成人骨髓间充质干细胞与造血细胞的发育级系的关系

目的：探讨骨髓间充质干细胞在细胞发育级系中与造血细胞的关系。方法：分离培养成年慢性髓性白血病患者骨髓的间充质干细胞，用FCM鉴定其纯度。应用RT－PCR检测细胞是否携带白血病特异性基因，结合流式细胞术观察多种造血调控因子刺激后是否具有造血细胞的表面分子标志及基因标志，结果：骨髓间充质干细胞不表达造血细胞特异性白血病基因，细胞因子作用2周后未出现血细胞表型，CD34及CD45均为阴性，结论：造血干细胞与间充质干细胞之间似乎不存在交叉分化现象，为解释其他细胞间的相互转化提出了新的线索。     

Studies of T cell reconstitution after hematopoietic stem cell transplant

Hematopoietic stem cell (HSC) transplantation, whatever its conditions, is associated with an increased risk of infections and tumoral complications, because of a delayed immune reconstitution. T-cell regeneration has been mostly investigated and appears to come more from graft and/or host mature T-cells, rather than from the differentiation/maturation of reinfused progenitors. In allogeneic setting, the immune defect is enhanced by the immune host/donor conflict and the use of prophylactic or curative immunosuppressive therapy. The tools used for studying post-transplant immunity are the following: immunophenotyping (kinetics and alterations of lymphocyte subset reconstitution), functional studies of T cell proliferation, cytokine production, cytotoxicity and signal transduction, as well as studies of T cell repertoire diversity. The CD4/CD8 cell immunophenotyping might be enough for routine clinical evaluation, allowing an adapted prophylaxis of opportunistic infections in those immune-suppressed patients, while functional assays might be useful to evaluate the persistence overtime of defects in immune reconstitution. These overall assays are useful both for basic and clinical research and allow better understanding in the mechanisms for T cell regeneration in the diverse types of HSC transplants performed nowadays particularly after graft of purified HSC where immune reconstitution remains a key question.     

自体骨髓间充质干细胞联合造血干细胞共移植治疗恶性血液病五例疗效观察

目的 研究自体骨髓间充质干细胞(MSCs)与造血干细胞共移植治疗恶性血液病的安全性和可行性,及其对移植后造血重建的影响.方法 从无骨髓浸润的恶性血液病患者本人骨髓中分离、培养间充质干细胞,经放化疗等预处理后,与造血干细胞共移植治疗恶性血液病患者5例,其中恶性淋巴瘤4例,粒细胞肉瘤1例,并观察其对移植后造血重建的影响.结果 MSCs联合造血干细胞共移植治疗恶性血液病患者5例,MSCs输注过程顺利,未见明显不良反应.移植后造血恢复过程中,中性粒细胞≥0.5×109/L的中位时间为9.4(8～11)d、血小板≥20×109/L的中位时间为12.2(10～14)d.结论 MSCs联合造血干细胞共移植治疗恶性血液病安全性好,未见明显副作用.结果 提示输注MSCs可促进造血恢复,但其远期疗效仍有待于进一步观察.     

小鼠骨髓造血干细胞转化为肝细胞的实验研究

为进一步明确向肝细胞分化的细胞为骨髓中的那一亚群细胞,我们将纯化后的雄性小鼠造血干细胞移植到雌性受体小鼠的体内,然后在受体小鼠的肝脏内进行检测,以确定有无供体来源的肝细胞存在。一、材料与方法1.实验动物、主要器材和试剂:健康清洁级雌雄性纯系BALB/C鼠,8～12周龄,均由第一军医大学动物实验中心提供。主要试剂及材料:磁性活化细胞分选(magnetic activated cell sorting,MACS)分离用MiniMACS缓冲液,     

Advances in understanding the leukaemia microenvironment

Dynamic interactions between leukaemic cells and cells of the bone marrow are a feature of haematological malignancies. Two distinct microenvironmental niches in the bone marrow, the ‘osteoblastic (endosteal)’ and ‘vascular’ niches, provide a sanctuary for subpopulations of leukaemic cells to evade chemotherapy‐induced death and allow acquisition of drug resistance. Key components of the bone marrow microenvironment as a home for normal haematopoietic stem cells and the leukaemia stem cell niches, and the molecular pathways critical for microenvironment/leukaemia interactions via cytokines, chemokines and adhesion molecules as well as hypoxic conditions, are described in this review. Finally, the genetic abnormalities of leukaemia‐associated stroma are discussed. Further understanding of the contribution of the bone marrow niche to the process of leukaemogenesis may provide new targets that allow destruction of leukaemia stem cells without adversely affecting normal stem cell self‐renewal.     

