Compartmentalization of a haematopoietic growth factor (GM-CSF) by glycosaminoglycans in the bone marrow microenvironment

Haematopoietic progenitor cells proliferate and mature in semisolid media when stimulated by exogenous haematopoietic cell growth factors (HCGFs) such as granulocyte-macrophage colony-stimulating factor (GM-CSF). They also proliferate in association with marrow-derived stromal cells although biologically active amounts of HCGFs cannot be detected in stromal culture supernatants. It is possible that HCGFs are synthesized in small amounts by stromal cells but remain bound to the stromal cells and/or their extracellular matrix (ECM). This interpretation accords with haematopoietic progenitor cell proliferation in close association with stromal layers in long-term cultures. Glycosaminoglycans (GAGs) are found in the ECM produced by stromal cells. They are prime candidates for selectively retaining HCGFs in the stromal layer; they influence embryonic morphogenesis and cyto-differentiation and they may regulate haematopoiesis. We now report that granulocyte-macrophage colony-stimulating activity can be eluted from cultured stromal layers and that exogenous GM-CSF binds to GAGs from bone marrow stromal ECM. Selective compartmentalization of HCGFs in this manner may be an important function of the marrow microenvironment and may be involved in haematopoietic cell regulation.     

Haemonectin, a bone marrow adhesion protein specific for cells of granulocyte lineage

There is substantial evidence that the haematopoietic microenvironment is crucial to the growth and differentiation of haematopoietic cells. This microenvironment is composed of stromal cells, soluble factors and extracellular matrix (ECM). We have shown that a complex extract of bone marrow ECM can stimulate the growth and differentiation of haematopoietic cells in vitro. Furthermore, the use of inhibitors or stimulators of ECM synthesis in long-term marrow culture affects cell proliferation. On a molecular level, however, the interactions between ECM and haematopoietic cells are not well understood. We have investigated the adhesion between specific bone marrow ECM components and haematopoietic cells, and found a protein, 'haemonectin', of relative molecular mass 60,000 in bone marrow ECM which is a lineage- and organ-specific attachment molecule for cells of granulocyte lineage. This specificity distinguishes haemonectin from previously described adhesion proteins which have a wider tissue distribution and cell type specificity.     

Transplanted bone marrow regenerates liver by cell fusion

Results from several experimental systems suggest that cells from one tissue type can form other tissue types after transplantation. This could be due to the presence of multipotential or several types of adult stem cells in donor tissues, or alternatively, to fusion of donor and recipient cells. In a model of tyrosinaemia type I, mice with mutations in the fumarylacetoacetate hydrolase gene (Fah-/-) regain normal liver function after transplantation of Fah+/+ bone marrow cells, and form regenerating liver nodules with normal histology that express Fah. Here we show that these hepatic nodules contain more mutant than wild-type Fah alleles, and that their hepatocytes express both donor and host genes, consistent with polyploid genome formation by fusion of host and donor cells. Using bone marrow cells marked with integrated foamy virus vectors that express green fluorescent protein, we identify common proviral junctions in hepatic nodules and haematopoietic cells. We also show that the haematopoietic donor genome adopts a more hepatocyte-specific expression profile after cell fusion, as the wild-type Fah gene was activated and the pan-haematopoietic CD45 marker was no longer expressed.     

Altered adhesive interactions with marrow stroma of haematopoietic progenitor cells in chronic myeloid leukaemia

Normal haematopoietic cell regulation involves interaction between marrow stromal cells and haematopoietic progenitor cells which may be facilitated by specific recognition and adhesion. Some leukaemogenic events might produce a selective growth advantage by altering this regulatory network, possibly by diminishing the capacities of cells to adhere to stromal elements. Using an in vitro culture system which allows investigation of adhesion to stromal layers and subsequent colony formation by blast colony-forming cells (B1-CFC) in normal marrow and Ph+ chronic myeloid leukaemic (CML) blood, we compared the adhesive properties of normal and malignant progenitor cells. We present evidence that altered adhesive interactions between primitive progenitor cells and marrow stromal cells occur in CML.     

