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.     

Introduction of new genetic material into pluripotent haematopoietic stem cells of the mouse

An infectious retrovirus vector has been used to transfer a bacterial gene encoding resistance to the neomycin analogue G418 into pluripotent haematopoietic stem cells present in explanted murine bone marrow tissue. Subsequent transplantation of the cells into lethally irradiated mice results in engraftment of the animals with donor haematopoietic tissue containing the bacterial gene. This approach affords an efficient and rapid means of re-introducing genetically modified tissue into intact organisms and provides a system whereby the expression and regulation of cloned genes can be followed within the context of a well characterized developmental programme.     

Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium

Under conditions of tissue injury, myocardial replication and regeneration have been reported. A growing number of investigators have implicated adult bone marrow (BM) in this process, suggesting that marrow serves as a reservoir for cardiac precursor cells. It remains unclear which BM cell(s) can contribute to myocardium, and whether they do so by transdifferentiation or cell fusion. Here, we studied the ability of c-kit-enriched BM cells, Lin- c-kit+ BM cells and c-kit+ Thy1.1(lo) Lin- Sca-1+ long-term reconstituting haematopoietic stem cells to regenerate myocardium in an infarct model. Cells were isolated from transgenic mice expressing green fluorescent protein (GFP) and injected directly into ischaemic myocardium of wild-type mice. Abundant GFP+ cells were detected in the myocardium after 10 days, but by 30 days, few cells were detectable. These GFP+ cells did not express cardiac tissue-specific markers, but rather, most of them expressed the haematopoietic marker CD45 and myeloid marker Gr-1. We also studied the role of circulating cells in the repair of ischaemic myocardium using GFP+-GFP- parabiotic mice. Again, we found no evidence of myocardial regeneration from blood-borne partner-derived cells. Our data suggest that even in the microenvironment of the injured heart, c-kit-enriched BM cells, Lin- c-kit+ BM cells and c-kit+ Thy1.1(lo) Lin- Sca-1+ long-term reconstituting haematopoietic stem cells adopt only traditional haematopoietic fates.     

Host origin of marrow stromal cells following allogeneic bone marrow transplantation

Although it is generally agreed that stromal cells are important in the regulation of haematopoietic cell development, the origin of these phenotypically diverse cells has been a subject for debate for more than 50 years. Data which support the concept of a separate origin for the haematopoietic stem cell and the marrow stroma are derived from cytogenetic or enzyme marker studies of explanted and expanded stromal cells grown under conditions that do not allow haematopoiesis in vitro. Recent evidence in man and in mouse suggesting that the stromal cells capable of transferring the haematopoietic microenvironment in vitro are transplantable seemingly questions this dichotomy, one interpretation being the existence of a common haematopoietic/stromal 'stem cell'. We used in situ hybridization to discriminate donor cells from host in blood and bone marrow samples obtained from patients with functioning sex-mismatched but HLA-identical allografts. Without exception, marrow-derived stromal cells that proliferate in long-term cultures were found to be of host genotype, whereas the macrophage component of the adherent layer in these cultures originated from the donor.     

Fusion of bone-marrow-derived cells with Purkinje neurons, cardiomyocytes and hepatocytes

Recent studies have suggested that bone marrow cells possess a broad differentiation potential, being able to form new liver cells, cardiomyocytes and neurons. Several groups have attributed this apparent plasticity to 'transdifferentiation'. Others, however, have suggested that cell fusion could explain these results. Using a simple method based on Cre/lox recombination to detect cell fusion events, we demonstrate that bone-marrow-derived cells (BMDCs) fuse spontaneously with neural progenitors in vitro. Furthermore, bone marrow transplantation demonstrates that BMDCs fuse in vivo with hepatocytes in liver, Purkinje neurons in the brain and cardiac muscle in the heart, resulting in the formation of multinucleated cells. No evidence of transdifferentiation without fusion was observed in these tissues. These observations provide the first in vivo evidence for cell fusion of BMDCs with neurons and cardiomyocytes, raising the possibility that cell fusion may contribute to the development or maintenance of these key cell types.     

VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche

The cellular and molecular mechanisms by which a tumour cell undergoes metastasis to a predetermined location are largely unknown. Here we demonstrate that bone marrow-derived haematopoietic progenitor cells that express vascular endothelial growth factor receptor 1 (VEGFR1; also known as Flt1) home to tumour-specific pre-metastatic sites and form cellular clusters before the arrival of tumour cells. Preventing VEGFR1 function using antibodies or by the removal of VEGFR1(+) cells from the bone marrow of wild-type mice abrogates the formation of these pre-metastatic clusters and prevents tumour metastasis, whereas reconstitution with selected Id3 (inhibitor of differentiation 3)-competent VEGFR1+ cells establishes cluster formation and tumour metastasis in Id3 knockout mice. We also show that VEGFR1+ cells express VLA-4 (also known as integrin alpha4beta1), and that tumour-specific growth factors upregulate fibronectin--a VLA-4 ligand--in resident fibroblasts, providing a permissive niche for incoming tumour cells. Conditioned media obtained from distinct tumour types with unique patterns of metastatic spread redirected fibronectin expression and cluster formation, thereby transforming the metastatic profile. These findings demonstrate a requirement for VEGFR1+ haematopoietic progenitors in the regulation of metastasis, and suggest that expression patterns of fibronectin and VEGFR1+VLA-4+ clusters dictate organ-specific tumour spread.     