Mesenchymal stem cells for the treatment of refractory pure red cell aplasia after major ABO-incompatible hematopoietic stem cell transplantation

Pure red cell aplasia (PRCA) is a well-known, although infrequent, hematological complication after allogeneic hematopoietic stem cell transplantation (HSCT). PRCA occurs in cases of major ABO mismatch between donor and recipient and is believed to be due to inhibition of donor erythroid progenitors by residual host isohemagglutinins. The purpose of our study was to further evaluate the efficacy of human adipose tissue-derived mesenchymal stem cells (AMSC) as the salvage therapy for refractory PRCA after major ABO-incompatible HSCT. Two patients with refractory pure red cell aplasia received intravenous infusions of AMSC at a dose of 1.5 × 10⁶/kg of the patients’ weight, and rapid recovery from PRCA without any side effects was observed. We conclude that AMSC seems to be a promising therapeutic option in patients with PRCA after ABO-mismatched HSCT, in whom conventional treatment fails.     

Effect of aged bone marrow microenvironment on mesenchymal stem cell migration

Mesenchymal stem cells (MSCs) are known to have many notable features, especially their multiple differentiation ability and immunoregulatory capacity. MSCs are important stem cells in the bone marrow (BM), and their characteristics are affected by the BM microenvironment. However, effects of the BM microenvironment on the properties of MSCs are not well understood. In this study, we found that BM from aged mice decreased MSC colony formation. Flow cytometry data showed that the proportion of B220⁺ cells in BM from aged mice was significantly lower than that in BM from young mice, while the proportion of CD11b⁺, CD3⁺, Gr-1⁺, or F4/80⁺ cells are on the contrary. CD11b⁺, B220⁺, and Ter119⁺ cells from aged mice were not the subsets that decreased MSC colony formation. We further demonstrated that both BM from aged mice and young mice exhibited similar effects on the proliferation of murine MSC cell line C3H10T1/2. However, when cocultured with BM from aged mice, C3H10T1/2 showed slower migration ability. In addition, we found that phosphorylation of JNK (c-Jun N-terminal kinases) in C3H10T1/2 cocultured with BM from aged mice was lower than that in C3H10T1/2 cocultured with BM from young mice. Collectively, our data revealed that BM from aged mice could decrease the migration of MSCs from their niche through regulating the JNK pathway.     

心肌微环境对骨髓间充质干细胞的诱导分化作用

目的通过与心肌细胞(CMs)共同培养和采用含有CMs裂解液的培养基两种方法体外模拟心肌微环境,探讨心肌微环境对骨髓间充质干细胞(MSCs)分化的诱导作用。方法自新生乳鼠的心脏分离CMs,自成年大鼠的骨髓分离MSCs,将MSCs与CMs按1∶4的比例共同培养1周,观察细胞形态的改变,并通过免疫荧光方法检测共培养后MSCs表达心脏特异性肌钙蛋白T(cTnT)及CD31的情况;将分离的CMs反复冻融制成CMs裂解液,将MSCs在含有4倍CMs裂解液的培养基中培养1周,观察细胞形态的改变,并通过免疫化学方法检测MSCs表达心脏特异性肌钙蛋白T(cTnT)及CD31的情况;以仅用普通培养基所培养的MSCs作为对照。结果与CMs共培养的MSCs和CMs裂解液培养的MSCs逐渐伸展变长,形成肌细胞形态,培养1周后经抗cTnT和抗CD31免疫染色均呈阳性;对照组MSCs没有明显形态变化,抗cTnT和抗CD31免疫染色成阴性。结论与CMs共培养和采用含有CMs裂解液的培养基两种方法均可在体外模拟心肌微环境,诱导MSCs向心肌样细胞和内皮样细胞方向分化。     