Formation of haematopoietic microenvironment and haematopoietic stem cells from single human bone marrow stem cells.

Haematopoietic stem cells are a population of cells capable both of self renewal and of differentiation into a variety of haematopoietic lineages. Enrichment techniques of human haematopoietic stem cells have used the expression of CD34, present on bone marrow progenitor cells. But most CD34+ bone marrow cells are committed to their lineage, and more recent efforts have focused on the precise characterization of the pluripotent subset of CD34+ cells. Here we report the characterization of two distinct subsets of pluripotent stem cells from human fetal bone marrow, a CD34+, HLA-DR+, CD38- subset that can differentiate into all haematopoietic lineages, and a distinct more primitive subset, that is CD34+, HLA-DR-, CD38-, that can differentiate into haematopoietic precursors and stromal cells capable of supporting the differentiation of these precursors. These data represent, to our knowledge, the first identification of a single cell capable of reconstituting the haematopoietic cells and their associated bone marrow microenvironment.     

Defective ability to self-renew in vitro of highly purified primitive haematopoietic cells

Stromal cells play a critical role in haematopoiesis, both in a permissive and, probably, in a directive manner. Study of the interactions between stromal cells and haematopoietic stem cells, however, is difficult to perform using whole bone marrow, in which stem cells are indistinguishable from precursor cells and maturing haematopoietic cells, and where stromal and haematopoietic cells co-exist in a heterogeneous mixture. We have purified primitive haematopoietic spleen colony-forming cells (CFU-S) using fluorescence-activated cell sorting (FACS) and produced CFU-S populations which approach 100% purity (ref. 6 and B.I.L. and E.S., in preparation). This cell population is devoid of significant stromal cells and mature haematopoietic cells. Here, we report that when purified CFU-S are seeded onto a stromal adherent layer in vitro, foci of haematopoietic cells develop within the stroma followed by production of a wave of maturing and mature progeny. However, self-renewal of CFU-S does not occur and haematopoietic activity rapidly declines, indicating that caution should be applied in the use of highly purified stem cells for human bone marrow transplantation.     

In vitro osteoclast generation from different bone marrow fractions, including a highly enriched haematopoietic stem cell population

It is well established that the osteoclast is formed by fusion of post-mitotic, mononuclear precursors derived from circulating progenitor cells. However, the precise haematopoietic origin of the osteoclast is unknown. We have investigated this here by fractionating mouse bone marrow and isolating haematopoietic stem cells using a three-step method combining equilibrium density centrifugation and two fluorescence-activated cell sortings (FACS), and have tested the ability of each bone marrow fraction, including highly purified haematopoietic stem cells, to generate osteoclasts during co-culture with preosteoclast-free embryonic long bones. The osteoclast-forming capacity was found to increase with increasing stem cell purity. On the other hand, the culture time needed for osteoclast formation also increased with purification, suggesting the presence of progressively more immature progenitor cells. The pluripotent haematopoietic stem cell fractions with the highest purity needed preincubation with a stem cell-activating factor (interleukin-3) to activate the predominantly quiescent stem cells in vitro.     

Osteoblastic cells regulate the haematopoietic stem cell niche

Stem cell fate is influenced by specialized microenvironments that remain poorly defined in mammals. To explore the possibility that haematopoietic stem cells derive regulatory information from bone, accounting for the localization of haematopoiesis in bone marrow, we assessed mice that were genetically altered to produce osteoblast-specific, activated PTH/PTHrP receptors (PPRs). Here we show that PPR-stimulated osteoblastic cells that are increased in number produce high levels of the Notch ligand jagged 1 and support an increase in the number of haematopoietic stem cells with evidence of Notch1 activation in vivo. Furthermore, ligand-dependent activation of PPR with parathyroid hormone (PTH) increased the number of osteoblasts in stromal cultures, and augmented ex vivo primitive haematopoietic cell growth that was abrogated by gamma-secretase inhibition of Notch activation. An increase in the number of stem cells was observed in wild-type animals after PTH injection, and survival after bone marrow transplantation was markedly improved. Therefore, osteoblastic cells are a regulatory component of the haematopoietic stem cell niche in vivo that influences stem cell function through Notch activation. Niche constituent cells or signalling pathways provide pharmacological targets with therapeutic potential for stem-cell-based therapies.     