小鼠非清髓性单倍体相合骨髓移植模型的建立

目的建立小鼠非清髓性单倍体相合骨髓移植模型,为研究移植前诱导免疫耐受或移植后输注供者细胞促进植入提供研究平台。方法以CB6F1雌性小鼠为受鼠,移植前1 d予450 cGy全身照射（TBI）后,随机分为2组,实验组移植0 d输注C57BL/6雄性小鼠骨髓有核细胞5×107/只,对照组不予移植。然后监测受鼠造血恢复、检测供鼠性别决定基因（SRY）判断植入情况,以及外周血供者细胞尤其CD3＋细胞嵌合状态,同时观察小鼠急性移植物抗宿主病（aGVHD）的发生情况。结果对照组小鼠均存活,仅表现轻度aGVHD,血象移植后30 d内基本恢复正常水平。实验组小鼠SRY基因在移植后＋14 d、＋30 d、＋60 d时检测PCR结果均阳性,供鼠外周血淋巴细胞、单核细胞、粒细胞嵌合率在移植后14 d分别为23.8%、36.9%%、19.4%;30 d分别为49.9%、53.2%、54.4%;60 d分别为67.6%、51.6%、56.9%,其中CD3＋细胞嵌合率分别为4.4%、21.2%、54.4%。结论 450 cGyTBI的非清髓性预处理方案,可以诱导受鼠免疫耐受、供者骨髓细胞植入,嵌合率处于中低水平混合嵌合状态。     

Dystrophin expression in the mdx mouse restored by stem cell transplantation.

The development of cell or gene therapies for diseases involving cells that are widely distributed throughout the body has been severely hampered by the inability to achieve the disseminated delivery of cells or genes to the affected tissues or organ. Here we report the results of bone marrow transplantation studies in the mdx mouse, an animal model of Duchenne's muscular dystrophy, which indicate that the intravenous injection of either normal haematopoietic stem cells or a novel population of muscle-derived stem cells into irradiated animals results in the reconstitution of the haematopoietic compartment of the transplanted recipients, the incorporation of donor-derived nuclei into muscle, and the partial restoration of dystrophin expression in the affected muscle. These results suggest that the transplantation of different stem cell populations, using the procedures of bone marrow transplantation, might provide an unanticipated avenue for treating muscular dystrophy as well as other diseases where the systemic delivery of therapeutic cells to sites throughout the body is critical. Our studies also suggest that the inherent developmental potential of stem cells isolated from diverse tissues or organs may be more similar than previously anticipated.     

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.     

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.     

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.     

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.     

Advances in studies on hepatic stem cells

The question whether hepatic stem cells exist or not has been debated for several decades. Current researches confirm that there are hepatic stem cells in the liver. Oval cells, putative bipotential hepatic stem cells, are probably located within canals of Hering, portal tracts or branches of biliary trees. Bone marrow is a potential source of oval cells, indicating that there exists a close relationship between liver and hematopoiesis in adulthood. Hepatic stem cells are able to proliferate in vitro and can be induced to differentiate into hepatocytes. This will provide a promising approach of cell transplantation, tissue engineering and gene therapy for liver diseases. In this review, the evidence of their presence, origin, identification, proliferation in vitro, differentiation by induction, application prospects of hepatic stem cells and future directions for the field are discussed.     

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.     

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.     

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.     

Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts

The mammalian heart has a very limited regenerative capacity and, hence, heals by scar formation. Recent reports suggest that haematopoietic stem cells can transdifferentiate into unexpected phenotypes such as skeletal muscle, hepatocytes, epithelial cells, neurons, endothelial cells and cardiomyocytes, in response to tissue injury or placement in a new environment. Furthermore, transplanted human hearts contain myocytes derived from extra-cardiac progenitor cells, which may have originated from bone marrow. Although most studies suggest that transdifferentiation is extremely rare under physiological conditions, extensive regeneration of myocardial infarcts was reported recently after direct stem cell injection, prompting several clinical trials. Here, we used both cardiomyocyte-restricted and ubiquitously expressed reporter transgenes to track the fate of haematopoietic stem cells after 145 transplants into normal and injured adult mouse hearts. No transdifferentiation into cardiomyocytes was detectable when using these genetic techniques to follow cell fate, and stem-cell-engrafted hearts showed no overt increase in cardiomyocytes compared to sham-engrafted hearts. These results indicate that haematopoietic stem cells do not readily acquire a cardiac phenotype, and raise a cautionary note for clinical studies of infarct repair.