Comparison of immuno-modulatory properties between bone-marrow derived MSCs and adipose derived MSCs

目的 比较脂肪源间充质干细胞(AMSCs)和骨髓来源间充质干细胞(BMSCs)的免疫调节能力的差异.方法 用流式细胞仪分析AMSCs和BMSCs的表型.通过MSCs和淋巴细胞共培养体系,检测MSCs对T细胞增殖、周期、活化、凋亡以及Th细胞分化的影响.结果 表型分析结果显示AMSCs和BMSCs的表型基本一致.AMSCs和BMSCs都能抑制T细胞的增殖和早期活化,并在调节辅助性T细胞亚群的分化上作用类似.但在MSC对活化后T细胞的凋亡影响方面,BMSCs能够抑制活化T细胞的凋亡,而AMSC没有这种作用.结论 AMSCs和BMSCs对T淋巴细胞的影响基本类似,其对活化T细胞凋亡影响的差异还有待进一步的机制研究.     

胎盘源间充质干细胞对T淋巴细胞体外增殖的影响

目的从人胎盘组织中分离培养获得间充质干细胞（mesenchymal stem cells,MSCs）,并进一步研究其对T淋巴细胞体外增殖的影响。方法首先从胎盘组织中分离培养胎盘间充质干细胞（PMSCs）,在体外观测其形态,并通过细胞表面抗原表达、分化潜能等特征进行鉴定;然后将体外分离培养扩增的PMSCs,按照不同比例加入双向混合淋巴细胞培养体系（mixed lymphocyte reaction,MLR）中,共同培养6d后,测定T淋巴细胞的增殖。结果从人胎盘组织中分离培养获得问充质干细胞,具有与骨髓间充质于细胞（BMSCs）极为相似的细胞形态及细胞表面标志,表达CD29、CD44和CD105,不表达CD34、CD45、CD106和HLA—DR,并且还具有与BMSCs相似的跨胚层分化能力,能在一定条件下被诱导分化出神经元样细胞。PMSCs与同种异体混合淋巴细胞共同培养能抑制T淋巴细胞的体外增殖。结论胎盘组织是MSCs的有效来源,PMSCs具有与BMSCs相似的生物学特性及免疫调节机制。它为问充质干细胞的研究提供了又一重要的细胞来源。     

骨髓和脂肪来源间充质干细胞的免疫调节作用

背景：脂肪来源的间充质干细胞是否具有和骨髓来源间充质干细胞类似的免疫调节作用? 目的：观察骨髓来源和脂肪来源间充质干细胞的免疫学特征。 方法：分离骨髓和脂肪来源的间充质干细胞,分别检测它们对T细胞周期、活化、抑制和增殖的作用情况。 结果与结论：骨髓来源和脂肪来源的间充质干细胞同样具有抑制T细胞增殖的能力,在有丝分裂原刺激和混合淋巴细胞反应的T细胞增殖中这种作用都是具有剂量依赖性的,在1︰2时有极强的抑制作用,但是在1︰100时这种作用基本消失,在共培养时骨髓来源和脂肪来源的间充质干细胞都可以使更多的T细胞被抑制在G0/G1期,同时也可以抑制T细胞的早期活化,但是上述作用脂肪来源的间充质干细胞均较骨髓来源间充质干细胞弱,且脂肪来源的间充质干细胞并不具有抑制T细胞凋亡的作用。     

间充质干细胞在T细胞免疫反应中的调节作用

间充质干细胞(MSCs)独特的免疫调节作用以T细胞为主,在混合淋巴细胞反应中,通过周期阻滞抑制T细胞增殖,但不引起T细胞凋亡增加、活化受抑。同时MSCs还能降低反应体系中的CD8+T细胞和Th1细胞,升高Th2细胞以抑制炎症反应,从而在T细胞介导的自身免疫性疾病中发挥治疗作用。     

