Developments in modern hematology.

In the past 40 years our concepts about hemopoiesis have been changed dramatically. The results of bone marrow transplantation into lethally irradiated mice since the mid-fifties suggested the existence of a hemopoietic stem cell, which was initially identified as a spleen colony forming cell (CFU-S). Later experiments showed that the stem cell compartment is rather heterogeneous and that the most primitive stem cell, unlike the CFU-S, has the ability for long-term engraftment of an irradiated recipient. Daughter cells of such primitive quiescent stem cells lose their capacity for self-generation gradually with each mitosis and become more and more committed to a specific differentiation lineage. In vitro culture techniques in a serum-free semi-solid medium enabled the establishment and analysis of specific hemopoietic growth factors. Such factors, which are essential for the maintenance, proliferation and differentiation of progenitor cells and the functional activity of mature cells can now be produced with recombinant DNA techniques in pure form and large quantities. Hemopoiesis requires an appropriate microenvironment, consisting of various stromal cell types and an extracellular matrix. Intercellular contacts, adhesion of cells and growth factors to the matrix molecules seem essential in the regulating action of this hemopoietic microenvironment. In long-term bone marrow cultures the development of a stromal hemopoietic microenvironment can facilitate long-term maintenance of stem cells and hemopoietic differentiation. For bone marrow transplantation and infusion of hemopoietic growth factors many clinical indications are well established and our possibilities to interfere in the regulation of hemopoiesis are still growing.     

Expression of bone morphogenetic proteins in stromal cells from human bone marrow long-term culture.

Highly purified primitive hemopoietic stem cells express BMP receptors but do not synthesize bone morphogenetic proteins (BMPs). However, exogenously added BMPs regulate their proliferation, differentiation, and survival. To further explore the mechanism by which BMPs might be involved in hemopoietic differentiation, we tested whether stromal cells from long-term culture (LTC) of normal human bone marrow produce BMPs, BMP receptors, and SMAD signaling molecules. Stromal cells were immunohistochemically characterized by the presence of lyzozyme, CD 31, factor VIII, CD 68, S100, alkaline phosphatase, and vimentin. Gene expression was analyzed by RT-PCR and the presence of BMP protein was confirmed by immunohistochemistry (IHC). The supportive role of the stromal cell layer in hemopoiesis in vitro was confirmed by a colony assay of clonogenic progenitors. Bone marrow stromal cells express mRNA and protein for BMP-3, -4, and -7 but not for BMP-2, -5, and -6 from the first to the eighth week of culture. Furthermore, stromal cells express the BMP type I receptors, activin-like kinase-3 (ALK-3), ALK-6, and the downstream transducers SMAD-1, -4, and -5. Thus, human bone marrow stromal cells synthesize BMPs, which might exert their effects on hemopoietic stem cells in a paracrine manner through specific BMP receptors.     

Multilineage hemopoiesis induced by cloned stromal cells

Long-term hemopoiesis in culture depends upon the presence of an adherent layer composed of a variety of stromal cells. A subtype of endothelial-adipocytes from the bone marrow stroma (clone 14F1.1) was previously shown to induce long-term myelopoiesis and renewal of pluripotent stem cells. One of a series of stromal cell lines and clones from mouse thymus stroma (STAC-1.2) has now been found to support long-term hemopoiesis. These marrow- and thymus-derived stromal cell clones also have lymphopoietic activities: precursor T cells, or pre-B cells accumulated in co-cultures of thymus cells and the stromal clones, as indicated by cell surface markers, T cell receptor and immunoglobulin gene rearrangements. The predominance of a cell type in these cultures depended upon the serum used to supplement the medium. Recombinant interleukin 2 (IL-2) and the 14F1.1 clone synergistically promoted the proliferation of thymocytes, while a thymus hormone, THF-gamma 2, shifted the population to a relatively mature phenotype. It is proposed that one major function of stromal cells, whether from the bone marrow or thymus, is to restrain the maturation flow and preferentially support the accumulation of cells at early differentiation stages.     

