Impairment of endothelial cell differentiation from bone marrow-derived mesenchymal stem cells: new insight into the pathogenesis of systemic sclerosis

OBJECTIVE: Systemic sclerosis (SSc) is a disorder characterized by vascular damage and fibrosis of the skin and internal organs. Despite marked tissue hypoxia, there is no evidence of compensatory angiogenesis. The ability of mesenchymal stem cells (MSCs) to differentiate into endothelial cells was recently demonstrated. The aim of this study was to determine whether impaired differentiation of MSCs into endothelial cells in SSc might contribute to disease pathogenesis by decreasing endothelial repair. METHODS: MSCs obtained from 7 SSc patients and 15 healthy controls were characterized. The number of colony-forming unit-fibroblastoid colonies was determined. After culture in endothelial-specific medium, the endothelial-like MSC (EL-MSC) phenotype was assessed according to the surface expression of vascular endothelial growth factor receptors (VEGFRs). Senescence, chemoinvasion, and capillary morphogenesis studies were also performed. RESULTS: MSCs from SSc patients displayed the same phenotype and clonogenic activity as those from controls. In SSc MSCs, a decreased percentage of VEGFR-2+, CXCR4+, VEGFR-2+/CXCR4+ cells and early senescence was detected. After culturing, SSc EL-MSCs showed increased expression of VEGFR-1, VEGFR-2, and CXCR4, did not express CD31 or annexin V, and showed significantly decreased migration after specific stimuli. Moreover, the addition of VEGF and stromal cell-derived factor 1 to cultured SSc EL-MSCs increased their angiogenic potential less than that in controls. CONCLUSION: Our data strongly suggest that endothelial repair may be affected in SSc. The possibility that endothelial progenitor cells could be used to increase vessel growth in chronic ischemic tissues may open up new avenues in the treatment of vascular damage caused by SSc.     

Interferon‐γ–dependent inhibition of B cell activation by bone marrow–derived mesenchymal stem cells in a murine model of systemic lupus erythematosus

Objective

Bone marrow–derived mesenchymal stem cells (BM‐MSCs) are multipotent cells characterized by immunomodulatory properties and are therefore considered a promising tool for the treatment of immune‐mediated diseases. This study was undertaken to assess the influence of murine BM‐MSCs on the activation of B cells in (NZB × NZW)F₁ mice as an animal model of systemic lupus erythematosus (SLE).

Methods

We evaluated the in vitro effects of BM‐MSCs on the proliferation and differentiation to plasma cells of splenic mature B cell subsets, namely follicular and marginal zone B cells isolated from (NZB × NZW)F₁ mice. Lupus mice were also treated with BM‐MSCs, and serum autoantibodies, proteinuria, histologic changes in the kidney, and survival rates were monitored.

Results

BM‐MSCs inhibited antigen‐dependent proliferation and differentiation to plasma cells of follicular and marginal zone B cells in vitro. This inhibitory effect was dependent on interferon‐γ (IFNγ) and was mediated by cell‐to‐cell contact, involving the programmed death 1 (PD‐1)/PD ligand pathway. In vivo treatment with BM‐MSCs did not affect the levels of anti–double‐stranded DNA antibodies or proteinuria. However, a reduction in glomerular immune complex deposition, lymphocytic infiltration, and glomerular proliferation was observed.

Conclusion

Our findings indicate that BM‐MSCs affect B cell receptor–dependent activation of both follicular and marginal zone B cells from lupus mice. This inhibitory effect is IFNγ‐dependent and cell contact–dependent. MSCs in vivo do not affect the production of autoantibodies, the level of proteinuria, or the mortality rates. Nonetheless, the significant improvement in histologic findings in the kidney supports the potential role of MSCs in the prevention of glomerular damage.