大鼠骨髓间充质干细胞对淋巴细胞增殖影响的体外实验

目的：探讨大鼠骨髓间充质干细胞对淋巴细胞增殖的影响。方法：常规分离培养大鼠的骨髓间充质干细胞（MSCs）及淋巴细胞,灭活后的骨髓间充质干细胞和淋巴细胞混合培养48h后,MTT法检测淋巴细胞的相对细胞数,Hoechst 33258染色观察细胞核变化。结果：MTT结果显示,加入MSCs组OD值明显低于未加MSCs的对照组（p〈0．05）。Hoechst 33258染色显示MSCs组淋巴细胞有核回缩及凋亡小体出现。结论：MSCs对淋巴细胞的增殖有抑制作用,其作用机制可能与细胞凋亡有关。     

骨髓和脂肪来源的间充质干细胞免疫调节作用的比较研究

本研究中我们分离骨髓和脂肪的间充质干细胞,分别检测它们对T细胞周期、活化、抑制和增殖的作用情况,研究脂肪来源(adipose-derived mesenchemal stem cell,AMSC)和骨髓来源(bone marrow derived mesenchemal stem cell,BMSC)间充质干细胞的免疫学特征,比较它们是否存在免疫功能的差异,结果显示AMSC和BMSC均具有抑制T细胞增殖的能力,在有丝分裂原刺激和MLR的T细胞增殖中均具有剂量依赖性的,在1∶2时有极强的抑制作用,但是在1∶100时这种作用基本消失,在共培养时AMSC和BMSC都可使更多的T细胞被抑制在G0/G1期,同时也可以抑制T细胞的早期活化,但是上述作用AMSC均较BMSC弱,但是AMSC并不具有抑制T细胞凋亡的作用,而两者在Th0向Th1或Th2分化中所起的作用是基本相似的,主要抑制Th0向Th1细胞(IL-2和IFN-γ生成细胞)的分化,而对向Th2细胞(IL-4和IL-10生成细胞)分化没有明显的影响。所以,AMSC和BMSC具有类似的免疫调节作用,因AMSC取材方便而成为更佳的材料来源。     

多发性骨髓瘤患者骨髓间充质干细胞免疫调节功能异常及其在骨髓瘤骨病中的作用

目的 探讨多发性骨髓瘤(MM)患者骨髓间充质干细胞(MSCs)免疫调节功能及其对骨髓瘤骨病的影响.方法 分离MM患者和正常对照MSCs,流式细胞技术比较二者的免疫表型.Real-time PCR检测实验组及对照组MSCs的TGF-β1、TGF-β2、TGF-β3、IL-6、IL-3、TNF-α、FasL和NF-κB配体的受体激活物(RANKL)表达量.共培养系统与流式细胞术检测MSCs对T细胞增殖、凋亡和早期活化标志CD25和CD69表达的影响.Von kossa染色、real-time PCR和Western blot等技术检测T细胞对正常对照MSCs向成骨细胞分化的影响.结果 MM来源和正常对照MSCs具有相似的形态学和免疫表型.MM来源的MSCs表达TGF-β1、IL-6、IL-3、TNF-α和RANKL较正常对照MSCs增加,而TGF-β2、TGF-β3和FasL的表达下降.MM来源的MSCs对T细胞增殖的抑制作用明显减弱.正常对照MSCs较MM来源MSCs使更多T细胞静止于G0/G1期.正常对照MSCs明显促进T细胞凋亡,而MM来源的MSCs对T细胞的促凋亡作用减弱.正常对照MSCs明显抑制T细胞活化分子表达,而MM患者的MSCs此抑制作用明显降低.与MM患者的MSCs共培养后的T细胞以及直接来源于MM患者的T细胞均可以明显抑制正常对照MSCs向成骨细胞分化.结论 MM患者的MSCs免疫调节功能较正常对照MSCs降低,主要表现为抑制T细胞活化的功能减弱,而活化的T细胞可抑制MSCs向成骨细胞分化,此可能为骨髓瘤骨病发病机制之一.     

