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.     

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.     

人胎肝基质细胞培养上清液中造血生长因子的分析

采用人胎肝造血基质细胞的体外液体培养技术,结合造血干细胞和祖细胞的体外测试方法,研究了造血基质细胞所释放的造血生长因子与造血干细胞和祖细胞之间的相互作用。结果表明,在适宜的条件下,人胎肝造血基质细胞可在体外传代培养达100d之久。培养过程中,对不同时间收集的培养上清液进行测试的结果表明,这些贴壁细胞可以不断地释放多种造血活性物质。在100d培养过程中,上清液中始终都可以检出CFU-S增殖刺激物活性。培养第24天的上清液中还可检出BPF和GM-CSF活性。这些造血活性物质对CFU-S的生理状态和祖细胞的增殖与分化有着深刻的影响。但是在培养上清液中未检出IL-3样活性物质。     

Association of reticular cells with CD34+/Sca-1+ apoptotic cells in the hemopoietic organ of grasshopper, Euprepocnemis shirakii

Hemopoiesis in orthopteran insects occurs in a hemopoietic organ that is located bilaterally along the aorta. This organ is also known as a reticulo-hemopoietic organ because of the rich presence of reticular cells. This study was performed to further elucidate hemopoiesis in the reticulo-hemopoietic organ of an orthopteran, Euprepocnemis shirakii. We focused on the question why reticular cells are so abundant (35% of cells in hemopoietic organ). Interestingly, 21% of these reticular cells surrounded hemocytes with their reticular cytoplasm. The surrounded hemocytes were distinguished by their different size and darkly stained nucleus. These cells were characterized by immunostaining using antibodies against several types of hemocytes: 45% of the surrounded hemocytes were CD34+, and these positive cells were double stained (over 85%) when immunostained by another hemopoietic pluripotent cell marker, Sca-1. Transmission electron microscopic analysis showed that reticular cells surrounded hemocytes containing large nuclei and poorly developed cytoplasmic organelles. This strongly suggests that the reticular cells surround hemopoietic stem cells. Additionally, surrounded hemopoietic progenitor cells are undergoing apoptosis as indicated by the TUNEL assay. The enclosed apoptotic cells are engulfed and then phagocytosed by reticular cells. Our results suggest that reticular cells are related to the differentiation and apoptosis of hemopoietic stem cells.     

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.     

The characteristics of the hematopoietic cells forming colonies on cellulose acetate membranes and their distinctions from other clonogenic cells]

In order to characterize hemopoietic cells forming colonies on membranes of cellulose acetate (CFU-acm) implanted into the peritoneal cavity of mice, we studied the effect of factors stimulating and inhibiting granulocytopoiesis on these cells. Proliferation of CFU-acm can be controlled by humoral factors and this allows us to conclude that they are not identical to CFUs and probably belong to the compartment of early hemopoietic cells of the granulocyte series. We also present evidence for fractional composition, ultrastructure and bone marrow origin of cells belonging to the layer providing for hemopoietic microenvironment for the resulting foci of hemopoiesis; we also present evidence for the role of fibroblasts (fibroblast-like cells) in the maintenance of hemopoiesis. Experiments on transplantation of bone marrow from several rodent species to syn-, allo- and xenogenic recipients allowed us to study interactions of hemopoietic elements of colonies with cells of the underlying layer.     

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.     

Effect of various factors on the differentiation of hematopoietic stem cells]

The treatment in vitro of bone marrow cells in mice by phytohemagglutinin, concanavaline, or antilimphocytic globulin resulted in the suppression of exogenous hemopoietic colonies in the spleen of lethally irradiated (830r) syngenic recipients, whereas lipopolysaccharide, tuberculin, anti-theta serum or nati-gamma-globulin serum exerted no influence on the colony-forming function of hemopoietic stem cells. The morphological analysis of the ratio and cell composition of hemopoietic colonies has revealed no marked differences between the experimental and control groups. The suppression of hemopoietic stem cells by mitogens might be due both to their direct effect and indirect one, possibly, through a humoral factor.     

Stromal cells from murine developing hemopoietic organs: comparison of colony-forming unit of fibroblasts and long-term cultures.

The adherent stromal layer in long-term marrow cultures is essential to the proliferation and differentiation of hemopoietic cells. Adhering cells are heterogeneous and morphologically not adequately characterized. Comparative morphological studies were conducted on adherent cells in short-term clonal assays and long-term cultures derived from liver and bone marrow. Liver and bone marrow at different developmental ages have different hemopoietic activities in vivo and in vitro, as tested via CFU-GM recovery in long-term cultures. Adherent cells from each organ were recovered at an age with high hemopoietic activity (fetal liver and adult bone marrow) and at an age with low hemopoietic activity (neonatal liver and bone marrow). The presence of macrophages, alkaline phosphatase, acid phosphatase, myeloperoxidase, sulfated and non-sulfated glycosaminoglycans (GAGs) and fibronectin was compared. For a given organ, CFU-f colonies showed characteristics similar to those of the confluent adherent stromal layer in long-term cultures. The presence of macrophages and GAGs (sulfated and non-sulfated) in the adherent layer were directly related to the hemopoietic activity. The amount of alkaline phosphatase-positive cells and the amount of fibronectin showed no correlation with the hemopoietic activity of the cultures.     

Levels of Ly-49 receptor expression are determined by the frequency of interactions with MHC ligands: evidence against receptor calibration to a "useful" level.

