造血干细胞移植条件对小鼠造血重建的影响

通过同种基因型小鼠构建造血干细胞移植模型,将预处理的全骨髓单个核细胞或c-Kit⁺造血干细胞移植至致死剂量照射的受体小鼠体内,动态监测移植2～16周后受体小鼠体内供体来源细胞造血重建以及嵌合情况,以期揭示不同群体的供体细胞以及预处理等因素对小鼠造血干细胞移植后造血重建的影响。实验结果显示,移植后早期(2周)全骨髓单个核细胞组髓系比例要高于c-Kit⁺细胞移植组,但全骨髓移植组受体小鼠呈现出较大的移植后不良反应,出现脱毛、食欲不振以及体重减轻的症状。c-Kit⁺细胞移植组在淋系重建上要早于全骨髓移植组,供体细胞的嵌合植入也早于全骨髓移植组,但两组实验组最终均能完成造血重建过程。实验结果表明c-Kit⁺细胞移植组在移植后能够较快地实现供体细胞植入,进而开始造血重建,且c-Kit⁺细胞移植组的不良反应要低于全骨髓移植组。结果说明在整体造血重建效果上c-Kit⁺细胞移植组要优于全骨髓移植组。     

Molecular mechanisms underlying adhesion and migration of hematopoietic stem cells

Hematopoietic stem cell transplantation is the most powerful treatment modality for a large number of hematopoietic malignancies, including leukemia. Successful hematopoietic recovery after transplantation depends on homing of hematopoietic stem cells to the bone marrow and subsequent lodging of those cells in specific niches in the bone marrow. Migration of hematopoietic stem cells to the bone marrow is a highly regulated process that requires correct regulation of the expression and activity of various molecules including chemoattractants, selectins and integrins. This review will discuss recent studies that have extended our understanding of the molecular mechanisms underlying adhesion, migration and bone marrow homing of hematopoietic stem cells.     

Molecular mechanisms underlying adhesion and migration of hematopoietic stem cells

Hematopoietic stem cell transplantation is the most powerful treatment modality for a large number of hematopoietic malignancies, including leukemia. Successful hematopoietic recovery after transplantation depends on homing of hematopoietic stem cells to the bone marrow and subsequent lodging of those cells in specific niches in the bone marrow. Migration of hematopoietic stem cells to the bone marrow is a highly regulated process that requires correct regulation of the expression and activity of various molecules including chemoattractants, selectins and integrins. This review will discuss recent studies that have extended our understanding of the molecular mechanisms underlying adhesion, migration and bone marrow homing of hematopoietic stem cells.     

The regulation of hematopoiesis following bone marrow transplantation

Allogeneic bone marrow transplantation requires that donor stem cells home to the recipient bone marrow, proliferate and differentiate under normal physiologic regulatory mechanisms. Recent observations that T cell depletion of donor bone marrow leads to a greatly increased incidence of graft failure mandate a detailed understanding of the engraftment process. Post-transplant hematopoietic deficiencies appear to be related to several sources: decreased number of stem cells, activation of donor hematopoietic suppressor cells, rejection of donor stem cells by residual recipient lymphocytes and abnormal function of accessory cells that produce hematopoietic growth factors. A better understanding of the relative roles of these factors should lead to a better understanding of engraftment as well as graft failure and its prevention.     

Young donor hematopoietic stem cells revitalize aged or damaged bone marrow niche by transdifferentiating into functional niche cells

The bone marrow niche maintains hematopoietic stem cell (HSC) homeostasis and declines in function in the physiologically aging population and in patients with hematological malignancies. A fundamental question is now whether and how HSCs are able to renew or repair their niche. Here, we show that disabling HSCs based on disrupting autophagy accelerated niche aging in mice, whereas transplantation of young, but not aged or impaired, donor HSCs normalized niche cell populations and restored niche factors in host mice carrying an artificially harassed niche and in physiologically aged host mice, as well as in leukemia patients. Mechanistically, HSCs, identified using a donor lineage fluorescence-tracing system, transdifferentiate in an autophagy-dependent manner into functional niche cells in the host that include mesenchymal stromal cells and endothelial cells, previously regarded as “nonhematopoietic” sources. Our findings thus identify young donor HSCs as a primary parental source of the niche, thereby suggesting a clinical solution to revitalizing aged or damaged bone marrow hematopoietic niche.     