Aging of hematopoietic stem cells is regulated by the stem cell niche

Adult stem cells provide the basis for regeneration of aging tissue. Their dual ability for self-renewal and multilineage differentiation is controlled by direct interaction with a specific microenvironment -- the so called "stem cell niche". Hematopoietic stem cells (HSC) reside in the bone marrow. It is still under debate if HSC can rejuvenate infinitively or if they do not possess "true" self-renewal and undergo replicative senescence such as any other somatic cell. Furthermore, the question arises to what extent age-related changes in HSC are due to intrinsic factors or regulated by external stimuli. There is growing evidence, that the stem cell niche is most important for the regulation of cellular aging in adult stem cells. It is the stem cell niche that (i) maintains HSC in a quiescent state that reduces DNA damage as well as replicative senescence, (ii) protects from radicals and toxic compounds, (iii) regulates cell intrinsic signal cascades and (iv) modulates gene expression and epigenetic modifications in HSC. Thus, the interplay with the stem cell niche controls HSC function including the aging process of the hematopoiesis.     

Induction of hematopoietic microenvironment by the extracellular matrix from long-term bone marrow cultures

Summary Extracellular matrix (ECM) plays an important role in the regulation of hematopoiesis. The ECM obtained from murine long-term bone marrow cultures (LTBMCs) induces hematopoietic foci formation within 3 months after implantation under the murine renal capsule. The foci consist of approximately 3×10⁶ hematopoietic cells and function for at least 11 months. The induced stroma contains transplantable precursors capable of transferring a hematopoietic microenvironment to secondary recipients, and is insensitive to the stroma-stimulating factor produced in recipient mice after irradiation. The ECM induces hematopoietic foci formation in chimeras irradiated by a dose which is lethal for most of the stromal precursors. These facts point to the differences observed between bone marrow stromal precursors and mesenchymal cells induced under the renal capsule. The foci contain bone, but its appearance is limited to early stages of foci growth, and depends on the dose of implanted ECM. Bone is not formed when the xenogeneic ECM from nonhematopoietic tissue is used as an inducer. In this case, the foci develop slowly and are observed only to the tenth month after implantation. The data obtained demonstrate a novel function of the ECM in the induction of a hematopoietic microenvironment.     

Pharmacogenomics in allogeneic hematopoietic stem cell transplantation: Implications on supportive therapies and conditioning regimens

Allogeneic hematopoietic stem cell transplantation mortality has declined over the years, though prevention and management of treatment-related toxicities and post-transplant complications remains challenging. Applications of pharmacogenomic testing can potentially mitigate adverse drug outcomes due to interindividual variability in drug metabolism and response. This review summarizes clinical pharmacogenomic applications relevant to hematopoietic stem cell transplantation, including antifungals, immunosuppressants, and supportive care management, as well as emerging pharmacogenomic evidence with conditioning regimens.     

大鼠胎血与骨髓间充质干细胞分化能力的体外研究

目的：比较大鼠胎血和骨髓中间充质干细胞（MSC）体外培养过程中的生长特性及体外诱导两者向神经元样细胞分化的异同。方法：采用标准Ficoll-hypague技术分离大鼠胎血骨髓的单个核细胞（MNC），收获MSC传代培养，流式细胞仪检测细胞的免疫表型。β-巯基乙醇、二甲基亚砜、叔丁基对羟基茴香醚诱导MSC向神经元分化，免疫细胞化学法检测其特异性标志巢蛋白（Nestin）、神经元特异性烯醇化酶（NSE）、胶原纤维酸性蛋白（GFAP）的表达。结果：2种MSC细胞形态、免疫表型无明显差异。定向诱导后，2种细胞均表达Nestin、NSE，但GFAP阴性。结论：大鼠胎血和骨髓MSC的细胞形态、生物学特性无明显差别；两者诱导分化为神经元碰细胞的能力无显著差异。胎血应是MSC的又一来源。     

Hematopoietic stem cell transplantation for the treatment of autoimmune diseases

Systemic autoimmune diseases that are resistant to conventional treatment cause considerable morbidity and mortality. Although aggressive new approaches to treating autoimmune diseases have been developed over the past decade, there are still patients with a severe, progressive, and life-threatening course. Based on animal studies and experience in the treatment of hematological disorders with preexisting autoimmune disease, hematopoietic stem cell transplantation has been proposed for the treatment of severe autoimmune diseases. Immunoablation and subsequent autologous peripheral blood stem cell transplantation using CD34⁺ hematopoietic cells with T cell depletion have been used for selected severe autoimmune diseases at many institutes in Australia, Europe, and the United States. However, it is necessary to assess the efficacy and safety of this therapy compared with conventional and other newly emerging therapies.     