人胎盘源与人骨髓源间充质干细胞的生物学特性的比较及负性协同刺激分子PD-L1的表达

目的:比较人胎盘源间充质干细胞(PMSCs)与人骨髓源间充质干细胞(BMSCs)的生物学特性,研究负性协同刺激分子PD-L1在胎盘源MSCs的表达和生物学意义.方法:采取酶消化法分离人胎盘组织,用密度梯度离心法分离骨髓单个核细胞,分别进行贴壁分离和传代培养,通过倒置相差显微镜观察细胞形态,并用免疫荧光标记和流式细胞仪检测细胞表面标志的表达做比较性分析.混合淋巴细胞反应,3H-TdR掺入和PD-L1单克隆抗体阻断实验进行免疫功能检测.结果:在贴壁生长、呈成纤维细胞样形态、表达CD29、CD105、CD166和不表达CD34、CD45、CD80、CD86及HLA-DR分子以及向成脂肪细胞方向诱导分化等方面,人PMSCs与BMSCs细胞生物学特性表现相同或相似.表型分析发现PMSCs高表达PD-L1,而BMSCs较低表达PD-L1.PMSCs表达的PD-L1具有对T细胞体外增殖的抑制作用.结论:人胎盘源MSCs与人骨髓源MSCs有相似的细胞生长特性,却有不同的表达负性协同分子PD-L1的特性.     

滑膜间充质干细胞分离培养、免疫表型鉴定及在混合淋巴反应体系中的抑制效应*

背景：滑膜组织中可分离出具有多向分化潜能干细胞特性的细胞。  目的：探索滑膜间充质干细胞体外分离、培养的可行性、免疫表型的鉴定及对混合淋巴细胞反应体系中淋巴细胞增殖的影响,评价其免疫学特性。  方法：关节镜下获取10例半月板损伤患者的膝关节滑膜组织,胶原酶消化获得有核细胞。挑选单细胞克隆,筛选获得滑膜间充质干细胞。检测其增殖能力、细胞活力,流式检测其细胞免疫表型,采用人双向混合淋巴细胞反应体系及植物血凝素刺激的淋巴细胞增殖反应体系,根据滑膜间充质干细胞与外周血单个核细胞的不同比例加入丝裂霉素处理滑膜间充质干细胞。MTT法检测并计算其抑制率。 结果与结论：10组滑膜间充质干细胞系增殖能力和细胞活力差异无显著性意义(P > 0.05)。10组细胞系免疫表型：CD44、CD90、CD105呈阳性,CD14、CD34、CD45和HLA-DR呈阴性。滑膜间充质干细胞抑制混合淋巴细胞反应体系中及植物血凝素刺激的淋巴细胞增殖反应体系中淋巴细胞的增殖、抑制率与加入的滑膜间充质干细胞数量成正比。提示关节滑膜组织可以分离、培养获得滑膜间充质干细胞,其具有间充质干细胞特异性表型,且滑膜间充质干细胞具有免疫调节作用。      

慢性粒细胞白血病肿瘤干细胞的免疫功能

背景：研究认为间充质干细胞可能是骨髓造血微环境的免疫保护位点。由于慢性粒细胞白血病存在造血微环境异常和免疫异常,所以推测间充质干细胞可能在慢性粒细胞白血病的病理过程中扮演了一个重要角色。 目的：观察慢性粒细胞白血病骨髓来源的肿瘤干细胞的免疫学特征,比较其与正常人来源的间充质干细胞是否存在免疫功能的异常。 方法：分离正常人和慢性粒细胞白血病患者的骨髓间充质干细胞,分别检测它们对T细胞周期、活化、抑制和增殖的作用。 结果与结论：慢性粒细胞白血病和正常志愿者骨髓来源的间充质干细胞形态和表型没有差异,慢性粒细胞白血病患者来源的间充质干细胞抑制T细胞增殖的作用减弱,抑制T细胞周期及活化的能力减弱,慢性粒细胞白血病患者抑制T细胞凋亡的作用增强。提示慢性粒细胞白血病患者骨髓来源的间充质干细胞存在明显的免疫调节功能缺陷,如果使用慢性粒细胞白血病患者自体的间充质干细胞移植治疗可能不是一种很好的选择,对于骨髓增生异常综合征患者最好是选用异基因的间充质干细胞移植。     