Ly-49 receptor expression was studied in NK cells that developed in fully MHC-mismatched mixed bone marrow chimeras, in which host and donor MHC ligands were expressed solely on various proportions of hemopoietic cells or on both hemopoietic and nonhemopoietic cells. When hemopoietic cells were the only source of MHC ligand, a strong correlation between the level of down-regulation of Ly-49A, Ly-49C, and Ly-49G2 and the number of hemopoietic cells expressing their MHC ligands was observed on both donor and host NK cells. In some animals with low levels of donor hemopoietic chimerism, NK cells of donor origin expressed Ly-49 receptors at higher levels than was observed in normal mice of the same strain. This unexpected observation is inconsistent with the receptor calibration theory, which states that expression of Ly-49 inhibitory receptors is calibrated to an optimal level to maintain an NK cell repertoire that is sensitive to perturbations in normal class I ligand expression. Our data suggest a model in which Ly-49 receptors down-modulate in accordance with the frequency of their interactions with ligand-bearing cells, rather than a model in which these receptors calibrate to a specific "useful" level in response to ligands present in their environment.     

Early and late effects of the cytogenetic action of doxorubicin

Cytogenetic disorders in hemopoietic cells of the bone marrow were studied on mice CBA at early and late periods after exposure to MIDs of doxorubicin, an anthracycline antibiotic. It was shown that at the early period doxorubicin induced aberrations, mainly of the chromatid type, in the hemopoietic cells of the bone marrow. Instability of the genetic apparatus of the hemopoietic cells observed for 3 months of the experiment was likely to be the immediate cause of the disorders in hemopoiesis at the late periods after exposure to doxorubicin.     

Wnt signaling regulates hemopoiesis through stromal cells.

Hemopoietic cells develop in a complex milieu that is made up of diverse components, including stromal cells. Wnt genes, which are known to regulate the fate of the cells in a variety of tissues, are expressed in hemopoietic organs. However, their roles in hemopoiesis are not well characterized. In this study, we examined the roles of Wnt proteins in hemopoiesis using conditioned medium containing Wnt-3a. This conditioned medium dramatically reduced the production of B lineage cells and myeloid lineage cells, except for macrophages in the long-term bone marrow cultures grown on stromal cells, although the sensitivity to the conditioned medium differed, depending on the hemopoietic lineage. In contrast, the same conditioned medium did not affect the generation of B lineage or myeloid lineage cells in stromal cell-free conditions. These results suggested that Wnt proteins exert their effects through stromal cells. Indeed, these effects were mimicked by the expression of a stabilized form of beta-catenin in stromal cells. In this study, we demonstrated that Wnt signaling regulates hemopoiesis through stromal cells with selectivity and different degrees of the effect, depending on the hemopoietic lineage in the hemopoietic microenvironment.     

A study of hemopoiesis on a sublayer of peritoneal cells in the presence of hemopoietic cytokines

We studied the effects of erythropoietin and thrombopoietin on the clonogenic capacity and direction of differentiation of the hemopoietic cells that form colonies on acetate cellulose membrane in the peritoneal cavity of mice. An increased level of erythropoietin in the blood of recipient mice after blood letting led to the appearance of erythroid colonies upon transplantation of syngeneic hemopoietic cells but did not affect the differentiation of transplanted xenogeneic (guinea pig) hemopoietic cells. Erythropoietin transported top the stromal sublayer by a polymeric carrier also induced erythroid differentiation, while thrombopoietin transported in a similar way somewhat enhanced megakaryocytopoiesis.     

A Study of Hemopoiesis on Sublayer of Peritoneal Cells in the Presence of Hemopoietic Cytokines

We studied the effects of erythropoietin and thrombopoietin on the clonogenic capacity and direction of differentiation of the hemopoietic cells that form colonies on acetate cellulose membrane in the peritoneal cavity of mice. An increased level of erythropoietin in the blood of recipient mice after blood letting led to the appearance of erythroid colonies upon transplantation of syngeneic hemopoietic cells but did not affect the differentiation of transplanted xenogeneic (guinea pig) hemopoietic cells. Erythropoietin transported top the stromal sublayer by a polymeric carrier also induced erythroid differentiation, while thrombopoietin transported in a similar way somewhat enhanced megakaryocytopoiesis.     

Effect of mouse age on the ability of hematopoietic stem cells to interact with thymus cells]

The effect of the thymus cells of the C57BL/6 mice on the colony forming ability of the stem hemopoietic cells of the embryonic liver and bone marrow of young (3 months) and old (2 years) mice was studied their joint transplantation into the mice (CBAXXC57BL/6) F1. The stimulating effect of the thymus cells on the colony forming ability of the stem hemopoietic cells of different age depends both on the dose of the stem hemopoietic cells of embryonic liver and the dose of T-lymphocytes. A suggestion is put forward that the stimulating effect of the thymus cells on the colony formation is due to their interaction with the stem cells in the G2 phase of the mitotic cycle.     

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.     

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.     

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.     

Origin of hematopoietic cells in a syngenous and semisyngenous focus of heterotopic hematopoiesis]

Heterotopic hemopoiesis foci were produced by the bone marrow of C57BL/6 or (CBA X C57BL)F1 mice grafted under the renal capsule of (CBAT6T6XC57BL)F1 mice, bearing the chromosomal translocation. The cytogenetic analysis of the hemopoietic cells in the foci 20 to 120 days after the transplantation showed that in 40% of the transplants only the recipient's hemopoietic cells proliferated, whereas the rest were mosaic and contained on the average less than 20% of donor's cells both in the syngeneic and in the semisyngeneic systems. These characteristics remained stable for at least 4 months. The data obtained suggest a single inflow of not less than 10 effective hemopoietic stem cells per graft. The clone stability indicated that during the steady-state hemopoiesis the cell exchange between various regions of the hemopoietic system was not great, if any.