骨髓间充质干细胞的培养及低氧条件对其影响

目的 骨髓间充质干细胞(BMMSC)的体外培养和低氧对于BMMSC增殖作用的影响.方法 成年雄性SD大鼠断颈处死后于75％酒精中浸泡5 min.用全骨髓贴壁法培养细胞,选取生长状态良好的第3代细胞进行鉴定.以单克隆抗体CD45、CD90行流式细胞术鉴定大鼠BMMSC.用四甲基偶氮唑蓝(MTT)法测定第3代BMMSC以及低氧处理的第3代BMMSC的增殖情况.结果 用全骨髓贴壁法分离并培养SD大鼠BMMSC;流式细胞仪检测显示:第3代BMMSC表面特异性标志CD90表达率为96.0％,而非BMMSC表面标志CD45仅为2.5％.MTT结果显示低氧条件下的BMMSC比常氧条件下增殖速率高,并且光镜下观察到细胞状态均一,折光性更好.结论 用全骨髓贴壁法可以在体外分离、培养出纯度较高的SD大鼠来源BMMSC,低氧环境可以刺激BMMSC的增殖.     

骨髓基质干细胞向肝细胞的定向分化及体内移植

目的:研究骨髓基质干细胞(BMSCs)在体外向肝细胞的定向分化和体内移植后对小鼠受损肝脏的修复作用.方法:在体外培养体系中用肝细胞提取液(HE)模拟体内肝脏微环境,诱导BMSCs向肝细胞定向分化,以免疫细胞化学和吲哚靛青绿(ICG)染色检测其分化程度.将5-溴脱氧尿嘧啶核苷(BrdU)标记的BMSCs移植入小鼠体内,免疫组织化学方法显示BMSCs在肝脏的分布,血清酶学检测肝脏受损小鼠移植后7,14,28d谷草转氨酶(AST,nkat/L)、谷丙转氨酶(ALT,nkat/L)和碱性磷酸酶(AKP,nkat/L)的含量变化.结果:在体外BMSCs可由HE诱导分化成肝细胞,但HE诱导组α1-抗胰蛋白酶(4d:52.5±3.9vs76.8±4.5,P<0.05;14d:60.3±6.1vs85.4±7.6,P<0.05;21d:80.5±8.0vs105.7±8.6,P<0.05)和白蛋白(4d:43.2±6.5vs71.6±7.6,P<0.05;14d:61.5±10.4vs93.6±13.9,P<0.05;21d:80.6±17.1vs128.3±22.2,P<0.05)表达量少于HGF诱导组;体内移植后BMSCs整合于肝实质内;肝脏受损小鼠移植BMSCs后,血清中ALT(7d:493.43±120.02vs696.81±140.03,P<0.01;14d:558.45±130.03vs780.16±151.7,P<0.01;28d:583.45±138.36vs880.18±170.53,P<0.01)、AST(7d:1521.97±186.7vs2342.14±208.38,P<0.01;14d:1590.32±200.04vs2692.21±238.38,P<0.01;28d:1625.33±208.38vs2872.24±281.72,P<0.01)和AKP(7d:1.24±0.22vs1.78±0.18,P<0.01;14d:1.21±0.21vs2±0.19,P<0.01;28d:1.32±0.19vs2.27±0.2,P<0.01)比损伤组小鼠相应值均有下降,但仍高于正常对照组.结论:BMSCs在体外可由HE诱导分化成肝细胞,在体内移植后可促进小鼠受损肝脏的修复.     

Treatment of autoimmune diseases in mice by a new method for allogeneic bone marrow transplantation

Remarkable advances have been made in bone marrow transplantation (BMT), which has become a powerful strategy for the treatment of leukemia, aplastic anemia, congenital immunodeficiency, and also autoimmune disease. Using various animal models, allogeneic (allo) BMT has been found to be useful in the treatment of various autoimmune diseases. In MRL/lpr mice, which are radiosensitive (<8.5 Gy) and are an animal model for autoimmune diseases, conventional BMT resulted in only transient effects; the manifestations of the autoimmune diseases recurred 3 months after BMT. Using MRL/lpr mice, we have very recently established a new strategy for allo BMT. We injected bone marrow cells (BMC) directly into the bone marrow cavity (intrabone marrow [IBM] injection) of recipients that had received fractionated irradiation. This 'IBM-BMT' was found to be effective in treating autoimmune diseases in radiation-sensitive and chimeric-resistant MRL/lpr mice. In addition, this strategy was found to be applicable for the transplantation of organs. We believe that these strategies for BMT and organ transplantation herald a new era in transplantation.     

