生长因子联合诱导大鼠间充质干细胞向软骨分化的实验研究

目的探索在体外培养条件下，转化生长因子（TGF）-β1、胰岛素样生长因子（IGF）-Ⅰ和骨形成蛋白（BMP）-2诱导间充质干细胞（MSCs）向软骨细胞分化的最佳组合。方法在体外单层培养条件下，利用TGF-β1、IGF-Ⅰ、BMP-2的组合作为诱导因子进行培养，用反转录聚合酶链反应（RT—PCR）检测细胞表达Ⅱ型前胶原mRNA水平，阿新蓝染色检测蛋白聚糖合成情况。结果BMP-2＋IGF-Ⅰ组、TGF-β1组、TGF-β1＋IGF-Ⅰ组、TGF-β1＋BMP-2组、TGF-β1＋IGF-Ⅰ＋BMP-2组Ⅱ型前胶原mRNA水平分别为0．147±0．062、0．478±0．069、0．489±0．022、0．653±0．013、0．807±0．051；而各组阿新蓝染色均阳性。结论TGF-β1、IGF-Ⅰ、BMP-2三者合用时诱导MSCs向软骨分化的效应最大。     

Premature induction of hypertrophy during in vitro chondrogenesis of human mesenchymal stem cells correlates with calcification and vascular invasion after ectopic transplantation in SCID mice

OBJECTIVE: Functional suitability and phenotypic stability of ectopic transplants are crucial factors in the clinical application of mesenchymal stem cells (MSCs) for articular cartilage repair, and might require a stringent control of chondrogenic differentiation. This study evaluated whether human bone marrow-derived MSCs adopt natural differentiation stages during induction of chondrogenesis in vitro, and whether they can form ectopic stable cartilage that is resistant to vascular invasion and calcification in vivo. METHODS: During in vitro chondrogenesis of MSCs, the expression of 44 cartilage-, stem cell-, and bone-related genes and the deposition of aggrecan and types II and X collagen were determined. Similarly treated, expanded articular chondrocytes served as controls. MSC pellets were allowed to differentiate in chondrogenic medium for 3-7 weeks, after which the chondrocytes were implanted subcutaneously into SCID mice; after 4 weeks in vivo, samples were evaluated by histology. RESULTS: The 3-stage chondrogenic differentiation cascade initiated in MSCs was primarily characterized by sequential up-regulation of common cartilage genes. Premature induction of hypertrophy-related molecules (type X collagen and matrix metalloproteinase 13) occurred before production of type II collagen and was followed by up-regulation of alkaline phosphatase activity. In contrast, hypertrophy-associated genes were not induced in chondrocyte controls. Whereas control chondrocyte pellets resisted calcification and vascular invasion in vivo, most MSC pellets mineralized, in spite of persisting proteoglycan and type II collagen content. CONCLUSION: An unnatural pathway of differentiation to chondrocyte-like cells was induced in MSCs by common in vitro protocols. MSC pellets transplanted to ectopic sites in SCID mice underwent alterations related to endochondral ossification rather than adopting a stable chondrogenic phenotype. Further studies are needed to evaluate whether a more stringent control of MSC differentiation to chondrocytes can be achieved during cartilage repair in a natural joint environment.     

Inhibition of P-glycoprotein facilitated glycosaminoglycan accumulation during chondrogenesis of human bone marrow mesenchymal stem cells

Aim: P‐glycoprotein (P‐gp) is an adenosine‐5‐triphosphate Binding Cassettes B 1 (ABCB1) transporter that exports various substrates on cellular membrane. Surface expression of P‐gp was decreased during chondrogenesis of human bone marrow mesenchymal stem cells (BM‐MSCs). We examined the role of P‐gp in extracellular matrix deposition during chondrogenesis of human BM‐MSCs. Method: BM‐MSCs were isolated from 16 volunteers after informed consent and incubated for 28 days using three‐dimensional culture methods in chondrogenic medium with and without P‐gp inhibitor (verapamil, 10 μmol/L). Results: Hematoxylin and eosin staining revealed a cartilaginous structure with chondrogenic cells in the lacunae after 2 weeks of culture. Total glycosaminoglycan (GAG) content was increased and rose during pellet culture. Hyaluronan (HA) content of the culture medium decreased with P‐gp inhibitor. Type II collagen deposition decreased with P‐gp inhibitor. Conclusion: Inhibition of P‐gp facilitated GAG accumulation via HA export inhibition during chondrogenic differentiation of human BM‐MSCs. Modulation of P‐gp expression during chondrogenesis would be a possible therapeutic approach for articular cartilage regeneration.     