     

兔骨髓间充质干细胞的鉴定及向血管内皮细胞的分化

目的探要诱导骨髓间充质干细胞(BMSCs)分化为血管内皮细胞(VECs)可行性,并比较传代对诱导率的影响。方法采用密度梯度离心法分离BMSCs,体外培养扩增并做形态学、细胞表型和成骨成脂诱导鉴定。细胞纯化后行内皮化诱导,诱导组分A、B两组,C组为对照组,A组于诱导第15天传代,B组常规诱导,24 d后终止培养,从形态学、CD31流式分析及一氧化氮(NO)分泌量检测三方面进行鉴定和比较。结果经鉴定,分离出来的细胞为纯度较高分化能力良好的BMSCs,内皮化诱导后,细胞呈现典型的内皮细胞形态,CD31和NO分泌量的检测证实诱导后的细胞为具有活性的内皮细胞,并且A组的诱导率高于B组。结论 BMSCs可以被诱导为VECs,诱导过程中传代可以提高诱导率。     

CCN 3对骨髓间充质干细胞向内皮细胞分化的影响

目的：探讨肾母细胞瘤过度表达基因（CCN3）对骨髓间充质干细胞（BM-MSCs）向内皮细胞分化的影响。方法：以含50ng/ml血管内皮生长因子培养基诱导 BM-MSCs向内皮细胞分化作为阳性对照组,以含100ng/ml重组CCN3蛋白并50ng/ml血管内皮生长因子培养基诱导BM-MSCs向内皮细胞分化作为CCN3组,并以单纯完全培养基培养BM-MSCs为阴性对照组,采用细胞免疫荧光化学法和半定量逆转录聚合酶链反应法（RT-PCR）检查诱导16 d后假性血友病因子（vWF）表达以评价内皮细胞分化,以半定量 RT-PCR法检测 BM-MSCs 诱导分化前后Notch1基因mRNA表达。结果：BM-MSCs 诱导分化16d 后,阳性对照组可见部分细胞 vWF 荧光染色阳性, CCN3组阳性染色细胞明显多于阳性对照组,且 vWF mRNA 表达明显强于阳性对照组[吸光度（OD）值比：（0.550±0.090）比（0.358±0.080）,P＜0.01],阴性对照组未见阳性染色细胞;此外,诱导分化前,三组Notch1基因mRNA均呈低表达,组间两两比较无明显差异（P 均＞0.05）,诱导分化16d后阳性对照组和 CCN3组 Notch1基因 mRNA表达明显强于阴性对照组[OD值比：（0.232±0.047）、（0.352±0.029）比（0.132±0.033）,P均＜0.01],但与阳性对照组相比,CCN3组 Notch1基因 mRNA表达明显更高（P＜0.01）。结论：肾母细胞瘤过度表达基因通过激活 Notch1信号通路可增强骨髓间充质干细胞向内皮细胞分化的能力。     

兔骨髓间充质干细胞体外成内皮细胞能力的研究

目的　探讨骨髓间充质干细胞体外扩增及其向内皮细胞定向分化能力。方法　无菌条件下采集兔骨髓 ,肝素抗凝 ,经淋巴细胞分层液密度梯度离心分离骨髓单个核细胞 ,通过贴壁培养法获得MSC ,然后在内皮细胞生长环境中进行诱导分化 ,最后对扩增的细胞特征进行鉴定 ,同时初步研究其体外成血管特性。结果　MSC每扩增一代 ,细胞数量增加 5～ 8倍 ,7.6 5× 10 3 个原代MSCs体外扩增 3代即获得 1.2 6× 10 8个细胞。在 70 %～80 %融合时呈现“铺路石”样形态 ,细胞免疫组化证实扩增细胞表达CD31和vonWillebrandfactor(vWF) ,具有吞噬ac LDL的功能 ,且扩增细胞在体外参与网状血管样结构形成。结论　MSC扩增能力强 ,在体外能诱导其向内皮细胞方向分化。     

Insights into the pathogenesis of systemic sclerosis based on the gene expression profile of progenitor‐derived endothelial cells

Objective

To determine the gene expression profile of endothelial cells derived from the endothelial progenitor cells (EPCs) of patients with systemic sclerosis (SSc).

Methods

Microarray experiments were performed on Affymetrix GeneChip Human Exon 1.0 ST Arrays in unstimulated and hypoxia‐stimulated EPC‐derived cells from patients with SSc and control subjects. Followup of the raised hypotheses was performed ex vivo by immunohistochemical analysis of skin tissue.