Culture and properties of adipose-derived mesenchymal stem cells: characteristics in vitro and immunosuppression in vivo

Objective: To compare the two sources of adipose and bone marrow derived mesenchymal stem cells (BMSCs and AMSCs) in immune regulation and to evaluate the therapeutic effects of AMSCs on Con A induced hepatitis and the possible mechanism involved in it. Methods: We isolated bone marrow and adipose derived mesenchymal stem cells respectively and compared their differences on T lymphocyte activation, proliferation and suppression. We also test the anti-apoptosis ability of AMSCs on LO2 cell line. The effects of intravenous infusion of AMSCs on liver damage were also tested and we detected donor AMSCs in liver of recipient and their effects on the activity of intrahepatic NKT cells. Results: BMSCs and AMSCs were similar in cell phenotype and the difference existed only in the expression of CD106. The results showed that the capacity of suppressing T cells proliferation and activation was weakened in AMSCs. AMSCs ameliorated liver damage and this effect was time and dose dependent. We detected donor AMSCs in liver of recipient which suggested tissue damage could be a clue for AMSCs migration. We also found AMSCs suppress the activity of intrahepatic NKT cells, but this suppress effects was not restricted in liver only, but the whole body. Conclusion: Cell origin and abundance are decisive factors in stem cells applications and with the same premise of AMSCs and BMSCs, adipose tissue is a more promising origin source of stem cells. The immunoregulatory features of MSCs might play an important role in various MSCs cellular therapies.     

Role for interferon-gamma in the immunomodulatory activity of human bone marrow mesenchymal stem cells

Mesenchymal stem cells (MSCs) inhibit the proliferation of HLA-unrelated T lymphocytes to allogeneic stimulation, but the mechanisms responsible for this activity are not fully understood. We show here that MSCs suppress the proliferation of both CD4+ and CD8+ T lymphocytes, as well as of natural killer (NK) cells, whereas they do not have an effect on the proliferation of B lymphocytes. The antiproliferative effect of MSCs was not associated with any effect on the expression of cell-activation markers, induction of cell apoptosis, or mimicry/enhancement of T regulatory cell activity. The suppressive activity of MSCs was not contact-dependent and required the presence of interferon (IFN)-gamma produced by activated T cells and NK cells. Accordingly, even activated B cells became susceptible to the suppressive activity of MSCs in the presence of exogenously added IFN-gamma. The suppressive effect of IFN-gamma was related to its ability to stimulate the production by MSCs of indoleamine 2,3-dioxygenase activity, which in turn inhibited the proliferation of activated T or NK cells. These findings suggest that the beneficial effect on graft-versus-host disease induced by in vivo coinfusion with the graft of MSCs may be due to the activation of the immunomodulatory properties of MSCs by T cell- derived IFN-gamma.     

Autocrine fibroblast growth factor 2 increases the multipotentiality of human adipose-derived mesenchymal stem cells

