Interleukin-3 promotes proliferation and differentiation of human hematopoietic stem cells but reduces their repopulation potential in NOD/SCID mice

In the present study we explored systematically the influence of human interleukin-3 (IL-3) on the cord blood (CB) cell-derived production of human hematopoietic cells in the bone marrow, blood, and spleen of chimeric nonobese/severe combined immunodeficient mice ((NOD/SCID) mice. CB mononuclear cells and MACS-enriched CB CD34(+) cells were injected into irradiated NOD/SCID mice. The mice were additionally transplanted with a stably transfected rat fibroblast cell line expressing the human IL-3 gene (Rat-IL-3) constitutively, or with the nontransfected rat fibroblast cell line as a control (Rat-1). Rat-IL-3 mice displayed a higher engraftment of human hematopoietic cells in bone marrow, spleen, and peripheral blood compared with mice with Rat-1 cotransplantation. When we transplanted their total bone marrow cell population into secondary mice, surprisingly, mice transplanted with bone marrow cells from Rat-1 mice displayed a higher proportion of human hematopoietic cells compared with Rat-IL-3 mice. As expected, bone marrow cultures (BMCs) from Rat-IL-3 mice contained a higher proportion of human cells than Rat-1 bone marrow cells. However, when BMCs were passaged to new flasks, we observed a higher proportion of human cells in BMCs from Rat-1 mice compared with BMCs from Rat-IL-3 mice. IL-3 promotes the proliferation and differentiation of hematopoietic stem cells in chimeric bone marrow. In addition, IL-3 may play a role in the depletion of hematopoietic stem cells in chimeric bone marrow. In the absence of IL-3, the hematopoietic stem cells may remain in a quiescent state and proliferation can be induced by stimuli, including secondary transplantation or cell passage.     

Choroidal neovascularization is provided by bone marrow cells

Choroidal neovascularization (CNV) is a known cause of age-related macular degeneration (ARMD). Moreover, the most common cause of blindness in the elderly in advanced countries is ARMD with CNV. It has recently been shown that bone marrow cells (BMCs) can differentiate into various cell lineages in vitro and in vivo. Adults maintain a reservoir of hematopoietic stem cells included in BMCs that can enter the circulation to reach various organs in need of regeneration. It has recently been reported that endothelial progenitor cells (EPCs) included in BMCs are associated with neovascularization. We examine the role of BMCs in CNV using a model of CNV in adult mice. Using methods consisting of fractionated irradiation (6.0 Gy x 2) followed by bone marrow transplantation (BMT), adult mice were engrafted with whole BMCs isolated from transgenic mice expressing enhanced green fluorescent protein (EGFP). Three months after BMT, we confirmed that the hematopoietic cells in the recipients had been completely replaced with donor cells. We then carried out laser photocoagulation to induce CNV in chimeric mice (donor cells >95%). Two weeks after the laser photocoagulation, by which time CNV had occurred, immunohistochemical examination was carried out. The vascular wall cells of the CNV expressed both EGFP and CD31. These findings indicate that newly developed blood vessels in the CNV are derived from the BMCs and suggest that the inhibition of EPC mobilization from the bone marrow to the eyes could be a new approach to the fundamental treatment of CNV in ARMD.     

Lack of correlation between an assay used to determine early marrow allograft rejection and long-term chimerism after murine allogeneic bone marrow transplantation: effects of marrow dose.

The acute rejection of bone marrow (BM) allografts by host effectors can occur within a short period after BM transplantation (BMT) in lethally irradiated mice. Common assays used to ascertain engraftment/resistance involve measuring the growth of granulocyte/monocyte progenitors (colony-forming unit-granulocyte-macrophage) in vitro or splenocyte proliferation assessed by radioisotope incorporation in vivo 5 to 8 days after BMT. However, the correlation of the long-term outcome of BMT with the kinetics of recovery by using the dose of allogeneic BM cells (BMCs) that leads to early rejection as determined by the in vitro assessment has not been extensively studied. Thus, to investigate whether the early rejection of donor BMCs is an indication of a long-term engraftment failure, C57BL/6 (H2b) mice were lethally irradiated and transplanted with various doses of BALB/c (H2d) BMCs. The short-term engraftment of donor precursors (colony-forming unit-granulocyte-macrophage), the kinetics of hematopoietic cell recovery, the extent of donor chimerism, and the proportion of the recipients with long-term survival were determined. The results show that the kinetics and extent of hematopoietic cell recovery were significantly delayed in mice receiving limiting doses of BMCs that were rejected or severely resisted at day 8 after BMT. However, a proportion of these mice survived up to 98 days after BMT with mixed chimerism or donor chimerism. This study demonstrates that early rejection of BM precursors, as assessed by measurement of myeloid progenitors in the spleen after BMT, does not always correlate with the long-term outcome of the marrow allograft and that significant variability is inherent in the extent of chimerism when threshold amounts of BMCs are used.     