Results

Signals from 92 probe sets and 188 probe sets were different in unstimulated and hypoxia‐stimulated cells, respectively, from patients with SSc compared with controls. Within the largest groups of genes related to cell–cell interaction and vascular remodeling, down‐regulation of tumor necrosis factor ligand superfamily member 10 (TNFSF10) and homeobox A9 (HOX‐A9) was confirmed by real‐time polymerase chain reaction and Western blots in EPC‐derived cells and by immunohistochemistry in SSc skin tissue. Signals from 221 and 307 probe sets were different in unstimulated and hypoxia‐stimulated cells, respectively, from patients with diffuse cutaneous SSc compared with patients with limited cutaneous SSc. Within the largest group of genes related to the inflammatory response, differential expression of TNFα‐induced protein 3 and prostaglandin‐endoperoxide synthase 2 was observed in EPC‐derived cells and skin tissue from patients with SSc.

Conclusion

Our data revealed important gene expression changes in EPC‐derived endothelial cells from patients with SSc, characterized by a proadhesive, proinflammatory, and activated phenotype. Differential expression in lesional SSc skin tissue of new targets, such as TNF family members and HOX‐A9, may contribute to the pathogenesis of SSc and deserves more in‐depth exploration.

     

成人骨髓间充质干细胞体外诱导分化为神经元样细胞的研究

目的 研究成人骨髓间充质干细胞(hMSC)体外定向诱导可否分化为神经元样细胞。方法 Percoll分离液离心分离hMSC，体外扩增，采用两种方法：①含碱性成纤维细胞生长因子(bFGF)预诱导24小时，甲氧酚(BHA)和二甲亚枫(DMSO)联合诱导6小时；②2-巯基乙醇(2-ME)预诱导24小时，BHA和DMSO诱导6小时。免疫组化检测神经元样细胞表达神经元烯醇化酶(NSE)、神经丝蛋白(NF)、胶质纤维酸性蛋白(GFAP)。结果 hMSC在体外扩增传至5代后，流式细胞仪显示hMSC表面抗原CD29、CD44、CD90表达阳性率分别为99．5％，97．8％，98．8％。采用两种方法诱导hMSC后，胞体收缩，突起伸出，较密的部分神经元拉成网状；免疫组化显示诱导的神经元样细胞能特异性表达NSE、NF，不表达GFAP。结论 成人骨髓hMSC在体外可以分化为神经元样细胞。     

骨髓间充质干细胞体外定向诱导内皮祖细胞的实验研究

目的:研究兔骨髓间充质干细胞(mensenchymal stem cell,MSC)在体外定向诱导分化为内皮祖细胞(endothelial progenitor cell,EPC)的状况。方法:取兔髂骨骨髓,经密度梯度离心法分离并培养骨髓间充质干细胞,实验组细胞在内皮细胞生长添加剂、血管内皮生长因子、成纤维细胞生长因子等诱导条件下定向分化为内皮祖细胞。对照组不干预,继续培养。结果:实验组细胞集落接近融合时形态呈现“鹅卵石”样或“铺路石”样外观,流式细胞仪检测发现实验组细胞较对照组高度表达CD133、CD34(P<0．05)。结论:体外诱导可使骨髓间充质干细胞分化为内皮祖细胞,并有效扩增。     

Treatment of murine lupus using nucleosomal T cell epitopes identified by bone marrow–derived dendritic cells

Objective

Systemic lupus erythematosus (SLE) is characterized by the existence of a heterogeneous group of autoantibodies directed against intact nuclear structures, such as nucleosomes. The most prominent of these autoantibodies are those directed against double‐stranded DNA (dsDNA) and histones. The majority are of the IgG isotype and show affinity maturation, both of which are known hallmarks of T cell help. Much evidence suggests that the nucleosome is a major candidate autoantigen in SLE. In this study, a novel strategy was used to identify the critical CD4+ T cell autoepitopes in nucleosomes. In addition, peptide‐based therapy was then performed in a lupus animal model.

Methods

Bone marrow (BM)–derived dendritic cells (DCs) were used to examine the self–T cell responses against nucleosomes and to characterize the T cell epitope(s) of nucleosomes in (NZB × NZW)F₁ (BWF₁) mice.

