Stromal cells of hemopoietic origin

Hemopoiesis is a multistep process involving stem cell renewal, commitment, differentiation, maturation and consequent positioning of the cells within the tissue. Stromal cells are a major component of the hemopoietic microenvironment. The in vitro culture of cloned stromal cells has enabled detailed analysis of their functions and has provided answers relating to the contribution of stromal cells to the control of hemopoiesis. Cultured stromal cells were found to support the renewal of stem cells through a mechanism that did not seem to involve already known cytokines. Cloned stromal cells from both marrow and thymus supported the in vitro accumulation of myeloid as well as T and B lymphoid cells. Thus, cloned stromal cells had the ability to induce multilineage hemopoiesis, irrespective of the organ from which they were derived. Invariably, stromal cells tended to select in culture for hemopoietic cells at early differentiation stages and restricted the accumulation of mature cells. These functions may be part of the mechanism that protects the stem cell pool from excess differentiation.     

Notch1 perturbation of hemopoiesis involves non-cell- autonomous modifications

To study the effects of Notch on hemopoiesis we used a bone marrow transduction/transplantation model and compared the transduced and nontransduced populations in reconstituted mice. While cells expressing a constitutively active form of murine Notch1 (Notch1IC) completely lacked B cells, a profound suppression of the B lineage was also seen in the nontransduced compartment. Experiments performed with retroviral supernatants of varying titers showed that the perturbations of B cell development among the nontransduced population correlated with the percentage of Notch1IC-transduced cells inoculated into the mice. The myeloid lineage of the Notch1IC-transplanted mice was altered as well, and this also affected the nontransduced population that had features of excessive maturation. To explore the basis of these non-cell-autonomous modifications we prepared conditioned medium from ex vivo cultures of Notch1IC-transplanted mice bone marrow and showed that it inhibited B cell maturation and promoted myeloid differentiation in a dose-dependent manner. Finally, we found that the T cell leukemia/lymphomas that occur in Notch1IC-transplanted mice were accompanied by abnormal maturation of nontransduced T cells in the bone marrow. These findings indicate that modifications of neighboring cells through non-cell-autonomous modifications take part in multiple facets of the activity of Notch on hemopoiesis.     

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.     

Emergence of T, B, and myeloid lineage-committed as well as multipotent hemopoietic progenitors in the aorta-gonad-mesonephros region of day 10 fetuses of the mouse.

We investigated the developmental potential of hemopoietic progenitors in the aorta-gonad-mesonephros (AGM) region, where the definitive type hemopoietic progenitors have been shown to emerge before the fetal liver develops. By using an assay system that is able to determine the developmental potential of individual progenitors toward the T, B, and myeloid lineages, we show that not only multipotent progenitors but also progenitors committed to the T, B, or myeloid lineage already exist in this region of day 10 fetuses. Bipotent progenitors generating myeloid and T cells or those generating myeloid and B cells were also detected, suggesting that the commitment to T and B cell lineages is in progress in the AGM region. The numbers of these progenitors, however, were only 1/200-1/1000 of those in fetal liver of day 12 fetuses. Such small numbers of progenitors suggest that hemopoiesis has just started in the AGM region of day 10 fetuses. Although most of T cell lineage-committed progenitors in the AGM region generated only a small number of immature T cells, some were able to generate a large number of mature T cells. The detection of various types of lineage-committed progenitors strongly suggests that the AGM region is not only the site of stem cell emergence, but also the site of hemopoiesis, including lineage commitment. The T cell progenitors found in the AGM region may represent the first immigrants to the thymus anlage.     

Activated macrophages induce hemopoietic islands in the adult rat liver

Focal hemopoiesis was induced in the liver of adult rats following treatment with glucan, a macrophage activator. Using endogenous peroxidase cytochemistry for identification of hemopoietic cell types, hemopoietic islands were studied by light and electron microscopy. Hemopoiesis in the islands was either restricted to the erythroid or myeloid lineage. Hemopoietic cells of various maturational stages frequently formed close contacts with macrophages. This indicates that activated macrophages exert a stimulatory effect on hemopoiesis by short-range action of humoral factors or by a close cell-to-cell-contact.     