Biological aspects of limb transplantation: I. Migration of transplanted bone marrow cells into recipient

The transplanted limb contains bone marrow tissue. The hematopoietic cells contained in the bone of the graft normally differentiate after transplantation and can be released to the recipient. The cells migrate to the recipient bone marrow cavities and lymphoid organs. This causes the immune reaction between the donor and the recipient, which develops not only in the graft itself but also in the recipient immune organs where donor bone marrow cells home. The purpose of this study was to investigate the process of migration of the hematopoietic cells from the donor limb to the recipient bone marrow cavities and lymphoid tissues. The questions the authors asked were: what is the rate of release of bone marrow cells from the transplanted bone, where do the released bone marrow cells home in the recipient, how fast are donor bone marrow cells rejected by the recipient, and can some bone marrow cells homing in the recipient tissues survive and create a state of microchimerism. Experiments were performed on Brown Norway and Lewis inbred rat strains (n = 30). Limb donors received intravenous chromium-51-labeled bone marrow cells. Twenty-four hours later, the limb with homing labeled bone marrow cells was transplanted to an allogeneic or syngeneic recipient. The rate of radioactivity of bone marrow cells released from the graft and homing in recipient tissues was measured after another 24 hours. To eliminate factors adversely affecting homing such as the "crowding effect" and allogeneic elimination of bone marrow cells by natural killer cells, total body irradiation and antiasialo-GM1 antiserum were applied to recipients before limb transplantation. In rats surviving with the limb grafts for 7 and 30 days, homing of donor bone marrow cells was studied by specific labeling of donor cells and flow cytometry as well as by detecting donor male Y chromosome. The authors found that transplantation of the limb with bone marrow in its natural spatial relationship with stromal cells and blood perfusion brings about immediate but low-rate release of bone marrow cells and their migration to recipient bone marrow and lymphoid tissues. Large portions of allogeneic bone marrow cells are rapidly destroyed in the mechanism of allogeneic elimination by radioresistant but antiasialo-GM1-sensitive natural killer cells. Some transplanted bone marrow cells remain in the recipient's tissues and create a state of cellular and DNA microchimerism. A low number of physiologically released donor bone marrow cells do not seem to adversely affect the clinical outcome of limb grafting. Quite the opposite, a slight prolongation of the graft survival time was observed.     

Evaluation of in vitro cytotoxic T lymphocyte assays as a predictive test for the occureence of graft vs host disease

The potential value of in vitro cytotoxic T lymphocyte (CTL) assays for predicting the occurrence of graft vs host disease (GVHD) following allogeneic bone marrow transplantation was evaluated in 12 mouse donor-host combinations associated with various degrees of GVHD. These donor-host combinations were selected after evaluation of GVHD triggered by minor histocompatibility antigens (MiHA) in 24 allogeneic strain combinations derived from six strains of H-2 ^b mice. Recipients (n=475), previously submitted to total body irradiation (9.5 Gy), were transplanted with 10⁷ bone marrow cells along with 5 x 10⁷ spleen cells. While lethal GVHD was observed in half of the strain combinations, it was possible to select 12 donor-host combinations characterized by severe, mild, or absent GVHD. When levels of anti-host CTL activity were assessed following in vivo priming and in vitro boosting, strong CTL-mediated cytotoxicity was observed in all combinations wheteer they developed GVHD or not. CTL frequency measured by limiting dilution analysis (LDA) ranged from 1/16880-1/306. The Spearman rank test revealed no positive correlation between GVHD intensity and donor anti-host CTL activity assayed either in bulk culture experiments or in LDA conditions. These results indicate that MiHA capable of triggering potent CTL responses in vitro do not necessarily initiate GVHD, and that in vitro measurement of donor CTL activity against host-type Con A blasts is not a predictive assay for anti-MiHA GVHD. However, the possibility to recruit CTL populations targeting host MiHA expressed specifically on hematopoietic cells suggests a novel therapeutic strategy for the cure of hematopoietic malignancies. Indeed, transplantation of donor hematopoietic stem cells supplemented with T cells aimed at MiHA specifically expressed by host hematopoietic cells, could possibly potentiate the desirable graft vs leukemia effect without increasing the risk of GVHD.     