Multipotent mesenchymal stem cells (MSCs), first identified in the bone marrow, have subsequently been found in many other tissues, including fat, cartilage, muscle, and bone. Adipose tissue has been identified as an alternative to bone marrow as a source for the isolation of MSCs, as it is neither limited in volume nor as invasive in the harvesting. This study compares the multipotentiality of bone marrow-derived mesenchymal stem cells (BMSCs) with that of adipose-derived mesenchymal stem cells (AMSCs) from 12 age- and sex-matched donors. Phenotypically, the cells are very similar, with only three surface markers, CD106, CD146, and HLA-ABC, differentially expressed in the BMSCs. Although colony-forming units-fibroblastic numbers in BMSCs were higher than in AMSCs, the expression of multiple stem cell-related genes, like that of fibroblast growth factor 2 (FGF2), the Wnt pathway effectors FRAT1 and frizzled 1, and other self-renewal markers, was greater in AMSCs. Furthermore, AMSCs displayed enhanced osteogenic and adipogenic potential, whereas BMSCs formed chondrocytes more readily than AMSCs. However, by removing the effects of proliferation from the experiment, AMSCs no longer out-performed BMSCs in their ability to undergo osteogenic and adipogenic differentiation. Inhibition of the FGF2/fibroblast growth factor receptor 1 signaling pathway demonstrated that FGF2 is required for the proliferation of both AMSCs and BMSCs, yet blocking FGF2 signaling had no direct effect on osteogenic differentiation. Disclosure of potential conflicts of interest is found at the end of this article.     

Mesenchymal stem cells derived from bone marrow favor tumor cell growth in vivo

Mesenchymal stem cells (MSCs) have generated a great deal of interest in clinical application because of their potential use in regenerative medicine and tissue engineering. However, the therapeutic application of MSCs still remain limited unless the favorable effect of MSCs for tumor growth in vivo and the long-term safety of the clinical applications of MSCs are better understood. In this study, MSCs derived from fetal bone marrow (FMSCs) and adult MSCs (AMSCs) alone or FMSCs and AMSCs with tumor cell line (F6 or SW480) together were transplanted subcutaneously into BALB/c-nu/nu mice to observe the outgrowth of tumor, and the characteristics of tumor cells were investigated by pathological and immunohistochemical methods, flow cytometry and real-time quantitative PCR. The results showed that both FMSCs and AMSCs could favor tumor growth in vivo. The pathologic examination revealed that tumor tissues had rich vessel distribution, extensive necrosis and invasion surrounding normal tissues, such as muscular tissue and subcutaneous tissue. In the immunohistochemical examination, tumor cells mixed with MSCs transplanted subcutaneously exhibited elevated capability of proliferation, rich angiogenesis in tumor tissues and highly metastatic ability. To understand whether MSCs affected the general properties of the tumor cells in vivo, the expression of some surface antigens and Bmi-1 gene of tumor tissue cells was detected in this study. The results indicated that these parameters were not affected after the interaction of MSCs with tumor cells in vivo. These findings suggested that MSCs could favor tumor growth in vivo. It is necessary to carry out a study for assurance of the long-term safety before MSCs were used as a therapy tools in regenerative medicine and tissue engineering.     

3种不同来源CD146阳性间充质干细胞亚群的生物学特性比较

目的比较脐带间充质干细胞(UCMSCs)、脂肪间充质干细胞(AMSCs)以及骨髓间充质干细胞(BM-MSCs)中CD146^+细胞亚群的生物学特性。方法用磁珠分选法分选不同组织来源的间充质干细胞,获得高纯度CD146^+亚群;用流式细胞计量术分析表型;透射电子显微镜观察细胞结构;成脂诱导分化后油红O染色;RT-qPCR检测成脂相关基因LPL、C/EBPα和PPARγ表达;成骨诱导分化后ALP染色,检测成骨相关基因ALP、OPN和RUNX2表达;检测细胞干性基因及血管生成相关基因表达;检测细胞体外成管能力。结果3种组织来源CD146^+MSCs具有相似的形态,表达除CD106外相似的细胞表面标志分子。与其他两种细胞比较,CD146^+AMSCs表达更高的干性基因OCT-4、SOX2和NANOG。在相同的诱导时间,CD146^+UCMSCs成脂和成骨能力较其他两种来源的CD146^+MSCs弱。3种组织来源的CD146^+MSCs均可表达血管内皮相关刺激因子BFGF、VEGF、Ang-1和EGF,但CD146^+BM-MSCs具有更强的体外成管能力。结论3种不同来源的CD146^+MSC具有不同的生物学特性,为进一步研究不同组织来源的间充质干细胞的特定应用提供了基础。     