Association between the abnormal expression of matrix-degrading enzymes by human osteoarthritic chondrocytes and demethylation of specific CpG sites in the promoter regions

OBJECTIVE: To investigate whether the abnormal expression of matrix metalloproteinases (MMPs) 3, 9, and 13 and ADAMTS-4 by human osteoarthritic (OA) chondrocytes is associated with epigenetic "unsilencing." METHODS: Cartilage was obtained from the femoral heads of 16 patients with OA and 10 control patients with femoral neck fracture. Chondrocytes with abnormal enzyme expression were immunolocalized. DNA was extracted, and the methylation status of the promoter regions of MMPs 3, 9, and 13 and ADAMTS-4 was analyzed with methylation-sensitive restriction enzymes, followed by polymerase chain reaction amplification. RESULTS: Very few chondrocytes from control cartilage expressed the degrading enzymes, whereas all clonal chondrocytes from late-stage OA cartilage were immunopositive. The overall percentage of non-methylated sites was increased in OA patients (48.6%) compared with controls (20.1%): 20% versus 4% for MMP-13, 81% versus 47% for MMP-9, 57% versus 30% for MMP-3, and 48% versus 0% for ADAMTS-4. Not all CpG sites were equally susceptible to loss of methylation. Some sites were uniformly methylated, whereas in others, methylation was generally absent. For each enzyme, there was 1 specific CpG site where the demethylation in OA patients was significantly higher than that in controls: at -110 for MMP-13, -36 for MMP-9, -635 for MMP-3, and -753 for ADAMTS-4. CONCLUSION: This study provides the first evidence that altered synthesis of cartilage-degrading enzymes by late-stage OA chondrocytes may have resulted from epigenetic changes in the methylation status of CpG sites in the promoter regions of these enzymes. These changes, which are clonally transmitted to daughter cells, may contribute to the development of OA.     

Human articular chondrocytes secrete parathyroid hormone–related protein and inhibit hypertrophy of mesenchymal stem cells in coculture during chondrogenesis

Objective

The use of bone marrow–derived mesenchymal stem cells (MSCs) has shown promise in cell‐based cartilage regeneration. A yet‐unsolved problem, however, is the unwanted up‐regulation of markers of hypertrophy, such as alkaline phosphatase (AP) and type X collagen, during in vitro chondrogenesis and the formation of unstable calcifying cartilage at heterotopic sites. In contrast, articular chondrocytes produce stable, nonmineralizing cartilage. The aim of this study was to address whether coculture of MSCs with human articular chondrocytes (HACs) can suppress the undesired hypertrophy in differentiating MSCs.

Methods

MSCs were differentiated in chondrogenic medium that had or had not been conditioned by parallel culture with HAC pellets, or MSCs were mixed in the same pellet with the HACs (1:1 or 1:2 ratio) and cultured for 6 weeks. Following in vitro differentiation, the pellets were transplanted into SCID mice.

Results

The gene expression ratio of COL10A1 to COL2A1 and of Indian hedgehog (IHH) to COL2A1 was significantly reduced by differentiation in HAC‐conditioned medium, and less type X collagen protein was deposited relative to type II collagen. AP activity was significantly lower (P < 0.05) in the cells that had been differentiated in conditioned medium, and transplants showed significantly reduced calcification in vivo. In mixed HAC/MSC pellets, suppression of AP was dose‐dependent, and in vivo calcification was fully inhibited. Chondrocytes secreted parathyroid hormone–related protein (PTHrP) throughout the culture period, whereas PTHrP was down‐regulated in favor of IHH up‐regulation in control MSCs after 2–3 weeks of chondrogenesis. The main inhibitory effects seen with HAC‐conditioned medium were reproducible by PTHrP supplementation of unconditioned medium.

Conclusion

HAC‐derived soluble factors and direct coculture are potent means of improving chondrogenesis and suppressing the hypertrophic development of MSCs. PTHrP is an important candidate soluble factor involved in this effect.

     

Association between the abnormal expression of matrix‐degrading enzymes by human osteoarthritic chondrocytes and demethylation of specific CpG sites in the promoter regions

Objective

To investigate whether the abnormal expression of matrix metalloproteinases (MMPs) 3, 9, and 13 and ADAMTS‐4 by human osteoarthritic (OA) chondrocytes is associated with epigenetic “unsilencing.”

Methods

Cartilage was obtained from the femoral heads of 16 patients with OA and 10 control patients with femoral neck fracture. Chondrocytes with abnormal enzyme expression were immunolocalized. DNA was extracted, and the methylation status of the promoter regions of MMPs 3, 9, and 13 and ADAMTS‐4 was analyzed with methylation‐sensitive restriction enzymes, followed by polymerase chain reaction amplification.

