column separation of peripheral blood leukocytes showed the stimulating cells to be actively adherent, unlike the majority of lymphocytes, and combined adherence column and density separation indicated that stimulating cells were present in hemopoietic tissue within the population of adherent lymphocytes or monocytes.     

In vitro inhibition of hemopoietic cell line growth by hepatitis B virus.

The effects of hepatitis B virus (HBV) on established human cell lines of various tissue origins were evaluated by clonal or colorimetric assays in methylcellulose culture. HBV exposure inhibited the growth of six hemopoietic cell lines, while similar incubation did not affect the growth of seven nonhemopoietic carcinoma cell lines of breast, colon, liver, and stomach origin. The inhibition of hemopoietic cell line colony formation was dependent on the presence of intact viral (Dane) particles and the ratio of exposure of virions to cells and was reversible with antibodies to pre-S1, pre-S2, and S envelope protein epitopes. Purified HBV DNA, surface antigen pre-S antigens, and core antigen did not inhibit cell line growth. These results further demonstrate the tropism of HBV for cells of hemopoietic origin, confirming our previous findings on the effects of HBV on the growth of normal bone marrow progenitor cells in vitro. Established human tissue culture cell lines may be used to study the interactions of hemopoietic cells with HBV.     

State of several rat hematopoietic organs following total and subtotal irradiation and after bone marrow autotransplantation]

Hemopoiesis was studied in rats after x-ray irradiation. Lethal doses of 800--820 R were applied totally, with screening the shin and with subsequent autotransplantation of bone marrow taken from noninjured hemopoietic tissue. Survival of the animals and status of hemopoietic organs (quantitative indices of the peripheral blood, bone marrow and the spleen, as well as morphological changes in hemopoietic organs) served as tests. All totally irradiated animals died by the 20th day, the 30th day in the group of screened animals 32% survived, in the group with autotransplantation of bone marrow--62%. According to the indices studied restoration of hemopoiesis proceeded more quickly and completely in the group with autotransplantation of bone marrow and somewhat slower in the group with screening the shin (but without autotransplantation); this was accompanied by repopulation of bone marrow comparing with the totally irradiated animals. Restoration of the hemopoietic organs was followed by a comparatively rapid increase in the number of myeloid cells, while the number of lymphoid cells increased more slowly.     

The radioprotective effect of hypothermia on the immune and hematopoietic systems in mammals

The functional activity of the synthetic apparatus (parameter alpha) in blood lymphocytes, bone marrow hemopoietic cells, and thymus cells, as well as the total number of blood and bone marrow cells in rats after y-irradiation at a dose of 8 Gy in the conditions of normothermia and hypothermia (16-18 degrees C) with hypoxia-hypercapnia were investigated after 2 h and on days 1 and 4. The recovery processes in blood in both groups of rats after acute X-irradiation at a dose of 7 Gy for 36 days were analyzed too. Under hypothermia, on days 1-4 after acute gamma-irradiation, a decrease in the synthetic activity in remaining cells and devastation in the hemopoietic system were pronounced to a lesser degree. After X-irradiation, the restoration of synthetic activity in blood lymphocytes was shown to begin earlier and to finish faster in "hypothermic" rats as compared with the animals irradiated in the state of normothermia. The survival of "hypothermic" rats was 100% as compared with 30% in "normothermic" animals. Thus, the data show that hypothermia exerts a radioprotective effect on the cells of the immune and hemopoietic systems, thus enhancing the resistance of the organism to radiation.     

Precursor cells of newly formed connective tissue in inflammatory foci in various organs in mice

The origin of fibroblast-like cells of the capsule around a foreign body in the spleen, liver, peritoneal cavity, subcutaneous connective tissue of mice, the localization of cells-precursors, their proliferative potencies and the ability to migrate through blood were studied using 3H-thymidine autoradiography. Precursors of the inflammation focus cells (irrespective of localization) reproduce intensively outside the limits of intraorganic connective tissue, supposedly, in hemopoietic organs of the bone marrow type and migrate, through the blood channel, into tissues (inflammation foci), where they terminate their differentiation.     

Cell surface peptidase CD26/dipeptidylpeptidase IV regulates CXCL12/stromal cell-derived factor-1 alpha-mediated chemotaxis of human cord blood CD34+ progenitor cells

CD26/dipeptidylpeptidase IV (DPPIV) is a membrane-bound extracellular peptidase that cleaves dipeptides from the N terminus of polypeptide chains. The N terminus of chemokines is known to interact with the extracellular portion of chemokine receptors, and removal of these amino acids in many instances results in significant changes in functional activity. CD26/DPPIV has the ability to cleave the chemokine CXCL12/stromal cell-derived factor 1alpha (SDF-1alpha) at its position two proline. CXCL12/SDF-1alpha induces migration of hemopoietic stem and progenitor cells, and it is thought that CXCL12 plays a crucial role in homing/mobilization of these cells to/from the bone marrow. We found that CD26/DPPIV is expressed by a subpopulation of CD34(+) hemopoietic cells isolated from cord blood and that these cells have DPPIV activity. The involvement of CD26/DPPIV in CD34(+) hemopoietic stem and progenitor cell migration has not been previously examined. Functional studies show that the N-terminal-truncated CXCL12/SDF-1alpha lacks the ability to induce the migration of CD34(+) cord blood cells and acts to inhibit normal CXCL12/SDF-1alpha-induced migration. Finally, inhibiting the endogenous CD26/DPPIV activity on CD34(+) cells enhances the migratory response of these cells to CXCL12/SDF-1alpha. This process of CXCL12/SDF-1alpha cleavage by CD26/DPPIV on a subpopulation of CD34(+) cells may represent a novel regulatory mechanism in hemopoietic stem and progenitor cells for the migration, homing, and mobilization of these cells. Inhibition of the CD26/DPPIV peptidase activity may therefore represent an innovative approach to increasing homing and engraftment during cord blood transplantation.     