Long-term (>1 year) analyses of chimerism and tolerance in mixed allogeneic chimeric mice using normal mouse combinations

We examined the induction of tolerance using pancreas allografts over the long term (>1 year) in mice for the human application of mixed allogeneic bone marrow transplantation (BMT). T cell-depleted BM cells (BMCs) of C57BL/6 (B6) and C3H/He (C3H) mice were transplanted at various ratios into lethally irradiated B6 mice. The percentages of C3H cells in the chimeric mice gradually decreased, finally declining to only a small percentage, except when the ratio of donor to recipient BMCs was 100:1. However, despite the marked decreases in C3H-type cells, all the pancreas allografts of C3H mice were accepted when more than 1% C3H cells were detected in the peripheral blood. To examine the relationships between percentages of transplanted donor cells and acceptance of pancreas allografts, various percentages of donor and recipient BMCs (5% to 30%) were transplanted. It was found that more than 10% donor cells were necessary for the pancreas allografts to be accepted. In vitro assays for mixed lymphocyte reaction and generation of cytotoxic T-lymphocytes revealed that spleen cells in chimeric mice accepting pancreas allografts are tolerant to both host-type and donor-type major histocompatibility complex (MHC) determinants, but show a vigorous responsiveness to third-party MHC determinants. Since donor-type hemopoietic stem cells (HSCs) were detected in the BM and the liver of the chimeric mice, donor-derived HSCs and donor-derived hematolymphoid cells are responsible for the induction of tolerance. It should be noted that the percentage of donor-type HSCs is higher in the liver (6.2%) than in the BM (0.9%).     

Bone marrow transplantation therapy using resistant donors for retrovirus-induced leukaemia in mice.

Infection with Rauscher leukaemia virus (RLV) causes erythroleukaemia, anaemia, viraemia, and splenomegaly, features which develop very quickly in mice of strains susceptible to RLV but more slowly or not at all in strains resistant to RLV. We compared the effects of bone marrow transplantation (BMT) of donor cells obtained from a mouse strain resistant to the virus with the effects of BMT of donor cells obtained from strains susceptible to the virus, in recipient virus-susceptible mice which had previously been inoculated with RLV. Our results indicated that bone marrow transplantation from a marrow-resistant donor, but not from a susceptible donor, delayed the expression of leukaemia and delayed death, although such treatment did not appear to inhibit viral infection in susceptible recipients.     

Crucial role of donor-derived stromal cells in successful treatment for intractable autoimmune diseases in mrl/lpr mice by bmt via portal vein

We have recently established a new bone marrow transplantation (BMT) method for the treatment of intractable autoimmune diseases in MRL/lpr mice; the method consists of fractionated irradiation (5.5 Gy x 2), followed by BMT of whole bone marrow cells (BMCs) from allogeneic C57BL/6 mice via the portal vein (abbreviated as 5.5 Gy x 2 + PV). In the present study, we investigate the mechanisms underlying the early engraftment of donor-derived cells in MRL/lpr mice by this method. In the mice treated with this method, the number of donor-derived cells possessing the mature lineage (Lin) markers rapidly increased in the BM, spleen, and liver; almost 100% were donor-derived cells by 14 days after the treatment. The number of donor-derived hemopoietic progenitor cells (defined as c-kit(+)/Lin(-) cells) increased in the BMCs, hepatic mononuclear cells, and especially spleen cells by 14 days after the treatment. Simultaneously, hemopoietic foci adjoining donor-derived stromal cells were observed in the liver when injected via the PV, but not via the peripheral vein (i.v.). When adherent cell-depleted BMCs were injected via the PV, recipients showed a marked reduction in the survival rate. However, when mice were transplanted with adherent cell-depleted BMCs with cultured stromal cells, all the recipients survived. These findings suggest that not only donor hematopoietic stem cells (HSCs) but also donor stromal cells administered via the PV were trapped in the liver, resulting in the early engraftment of donor HSCs in cooperation with donor-derived stromal cells. This new strategy to facilitate the early recovery of hemopoiesis would therefore be of great advantage in human application.     

