骨髓间充质干细胞向肝细胞分化的相关细胞因子及信号通路的研究进展

骨髓干细胞包括造血干细胞（HSCs）和间充质干细胞（MSCs）,骨髓间充质干细胞（BMSCs）是一类具有自我更新、增殖和多向分化能力的细胞,具有不对称分裂和无限增殖的特点。在肝细胞生长因子（HGF）的作用下,BMSCs可以分化为肝细胞,参与诱导这一分化过程的相关信号通路包括NF-kB信号通路、Notch信号通路、MAPK信号通路、Wnt信号通路和STAT3信号通路。文章主要就BMSCs分化为肝细胞的相关信号通路进行了综述。     

脱细胞神经移植物对骨髓间充质干细胞的诱导分化

目的探讨脱细胞神经移植物诱导大鼠骨髓间充质干细胞分化为施旺细胞样细胞的可行性。方法将分离纯化的SD大鼠骨髓间充质干细胞进行体外培养扩增,行表型鉴定后,取第5代细胞,诱导组采用脱细胞神经移植物匀浆进行诱导,非诱导组加入等量无血清培养基,倒置相差显微镜观察诱导后细胞形态变化,免疫细胞化学染色检测诱导后细胞S-100,神经胶质纤维酸性蛋白(glial fibrillary acidic protein GFAP)的表达情况。结果BMSCs表型鉴定为CD44+、CD54+、CD34-,免疫细胞化学染色GFAP、S-100的阳性表达率分别为为(42±4)%和(64±5)%。结果 脱细胞神经移植物可诱导骨髓间充质干细胞分化为施旺细胞样细胞。     

小鼠骨髓间充质干细胞的培养、鉴定和Survivin的表达研究

为培养及鉴定小鼠来源骨髓间充质干细胞,并测定细胞中Survivin的表达情况,采用全骨髓培养法获取骨髓间充质干细胞,绘制生长曲线,流式细胞仪检测细胞表面标志物,行成骨、成脂检测,RT-PCR测定Survivin表达情况.结果表明培养出的细胞呈长梭状成纤维细胞样,经流式细胞仪检测细胞表面高表达CD29、CD34、CD44、SCA-1,低表达CD117;细胞曲线显示传代细胞培养1～3d生长缓慢,第4d生长加快并于第7d达到高峰;成骨诱导20d经茜素红染色呈红色结节,成脂诱导14d油红O染色显示有大量脂质沉淀;RT-PCR结果显示Survivin mRNA阳性表达.经全骨髓培养法可以培养出大量骨髓间充质干细胞,同时Survivin在小鼠骨髓间充质干细胞中正常表达,提示可能参与骨髓间充质干细胞抗凋亡过程.     

诱导体外培养的骨髓间充质干细胞向心肌样细胞的分化

选用Wistar大鼠分离骨髓间充质干细胞作体外培养及鉴定其表达抗原CD44、CDw90;采用10μmol／L 5-氮胞苷诱导第1代的骨髓间充质干细胞,于诱导后2、4周进行免疫细胞化学反应检测α-横纹肌肌动蛋白、肌钙蛋白T。证实体外培养的第1代骨髓间充质干细胞经5-氮胞苷诱导可分化为心肌样细胞,为指导体外诱导的心肌细胞应用于。临床提供一定的理论依据和技术手段。     

Wnt 信号通路与骨髓间充质干细胞成骨之间的关系

Wnt信号通路是由Wnts诱发的一系列相互作用的分子组成。Wnt信号对骨髓间充质干细胞的影响在所有研究中均证实有明显作用,其可调节干细胞增殖、分化及凋亡。研究表明,抑制Wnt信号通路转导可使成骨细胞分化进程受阻,从而抑制骨形成;若诱导Wnt家族成员表达则可使成骨细胞特异性基因表达增加,促进骨形成。本文就Wnt信号通路的作用过程及其与骨髓间充质干细胞成骨诱导的关系做一综述。     

间充质干细胞向成骨细胞分化的信号通路

间充质干细胞具有向成骨细胞分化的潜能,可体外分离、培养和扩增,是骨组织工程中理想的种子细胞。近年的研究表明间充质干细胞的成骨分化受到多种信号通路的调控,现就其中研究较为深入的MAPK和Notch通路的情况作一简要综述。     