Cell fusion is the principal source of bone-marrow-derived hepatocytes

Evidence suggests that haematopoietic stem cells might have unexpected developmental plasticity, highlighting therapeutic potential. For example, bone-marrow-derived hepatocytes can repopulate the liver of mice with fumarylacetoacetate hydrolase deficiency and correct their liver disease. To determine the underlying mechanism in this murine model, we performed serial transplantation of bone-marrow-derived hepatocytes. Here we show by Southern blot analysis that the repopulating hepatocytes in the liver were heterozygous for alleles unique to the donor marrow, in contrast to the original homozygous donor cells. Furthermore, cytogenetic analysis of hepatocytes transplanted from female donor mice into male recipients demonstrated 80,XXXY (diploid to diploid fusion) and 120,XXXXYY (diploid to tetraploid fusion) karyotypes, indicative of fusion between donor and host cells. We conclude that hepatocytes derived form bone marrow arise from cell fusion and not by differentiation of haematopoietic stem cells.     

Erythropoietin-stimulated erythropoiesis in long-term bone marrow culture.

The proliferation of multipotential haematopoietic stem cells (CFU-S) is possible in some long-term bone marrow cultures. Granulocyte and macrophage progenitor (CFU-C) and megakaryocyte precursor cells (CFU-M) are present in these cultures and undergo full development into mature cells. In contrast, while immature erythroid progenitors ('early' BFU-E) are maintained in long-term culture, none of the more differentiated progeny (CFU-E) have been detected, and no morphologically recognisable erythroid cells have been observed. We now describe a modified culture system in which the 'early' BFU-E develop into 'late' BFU-E in response to added erythropoietin. Further maturation of these cells into CFU-E and non-nucleated erythrocytes can be achieved by mechanical agitation of the long-term cultures or by transferring the cells into dishes which do not allow cell attachment to occur.     

Pluripotency of mesenchymal stem cells derived from adult marrow

We report here that cells co-purifying with mesenchymal stem cells--termed here multipotent adult progenitor cells or MAPCs--differentiate, at the single cell level, not only into mesenchymal cells, but also cells with visceral mesoderm, neuroectoderm and endoderm characteristics in vitro. When injected into an early blastocyst, single MAPCs contribute to most, if not all, somatic cell types. On transplantation into a non-irradiated host, MAPCs engraft and differentiate to the haematopoietic lineage, in addition to the epithelium of liver, lung and gut. Engraftment in the haematopoietic system as well as the gastrointestinal tract is increased when MAPCs are transplanted in a minimally irradiated host. As MAPCs proliferate extensively without obvious senescence or loss of differentiation potential, they may be an ideal cell source for therapy of inherited or degenerative diseases.     

Continuous in vitro generation of multipotential stem cell clones from src-infected cultures

A molecular recombinant of Rous sarcoma virus and murine amphotropic leukaemia virus, src(MoMuLV), where the avian src oncogene has been placed under the influence of a murine virus promoter sequence, has been reported. Infection of long-term marrow cultures with this virus led to a dramatic change in the relative numbers of stem cells, granulocyte-macrophage progenitor cells and mature cells found in normal haematopoietic cell development. However, although the balance between self-renewal, differentiation and development was disturbed, injection of the cultured cells into irradiated syngeneic recipients did not lead to the development of leukaemia. Thus, although the control had been 'loosened', the host regulatory mechanisms were sufficient to impose a restraint on unlimited growth of the cells. We now show that the stem cells from the src-infected cultures show a remarkably increased capacity for self-renewal in vitro in situations which are inimical to the maintenance of self-renewal in normal uninfected stem cells and that self-renewal/differentiation can be modified by the culture conditions.     