Restrictins: stromal cell associated factors that control cell organization in hemopoietic tissues

Hemopoiesis is a sequence of events initiated by the self-renewal of pluripotent stem cells followed by a series of differentiation steps and completed in the formation of distinct tissue patterns. Differentiation and self-renewal are antagonistic processes. A mechanism that attenuates the differentiation flow is obligatory to prevent the exhaustion of the stem cell pool. We suggest that stromal cells from the bone marrow control stem cell renewal through a mechanism that does not require colony-stimulating factors. The organization of cells within the tissue and their specific localization is suggested to be directed by stromal cell activities other than differentiation inducers. These stromal cell activities restrict differentiation or accumulation of mature cells. They are therefore designated as 'Restrictins'.     

Genetic approach and phenotype-based complementation screening for identification of stroma cell-derived proteins involved in cell proliferation.

The functional capacities of stromal cell lines to support stem cell activity are heterogeneous and the mechanism of how they support bone marrow cultures remains unclear. Recently, we reported a strategy of functional analysis in which a genetic approach is combined with phenotype-based complementation screening to search for a novel secreted growth factor from mouse bone marrow stroma called ShIF that supported proliferation of bone marrow cells. To investigate the role of stromal cells in hemopoiesis, we extended this strategy to search for stroma-derived proteins that induce cell proliferation by establishing stroma-dependent Ba/F3 mutants of three stroma cell lines from two mouse tissues. Seven stroma-dependent Ba/F3 mutants were used as responder cells to identify cDNAs from stroma cell lines whose products supported proliferation not only to the mutant cells but also to hemopoietic progenitor cells in vitro.     

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.     

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。     

Long-term marrow cultures: human and murine systems

The intramedullary control of marrow cell production has been a difficult area to approach experimentally. The introduction by Dr. Dexter and colleagues of long-term stromal dependent culture systems for murine marrow and the adaptation of these systems to human marrow growth have allowed for in-vitro studies of stromal dependent hemopoiesis. Despite some controversy in this area, most studies appear to show that adherent murine or human stromal cells are capable of producing a relatively large number of hemopoietic growth factors including G-CSF, GM-CSF, CSF-1, IL-6 and, at least by PCR analysis, IL-3. Other work indicates that the most primitive hemopoietic cells which appear to be multifactor responsive adhere directly to these stromal cells presumably through mediation of various adherence proteins. An early acting, multilineage factor termed hemolymphopoietic growth factor-1 (HLGF-1) has been isolated from a murine stromal cell line and may be identical to the recently described ligand for the c-kit receptor. This may represent an important early survival/maintenance factor for stem cells in this system. Studies on primitive stem cells, especially the high proliferative potential colony forming cell (HPP-CFC), indicate that they are responsive to varying combinations of growth factors and that with increasing numbers of growth factors, as studied in serum-free systems, decreasing concentrations of the factors may be biologically active. These observations altogether suggest that intramedullary hemopoiesis may be regulated by the positioning of early multifactor responsive stem cells via adherent proteins in juxtaposition to synergistically acting combinations of growth factors attached to stromal cell surfaces or the extracellular matrix.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Unique pathway of IL-3-driven hemopoietic differentiation

The results of this study support a proposed sequence of IL-3-induced hemopoietic cell proliferation and differentiation. Specifically, IL-3 uniquely induces the transient expression of Thy-1 Ag on Thy-1- bone marrow cells during a 2-wk culture period. Thy-1 Ag is expressed on immature myeloid cells that are undergoing lineage restrictions to granulocytes, macrophages, and mast cells. Flow microfluorimetry-separated Thy-1+ cells require the addition of IL-3 or granulocyte/macrophage-CSF to the culture medium for continued growth and, as these cells divide and undergo terminal differentiation they gradually lose Thy-1 Ag expression. The loss of Thy-1 expression is not strictly correlated with cellular proliferation since the expression of Thy-1 decreases on proliferating cells. Last, IL-3 does not maintain the Thy-1- stem cell population that can give rise to Thy-1+ cells in vitro. The relevance of this scheme of differentiation to normal hemopoiesis and to differentiation-arrested IL-3-dependent leukemic cell populations is discussed.     