Results

Very few chondrocytes from control cartilage expressed the degrading enzymes, whereas all clonal chondrocytes from late‐stage OA cartilage were immunopositive. The overall percentage of nonmethylated sites was increased in OA patients (48.6%) compared with controls (20.1%): 20% versus 4% for MMP‐13, 81% versus 47% for MMP‐9, 57% versus 30% for MMP‐3, and 48% versus 0% for ADAMTS‐4. Not all CpG sites were equally susceptible to loss of methylation. Some sites were uniformly methylated, whereas in others, methylation was generally absent. For each enzyme, there was 1 specific CpG site where the demethylation in OA patients was significantly higher than that in controls: at −110 for MMP‐13, −36 for MMP‐9, −635 for MMP‐3, and −753 for ADAMTS‐4.

Conclusion

This study provides the first evidence that altered synthesis of cartilage‐degrading enzymes by late‐stage OA chondrocytes may have resulted from epigenetic changes in the methylation status of CpG sites in the promoter regions of these enzymes. These changes, which are clonally transmitted to daughter cells, may contribute to the development of OA.

     

肝癌细胞中OY-TES-1基因启动子的甲基化状态

目的:研究肝癌细胞株OY-TES-1启动子的甲基化状态及甲基转移酶抑制剂对其启动子甲基化的影响.方法:运用生物信息学在线软件预测OY-TES-1启动子区域及转录因子结合位点;应用亚硫酸氢盐测序法(bisulfite-sequencing PCR,BSP)分析目的基因CpG位点的甲基化状态;用甲基转移酶抑制剂5-Aza-...     

体外诱导间充质干细胞向心肌细胞方向分化的研究

目的证实来源于胎儿肝脏的间充质干细胞(FMSCs)具有向心肌细胞方向分化的潜能。方法取6~9代细胞,用不同浓度的诱导剂组合诱导细胞,分别置于不同的条件下进行培养,包括不同温度、不同氧浓度及不同培养液。结果在使用心肌分化培养液及常规培养条件下(37℃、20%O2),5-氮胞苷(50μmol/L)、维甲酸(10-3μmol/L)、二甲基亚砜(0.8%)的联合应用,诱导了FMSCs发生向心肌方向的分化。分化细胞呈小圆形细胞,具有相互聚集并形成球样细胞团结构的趋势,同时表达结蛋白及心肌肌钙蛋白Ⅰ。结论FMSCs具有向心肌细胞分化的潜能,FMSCs发生向心肌方向的分化所需条件与来源于其他动物种属的间充质干细胞的分化条件不同。     

Co-culture of hematopoietic stem cells with mesenchymal stem cells increases VCAM-1-dependent migration of primitive hematopoietic stem cells

Hematopoietic stem cells (HSC) lose their capacity for engraftment during ex vivo cytokine expansion. It has been shown that mesenchymal stem cells (MSC) improve HSC transplantability; however, the molecular mechanisms responsible for this effect have not yet been completely elucidated. This paper reports that expanding HSC in co-culture with MSC enhances a vascular cell adhesion molecule (VCAM-1)-dependent pro-migratory phenotype. MSC did not regulate the HSC expression of CD49d (VCAM-1 counter-receptor molecule), but did decrease the cytokine-induced HSC VCAM-1-mediated pro-adhesive phenotype. Co-culture with MSC reduced the expression of the inactive conformation of lymphocyte function-associated antigen (LFA-1) at the HSC uropod, and induced higher expression of an LFA-1 activation epitope. Interestingly, VCAM-1-dependent HSC migration was modulated by targeting this LFA-1 high affinity form, suggesting integrin cross-regulation. VCAM-1-mediated HSC transmigration appeared to favor the more primitive HSC immunophenotype. Our results suggested that co-culture with MSC improved VCAM-1-dependent migration of primitive HSC, which was affected in ex vivo cytokine-expanded HSCs by a mechanism involving LFA-1 modulation.     

心肌细胞裂解液诱导骨髓间充质干细胞向心肌细胞的分化作用

目的:用心肌细胞裂解液体外诱导分化骨髓间充质干细胞(SMCs),观察SMCs能否表达脑钠肽(BNP)及β1受体mRNA,是否具有受体后信号转导通路.方法:分离新生乳鼠的心肌细胞并制成心肌细胞裂解液,自成年小鼠长骨骨髓中分离MSCs,并用含有心肌细胞裂解液的培养基培养1周.用逆转录聚合酶链反应方法定性检测MSCs中BNP及β1受体mRNA水平,用放射免疫方法测定MSCs中环磷酸腺苷(cAMP)的含量.结果:诱导组SMCs能表达BNP及β1受体mRNA,对照组不表达BNP和β1受体mRNA.诱导的SMCs经10-8,10-7,10-6,10-5mol/L异丙肾上腺素(ISO)作用2 h后,均能增加细胞内cAMP含量,且具有ISO浓度依赖性(P＜0.05或P＜0.01).诱导的SMCs经不同浓度的美托洛尔(MET)10-6,10-5 mmol/L作用10 min后,和1×10-7mol/L ISO再一起作用110 min后,测定细胞内cAMP含量与空白组比较,差异无统计学意义;与10-7mol/L ISO作用结果比较,差异有统计学意义(P＜0.01),表明MET能完全阻断ISO升高细胞内cAMP作用.结论:心肌细胞裂解液能体外模拟心肌微环境诱导分化SMCs表达β1受体及BNP mRNA,并具有受体后活性的信号转导通路.     