IL-1 inhibits B cell differentiation in long term bone marrow cultures

There is evidence that stromal cells are responsive to changes in their external milieu and that this can affect their function. IL-1 has been identified as one mediator that can affect stromal cells by increasing their secretion of CSF. The monokine has also been reported to be a B cell differentiation factor. The purpose of this study was to test the effects of IL-1 on the pattern of hemopoietic cell differentiation by adding IL-1 alpha to myeloid long term bone marrow cultures (MBMC) at the time of their transfer to lymphoid bone marrow culture conditions. This usually results in the cessation of myelopoiesis and the induction of B lymphopoiesis. The addition of 50 U/ml of rIL-1 alpha, but not 10 U/ml, to MBMC at the time of their transfer to lymphoid conditions resulted in a complete inhibition of B cell differentiation and sustained myelopoiesis. To determine whether adherent layer cells contributed to this effect, conditioned medium (CM) was collected from adherent layers treated previously with the antibiotic mycophenolic acid. This depletes the hemopoietic cells from the cultures and retains a purified population of stromal cells. CM from mycophenolic acid- treated adherent layers exposed for 24 h to 50 U/ml of IL-1 was added at volume concentrations of 5, 10, and 25% to MBMC at the time of transfer to lymphoid bone marrow culture conditions and at each feeding thereafter. Expression of the B lineage associated 14.8 Ag and IgM was inhibited on a dose dependent basis, and myelopoiesis was sustained in cultures to which 25% CM had been added. Induction of B lymphopoiesis occurred in cultures to which adherent cell CM not exposed to IL-1 had been added. The CM from the IL-1-treated adherent cells contained CSF, because it promoted the growth of myeloid colonies from fresh marrow or MBMC cells and stimulated the granulocyte-macrophage-CSF sensitive FDC-P1 cell line to proliferate. IL-3 was not present in the CM, because stimulation of the IL-3 sensitive 32D cell line was not observed. The CM from the IL-1-treated adherent cells stimulated thymocytes to proliferate in the presence of PHA. This raised the possibility that the induced CSF may have required IL-1 to mediate their effects in the cultures. However, B lymphopoiesis was inhibited and myelopoiesis maintained upon addition of recombinant granulocyte-, macrophage-, and granulocyte-macrophage-CSF to cultures, indicating that IL-1 or other non-CSF molecules induced by it need not be present.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of a murine mammary tumor on in vivo and in vitro hemopoiesis

The effects of an autologous transplanted mammary tumor (RIII-T3) on hemopoiesis in RIII mice are described. Tumor-bearing animals died 30 to 40 days after inoculation and displayed splenomegaly, extreme neutrophilia, and moderately increased monocyte levels in the spleen, peripheral blood, and bone marrow. The precursors of neutrophils and monocytes, granulocyte/macrophage colony-forming cells (GM-CFC) were elevated in the spleen, bone marrow, and peripheral blood. RIII-T3-conditioned medium stimulated bone marrow GM-CFC and caused the myelomonocytic cell line, WEHI-3B, to differentiate in vitro. The conditioned medium did not stimulate erythroid, megakaryocyte, or eosinophil colony formation. When conditioned medium was fractionated, two peaks of activity corresponding to GM-CSF and G-CSF were observed, suggesting that the extreme neutrophilia observed in tumor-bearing animals may result from chronic exposure of the hemopoietic system to these hemopoietic hormones.     

Postnatal liver hemopoiesis in mice: generation of pre-B cells, granulocytes, and erythrocytes in discrete colonies

Morphologic analysis of hemopoietic tissue in mouse liver reveals the persistence of erythropoietic, granulopoietic, and lymphopoietic activity for approximately 2 wk after birth. Near the end of the first postnatal week, we noted a remarkable reorganization of the hemopoietic cells that was characterized by a transition from a diffuse distribution of mixed erythroid, myeloid, and lymphoid elements to a focal pattern of discrete hemopoietic colonies scattered among the cords of hepatic parenchymal cells. Each hemopoietic focus contained cells progressing along a single differentiation pathway (i.e., erythroid, myeloid, or lymphoid cells). Megakaryocytes were seen as solitary cells surrounded by hepatocytes. This pattern of colonization was observed in all strains of mice examined. In the livers of mice with known hemopoietic defects, however, differences were found in the duration of postnatal hemopoiesis. Accessory cells with macrophage-like features were consistently observed in erythropoietic foci, but were rarely seen in lymphoid foci. The latter were formed by pre-B cells identifiable by the presence of cytoplasmic mu-heavy chains and the absence of light chain expression. The occurrence of discrete colonies of erythroid, myeloid, and pre-B lymphoid cells in the postnatal liver suggests that each is derived from a single, committed precursor cell. This anatomical compartmentalization according to cell type offers a useful model system for analysis of hemopoietic differentiation and of the generation of clonal diversity among B lineage cells.     