A new method for tolerance induction: busulfan administration followed by intravenous injection of neuraminidase-treated donor bone marrow

The portal venous (p.v.) administration of foreign cells induces donor-specific tolerance. Recently, we have demonstrated that the p.v. administration of donor cells elicits donor-specific tolerance across major histocompatibility complex barriers. In the present study, utilizing the intrahepatic tolerance-inducing system, we have established a new method for organ transplantation using both busulfan ([Bu] to provide a sufficient "space" for the donor hematopoietic cells to expand in the recipient) and neuraminidase ([Neu] to enhance the trapping of i.v.-injected cells in the liver). Radiolabeled bone marrow cells (BMCs) were found to exclusively accumulate in the livers of the recipients as a result of the Neu treatment. Furthermore, hematopoietic progenitors (forming hematopoietic foci) in the accumulated BMCs were retained in the recipient livers for at least 18 days. C57BL/6 (B6) mice that had been transplanted with skins of BALB/c mice immediately after the injection of BALB/c BMCs showed a 90% skin graft survival rate over 400 days as a result of using the combination of injecting 50 mg/kg Bu into the B6 mice and treatment of the BALB/c BMCs with 0.25 U/ml Neu (50 Bu + 0.25 Neu). However, the survival rate significantly decreased when either the Bu or Neu treatment was omitted. In tolerant recipients, microchimerism was observed in the various hematolymphoid organs. T cells collected from the tolerant recipients suppressed proliferative responses to the donor-alloantigens but enhanced the production of Th2 and Th3 cytokines. These findings suggest that the enhanced retention of donor BMCs in the recipient livers as a result of the Bu and Neu treatments efficiently induces tolerance induction. Therefore, this "single-day protocol" would be of great advantage for human organ transplantation.     

Intra-bone marrow injection of donor bone marrow cells suspended in collagen gel retains injected cells in bone marrow, resulting in rapid hemopoietic recovery in mice

We have recently developed an innovative bone marrow transplantation (BMT) method, intra-bone marrow (IBM)-BMT, in which donor bone marrow cells (BMCs) are injected directly into the recipient bone marrow (BM), resulting in the rapid recovery of donor hemopoiesis and permitting a reduction in radiation doses as a pretreatment for BMT. However, even with this IBM injection, some of the injected BMCs were found to enter into circulation. Therefore, we attempted to modify the method to allow the efficient retention of injected BMCs in the donor BM. The BMCs of enhanced green fluorescent protein transgenic mice (C57BL/6 background) were suspended in collagen gel (CG) or phosphate-buffered saline (PBS), and these cells were then injected into the BM of irradiated C57BL/6 mice. The numbers of retained donor cells in the injected BM, the day 12 colony-forming units of spleen (CFU-S) counts, and the reconstitution of donor cells after IBM-BMT were compared between the CG and PBS groups. The number of transplanted cells detected in the injected BM in the CG group was significantly higher than that in the PBS group. We next carried out CFU-S assays. The spleens of mice in the CG group showed heavier spleen weight and considerably higher CFU-S counts than in the PBS group. Excellent reconstitution of donor hemopoietic cells in the CG group was observed in the long term (>100 days). These results suggest that the IBM injection of BMCs suspended in CG is superior to the injection of BMCs suspended in PBS.     

