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.     

Localization of hematopoietic 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.     

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.     

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.     

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

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

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.     

Experimental research on the cellular interactions during hemopoiesis

We present a review of experimental studies performed at the Laboratory of Histogenesis of the Institute of Developmental Biology, Russian Academy of Sciences, on the problem of cell interactions during hemopoiesis. Special attention has been given to original experimental models, such as production of hemopoietic foci on underlayers of fibroblasts encapsulating a foreign body in the peritoneal cavity of rodents (after intraperitoneal transplantation of hemopoietic cells) and repopulation of ectopic hemopoietic territories under the kidney capsule of mice by syngeneic or xenogeneic hemopoietic cells. We describe the competitive interactions of genetically different hemopoietic cells after the transplantation of their mixtures to irradiated mice (multicomponent radiation chimeras). Xenogeneic and multicomponent chimeras have also been obtained in long-term bone marrow culture. We have examined characteristics of hemopoiesis on stromal cell underlayers produced by cells of various origins in vitro and then transplanted into the peritoneal cavity of irradiated mice. We discuss the results obtained and possible mechanisms of these phenomena.     

Effect of chronic administration of vitamin E on the hemopoietic system in hypercholesterolemia

Hypercholesterolemia induces oxidative stress, which is known to have adverse effects on the integrity of cells. Hence, hypercholesterolemia may have adverse effects on the hemopoietic system. Vitamin E, an antioxidant, is being used by normo- and hypercholesterolemic subjects. It is, however, not known if vitamin E has any beneficial or adverse effects on the hemopoietic system. The objectives of this study are to determine if (i) hypercholesterolemia has any adverse effects on the hemopoietic system [red blood cell (RBC) count, mean corpuscular volume (MCV), red blood cell distribution width (RDW), hematocrit (Hct), hemoglobin (Hb), mean corpuscular hemoglobin (MCH), MCH concentration (MCHC), white blood cell (WBC), and platelet counts, and mean platelet volume (MPV)], and (ii) vitamin E has any effect on the hemopoietic system in hypercholesterolemia. Blood samples were collected before and at various intervals during a high cholesterol diet (0.25% cholesterol) for 2 and 4 months, and while on high cholesterol diet with vitamin E (2 months) following a high cholesterol diet (2 months). Serum cholesterol was measured on an automated Clinical System Analyzer and hemopoietic parameters were measured on an automated Cell-dyn-4000. The results show that hypercholesterolemia decreased RBC count, Hct and Hb, increased MCV, RDW, MCH, and MCHC, and had no effect on WBC and platelet counts, and MPV. Vitamin E did not affect any of the parameters of the hemopoietic system. In conclusion, hypercholesterolemia of short duration has adverse effects on certain elements of the hemopoietic system. Vitamin E does not affect the hemopoietic system during hypercholesterolemia.     

Clonogenic fibroblasts from the hematopoietic organs of the human embryo and fetus at different gestational ages

Fibroblast precursors of hemopoietic organs of 72 embryos and fetuses 5-27 weeks of age have been studied. The study has shown that the increase in the number of clonogenic fibroblasts took place in the bone marrow and spleen 2-3 weeks before the beginning of hemopoiesis, that is during the period of the highest hemopoietic stem cell concentration. These data suggest possible participation of stromal fibroblasts of hemopoietic organs in the formation of microenvironment for hemopoietic stem cell functioning.     

Cytological study of the hemopoietic organ in the crab: Carcinus maenas (L.) (Crustacea, Decapoda). (author's transl)

A cytological observation, using conventional fixing and staining, is made on the hemopoietic tissue in the crab, Carcinus maenas. The hemopoietic organ is formed by nodules grouping different cell types; nodules are surrounded by a limiting layer including collagenous filaments and material looking like basal lamina. Some fibrocytes and semi-granulous hemocytes are lining this limited layer. These hemocytes, more or less flattened, are transforming in fibrocytes. Fibroblast-like cells, with well developed intercellular junctions, are the first cell type: their dedifferentiation gives rise to isolated mitoting cells. We have named these mitoting cells "hemocytoblast". They are stem cells for hyaline hemocytes. Fibroblast-like cells can be compared with "reticular cells" in Insects. Uncertainty exists as to the formation and evolution of nodules.     

Microcolonies of hemopoietic cells in the rat choroid plexus during the neonatal period

Microcolonies of hemopoietic cells have occasionally been found in the choroidal stroma of the rat myelencephalic choroid plexus during neonatal life. These hemopoietic foci are mixed colonies mainly composed of erythroblasts and maturing megakaryocytes; granulocyte precursors were not identified. The morphological data indicate that both erythro- and magakaryopoiesis occur in these microcolonies. With respect to their origin, we suggest that circulating pluripotential stem cells may colonize the choroidal stroma and produce erythro- and megakaryocyte cell lines.     