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.     

Contrasting responses of lymphoid progenitors to canonical and noncanonical Wnt signals

The Wnt family of secreted glycoproteins has been implicated in many aspects of development, but its contribution to blood cell formation is controversial. We overexpressed Wnt3a, Wnt5a, and Dickkopf 1 in stromal cells from osteopetrotic mice and used them in coculture experiments with highly enriched stem and progenitor cells. The objective was to learn whether and how particular stages of B lymphopoiesis are responsive to these Wnt family ligands. We found that canonical Wnt signaling, through Wnt3a, inhibited B and plasmacytoid dendritic cell, but not conventional dendritic cell development. Wnt5a, which can oppose canonical signaling or act through a different pathway, increased B lymphopoiesis. Responsiveness to both Wnt ligands diminished with time in culture and stage of development. That is, only hematopoietic stem cells and very primitive progenitors were affected. Although Wnt3a promoted retention of hematopoietic stem cell markers, cell yields and dye dilution experiments indicated it was not a growth stimulus. Other results suggest that lineage instability results from canonical Wnt signaling. Lymphoid progenitors rapidly down-regulated RAG-1, and some acquired stem cell-staining characteristics as well as myeloid and erythroid potential when exposed to Wnt3a-producing stromal cells. We conclude that at least two Wnt ligands can differentially regulate early events in B lymphopoiesis, affecting entry and progression in distinct differentiation lineages.     

A stromal cell line from rainbow trout spleen, RTS34st, that supports the growth of rainbow trout macrophages and produces conditioned medium with mitogenic effects of leukocytes

Summary A rainbow trout spleen cell line, RTS34, was developed from a long-term hemopoietic culture. This cell line consisted of a mixed stromal cell layer with an associated cell population of macrophage-like cells that formed proliferative foci and released nonadherent progeny cells into the culture medium. A stromal cell line, RTS34st, was isolated from the RTS34 cell line. RTS34st cultures contained cells with fibroblast-like and epithelial-like morphologies and showed enhanced [³H]thymidine incorporation in response to either FBS or rainbow trout serum. The combination of FBS and trout serum was synergistic. Conditioned medium from RTS34st stimulated thymidine incorporation by peripheral blood and head kidney leukocytes, but not by leukocytes from the spleen. In addition, RTS34st provided a hemopoietic inductive microenvironment for immature precursor cells, selectively supporting the growth of macrophage-like cells. Therefore, RTS34st appears useful for studying the different roles of the stroma in regulating hemopoiesis in fish.     

Concentrative uptake of cyclic ADP-ribose generated by BST-1+ stroma stimulates proliferation of human hematopoietic progenitors

Cyclic ADP-ribose (cADPR) is an intracellular calcium mobilizer generated from NAD(+) by the ADP-ribosyl cyclases CD38 and BST-1. cADPR, both exogenously added and paracrinally produced by a CD38(+) feeder layer, has recently been demonstrated to stimulate the in vitro proliferation of human hemopoietic progenitors (HP) and also the in vivo expansion of hemopoietic stem cells. The low density of BST-1 expression on bone marrow (BM) stromal cells and the low specific activity of the enzyme made it unclear whether cADPR generation by a BST-1(+) stroma could stimulate HP proliferation in the BM microenvironment. We developed and characterized two BST-1(+) stromal cell lines, expressing an ectocellular cyclase activity similar to that of BST-1(+) human mesenchymal stem cells, the precursors of BM stromal cells. Long term co-culture of cord blood-derived HP over these BST-1(+) feeders determined their expansion. Influx of paracrinally generated cADPR into clonogenic HP was mediated by a concentrative, nitrobenzylthioinosine- and dipyridamole-inhibitable nucleoside transporter, this providing a possible explanation to the effectiveness of the hormone-like concentrations of the cyclic nucleotide measured in the medium conditioned by BST-1(+) feeders. These results suggest that the BST-1-catalyzed generation of extracellular cADPR, followed by the concentrative uptake of the cyclic nucleotide by HP, may be physiologically relevant in normal hemopoiesis.     