Notch signaling in hematopoietic cell transplantation and T cell alloimmunity

Notch signaling can regulate both hematopoietic progenitors and alloimmune T cells in the setting of allogeneic bone marrow or hematopoietic cell transplantation (allo-HCT). Ex vivo culture of multipotent blood progenitors with immobilized Delta-like ligands induces supraphysiological Notch signals and can markedly enhance progenitor expansion. Infusion of Notch-expanded progenitors shortened myelosuppression in preclinical and early clinical studies, while accelerating T cell reconstitution in preclinical models. Notch also plays an essential role in vivo to regulate pathogenic alloimmune T cells that mediate graft-versus-host disease (GVHD), the most severe complication of allo-HCT. In mouse allo-HCT models, Notch inhibition in donor-derived T cells or transient blockade of Delta-like ligands after transplantation profoundly decreased GVHD incidence and severity, without causing global immunosuppression. These findings identify Notch in T cells as an attractive therapeutic target to control GVHD. In this review, we discuss these contrasting functions of Notch signaling with high translational significance in allo-HCT patients.     

Selection, enrichment, and culture expansion of murine mesenchymal progenitor cells by retroviral transduction of cycling adherent bone marrow cells

It has been difficult to characterize murine bone marrow (BM)-derived mesenchymal progenitor cells (MPCs) because of contamination with hematopoietic cells. We took advantage of the rapid proliferation of MPCs after replating to enrich murine MPCs by transfection with a retroviral vector carrying both LacZ and the selective neomycin resistance (neoR) gene.Freshly harvested BM cells from mice were incubated with BAG retroviral vector produced by amphotropic psi-CRIP or ecotropic psi-CRE producer cells for 48 hours and grown in the presence of G418.Cells incubated in psi-CRIP supernatant formed colonies composed of large homogeneous cells that were free of CD45(+) cells, but cells incubated in psi-CRE supernatant did not form stromal cell colonies. In the undifferentiated state, the cells displayed a fibroblast-like phenotype with low alkaline phosphatase activity. However, upon treatment with dexamethasone or 5-azacytidine, the retrovirally transduced cells differentiated into oil-red-O-positive adipocytic cells and osteogenic cells generating von Kossa-positive bone nodules. Osteogenic supplements composed of beta-glycerophosphate, dexamethasone, and ascorbic acid induced an increase in alkaline phosphatase activity and acute osteogenesis associated with early cell detachment. Subcutaneous injection with retrovirally transduced cells into day 1 newborn mice of the same strain produced ectopic calcium depositions surrounded by X-gal(+) cells.Retroviral selection of cycling adherent cells is an effective approach for enrichment of MPCs.     

Hyperthyroidism After Allogeneic Hematopoietic Stem Cell Transplantation: A Report of Four Cases

Hematopoietic stem cell transplantation (HSCT) is the only curative treatment for many hematological disorders, primary immunodeficiencies, and metabolic disorders. Thyroid dysfunction is one of the frequently seen complications of HSCT. However, hyperthyroidism due to Graves’ disease, autoimmune thyroiditis, and thyrotoxicosis are rare. Herein, we report a series of 4 patients who were euthyroid before HSCT but developed hyperthyroidism (3 of them developed autoimmune thyroid disease) after transplantation.     

Implantable microenvironments to attract hematopoietic stem/cancer cells

The environments that harbor hematopoietic stem and progenitor cells are critical to explore for a better understanding of hematopoiesis during health and disease. These compartments often are inaccessible for controlled and rapid experimentation, thus limiting studies to the evaluation of conventional cell culture and transgenic animal models. Here we describe the manufacture and image-guided monitoring of an engineered microenvironment with user-defined properties that recruits hematopoietic progenitors into the implant. Using intravital imaging and fluorescence molecular tomography, we show in real time that the cell homing and retention process is efficient and durable for short- and long-term engraftment studies. Our results indicate that bone marrow stromal cells, precoated on the implant, accelerate the formation of new sinusoidal blood vessels with vascular integrity at the microcapillary level that enhances the recruitment hematopoietic progenitor cells to the site. This implantable construct can serve as a tool enabling the study of hematopoiesis.