白细胞介素-10基因修饰对骨髓间充质干细胞白细胞介素-10表达及增殖能力的影响

我们对白细胞介素-10(IL-10)转染对骨髓间充质干细胞(BMSC)增殖能力及IL-10表达的影响进行了探讨,以期为进一步研究IL-10修饰的BMSC治疗肝纤维化提供实验基础.     

心肌细胞裂解液对骨髓间充质干细胞向心肌细胞分化诱导作用的研究

目的通过向骨髓间充质干细胞(MSCs)培养体系中添加心肌细胞裂解液的方法,体外模拟心肌微环境,观察MSCs向心肌细胞分化的诱导作用,并与诱导分化剂5-氮杂胞苷(5-aza)比较。方法分离新生乳鼠的心肌细胞并制成心肌细胞裂解液,自成年大鼠骨髓中分离MSCs,用含有心肌细胞裂解液的培养基(A组)、含有5-aza的培养基(B组)、含有5-aza和心肌细胞裂解液的培养基(c组)以及普通培养基(对照组)培养。观察细胞形态的改变,并通过免疫组化分析分化后细胞表达α-肌动蛋白、心脏特异性肌钙蛋白T(cTnT)、连接蛋白43及CD31的情况。结果A、B组的MSCs在培养1周后均形成肌细胞形态,并且均表达α-肌动蛋白和cTnT;A组MSCs分化的肌样细胞所含的肌纤维较B组更丰实,细胞生长趋势也优于B组,并且可以表达CD31;B组MSCs分化的肌样细胞不表达CD31;对照组细胞仅表达α-肌动蛋白。结论心肌细胞裂解液是体外诱导MSCs分化为心肌样细胞的理想条件,优于传统的5-aza,在心肌细胞移植技术中可以用于体外模拟心肌细胞微环境。     

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…     

骨髓间质干细胞体外诱导为心肌细胞的实验研究

目的:探讨骨髓间质干细胞(MSCs)体外诱导分化为心肌细胞的可行性,为心肌梗死治疗提供理想的细胞材料。方法:分离大鼠下肢骨获取MSCs进行培养;5-氮胞苷(5-aza)诱导24h后继续培养;免疫细胞化学检测细胞对连接蛋白-43和α横纹肌肌动蛋白的表达;逆转录-聚合酶链反应(RT-PCR)进一步了解心肌细胞特异性蛋白的表达。结果:MSCs经5-aza诱导后,细胞形态不规则;诱导后3周10μmol/L 5-aza组细胞连接蛋白-43、α横纹肌肌动蛋白表达阳性。RT-PCR显示10μmol/L 5-aza诱导后3周的细胞可表达心肌肌钙蛋白I、α心肌肌动蛋白。结论:MSCs体外经5-aza诱导后可分化为心肌细胞。     

A paradoxical role for IFN-gamma in the immune properties of mesenchymal stem cells during viral challenge

OBJECTIVE: The functional "plasticity" and immune-suppressive effects of human bone marrow (BM)-derived mesenchymal stem cells (MSC) provide them with the potential to be used across allogeneic barriers. The immunosuppressive properties of MSC may be detrimental in a clinical setting in which viral exposure is common. The study hypothesizes that MSC-derived IFN-gamma could offset the immune-suppressive functions of MSC and mediate partial CTL responses during viral infection. METHODS: CTL responses were studied in bioassays with (51)Cr-P815 targets and PBMC (uninfected or infected) as effectors. Immunofluorescence studied the relative expression of CD8(+) cells. Cytokine analyses were performed with microarrays. Roles for IFN-gamma in CTL responses were studied with IFNgammaRI mAb or with MSC knockdown for IFN-gamma by siRNA (pPMSKH1-IFNgamma). RESULTS: MSC showed no significant effect on circulating CTL of healthy subjects. For virus-induced CTL, MSC demonstrated approximately 50% suppression. CD8(+) cell expansion could not explain the suppressive effects of MSC. Soluble factors produced by MSC were responsible for the retention of 50% CTL responses. Cytokine microarray analyses, noncontact cultures, and functional assays identified a role for IFN-gamma. MSC were identified as the relevant source of IFN-gamma. CONCLUSION: The results show a facilitating role of IFN-gamma on CTL responses, although paradoxical in light of the veto properties of MSC. This report shows that in cases where MSC are used in transplantation for repair of damaged tissue, they can exert an additional role by protecting the host to viral challenges and thereby protect from its immunosuppressive properties.