Endothelial and perivascular cells maintain haematopoietic stem cells

Several cell types have been proposed to create niches for haematopoietic stem cells (HSCs). However, the expression patterns of HSC maintenance factors have not been systematically studied and no such factor has been conditionally deleted from any candidate niche cell. Thus, the cellular sources of these factors are undetermined. Stem cell factor (SCF; also known as KITL) is a key niche component that maintains HSCs. Here, using Scf(gfp) knock-in mice, we found that Scf was primarily expressed by perivascular cells throughout the bone marrow. HSC frequency and function were not affected when Scf was conditionally deleted from haematopoietic cells, osteoblasts, nestin-cre- or nestin-creER-expressing cells. However, HSCs were depleted from bone marrow when Scf was deleted from endothelial cells or leptin receptor (Lepr)-expressing perivascular stromal cells. Most HSCs were lost when Scf was deleted from both endothelial and Lepr-expressing perivascular cells. Thus, HSCs reside in a perivascular niche in which multiple cell types express factors that promote HSC maintenance.     

Epidermal Langerhans cells are derived from cells originating in bone marrow

Langerhans cells constitute a morphologically well characterised subpopulation (3--8%) of mammalian epidermal cells which, in contrast to the bulk of epidermal cells, bear Fc-IgG and C3 receptors, express immune response-associated (Ia) antigens and function as antigen-presenting cells and allogeneic stimulatory cells to primed T lymphocytes. The ontogeny of Langerhans cells has been a subject of considerable debate since their discovery. Although some studies suggest that Langerhans cells are of mesenchymal as opposed to neural or melanocytic origin, direct evidence for this has not been presented. In this study we demonstrate that, after 3 weeks, most of the Langerhans cells (LC) in parenteral skin which had been transplanted on to F1 hybrids were of recipient origin whereas keratinocytes remained of donor origin; this indicates that the LC are derived from a mobile pool of cells. Furthermore, in studies of skin from radiation-induced bone marrow chimaeric animals we found that, depending on the strain combination, up to 80% of the epidermal LC were derived from the bone marrow of the donor animals.     

Retrovirus transfer of a bacterial gene into mouse haematopoietic progenitor cells

The haematopoietic system is made up of a hierarchy of cells with different developmental, functional and proliferative capacities. Although cellular diversity appears to arise from the commitment and maturation of stem cells, the molecular basis for this differentiation process is unknown. The introduction of cloned DNA sequences into haematopoietic progenitor cells would provide a novel approach for studying this differentiating in vivo system. One laboratory has reported DNA-mediated transfer of genes into mouse bone marrow cells. However, retroviruses offer a number of advantages over DNA-mediated gene transfer procedures, including high efficiency infection of a wide range of cell types in vitro and in vivo, stable and low copy integration into the host chromosome, and a defined integrated provirus structure. For these reasons recombinant DNA techniques have been utilized to construct high efficiency retrovirus vectors expressing foreign genes. We demonstrate here, using such a retrovirus vector, the transfer of a dominant selectable drug-resistance gene into defined classes of mouse haematopoietic progenitor cells. These observations should facilitate the development of molecular genetic approaches to fundamental and clinical problems in haematopoiesis.     

Granulocyte- and granulocyte-macrophage-colony stimulating factors induce human endothelial cells to migrate and proliferate

Granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage-colony stimulating factor (GM-CSF) belong to a family of glycoprotidic growth factors required for the survival, growth and differentiation of haematopoietic precursors and which affect the function of circulating mature cells. They are produced by resting or stimulated stromal cells of the haematopoietic microenvironment (fibroblasts and endothelium) and by immunocompetent cells (T cells and monocytes/macrophages). The action of these CSF molecules was thought to be restricted to cells of haematopoietic origin. Here, we report that G-CSF and GM-CSF influence the migration and proliferation of human endothelial cells suggesting that these molecules may act as regulatory signals outside the haematopoietic system.     