Ex vivo expansion of primitive hematopoietic cells for cellular therapies: An overview

Sources of hematopoietic cells for bone marrow transplantation are limited by the supply of compatible donors, the possibility of viral infection, and autologous (patient) marrow that is depleted from prior chemo- or radiotherapy or has cancerous involvement. Anex vivo system to amplify hematopoietic progenitor cells could increase the number of patients eligible for autologous transplant, allow use of cord blood hematopoietic cells to repopulate an adult, reduce the amount of bone marrow and/or mobilized peripheral blood stem and progenitor cells required for transplantation, and reduce the time to white cell and platelet engraftment. The cloning of hematopoietic growth factors and the identification of appropriate conditions has enabled the development of successfulex vivo hematopoietic cell cultures. Purification systems based on the CD34 marker (which is expressed by the most primitive hematopoietic cells) have proven an essential tool for research and clinical applications. Present methods for hematopoietic cultures (HC) on stromal (i.e. accessory cells that support hematopoiesis) layers in flasks lack a well-controlled growth environment. Several bioreactor configurations have been investigated, and a first generation of reactors and cultures has reached the clinical trial stage. Our research suggests that perfusion conditions improve substantially the performance of hematopoietic reactors. We have designed and tested a perfusion bioreactor system which is suitable for the culture of non-adherent cells (without stromal cells) and readily scaleable for clinical therapies. Eliminating the stromal layer eliminates the need for a stromal cell donor, reduces culture time, and simplifies the culture system. In addition, we have compared the expansion characteristics of both mononuclear and CD34⁺ cells, since the latter are frequently assumed to give a superior performance for likely transplantation therapies.Abbreviations BFU0-E burst forming unit-erythroid - BM bone marrow - CB cord blood - CFU-C colony forming unit-culture - CFU-E colony forming unit-erythroid - CFU-F colony forming unit-fibroblast - CFU-GEMM colony forming unit-granulocyte, erythroid, macrophage, megakaryocyte - CFU-GM colony forming unit-granulocyte, macrophage - CFU-Mix colony forming unit-mixed (also known as CFU-GEMM) - CML chronic myeloid leukemia - CSF colony stimulating factor - DMSO dimethyl sulfoxide - ECM extracellular matrix - EPO erythropoietin - FL fetal liver - HC hematopoietic culture - LTBMC long-term bone marrow culture - LTC-IC long-term culture initiating cell - LTHC long-term hematopoietic culture - MNC mononuclear cells - PB peripheral blood     

Donor APCs are required for maximal GVHD but not for GVL

Graft-versus-host disease (GVHD) is a major source of morbidity in allogenic stem cell transplantation. We previously showed that recipient antigen-presenting cells (APCs) are required for CD8-dependent GVHD in a mouse model across only minor histocompatibility antigens (minor H antigens). However, these studies did not address the function of donor-derived APCs after GVHD is initiated. Here we show that GVHD develops in recipients of donor major histocompatibility complex class I-deficient (MHC I(-)) bone marrow. Thus, after initial priming, CD8 cells caused GVHD without a further requirement for hematopoietic APCs, indicating that host APCs are necessary and sufficient for GHVD. Nonetheless, GVHD was less severe in recipients of MHC I(-) bone marrow. Therefore, once initiated, GVHD is intensified by donor-derived cells, most probably donor APCs cross-priming alloreactive CD8 cells. Nevertheless, donor APCs were not required for CD8-mediated graft-versus-leukemia (GVL) against a mouse model of chronic-phase chronic myelogenous leukemia. These studies identify donor APCs as a new target for treating GVHD, which may preserve GVL.     