Divergent effects of Wnt5b on IL-3- and GM-CSF-induced myeloid differentiation

The multiple specialized cell types of the hematopoietic system originate from differentiation of hematopoietic stem cells and progenitors (HSPC), which can generate both lymphoid and myeloid lineages. The myeloid lineage is preferentially maintained during ageing, but the mechanisms that contribute to this process are incompletely understood. Here, we studied the roles of Wnt5a and Wnt5b, ligands that have previously been linked to hematopoietic stem cell ageing and that are abundantly expressed by both hematopoietic progenitors and bone-marrow derived niche cells. Whereas Wnt5a had no major effects on primitive cell differentiation, Wnt5b had profound and divergent effects on cytokine-induced myeloid differentiation. Remarkably, while IL-3-mediated myeloid differentiation was largely repressed by Wnt5b, GM-CSF-induced myeloid differentiation was augmented. Furthermore, in the presence of IL-3, Wnt5b enhanced HSPC self-renewal, whereas in the presence of GM-CSF, Wnt5b accelerated differentiation, leading to progenitor cell exhaustion. Our results highlight discrepancies between IL-3 and GM-CSF, and reveal novel effects of Wnt5b on the hematopoietic system.     

Long-term maintenance of multilineal hemopoiesis in collagen gel culture.

We examined the long-term maintenance of multilineal hemopoiesis in a collagen gel culture of mouse bone marrow cells. When cells were inoculated into the gel, stromal cells formed foci that were composed of sinusoidlike capillary structures, fibroblastic cells, adipocytes and macrophages. Many small hemopoietic foci similar to granulocyte-macrophage colonies (CFU-GM) appeared within a week and disappeared after two weeks. Several large hemopoietic foci appeared after two to three weeks of culture, without a second challenge of marrow cells. These large hemopoietic foci were composed mainly of myeloid cells. Megakaryocytes and mast cells were also observed. When erythropoietin (EPO) was added to the culture at the beginning, the erythroid focus appeared after 3 weeks and the number of megakaryocytes was greater than that in the culture without EPO. However, when EPO was added to the cultures after 6 or 12 weeks, erythroid cells appeared after 1 week and the number of megakaryocytes increased. This hemopoiesis lasted more than 6 months.     

Colony-stimulating factors and cytokine receptor network.

Cytokines play a vital role in coordinating immune and inflammatory responses. As many cytokine gene hunters have begun to focus their efforts on receptors, novel aspects of hemopoietic growth factor receptors have emerged. Two types of growth factor receptors have been classified--the cytokine receptor family and growth factor receptor family with tyrosine kinase activity. The two types of receptors may have unique roles in 'inducible' and 'constitutive' hemopoiesis which are controlled by immunological stimuli and by interaction with stromal cells, respectively.     

Activation of vascular endothelial growth factor receptor-2 in bone marrow leads to accumulation of myeloid cells: role of granulocyte-macrophage colony-stimulating factor

Vascular endothelial growth factor (VEGF) is a secreted cytokine that plays a major role in the formation and maintenance of the hemopoietic and vascular compartments. VEGF and its receptors, VEGFR-1 and VEGFR-2, have been found to be expressed on subsets of normal and malignant hemopoietic cells, but the role of the individual receptors in hemopoiesis requires further study. Using a VEGFR-2 fusion protein that can be dimerized with a synthetic drug, we were able to specifically examine the effects of VEGFR-2 signaling in hemopoietic cells in vivo. Mice transplanted with bone marrow transduced with this inducible VEGFR-2 fusion protein demonstrated expansion of myeloid cells (Gr-1+, CD11b+). Levels of myeloid progenitors were also increased following VEGFR-2 activation, through autocrine and paracrine mechanisms, as measured by clonogenic progenitor assays. VEGFR-2 activation induced expression of GM-CSF and increased serum levels in vivo. Abrogation of GM-CSF activity, either with neutralizing Abs or by using GM-CSF-null hemopoietic cells, inhibited VEGFR-2-mediated myeloid progenitor activity. Our findings indicate that VEGF signaling through VEGFR-2 promotes myelopoiesis through GM-CSF-dependent and -independent mechanisms.     