Reconstitution with syngeneic plus allogeneic or xenogeneic bone marrow leads to specific acceptance of allografts or xenografts

Clinical organ transplantation between genetically disparate individuals currently requires the use of chemotherapeutic agents to suppress the rejection reaction. The deleterious side effects of these reagents and their inability to prevent rejection completely has led to a continuing search for methods to induce specific transplantation tolerance in adult recipients. Numerous experimental animal models utilizing irradiation and bone marrow transplantation coincident with organ transplantation have been proposed. Bone marrow transplantation, however, has its own major complications, including graft-versus-host reactions and immunoincompetence, probably resulting from a failure of appropriate immune cell interactions in the reconstituted host. We have now attempted to overcome these difficulties by reconstituting the irradiated host with T-cell depleted bone marrow containing both host (syngeneic) and donor (allogeneic or xenogeneic) components. This technique leads to long-term survival of the reconstituted animals and specific prolongation of subsequent skin grafts of donor type. Animals reconstituted in this fashion are fully reactive to third-party allografts and xenografts and do not appear to manifest signs of graft-versus-host disease.     

Long-term culture of murine erythropoietic progenitor cells in the absence of an adherent layer

Replication of multipotential stem cells in long-term murine bone marrow cell culture is known to depend on the development of an adherent stromal cell layer. In these conditions, restricted haematopoietic progenitor cells have also been generated for up to several months1-3. However, maturation is observed only in the granulocyte/macrophage and megakaryocyte lineages; erythropoiesis appears to be blocked at the earliest burst-forming unit (BFU-E) stage. Addition of exogenous erythropoietin (Epo) or anaemic mouse serum results in full erythropoietic maturation, but it is transient. We describe here a culture system in which production of erythropoietic progenitor cells can be maintained for over 6 months in the absence of an adherent stromal layer and in the absence of added Epo, but in the presence of pokeweed mitogen-stimulated spleen cell conditioned medium (PWSCM). The data indicate that restricted erythroid progenitor cells exist which are capable of extensive self-renewal.     

Fibronectin and VLA-4 in haematopoietic stem cell-microenvironment interactions

The self-renewal and differentiation of haematopoietic stem cells occurs in vivo and in vitro in direct contact with cells making up the haematopoietic microenvironment. In this study we used adhesive ligands and blocking antibodies to identify stromal cell-derived extracellular matrix proteins involved in promoting attachment of murine haematopoietic stem cells. Here we report that day-12 colony-forming-unit spleen (CFU-S12)5 cells and reconstituting haematopoietic stem cells attach to the C-terminal, heparin-binding fragment of fibronectin by recognizing the CS-1 peptide of the alternatively spliced non-type III connecting segment (IIICS) of human plasma fibronectin. Furthermore, CFU-S12 stem cells express the alpha 4 subunit of the VLA-4 integrin receptor, which is known to be a receptor for the CS-1 sequence, and monoclonal antibodies against the integrin alpha 4 subunit of VLA-4 block adhesion of CFU-S12 stem cells to plates coated with the C-terminal fibronectin fragment. Finally, polyclonal antibodies against the integrin beta 1 subunit of VLA-4 inhibit the formation of CFU-S12-derived spleen colonies and medullary haematopoiesis in vivo following intravenous infusion of antibody-treated bone marrow cells.     

A novel human lymphokine that inhibits haematopoietic progenitor cell proliferation

T lymphocytes in culture synthesize and secrete a variety of factors that activate and guide the differentiation, replication and maturation of haematopoietic cells in vitro. Malignant T-cell lines as well as T-cell hybridomas producing several of these factors have been established. We report here a factor produced by a human cell line that exerts a potent inhibitory effect on the growth of bone marrow progenitor cells. The properties of this factor, which we have termed colony-inhibiting lymphokine ( CIL ), differ from other inhibitors of haematopoietic progenitor cell proliferation, but resemble those of a T-cell-derived factor causally linked with some cases of severe aplastic anaemia in humans. Sensitivity of cells to this factor appears to correlate positively with expression of HLA-DR surface antigens.