Role of MAdCAM-1 and its ligand on the homing of transplanted hematopoietic cells in irradiated mice

We examined the expression of VCAM-1 and MAdCAM-1 after bone marrow transplantation (BMT). We also examined the influence of alpha(4)beta(7) integrin blockade on the homing of cells to the bone marrow and spleen. The expression of VCAM-1 and MAdCAM-1 by endothelial cells in the spleen and bone marrow was examined by immunoelectron microscopy using colloidal gold and was analyzed semiquantitatively. To examine the role of alpha(4)beta(7) integrin in donor cells, a homing assay was conducted following alpha(4)beta(7) integrin blockade in bone marrow-derived hematopoietic cells or spleen colony cells. Immediately after BMT, the expression of VCAM-1 and MAdCAM1 markedly decreased, but expression recovered significantly between 12 and 24 h after BMT. VCAM-1 recovered more acutely than MAdCAM-1 from 12 h onward. In the group transplanted with anti-alpha(4)beta(7) integrin antibody-treated bone marrow cells, the numbers of homing cells in the spleen and bone marrow were significantly decreased in an antibody dose-dependent manner. However, the number of homing cells was not different in either the spleen or bone marrow between anti-alpha(4)beta(7) integrin antibody treated and untreated spleen colony cells. It has been reported that alpha(4)beta(1) integrin and its receptor VCAM-1 play major roles in the homing of hematopoietic cells to bone marrow. Our study indicates the importance of MAdCAM-1 and its ligand, alpha(4)beta(7) integrin, in the homing of bone marrow-derived hematopoietic cells, but not spleen colony-derived cells, to both the spleen and bone marrow.     

Gadd45a基因对小鼠造血干细胞功能的影响

目的 Gadd45a基因对小鼠造血干细胞功能的影响。方法流式细胞仪分选小鼠骨髓造血干细胞、体外单克隆培养,竞争性骨髓移植,放射线照射观察生存曲线。结果 Gadd45a基因缺失的小鼠造血干细胞克隆形成能力增强,短期造血重建能力无差异,8.5Gy放射线照射后生存情况无差异。结论 Gadd45a基因对小鼠造血干细胞功能起重要作用。     

Allogeneic bone marrow grafts with high levels of CD4(+) CD25(+) FoxP3(+) T cells can lead to engraftment failure

Regulatory CD4(+) CD25(+) FoxP3(+) T cells (T(regs) ) suppress immunological reactions. However, the effect of adding T(regs) to hematopoietic stem cell grafts on recovery and graft versus host disease (GvHD) is unknown. T(regs) from splenocytes of C57Bl/6 and Balb/c wild-type mice were isolated by MACS separation and analyzed by flow cytometry. Using a murine syngeneic transplantation model that clearly distinguishes between donor and host hematopoiesis, we showed that co-transplantation of bone marrow cells (BMCs) with high levels of T(regs) leads to a 100% survival of the mice and accelerates the hematopoietic recovery significantly (full donor chimerism). In allogeneic transplantation, bone marrow and T(regs) co-transplantation were compared to allogeneic bone marrow transplantation with or without the addition of splenocytes. Survival, leukocyte recovery, chimerism at days -2, 19, 33, and 61 for murine CD4, human CD4, HLA-DR3, murine CD3, murine CD8, murine Balb/c-H2K(d) , murine C57Bl/6-H2K(b) , and GvHD appearance were analyzed. Allogeneic bone marrow transplantation requires the addition of splenocytes to reach engraftment. Mice receiving grafts with bone marrow, splenocytes and high levels of allogeneic T(regs) died within 28 days (hematopoietic failure). Here, we show also detailed flow cytometric data reagarding analysis of chimerism after transplantation in unique murine hematopoietic stem cell transplantation models. Our findings showed that the syngeneic co-transplantation of CD4(+) , CD25(+) , FoxP3(+) T-cells and BMCs induced a stimulating effect on reconstitution of hematopoiesis after irradiation. However, in the allogeneic setting the co-transplantation of T(regs) aggravates the engraftment of transplanted cells.     

The use of CD 34(+) mobilized peripheral blood as a donor cell source does not improve chimerism after in utero hematopoietic stem cell transplantation in non-human primates

In utero hematopoietic stem cell transplantation is a therapeutic procedure that could potentially cure many developmental diseases affecting the immune and hematopoietic systems. In most clinical and experimental settings of fetal hematopoietic transplantation the level of donor cell engraftment has been low, suggesting that even in the fetus there are significant barriers to donor cell engraftment. In postnatal hematopoietic transplantation donor cells obtained from mobilized peripheral blood engraft more rapidly than cells derived from marrow. We tested the hypothesis that use of donor hematopoietic/stem cells obtained from mobilized peripheral blood would improve engraftment and the level of chimerism after in utero transplantation in non-human primates. Despite the potential competitive advantage from the use of CD 34(+) from mobilized peripheral blood, the level of chimerism was not appreciably different from a group of animals receiving marrow-derived CD 34(+) donor cells. Based on these results, it is unlikely that this single change in cell source will influence the clinical outcome of fetal hematopoietic transplantation.     

