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.     

Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion

Recent studies have demonstrated that transplanted bone marrow cells can turn into unexpected lineages including myocytes, hepatocytes, neurons and many others. A potential problem, however, is that reports discussing such 'transdifferentiation' in vivo tend to conclude donor origin of transdifferentiated cells on the basis of the existence of donor-specific genes such as Y-chromosome markers. Here we demonstrate that mouse bone marrow cells can fuse spontaneously with embryonic stem cells in culture in vitro that contains interleukin-3. Moreover, spontaneously fused bone marrow cells can subsequently adopt the phenotype of the recipient cells, which, without detailed genetic analysis, might be interpreted as 'dedifferentiation' or transdifferentiation.     

GM-CSF and TNF-alpha cooperate in the generation of dendritic Langerhans cells.

Dendritic cells comprise a system of highly efficient antigen-presenting cells which initiate immune responses such as the sensitization of T cells restricted by major histocompatibility complex molecules, the rejection of organ transplants and the formation of T-cell-dependent antibodies. Dendritic cells are found in many non-lymphoid tissues, such as skin (Langerhans cells) and mucosa, and they migrate after antigen capture through the afferent lymph or the bloodstream to lymphoid organs, where they efficiently present antigen to T cells. Dendritic cells are difficult to isolate and, although they originate from bone marrow their site of maturation and the conditions that direct their growth and differentiation are still poorly characterized. Granulocyte macrophage-colony stimulating factor (GM-CSF) favours the outgrowth of dendritic cells from mouse peripheral blood. Here we extend this finding to man and demonstrate that cooperation between GM-CSF and tumour necrosis factor-alpha (TNF-alpha) is crucial for the generation of human dendritic/Langerhans cells from CD34+ haematopoietic progenitors. The availability of large numbers of these cells should now facilitate the understanding of their role in immunological regulation and disorder.     

T-cell specificity for H-2 and Ir gene phenotype correlates with the phenotype of thymic antigen-presenting cells

Experiments with chimaeric animals have demonstrated that the H-2 restriction specificity and immune response (Ir) gene phenotype of the T cell is acquired during development in the thymus. The mechanism by which this process occurs is unclear. One level of obligate expression of H-2 and Ir gene products is on the surface of antigen-presenting cells (APCs) which come from bone marrow precursors. We have now examined the turnover of APCs in the thymuses of F1 leads to parent (P) radiation-induced bone marrow chimaeras and found that APCs of donor phenotype appear at about 2 months after reconstitution. If the peripheral T-cell population is depleted after this time, new T cells emerging from the parental thymus (containing F1 APCs) behaving like F1 T cells, suggesting that cells from the bone marrow can influence thymic-directed T-cell differentiation. The thymic APC is an attractive condidate to play such a part in the development of the T-cell repertoire.     

Origin of Thy-1+ dendritic epidermal cells of adult mice from fetal thymic precursors

The skin of mice contains dendritic epidermal cells carrying the Thy-1 antigen (Thy-1+ dEC) which express antigen receptors composed of the T-cell antigen receptor (TCR) gamma- and delta-chains. Although the role of the thymus in the generation of most T cells is well established, the involvement of the thymus in the generation of Thy-1+ dEC is not clear. Because bone marrow cells can give rise in Thy-1+ dEC in chimaeric mice and Thy-1+ dEC are detected in the skin of athymic nude nice, it has been proposed that Thy-1+ dEC arise continuously from bone marrow precursors by a thymus-independent mechanism. But it has recently been determined that Thy-1+ dEC in nude mice do not express TCR at the cell surface, and that the gamma- and delta-chain genes are in germ-line configuration, leaving the role of the thymus in the generation of Thy-1+ dEC uncertain. Most Thy-1+ dEC in all normal mouse strains examined express TCR containing the V gamma 3 gene product. This V gene segment is expressed on the first wave of TCR-expressing cells to emerge during fetal development, and in adult mice is detectable only on cells in the epidermis. In addition to use of this 'fetal' V gamma segment, other features of the Thy-1+ dEC TCR genes, including absence or minimal presence of nongerm-line-encoded nucleotides at the junctions and use of a single D element in the rearranged delta-chain gene are typical of rearrangements found in fetal, and not adult, thymus. Here we demonstrate that precursors that are present only in the fetal thymus give rise to Thy-1+ dEC in the skin of adult mice.     

Regulation of T-cell receptor gamma-chain RNA expression in murine Thy-1+ dendritic epidermal cells

The epidermis of normal mice contains two distinct populations of dendritic cells derived from the bone marrow, Ia+ Langerhans cells and Ia- cells that express the Thy-1 alloantigen. The Thy-1-bearing dendritic epidermal cells (Thy-1+ dEC) have a surface phenotype similar to that of very early T-lineage cells, produce IL-2-like growth factors and exhibit cytotoxicity which is not restricted by the major histocompatibility complex (MHC). The relationship of Thy-1+ dEC to the T-cell lineage is unclear. Most T lymphocytes bear a receptor for antigen composed of an alpha chain and a beta chain associated with a nonpolymorphic complex termed CD3 (T3). A minor population carries a receptor in which CD3 is associated with a gamma/delta complex. We have analysed clones of Thy-1+ dEC for rearrangement and expression of the genes for the alpha-, beta- and gamma-chains of the T-cell receptor (TCR). They do not express alpha or beta but do carry a gamma/delta complex. Activation of the cells with Con A is associated with a rapid decrease in the steady-state level of gamma-chain RNA. Because Thy-1+ dEC resemble early stage T lymphocytes, down-regulation of TCR expression may reflect a necessary event during T cell differentiation.     