Enhancement of allogeneic hematopoietic stem cell engraftment and prevention of GVHD by intra-bone marrow bone marrow transplantation plus donor lymphocyte infusion

We examined the effect of intra-bone marrow (IBM)-bone marrow transplantation (BMT) in conjunction with donor lymphocyte infusion (DLI) on the engraftment of allogeneic bone marrow cells (BMCs) in mice. Recipients that had received 6 Gy of radiation completely rejected donor BMCs, even when IBM-BMT was carried out. However, when BMCs were IBM injected and donor peripheral blood mononuclear cells (PBMNCs) were simultaneously injected intravenously (DLI), donor cell engraftment was observed 7 days after BMT and complete donor chimerism continued thereafter. It is of interest that the cells of recipient origin did not recover, and that the hematolymphoid cells, including progenitor cells (Lin-/c-kit+ cells) in the recipients, were fully reconstituted with cells of donor origin. The cells in the PBMNCs responsible for the donor BMC engraftment were CD8+. Recipients that had received 6 Gy of radiation, IBM-BMT, and DLI showed only a slight loss of body weight, due to radiation side effects, and had no macroscopic or microscopic symptoms of graft-versus-host disease. These findings suggest that IBM-BMT in conjunction with DLI will be a valuable strategy for allogeneic BMT in humans.     

Cognate interaction plays a key role in the surveillance of autoreactive B cells in induced mixed bone marrow chimerism in BXSB lupus mice

The effects of bone marrow transplantation (BMT) as a treatment for and/or preventive measure against autoimmune diseases in mice were investigated extensively. The reconstitution of the hematopoietic system with a mixture of autologous and heterologous bone marrow cells was reported to suppress the development of autoimmune diseases. However, the pathological mechanism through which mixed chimerism results in the suppression of disease development is still unknown. We have previously reported that the induction of fully major histocompatibility complex (MHC)-mismatched allogeneic mixed chimerism can prevent the disease development in BXSB mice. Interestingly, serum anti-dsDNA IgM antibody (anti-DNA IgM) levels were not significantly decreased in these chimeric mice, though other symptoms of autoimmune disease were ameliorated. In this study, we showed that self-reactive anti-DNA IgM production was mainly attributable to genetically normal B cells from the donor rather than genetically deficient B cells from the host. Host-type B cells responded normally to foreign antigens and produced the appropriate antibodies. BMT from fully MHC-matched or haplo-identical donors could suppress the production of anti-DNA antibodies. Our present study suggests the existence of a surveillance system dependent on the recognition of MHC molecules on B cells.     

Facilitation of hematopoietic recovery by bone grafts with intra-bone marrow-bone marrow transplantation

We have previously shown that T cells can acquire donor-type major histocompatibility complex (MHC) restriction and can interact with both donor-type antigen-presenting cells (APCs) and B cells, when adult donor bones are co-grafted with intravenous (IV) injection of bone marrow cells (BMCs) in order to supply donor bone marrow (BM) stromal cells. We have also found that the direct injection of donor BMCs into recipient BM (intra-bone marrow-bone marrow transplantation: IBM-BMT) produces more rapid reconstitution (including T-cell functions) and higher survival rates than IV injection (IV-BMT) even in chimerism-resistant combinations. In the present study, we show that the co-administration of bones from suckling (2-3 days old) donor mice is also effective in the IBM-BMT system. Even when a relatively low number of BMCs were injected into adult (more than 15 weeks old) mice, complete reconstitution was achieved in the mice that had received IBM-BMT+bone grafts, but not in the mice that had received IBM-BMT alone. Most BM and splenic adherent cells obtained from the recipients that had received IBM-BMT+bone grafts were reconstituted by donor-type cells. Both T-cell proliferation and plaque-forming cell assays indicated that the T cells of such mice showed donor-type MHC restriction. Moreover, the analyses of thymic sections using confocal microscopy revealed that donor BM stromal cells had migrated into the thymus. Thus, the co-administration of donor bones has great advantages for allogeneic BMT in adult mice.     