Adipogenesis in a myeloid supporting bone marrow stromal cell line.

The bone marrow stroma contains pre-adipocyte cells which are part of the hemopoietic microenvironment. Cloned stromal cell lines differ both in their ability to support myeloid and lymphoid development and in their ability to undergo adipocyte differentiation in vitro. These processes have been examined in the +/+2.4 murine stromal cell line and compared to other stromal and pre-adipocyte cell lines. In long-term cultures, the +/+2.4 stromal cells support myeloid cell growth, consistent with their expression of macrophage-colony stimulating factor mRNA. However, despite the presence of mRNA for the lymphoid supportive cytokines interleukins 6 and 7, +/+2.4 cells failed to support stromal cell dependent B lineage lymphoid cells in vitro, suggesting that these stromal cells exhibit only a myelopoietic support function. The +/+2.4 cells differentiate into adipocytes spontaneously when cultured in 10% fetal bovine serum. The process of adipogenesis can be accelerated by a number of agonists based on morphologic and gene marker criteria. Following induction with hydrocortisone, methylisobutylxanthine, indomethacin, and insulin in combination, a time dependent increase in the steady state mRNA and enzyme activity levels of the following adipocyte specific genes was observed: adipocyte P2, adipsin, CAAT/enhancer binding protein, and lipoprotein lipase. In contrast, adipogenesis was accompanied by a slight decrease in the signal intensity of the macrophage-colony stimulating factor mRNA level, similar to that which has been reported in other bone marrow stromal cell lines. These data demonstrate that although the lympho-hematopoietic support function of pre-adipocyte bone marrow stromal cell lines is heterogeneous, they share a common mechanism of adipogenesis.     

Role of stromal microenvironment in the regulation of bone marrow hemopoiesis after curantyl administration

Role of the stromal microenvironment in regulation of bone marrow hemopoiesis at the administration of the thrombocyte disaggregant curantyl was studied by the method of heterotopic transplantation of the mice bone marrow. It is shown that the action of curantyl on hemopoiesis is realised through the stem stromal cells of the bone marrow. It is noted that the inhibitory action of the preparation on proliferation of osteogenic precursor-cells is followed by activation of bone resorption processes in regenerating ectopic hemopoietic organ. Under the action of curantyl at low bone marrow cellularity in the focus of heterotopic hemopoiesis and femur an increase of mitotic activity in hemopoietic elements is noted. It is revealed that a phenomenon of ineffective megakaryocytopoiesis with intramedullary destruction of megakaryocytes leads to the local excretion of the thrombocyte released growth factor (TRGF) which has a compensatory character.     

In situ visualization of hemopoietic cell subsets and stromal elements in rat and mouse bone marrow by immunostaining of frozen sections

We have developed a method to section frozen long bones of rat and mouse and stained bone marrow (BM) by (double) immunofluorescence and immunoperoxidase. Here we report this method and reveal the location of early hemopoietic progenitors (Thy-1) and myeloid cells (Mac-1) in mouse BM, and early hemopoietic progenitors and lymphoid cells (Thy-1), erythroid cells (HIS49), and macrophages (ED2) in rat BM. In mouse BM our new findings include (a) the scattered localization of early hemopoietic progenitors (Thy-1low) all over the marrow, and (b) the presence of Thy-1+ stromal cells, mainly subendosteally. In rat BM an important finding is that of (a) a subendosteal region of 12-14 hemopoietic cell layers characterized by an abundance of Thy-1 and the virtual absence of erythroid cells, and (b) the scattering of Thy-1very bright cells which are candidates for the earliest hemopoietic progenitors in this species. The results illustrate that the technique is an excellent tool for studying the topology of BM as an organ of hemopoiesis.     

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.