Results

Several potential auto–T cell epitopes of core histone proteins (H2A, H2B, H3, and H4) were identified. Nucleosome‐pulsed BM‐derived DCs elicited release of interleukin‐4 and interferon‐γ, representing a Th0 (i.e., mixed Th1 and Th2) pattern of cytokine production. In addition, intradermal immunization of BWF₁ mice with the H3^111–130 peptide not only suppressed the level of anti‐dsDNA and anti–single‐stranded DNA IgG, but also significantly delayed the progress of glomerulonephritis in lupus‐prone BWF₁ mice.

Conclusion

These results will help in understanding how pathogenic autoimmune responses develop in spontaneous SLE. This may potentially open the way to T cell–based immunotherapy for lupus.

     

骨髓间充质干细胞向肝细胞的诱导分化

目的:探索肝细胞生长因子(HGF)、制瘤素M(OSM)在体外诱导骨髓间充质干细胞(MSCs)向肝细胞分化的能力及效果.方法:分离、培养大鼠MSCs,取第3代按以下分组诱导其向肝细胞分化:A组:低糖杜氏改良培养基(DMEM-LG) 100 mL/L胎牛血清(fetal calf,serum,FCS);B组:肝细胞生长培养基(HGM);C组:HGM 20μg/L HGF;D组:HGM 20μg/L OSM;E组:HGM 20μg/L HGF 20μg/L OSM,于不同时间点用免疫细胞化学检测甲胎蛋白(AFP)、细胞角蛋白18(CK18)表达,高碘酸-希夫氏(PAS)染色检测糖原表达,谷氨酰胺脱氢酶法检测上清液尿素含量.结果:C,E组于诱导第7天出现AFP阳性表达,以后其阳性表达率随诱导时间延长逐渐降低,诱导第7天E组阳性表达率高于C组(x~2=6.322,P<0.05).C组、E组分别于诱导第7、14天出现CK18阳性表达,于诱导第7天开始出现糖原阳性表达,随诱导时间延长表达率逐渐增高,同一时间点E组CK18(14 d:x~2=4.811,P<0.05;21 d:x~2=6.902,P<0.01;28 d:x~2=5.771,P<0.05)及糖原(14 d:x~2=6.902,P<0.01;21 d:x~2=6.818,P<0.01;28 d:x~2=6.818,P<0.01)阳性表达率高于C组.C,E组培养上清液中尿素浓度随诱导时间延长逐渐增高,E组增长幅度比C组高.A,B,D组各时间点均未见AFP、CK18、糖原表达及尿素浓度变化.结论:MSCs具有向肝细胞分化的能力,HGF能够诱导MSCs分化肝样细胞,OSM与HGF联合使用,能促进MSCs的分化,明显提高分化率.     

人骨髓间充质干细胞体外分化为肝细胞样细胞

目的 探讨人骨髓间充质干细胞(MSCs)的体外培养及特异性诱导为肝细胞样细胞的能力。方法 骨髓标本来源于健康志愿者的胸骨,年龄2～35岁,采用淋巴细胞分离液(密度1.077)分离人MSCs,并分别采用HGF、FGF4、HGF FGF4以及无生长因子四种处理因素体外诱导第三代人MSC向肝细胞样细胞分化。通过流式细胞术分析鉴定.MSCs的纯度,于诱导培养的0、7、14、21、28天时留取细胞检测CK18、AFP和白蛋白的表达情况,同时进行糖原染色验证细胞功能。结果 用淋巴细胞分离液分离出的人MSCs纯度可达90％,采用HGF、FGF4及HGF FGF4三种处理因素均可在体外诱导人MSCs特异分化为具有肝细胞样细胞表型和功能的细胞。结论 人MSCs能在体外扩增并定向诱导为肝细胞样细胞。     

Hepatogenic differentiation of human mesenchymal stem cells from adipose tissue in comparison with bone marrow mesenchymal stem cells

INTRODUCTIONMost liver diseases lead to hepatocyte dysfunction with the possibility of eventual organ failure. The replacement of diseased hepatocytes and the stimulation of endogenous or exogenous regeneration by stem cells are the main aims of liver-dir…