Variants of autophagy-related gene 5 are associated with neuromyelitis optica in the Southern Han Chinese population

Using animal models for autoimmune diseases, we have previously shown that allogeneic bone marrow transplantation (allo BMT) can be used to treat autoimmune diseases. Using cynomolgus monkeys, we have recently developed new BMT methods for the treatment of autoimmune diseases. The methods include the perfusion method (PM) for the collection of bone marrow cells (BMCs), and intra-bone marrow (IBM)-BMT for the direct injection of collected whole BMCs into the bone marrow cavity. The PM, in comparison with the conventional aspiration method, can minimize the contamination of BMCs with T cells from the peripheral blood. Therefore, without removing T cells, no graft-versus-host disease (GvHD) develops in the case of the PM. Since BMCs collected by the PM contain not only hemopoietic stem cells (HSCs) but also mesenchymal stem cells (MSCs), the injection of both cells directly into the bone marrow cavity (IBM–BMT) facilitates the engraftment of donor hemopoietic cells. In organ allografts with IBM–BMT, no graft failure occurs even if the radiation dose is reduced. In addition, IBM–BMT is applicable to regeneration therapy and various age-associated diseases such as osteoporosis, since it can efficiently recruit donor-derived normal MSCs.

We have also found that IBM–BMT in conjunction with donor lymphocyte infusion can prevent GvHD, but suppress tumor growth. We believe that this strategy heralds a revolution in the field of transplantation (BMT and organ allografts) and regeneration therapy.     

Analyses of very early hemopoietic regeneration after bone marrow transplantation: comparison of intravenous and intrabone marrow routes

In bone marrow transplantation (BMT), bone marrow cells (BMCs) have traditionally been injected intravenously. However, remarkable advantages of BMT via the intra-bone-marrow (IBM) route (IBM-BMT) over the intravenous route (IV-BMT) have been recently documented by several laboratories. To clarify the mechanisms underlying these advantages, we analyzed the kinetics of hemopoietic regeneration after IBM-BMT or IV-BMT in normal strains of mice. At the site of the direct injection of BMCs, significantly higher numbers of donor-derived cells in total and of c-kit(+) cells were observed at 2 through 6 days after IBM-BMT. In parallel, significantly higher numbers of colony-forming units in spleen were obtained from the site of BMC injection. During this early period, higher accumulations of both hemopoietic cells and stromal cells were observed at the site of BMC injection by the IBM-BMT route. The production of chemotactic factors, which can promote the migration of a BM stromal cell line, was observed in BMCs obtained from irradiated mice as early as 4 hours after irradiation, and the production lasted for at least 4 days. In contrast, sera collected from the irradiated mice showed no chemotactic activity, indicating that donor BM stromal cells that entered systemic circulation cannot home effectively into recipient bone cavity. These results strongly suggest that the concomitant regeneration of microenvironmental and hemopoietic compartments in the marrow (direct interaction between them at the site of injection) contributes to the advantages of IBM-BMT over IV-BMT. Disclosure of potential conflicts of interest is found at the end of this article.     

人源化NOD/SCID小鼠免疫细胞的动态变化与鉴定

目的： 比较脐血干细胞与单个核细胞移植NOD/SCID鼠所建立的人源化SCID模型，分析人源化淋巴细胞重建。方法： 磁珠分选法分离脐血中CD34⁺细胞，淋巴细胞分层液分离脐血单个核细胞，分别经尾静脉输入NOD/SCID小鼠。每隔2周采血至10周，流式细胞术动态检测人源淋巴细胞CD45、CD19、CD3抗原。第10周处死小鼠收集外周血、骨髓、胸腺组织，RT-PCR检测模型鼠组织中人β₂M基因及RAG2基因。结果： 两种类型细胞移植均可重建人源免疫细胞，人源淋巴细胞表达水平均在第8周达高峰。骨髓中人源淋巴细胞表达水平明显高于外周血。RT-PCR在外周血与骨髓检测到人β₂M基因及RAG2基因标志。结论： CD34⁺细胞移植重建人源化NOD/SCID免疫系统模型效果要好于脐血单个核细胞。人源T淋巴细胞在模型鼠骨髓中分化成熟。     

Long-term immunologic tolerance induction in chimeric mice after bone marrow transplantation across major histocompatibility barriers: persistent or redeveloping immunologic responsiveness after prolonged survival