Murine Mesenchymal Stem Cells Suppress T Lymphocyte Activation Through IL-2 Receptor α (CD25) Cleavage by Producing Matrix Metalloproteinases

Mesenchymal stem cells (MSCs) are multipotent, non-hematopoietic stem cells that exhibit the capacity to inhibit the proliferation of a variety of immune cells. However, the underlying mechanisms of the immunosuppressive effects of MSCs are still obscure. Therefore, we attempted to identify the mechanisms underlying immunosuppression toward the activated T lymphocytes by MSCs in a murine model. In particular, we aimed to find possible factors derived from MSCs that drive this phenomenon. We found that T lymphocytes incubated with conditioned media of MSCs (MSC CM) entered into apoptosis and were subjected to cell cycle arrest during the course of activation, and these phenomena were accompanied by the reduction of IL-2 production. Specifically, matrix metalloproteinases (MMPs) derived from MSCs caused cleavage of IL-2 receptor α (CD25) from the surface of activated T cells, and as a consequence, IL-2 signaling in response to engagement of the IL-2 receptor (IL-2R) was downregulated. The inhibition of MMP activity in the MSC CM by GM6001 abrogated CD25 cleavage and restored IL-2 production from the activated splenocytes. However, the blockade of MMP activity could not fully restore the proliferative response and apoptosis of T cells altered by MSC CM. In conclusion, MSC-derived MMPs have a significant role in the suppression of IL-2 production through induction of CD25 cleavage and have a partial role in the suppression of T cell proliferation.     

Poly I:C primes the suppressive function of human palatine tonsil-derived MSCs against Th17 differentiation by increasing PD-L1 expression

It has been established that mesenchymal stem cells (MSCs) can have a suppressive effect on T cells, yet much remains unknown about the underlying mechanisms that support this effect. The T cell co-stimulatory pathway involving the programmed death-1 (PD-1) receptor and its ligand PD-L1 regulates T cell activation, tolerance, and subsequent immune-mediated tissue damage. In this study, human palatine tonsil-derived MSCs (T-MSCs) constitutively expressed PD-L1 and exhibited a suppressive activity that specifically targeted murine Th17 differentiation. Additionally, polyinosinic–polycytidylic acid (poly I:C), a Toll-like receptor 3 (TLR3) ligand, increased PD-L1 expression on T-MSCs. The elevated PD-L1 levels enhanced the suppressive functions of T-MSCs on Th17 differentiation. Therefore, pre-stimulation of T-MSCs with poly I:C may serve as an effective therapeutic priming step for modulating Th17-dominant immune responses.     

慢性粒细胞白血病肿瘤干细胞的免疫调节功能

目的 研究慢性粒细胞白血病(CML)骨髓来源的肿瘤干细胞的免疫学特征,比较其与正常人来源的间充质干细胞(MSC)是否存在免疫功能的异常.方法 分离正常人和CML患者骨髓中的MSC,MLR法检测其对T细胞增殖的影响,流式细胞术检测其对T细胞周期、凋亡的作用情况.结果 CML和正常志愿者骨髓来源的MSC的细胞形态和表型没有差异,CML患者来源的MSC抑制T细胞增殖能力和抑制T细胞停留在G0/G1期的作用均减弱(CML MSC组74.5%±1.2%,BMSC组94.0%±1.9%,P＜0.05),CML患者抑制T细胞凋亡的作用增强(CMLMSC组8.36%±1.31%,BMSC组14.10%±0.65%,P＜0.05).结论 CML患者骨髓来源的MSC存在明显的免疫调节功能缺陷,如果使用CML患者自体的MSC移植治疗可能不是一种很好的选择,对于CML患者最好是选用异基因的MSC移植.