Self-renewal and differentiation of interleukin-3-dependent multipotent stem cells are modulated by stromal cells and serum factors

Interleukin-3 (IL-3)-dependent cell lines (FDCP-mix) were cloned and isolated from long-term bone-marrow cultures infected with src-MoMuLV. These cell lines have many of the characteristics of hematopoietic stem cells. Early isolates of the FDCP-mix cells form spleen colonies in irradiated mice and establish long-term hematopoiesis on irradiated marrow stroma in vitro in the absence of IL-3. These two properties of the cells are lost within 15 weeks of establishing the cell lines, but the cell lines retain their ability to differentiate in a multilineage response to hematopoietic growth factors and to hematopoietic stromal cells, as well as to self-renew in the presence of IL-3. The choice between differentiation and self-renewal in FDCP-mix cells can clearly be modified by culture conditions: in particular, cultures containing horse serum preferentially promote self-renewal, whereas cultures containing fetal calf serum preferentially promote differentiation. The FDCP-mix cell lines are not leukemic, nor do they contain the src oncogene. Their ability to respond to hematopoietic growth factors and stroma in a similar manner to normal hematopoietic cells makes them a valuable model for studying the regulation of hemopoietic cell self-renewal and differentiation.     

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.     

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.     

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.     

Characterization of the in vitro stromal microenvironment of human bone marrow

Utilizing long-term in vitro culture techniques, we characterized the cellular composition and functional attributes of the human in vitro bone marrow stromal microenvironment. Morphologic, specific cytochemical and immunologic methods demonstrated that the marrow stromal adherent layer (AL) reached confluency at two to three weeks, and was comprised of 60%-70% fibroblastic cells, 10%-20% endothelial cells, 10%-20% monocyte/macrophages and 5%-10% fat-laden adherent cells. These proportions of cell types persisted for at least three months concomitant with proliferation of CFU-gm and BFU-e. In contrast, umbilical cord blood cells did not form a stromal AL despite persistence of hemopoietic progenitor cell proliferation. These findings provide a basis for improved understanding of cellular interactions regulating 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     

Effect of dipyridamole on the interrelations of the stromal and hematopoietic tissues of the bone marrow in experimental studies]

By means of heterotopic transplantation of the bone marrow interrelations of the stromal and hemopoietic tissues of the mice bone marrow have been studied at administration of dipiridamol. Effect of the drug to the hemopoiesis is realized via stem stromal cells of the bone marrow. Under the influence of dipiridamol a focus of heterotopic hemopoiesis the osteogenic component in it is present only in 30% of cases in comparison with the control. Inhibition of the stromal component proliferation is accompanied with increasing mitotic activity of the hemopoietic elements against the background of the bone marrow cellularity decrease both in the femoral bone and in the focus of heterotopic hemopoiesis. At administration of dipiridamol a phenomenon of noneffective megakaryocytopoiesis with the intrabone marrow destruction of megakaryocytes, resulting in local release of thrombocyte growth factor, which has a compensatory character.     

Isolation of a human stromal cell strain secreting hemopoietic growth factors

A diploid fibroblastoid cell strain, termed "ST-1," has been established from a long-term liquid culture of human fetal liver cells. ST-1 cells are nonphagocytic, nonspecific esterase negative and do not possess factor VIII-related antigen but stain with antibodies specific for fibronectin and type I collagen. The ST-1 cells produce nondialyzable hemopoietic growth factors capable of stimulating the development of erythroid bursts, mixed granulocyte-macrophage colonies, pure granulocyte colonies, and pure macrophage colonies. These factors are active on both human fetal liver and human adult bone marrow progenitors. When liquid cultures of human fetal liver hemopoietic progenitors are established with a preformed monolayer of ST-1 cells, the yields of nonadherent cells, erythroid progenitors, and myeloid progenitors are greatly increased. These studies demonstrate that the fibroblastoid ST-1 cells support hemopoiesis in vitro and may be a critical element in the stromal microenviroment in vivo.