Employing allogeneic bone marrow chimeras [B6----C3H] (C3H/HeN mice lethally irradiated and then reconstituted with T cell-depleted C57BL/6J bone marrow), we investigated the kinetics of immunological reconstitution and the functional characteristics of the immune system at regular intervals following bone marrow transplantation (BMT). In chimeric mice, almost all spleen cells and peritoneal macrophages showed donor H-2 haplotype within 2 weeks after BMT. The differentiation and maturation of B lymphocytes and macrophages was largely complete by 2 weeks following BMT, whereas T cell functions such as Con A responsiveness and alloreactivity were not restored within 4 weeks. Newly developed T cells were found to be exclusively of donor origin but were tolerant of both donor type and host type major histocompatibility complex (MHC) determinants in assays of mixed lymphocyte reactions and capacity to generate cytotoxic T lymphocytes. Fully allogeneic chimeric mice survived up to one year after bone marrow transplantation without significant reactions or serious diseases. Neither graft-versus-host reaction (GVHR), host-versus-graft reaction (HVGR), nor the wasting diseases described. As compared to age-matched control mice, such fully allogeneic chimeras exhibited one year following BMT somewhat reduced but quite significant plaque forming cell responses to in vitro stimulation with a T-dependent antigen, SRBC. These findings suggest that BMT across MHC barriers may ultimately be useful in humans if all manifestations of GVHR are eliminated by completely removing all T cells from the bone marrow and if all hematopoietic resistance and all aspects of HVGR are eliminated by using sufficient immunosuppression and myeloablation.     

异基因骨髓细胞在致敏模型体内归巢示踪与植入分析

目的: 探讨异基因供者骨髓细胞在致敏模型体内的归巢示踪与植入分析。方法: 以异基因脾细胞输注方法建立致敏的BALB/c小鼠模型,同时取正常BABL/c小鼠作为非致敏模型。致敏或非致敏模型经8 Gy 照射后分别经尾静脉移植1×10⁷ C57BL/6小鼠骨髓细胞。用绿色荧光染料CFSE标记供者骨髓细胞,并分别在移植后不同时点(2 h、12 h及48 h),通过组织细胞悬液动态示踪供者细胞在致敏受者各组织的分布。移植后记录各组的生存情况,每周监测造血重建与骨髓恢复情况。予H-2D^b进行标记移植后受者骨髓细胞,检测供者嵌合百分比。结果: CFSE能标记供者骨髓细胞并用于体内示踪实验。动物体内归巢示踪实验表明,与非致敏组相比,异基因供者骨髓细胞在致敏受者体内的外周血、脾脏及股骨的分布均明显减少。植入分析结果发现,非致敏受者于移植后能长期存活,外周血及骨髓细胞均能迅速恢复;而致敏组中,小鼠均于移植后2周左右全部死亡,生存中位数为13 d,外周血及骨髓细胞均随时间推移呈进行性减少。嵌合分析显示移植后第7 d,非致敏受者与致敏受者的供者骨髓细胞百分比分别为(48.07±4.70)％和(0.77±0.11)％,两者差异显著(P<0.01)。结论: 异基因供者骨髓细胞在致敏受者体内脾脏及股骨等部位被清除,不能有效植入。     

Allogeneic bone marrow transplantation in models of experimental autoimmune encephalomyelitis: evidence for a graft-versus-autoimmunity effect.

Autologous hematopoietic stem cell transplantation (HSCT) is being explored in the treatment of severe multiple sclerosis (MS), and is based on the concept of "resetting" the immune system. The use of allogeneic HSCT may offer additional advantages, such as the replacement of the autoreactive immune compartment by healthy allogeneic cells and development of a graft-versus-autoimmunity (GVA) effect. However, in clinical practice, the genetic susceptibility to MS of allogeneic stem cell donors is generally unknown, and GVA may therefore be an important mechanism of action. Experimental autoimmune encephalomyelitis (EAE)-susceptible and -resistant mouse strains were used to determine the roles of genetic susceptibility, level of donor-chimerism, and alloreactivity in the therapeutic potential of syngeneic versus allogeneic bone marrow transplant (BMT) for EAE. After transplantation and EAE induction, animals were evaluated for clinical EAE and ex vivo myelin oligodendrocyte glycoprotein-specific proliferation. Early after BMT, both syngeneic and allogeneic chimeras were protected from EAE development. On the longer term, allogeneic but not syngeneic BMT conferred protection, but this required high-level donor-chimerism from EAE-resistant donors. Importantly, when EAE-susceptible donors were used, robust protection from EAE was obtained when active alloreactivity, induced by donor lymphocyte infusions, was provided. Our findings indicate the requirement of a sufficient level of donor-chimerism from a nonsusceptible donor in the therapeutic effect of allogeneic BMT. Importantly, the data indicate that, independently of genetic susceptibility, active alloreactivity is associated with a GVA effect, thereby providing new evidence to support the potential role of allogeneic BMT in the treatment of MS.     