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.     

The renewal and differentiation of hemopoietic stem cells.

Blood-forming tissues are organized in well-defined microenvironments composed of hemopoietic cells and a supportive stroma of connective tissue and endothelium. Hemopoietic cells segregate to various lineages, all derived from a small population of pluripotent stem cells residing in the bone marrow. Regulation of growth and differentiation, particularly under conditions of perturbations, damage, and disease, is mediated by inducer colony-stimulating factors and interleukins counteracted by inhibitory cytokines. Whereas much is known about the mode of induction of differentiation, insufficient information is available to explain the process of stem cell renewal that is crucial for the longevity of the hemopoietic system. It is also only partially known how inhibition of hemopoietic processes occurs, and what molecules in blood-forming tissues signal organization into discrete patterns. This paper reviews recent progress that has opened new avenues to a better understanding of this highly complex issue.     

Morphological and histochemical studies on a PAS-positive granular leukocyte in blood and connective tissues of Catostomus commersonii Lacépède (teleostei:pisces).

This study was undertaken to identify an ubiquitous granular leukocyte found in Catostomus commersonni Lacépède. The cell contains large, numerous, strongly PAS-positive cytoplasmic granules, an eccentric nucleus and prominent, persistent juxtanuclear space. It develops in the hemopoietic tissue of the kidney, and mature cells are found not only in kidney and peripheral blood but also in areas of connective tissue where mast cells are usually located. Electron microscopy confirms the presence of a large Golgi apparatus, unlamellated cytoplasmic granules and extensive rough-surfaced endoplasmic reticulum. Histochemical studies show that the cytoplasmic granules are alcianophobic, non-metachromatic and unstained by acridine orange. Histamine is detectable spectrophotometrically in kidney tissue, but the PAS-positive granular leukocyte does not consistently degranulate after treatment with histamine liberator 48/80. The authors suggest that while the PAS-positive granular leukocyte is not identical with classical basophils/mast cells, which are absent in C. commersonnii, it may represent an evolutionary precursor of these 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.     

Differential contribution of dorsal and ventral lateral plate mesoderm to hemopoiesis during Rana pipiens embryogenesis

Data obtained from studies on the origin and development of hemopoietic cells in several classes of vertebrate embryos argue for two distinct sources of hemopoietic cells, the intraembryonic dorsal lateral plate and the extraembryonic ventral blood island/yolk sac. In the present study, a stage by stage comparison of the hemopoietic potential of both of these regions was made during development of the frog, Rana pipiens. Either dorsal lateral plate or ventral blood island mesoderm was reciprocally transplanted between cytogenetically labeled triploid and diploid embryos. The ratio of donor-derived cells to host-derived cells (labeling index) was determined from Feulgen-stained DNA measurements of cells harvested from hemopoietic organs of young larvae. Blood island transplants consistently resulted in larvae with positive labeling of the circulating blood. Transplanted dorsal mesoderm supplied mesonephric granulocytes and thymocytes, but not circulating erythrocytes to larvae. However, the contribution of dorsal mesoderm to larval hemopoiesis fluctuated with respect to embryonic stage at transplantation.     

Functional Morphology of the White Bodies of the Cephalopod Mollusc Sepia officinalis

Abstract The white bodies and blood cells of the cuttlefish, Sepia officinalis, were investigated using light microscopy and electron microscopy. Each white body is supplied with arterial blood by two branches of the ophthalmic artery. These two branches arborize into the tissue and form an extensive capillary network that appears to be confluent with a system of thin-walled venous channels in the white bodies. This vascular arrangement appears to allow maturing cells to leave the white body tissue and enter the general circulation. The putative hemopoietic cells are embedded in an internal connective tissue network. In addition to an extensive extracellular matrix system, this network contains many obliquely striated muscle fibers that probably aid in microcirculation. The white body cells were grouped into four categories that, ultrastructural evidence suggests, represent developmental stages of a single cell line. Morphological similarities between the putative final developmental stage of this cell line and circulating blood cells strongly support the hypothesis that white body cells are hemopoietic in function. Several lines of evidence indicate that proliferation within this cell line occurs at the first two stages of development. Thus a new developmental scheme is proposed. Quantitative analyses have shown that the white bodies are structurally and functionally uncompartmentalized. A comparison of developmental stages suggests that the physiology of the white bodies shifts from proliferation to differentiation as the animals mature. Blood cell densities decrease as the animals mature. Finally, cytological evidence indicates that the white bodies of Sepia officinalis are involved in reticuloendothelial functions in addition to hemopoiesis.