The expression of fetuin in the development and maturation of the hemopoietic and immune systems

 The distribution and expression of fetuin, a fetal plasma protein that has been shown to have a widespread intracellular presence in many developing tissues including the central nervous system, has been studied in the developing immune and hemopoietic organs of fetal and adult sheep. The presence of fetuin was demonstrated using immuno-cytochemistry and expression of fetuin was studied using northern blot analysis and in situ hybridization. In the developing sheep fetus, fetuin was shown to be expressed first in the hemopoietic cells of the fetal liver and subsequently in the forming spleen. The very first stromal, bone marrow-forming cells, also expressed fetuin mRNA. These cells became more numerous during gestation and by embryonic day (E)115 (term is 150 days), fetuin-expressing cells were identified morphologically to be monocytes/macrophages. Fetuin protein, on the other hand, was present in all hemopoietic and immune organs from the earliest age studied (E30) but was confined initially to matrix, mesenchymal tissue. Fetuin-positive cells could be identified in the spleen at E60 as early hemopoietic cells, in the lymph nodes at E60 as stromal cells and macrophages, and at E115 in the thymus as macrophages and squamous cells. In the adult, fetuin mRNA was only detectable by northern blot in the liver and the bone marrow. Using in situ hybridization in adult tissue, fetuin mRNA-positive cells were identified in the bone marrow to be monocytes/macrophages. Additionally, in the spleen germinal centres, fetuin mRNA was identified in cells with the morphology of dendritic cells. Using three separate cellular markers: lysozyme, S-100, and α₁-antitrypsin, the cellular identification of fetuin-positive cells was confirmed to be in the monocyte/macrophage lineage. Accepted: 3 May 1996     

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.     

Isolation and analysis of primitive hemopoietic progenitor cells on the basis of differential expression of Qa-m7 antigen

In the presence of the hemopoietic growth factor CSF-1, the later committed cells of the macrophage lineage can be detected by their ability to form small colonies in clonal agar culture (CFCCSF-1). Synergistic factors have been described that in combination with CSF-1 stimulate developmentally early hemopoietic progenitor cells of high proliferative potential (HPP-CFC). By using a monoclonal antibody to the Qa-m7 antigenic determinant, we investigated and compared the expression of Qa-m7 on CFCCSF-1 and on HPP-CFC of two types that grow in response to either 1) CSF-1 plus synergistic factor from human placenta-conditioned medium (HPP-CFCHplac+CSF-1) or 2) CSF-1 plus synergistic factor from conditioned medium of the WEHI-3 myelomonocytic cell line (HPP-CFCW+CSF-1). We have shown that HPP-CFC of both types express relatively more Qa-m7 antigen than CFCCSF-1 and can be separated and enriched on this basis by discontinuous buoyant density centrifugation and fluorescence-activated cell sorting of normal bone marrow. Significant enrichments of HPP-CFCHPlac+CSF-1 (43.5-fold) and HPP-CFCW+CSF-1 (28.8-fold) have been achieved with cloning efficiencies of HPP-CFC in the most enriched fractions reaching 4 to 5%. These results clearly illustrate the fact that there are populations of progenitor cells from normal, unperturbed bone marrow that strictly require a combination of two hemopoietic growth factors (CSF-1 plus synergistic factor) in order to be detected.     

A human GM-CSF receptor expressed in transgenic mice stimulates proliferation and differentiation of hemopoietic progenitors to all lineages in response to human GM-CSF.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) mainly stimulates proliferation and maturation of myeloid progenitor cells. Although the signal transduction pathways triggered by GM-CSF receptor (GMR) have been extensively characterized, the roles of GMR signals in differentiation have remained to be elucidated. To examine the relationship between receptor expression and differentiation of hemopoietic cells, we used transgenic mice (Tg-mice) that constitutively express human (h) GMR at almost all stages of hemopoietic cell development. Proliferation and differentiation of hemopoietic progenitors in bone marrow cells from these Tg-mice were analyzed by methylcellulose colony formation assay. High affinity GMR interacts with GM-CSF in a species-specific manner, therefore one can analyze the effects of hGMR signals on differentiation of mouse hemopoietic progenitors using hGM-CSF. Although mouse (m) GM-CSF yielded only GM colonies, hGM-CSF supported various types of colonies including GM, eosinophil, mast cell, erythrocyte, megakaryocyte, blast cell, and mixed hemopoietic colonies. Thus, the effects of hGM-CSF on colony formation more closely resembled mIL-3 than those of mGM-CSF. In addition, hGM-CSF generated a much larger number of blast cell colonies and mixed cell colonies than did mIL-3. hGM-CSF also generated erythrocyte colonies in the absence of erythropoietin. Therefore, GM-CSF apparently has the capacity to promote growth of cells of almost all hemopoietic cell lineages, if functional hGMR is present.     

Immunofluorescent study of the hemopoietic organs of xenogeneic mouse radiation chimeras]

An immunofluorescent study of hemopoietic organs in xenogenic (mouse-rat) radiation chimaeras has been carried out by means of specific antiserum against hemopoietic cells of the rat bone marrow. The presence of donor cells was tested at different times after the transplantation in the bone marrow, spleen, lymph nodes, thymus and liver of radiochimaeras. The transplanted cells were shown to populate all hemopoietic organs of the recipient, first of all tissues of the bone marrow type and, then, lymphoid organs. The donor (bone marrow) origin of the extramedullar foci of hemopoiesis in the liver was established.     

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

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