Monoclonal antibodies to promote marrow engraftment and tissue graft tolerance

Allogeneic reactions are the major limitation to organ transplantation. These are manifested as rejection of the grafted tissue, and also, in the case of bone marrow transplantation (BMT), graft-versus-host disease (GVHD). Recent methods of avoiding GVHD, by depleting T cells from donor marrow, have led to an increased incidence of marrow graft rejection. Current recipient conditioning protocols involving drugs or irradiation cannot safely be increased, so alternatives must be found. Monoclonal antibodies can be used to control immune responses in vivo, and would be useful in this context if we could define and deplete the cells responsible for marrow rejection. We show here that elimination of residual L3T4+ and Lyt-2+ cells from mice receiving fully mismatched bone marrow abrogates rejection and promotes tolerance to donor-type skin grafts, even in sub-lethally irradiated recipients.     

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.     

造血重建小鼠骨髓中成纤维细胞的起源

选择Y染色体特异的性别决定基因(Sry)作为新的细胞遗传标志,采用PCR技术对小鼠骨髓细胞重建造血的性能和造血重建小鼠骨髓中成纤维细胞的起源进行研究.将正常雄鼠骨髓细胞输注给经致死剂量射线照射的受体雌性小鼠,PCR技术检测结果表明,在活存小鼠的骨髓、脾脏、胸腺和淋巴结中均具有供体起源的细胞.而正常雄鼠或5-氟尿嘧啶处理的雄鼠骨髓细胞输注给受体雌性小鼠后,造血重建小鼠骨髓中的成纤维细胞则为受体起源.由此可见,小鼠骨髓中的成纤维细胞与造血细胞具有不同的起源.     

Variable expression of Ia antigens on the vascular endothelium of mouse skin allografts

Ia antigens are membrane-bound glycoproteins that play a part in antigen recognition and subsequent cell-cell interactions in the immune response. In the mouse they are coded for by the I region of the major histocompatibility complex H-2 and have been demonstrated on B lymphocytes, monocytes, activated T cells, macrophages and dendritic cells, including Langerhans cells. Ia-like antigens have also been detected on the vascular endothelium in man and on epidermal keratinocytes in rats but expression on the latter cells was induced by a graft-versus-host reaction or by contact hypersensitivity. In the mouse, previous studies have suggested that Ia antigens in skin are restricted to epidermal Langerhans cells and it was thought that these were the targets for Ia-dependent rejection of skin allografts. The results presented here show that Ia antigens in mouse allografts are also present on the vascular endothelium but their expression is variable and dependent on the immunological status of the recipient. These findings suggest that vascular endothelial cells can act as targets in Ia-incompatible skin allograft rejection.     

Clonal nature of mast-cell clusters formed in W/Wv mice after bone marrow transplantation.

We have recently found that the number of mast cells in the skin of adult W/Wv mice is less than 1% of that observed in congeneic +/+ mice, and that no mast cells are detected in other tissues of W/Wv mice. After the transplantation of bone marrow cells from congeneic +/+ mice, the number of mast cells in the skin, stomach, caecum and mesentery of the W/Wv mice increased to levels similar to those of the +/+ mice. Study of the mast-cell number in the W/Wv mice at various times after transplantation suggested to use that mast cells might develop in groups, particularly in the skin and mesentery. In this report, we have attempted to elucidate the possible clonal origin of such mast-cell clusters from a single precursor cell, using giant granules of beige (C57BL/6-bgJ/bgJ, Chediak-Higashi syndrome) mice as a marker to identify the origin of the mast cells (Fig. 1). We found that when WB-W/+xC57BL/6-Wv (WBB6F1)-W/Wv mice were injected with a mixture of bone marrow cells from beige C57BL/6 mice and normal C57BL/6 mice, more than 95% of mast-cell clusters consisted of either beige-type cells alone or normal-type cells alone. We conclude, therefore, that the cluster of mast cells originated from a single precursor cell.     