Post-Irradiation Thymocyte Regeneration After Bone Marrow Transplantation I. Regeneration and Quantification of Thymocyte Progenitor Cells In the Bone Marrow

Growth kinetics of the donor-type thymus cell population after transplantation of bone marrow into irradiated syngeneic recipient mice is biphasic. During the first rapid phase of regeneration, lasting until day 19 after transplantation, the rate of development of the donor cells is independent of the number of bone marrow cells inoculated. the second slow phase is observed only when low numbers of bone marrow cells (2.5 × 10⁴) are transplanted. the decrease in the rate of development is attributed to an efflux of donor cells from the thymus because, at the same time, the first immunologically competent cells are found in spleen. After bone marrow transplantation the regeneration of thymocyte progenitor cells in the marrow is delayed when compared to regeneration of CFUs. Therefore, regenerating marrow has a greatly reduced capacity to restore the thymus cell population. One week after transplantation of 3 × 10⁶ cells, 1% of normal capacity of bone marrow is found. It is concluded that the regenerating thymus cells population after bone marrow transplantation is composed of the direct progeny of precursor cells in the inoculum.     

Induction of histiocytic sarcoma in mouse skeletal muscle

Myeloid sarcomas are extramedullary accumulations of immature myeloid cells that may present with or without evidence of pathologic involvement of the bone marrow or peripheral blood, and often coincide with or precede a diagnosis of acute myeloid leukemia (AML). A dearth of experimental models has hampered the study of myeloid sarcomas and led us to establish a new system in which tumor induction can be evaluated in an easily accessible non-hematopoietic tissue compartment. Using ex-vivo transduction of oncogenic Kras(G12V) into p16/p19(-/-) bone marrow cells, we generated transplantable leukemia-initiating cells that rapidly induced tumor formation in the skeletal muscle of immunocompromised NOD.SCID mice. In this model, murine histiocytic sarcomas, equivalent to human myeloid sarcomas, emerged at the injection site 30-50 days after cell implantation and consisted of tightly packed monotypic cells that were CD48+, CD47+ and Mac1+, with low or absent expression of other hematopoietic lineage markers. Tumor cells also infiltrated the bone marrow, spleen and other non-hematopoietic organs of tumor-bearing animals, leading to systemic illness (leukemia) within two weeks of tumor detection. P16/p19(-/-); Kras(G12V) myeloid sarcomas were multi-clonal, with dominant clones selected during secondary transplantation. The systemic leukemic phenotypes exhibited by histiocytic sarcoma-bearing mice were nearly identical to those of animals in which leukemia was introduced by intravenous transplantation of the same donor cells. Moreover, murine histiocytic sarcoma could be similarly induced by intramuscular injection of MLL-AF9 leukemia cells. This study establishes a novel, transplantable model of murine histiocytic/myeloid sarcoma that recapitulates the natural progression of these malignancies to systemic disease and indicates a cell autonomous leukemogenic mechanism.     

Allogeneic bone marrow transplantation as primary therapy for chronic myelomonocytic leukemia

This is the first report of a successful bone marrow transplantation for chronic myelomonocytic leukemia. A 41-year-old woman with chronic myelomonocytic leukemia received, as primary treatment, a novel preparatory regimen consisting of high dose fractionated total body irradiation and high dose VP-16 chemotherapy followed by allogeneic marrow transplantation from her histocompatible brother. The patient is now more than two years after marrow transplantation with normal blood counts and a normal bone marrow which is of donor type. For younger patients with this disease who have a histocompatible sibling donor, bone marrow transplantation may represent a valid therapeutic option with curative potential.     