Tolerance induction through simultaneous double bone marrow transplantation with two-signal blockade

Cotransplantation of donor bone marrow cells (BMCs) in allograft recipients is currently the most promising concept for clinical tolerance induction; however, it still has many difficulties in its successful performance due to the toxicity of the required host conditioning, the risk of engraftment failure, and the problem of graft-versus-host disease (GVHD), as well as the limited accessibility of donor bone marrow cells. Therefore, we performed the studies to determine whether BMCs from multi-donors are simultaneously engrafted and lead to induction of chimerism-based tolerance through the tolerogenic protocol of whom effectiveness we have shown in a previous study. Using a murine model, it was demonstrated that grafted BMCs from BALB/c (H-2(d)) and CBA mice (H-2(k)) establish mixed type and multi-lineage double chimerism and induce immunological donor-specific tolerance to fully MHC-mismatched skin allografts in host C57BL/6 mice (H-2(b)) receiving conditioning with Busulfan and treatment with the two-signal blockade comprised of anti-CD45RB and anti-CD154 monoclonal antibodies.     

同基因造血干细胞移植延长同种异体小鼠皮肤移植物存活时间的实验研究

为了研究同基因造血干细胞移植诱导器官移植免疫耐受的可行性。建立小鼠异基因皮肤移植模型,术后2周给予FK506腹腔注射,3周起行全身照射及同基因骨髓移植,观察记录小鼠和移植物存活情况,以流式细胞检测受体GFP嵌合表达,混合淋巴细胞反应、迟发型超敏反应检测诱导耐受的特异性和效能。实验组小鼠移植物存活时间达(29.14±4.92)d,显著长于对照组(P<0.05);GFP在BMT后4周、6周嵌合程度达到82%、91%;实验组MLR、DTH结果与对照组差异显著,提示诱导耐受具有高度特异性和高效性。同基因造血干细胞移植联合免疫抑制剂治疗可以有效诱导小鼠皮肤移植的免疫耐受。     

Stem cell transplantation for autoimmune diseases: what can we learn from experimental models?

Using animal models for autoimmune diseases, we have previously shown that allogeneic bone marrow transplantation (allo BMT) can be used to treat autoimmune diseases. Using cynomolgus monkeys, we have recently developed new BMT methods for the treatment of autoimmune diseases. The methods include the perfusion method (PM) for the collection of bone marrow cells (BMCs), and intra-bone marrow (IBM)-BMT for the direct injection of collected whole BMCs into the bone marrow cavity. The PM, in comparison with the conventional aspiration method, can minimize the contamination of BMCs with T cells from the peripheral blood. Therefore, without removing T cells, no graft-versus-host disease (GvHD) develops in the case of the PM. Since BMCs collected by the PM contain not only hemopoietic stem cells (HSCs) but also mesenchymal stem cells (MSCs), the injection of both cells directly into the bone marrow cavity (IBM-BMT) facilitates the engraftment of donor hemopoietic cells. In organ allografts with IBM-BMT, no graft failure occurs even if the radiation dose is reduced. In addition, IBM-BMT is applicable to regeneration therapy and various age-associated diseases such as osteoporosis, since it can efficiently recruit donor-derived normal MSCs. We have also found that IBM-BMT in conjunction with donor lymphocyte infusion can prevent GvHD, but suppress tumor growth. We believe that this strategy heralds a revolution in the field of transplantation (BMT and organ allografts) and regeneration therapy.