Combination of<Emphasis Type="Italic">CTLA4-FasL</Emphasis> gene transfer and allogeneic bone marrow transplantation led to durable macrochimerism and donor-specific tolerance in mouse model

Mixed hemopoietic chimerism is capable of inducing donor specific tolerance, thus eliminating the chronic immunosuppressive therapy following organ transplantation. As yet no safe and effective tolerance protocol is available for clinical implementation. Here we describe an alternative nonmyeloablative based strategy of using a single injection of recombination adenovirus vector encoding CTLA4-FasL fusing gene and donor bone marrow cells to promote durable mixed macrochimerism （〉20% on 140 d）. Chimeras exhibited robust donor-specific tolerance, as evidenced by acceptance of fully allogeneic skin grafts （the mean survival time （MST）〉200 d） and rejection of third-party skin grafts in a normal manner （MST〈10 d）. In this model, the frequencies of helper T lymphocyte precursor （HTLp） and cytotoxic T lymphocyte precursor （CTLp） were greatly reduced on day 14 after transplantation, suggesting that CTLA4-FasL led to rapid systemic peripheral tolerance to facilitate the bone marrow engraftment, while both HTLp and CTLp remained at low level only in recipient mice with mixed chimerism on day 140 after transplantation, demonstrating that long-term skin grafts tolerance was associated with stable mixed chimerism, and central deletion of donor specific T cell may be the main mechanism for tolerance maintenance.     

Advances in tissue engineering

Tissue engineering is a newly developed specialty involved in the construction of tissues and organs either in vitro or in vivo. Tremendous progress has been achieved over the past decade in tisse construction as well as in other related areas, such as bone marrow stromal cells, embryonic stem cells and tissue progenitor cells. In our laboratory, tissues of full-thickness skin, bone, cartilage and tendon have been successfully engineered, and the engineered tissues have repaired full-thickness skin wound, cranial bone defects, articular cartilage defects and tendon defects in animals. In basic research areas, bone marrow stromal cells have been induced and transformed into osteoblasts and chondrocytes in vitro. Mouse embryo stem cell lines we established have differentiated into neuron precursor, cardiac muscle cells and epithelial cells. Genetic modifications of seed cells for promoting cell proliferation, delaying cell aging and inducing immune tolerance have also been investigated.     

I-J epitopes are adaptively acquired by T cells differentiated in the chimaeric condition

I-J has been defined as a locus mapped in the murine major histocompatibility complex (MHC) which encodes serological markers found primarily on the surface of suppressor T cells (TS) and soluble suppressor factors (TSF). Recent studies have, however, revealed that there is no such specialized locus within the MHC at the DNA level. As the existence of I-J determinants at the protein level on functional T cells, T-cell clones and hybridomas has been confirmed by several serological and biochemical studies, this contradiction has raised serious arguments in the immunological community concerning the nature, origin and expression of I-J determinants. We have raised a number of monoclonal antibodies against the polymorphic structure of I-J molecules, and have studied the expression of I-J epitopes on T cells derived from irradiated bone marrow chimaeras in which stem cells of different genotype differentiated into T cells under the foreign host MHC environment. The results, presented here, indicate that I-J epitopes are not primarily determined by the MHC genes of the stem cells themselves, but are adaptively acquired by T cells differentiated in the chimaeric condition according to the environmental MHC phenotype. Thus, the serologically detectable I-J epitopes are found to be associated with inducible T-cell receptors recognizing self class II MHC antigens.     

A minor transplantation antigen detected by MHC-restricted cytotoxic T lymphocytes during graft-versus-host disease

Transplantation of bone marrow can give rise to graft-versus-host disease when donor T lymphocytes, mismatched with the host for major histocompatability (MHC) antigens, become sensitized and attack host tissues. However, graft-versus-host disease can also arise between donor and host with compatible MHC antigens but mismatched for a minor histocompatability antigen. We report here on the occurrence of severe acute graft-versus-host disease in a male patient with acute myeloid leukaemia who had received bone marrow matched for MHC (HLA) antigens from his sister. Strong cytotoxicity of the posttransplantation (that is, donor) lymphocytes against the patient's pretransplantation lymphocytes was found. Thus, the transplanted lymphocytes differed in a non-HLA antigen from the patient. The possible role of this strong cytotoxic minor histocompatability antigen in the development of graft-versus-host disease in man is being evaluated. Furthermore, with the use of cytotoxic T-cell lines, derived from the patient's 6 day effector cells, we are now able to type for it before grafting.     

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.     

Bone marrow cells regenerate infarcted myocardium

Myocardial infarction leads to loss of tissue and impairment of cardiac performance. The remaining myocytes are unable to reconstitute the necrotic tissue, and the post-infarcted heart deteriorates with time. Injury to a target organ is sensed by distant stem cells, which migrate to the site of damage and undergo alternate stem cell differentiation; these events promote structural and functional repair. This high degree of stem cell plasticity prompted us to test whether dead myocardium could be restored by transplanting bone marrow cells in infarcted mice. We sorted lineage-negative (Lin-) bone marrow cells from transgenic mice expressing enhanced green fluorescent protein by fluorescence-activated cell sorting on the basis of c-kit expression. Shortly after coronary ligation, Lin- c-kitPOS cells were injected in the contracting wall bordering the infarct. Here we report that newly formed myocardium occupied 68% of the infarcted portion of the ventricle 9 days after transplanting the bone marrow cells. The developing tissue comprised proliferating myocytes and vascular structures. Our studies indicate that locally delivered bone marrow cells can generate de novo myocardium, ameliorating the outcome of coronary artery disease.     

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.     

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.     

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.