A Study of the Regenerative Potential of Bone Marrow Cells of Donor Mice that Carry the <Emphasis Type="Italic">egfp</Emphasis> Gene in Irradiated Mice

A study of the regenerative potential of bone marrow cells of donor mice that express the enhanced green fluorescent protein was conducted in mice irradiated at a dose of 7 Gy. Expression of this protein allowed us to carry out monitoring of the presence of donor cells in recipient blood over the entire lifespan of the recipient. The lifespan of young recipients increased by 93% after transplantation; for old recipients it increased by 15%. Total acceptance of the bone marrow, spleen, thymus, and blood of the recipient with donor bone marrow cells was demonstrated over the entire life of the recipient. Only the donor colonies were detected with the studied irradiation dose and number of transplanted cells (11.7 ± 0.4) · 10⁶ on the spleen surface. The percentage of bone marrow and spleen cells that expressed the CD117 and CD34 stem cell markers in the recipient mice was above the control level for a long period of time after the irradiation. More than half of the cells with CD117, CD34, CD90.2, and CD45R/B220 phenotypes in the studied organs were donor cells. Further detailed study of the peculiarities of the engraftment of bone marrow cells, both without preliminary treatment of recipients and after the effects of extreme factors, will allow improvement of the methods of cell therapy.     

Effect of graft-versus-host disease (GVHD) on host hematopoietic progenitor cells is mediated by Fas-Fas ligand interactions but this does not explain the effect of GVHD on donor cells.

The acute graft-versus-host disease (GVHD) generated in BDF1 mice by the injection of spleen cells from the C57BL/6 parental strain induces a direct cell-mediated attack on host lymphohematopoietic populations, resulting in the reconstitution of the host with donor hematopoietic stem cells. We examined the effect of GVHD on the donor and host hematopoiesis in parental-induced acute GVHD. The bone marrow was hypoplastic and the number of hematopoietic progenitor cells significantly decreased at 4 weeks after GVHD induction. However, extramedullary splenic hematopoiesis was present and the number of hematopoietic progenitor cells in the spleen significantly increased at this time. Fas expression on the host spleen cells and bone marrow cells significantly increased during weeks 2 to 8 of GVHD. Host cell incubation with anti-Fas Ab induced apoptosis, and the number of hematopoietic progenitor cells decreased during these weeks. A significant correlation between the augmented Fas expression on host bone marrow cells and the decreased number of host bone marrow cells by acute GVHD was observed. Furthermore, the injection of Fas ligand (FasL)-deficient B6/gld spleen cells failed to affect host bone marrow cells. Although Fas expression on repopulating donor cells also increased, Fas-induced apoptosis by the repopulating donor cells was not remarkable until 12 weeks, when more than 90% of the cells were donor cells. The number of hematopoietic progenitor cells in the bone marrow and the spleen by the repopulating donor cells, however, decreased over an extended time during acute GVHD. This suggests that Fas-FasL interactions may regulate suppression of host hematopoietic cells but not of donor hematopoietic cells. Hematopoietic dysfunctions caused by the reconstituted donor cells are independent to Fas-FasL interactions and persisted for a long time during parental-induced acute GVHD.     

Magnetically empowered bone marrow cells as a micro-living motor can improve early hematopoietic reconstitution

Background aimsBone marrow-derived hematopoietic stem cell transplantation/hematopoietic progenitor cell transplantation (HSCT/HPCT) is widely used and one of the most useful treatments in clinical practice. However, the homing rate of hematopoietic stem cells/hematopoietic progenitor cells (HSCs/HPCs) by routine cell transfusion is quite low, influencing hematopoietic reconstitution after HSCT/HPCT.MethodsThe authors developed a micro-living motor (MLM) strategy to increase the number of magnetically empowered bone marrow cells (ME-BMCs) homing to the bone marrow of recipient mice.ResultsIn the in vitro study, migration and retention of ME-BMCs were greatly improved in comparison with non-magnetized bone marrow cells, and the biological characteristics of ME-BMCs were well maintained. Differentially expressed gene analysis indicated that ME-BMCs might function through gene regulation. In the in vivo study, faster hematopoietic reconstitution was observed in ME-BMC mice, which demonstrated a better survival rate and milder symptoms of acute graft-versus-host disease after transplantation of allogeneic ME-BMCs.ConclusionsThis study demonstrated that ME-BMCs serving as MLMs facilitated the homing of HSCs/HPCs and eventually contributed to earlier hematopoietic reconstitution in recipients. These data might provide useful information for other kinds of cell therapies.