心肌微环境对诱导大鼠骨髓间充质干细胞分化为心肌样细胞的影响

目的:探讨体外心肌微环境对骨髓间充质干细胞(BMSCs)分化为心肌样细胞的诱导作用。方法:分离大鼠骨髓间充质干细胞(BMSCs)和心肌细胞并在体外培养纯化。以2×10~4/mL的细胞密度种植心肌细胞,培养72h后收集上清,在BMSCs的培养基内加入心肌细胞培养上清诱导BMSCs分化为心肌样细胞,实验共分6组:10%、20%、30%、40%及50%心肌细胞培养上清组及含10%胎牛血清的对照组,均诱导BMSCs7d,每天在倒置显微镜下观察细胞的形态变化;应用免疫细胞化学技术检测不同浓度心肌细胞培养上清组诱导7d后BMSCs中a-平滑肌肌动蛋白(a-sMA)、β-肌动蛋白(β-actin)、心肌特异性肌钙蛋白T(Troponin-T)、CD31以及FactorⅧ的表达。结果:心肌细胞培养上清组诱导BMSCs7d后,细胞的形态没有改变。10%、20%、30%、40%及50%心肌细胞培养上清组a-sMA、。-actin以及Troponin-T的表达均呈阳性,以30%心肌细胞培养上清组的蛋白表达量最高(P<0.01),而CD31和FactorⅧ的表达呈阴性。结论:心肌细胞的培养上清能够诱导BMSCs分化为心肌样细胞,且30%心肌细胞培养上清是诱导BMSCs向心肌样细胞分化的最适浓度。     

猫骨髓间充质干细胞的体外分离培养、纯化、鉴定

目的:分离、培养、纯化家猫的骨髓间充质干细胞,并对获得细胞的表面标志物进行鉴定,为进一步利用骨髓间充质干细胞的细胞移植实验奠定基础。方法:采用全骨髓贴壁法体外分离、培养、纯化家猫骨髓间充质干细胞,通过多次更换培养液获得较纯化的骨髓间充质干细胞,倒置相差显微镜下对细胞形态进行观察;根据第1、3、5、7、9代细胞的镜下增殖情况绘制出生长曲线;通过流式细胞仪检测细胞表面标志抗原CD34、CD44和CD90的表达率。结果:在倒置相差显微镜下观察,分离培养的骨髓间充质干细胞贴壁呈梭形或纺锤形;原代细胞生长丛集成片,5~7 d达到融合,进行传代;培养到第三代以后,细胞出现相对均匀的梭形扁平外观,迅速增殖的细胞呈涡流样排列;第3、5代骨髓间充质干细胞增殖能力强于第7、9代;采用流式细胞仪分析结果显示细胞的CD34、CD44和CD90阳性率分别为17.5%、97.9%和91%,这与骨髓间充质干细胞表面抗原的表达一致。结论:分离培养的细胞具有骨髓间充质干细胞特性,成分相对单一,第3、5代细胞纯度高,增殖能力强,适用于进一步的实验研究。     

猪骨髓间充质干细胞的分离培养与鉴定

目的:建立猪骨髓间充质干细胞(pMSCs)体外分离培养、纯化和鉴定的方法,为下一步实验研究奠定基础.方法:采用密度梯度离心法获得骨髓单核细胞,接种后形成单层贴壁的成纤维样细胞.免疫荧光及PCR检测细胞表面标志及多能性基因的表达,并鉴定分离细胞的多向诱导分化潜能.结果:体外培养的原代细胞10天达到融合,传代后仍具有成纤维样的形态;免疫荧光结果见波形蛋白(Vimention)和Oct4标记阳性,CD45阴性;PCR分子检测见多能性基因OCT-4,nanog的表达;细胞具有分化为成骨细胞和成脂细胞的能力.结论:采用密度梯度离心法获得的pMSCs体外增殖能力强,纯度高,具有间充质干细胞的特性,pMSCs分离培养体系的成功建立为下一步实验研究奠定基础.     

人骨髓间充质干细胞向神经细胞分化过程中Notch通路信号分子表达的变化

本研究探讨Noah信号通路在人骨髓间充质干细胞（human mesenchymal stem cells,hMSCs）体外增殖及向神经细胞分化过程中的作用。采集健康自愿者骨髓,体外培养获得hMSCs,取第3代hMSCs,在诱导剂（β-ME,DMSO,BHA）作用下向神经细胞分化。诱导后用免疫细胞化学鉴定神经元特异性烯醇化酶（neuron-specific enolase,NSE）和尼氏体的表达以确定诱导效果：用流式细胞术检测细胞生长周期时相的变化。在诱导前后,用免疫荧光和RT-PCR方法检测Notch通路中Notch1受体蛋白、配体Jagged1（JAG1）、调节蛋白活化相关物早老素1（presenilin 1,PS1）、靶基因hairy and enhancer of split1（HES1）信号分子表达的变化。结果显示：诱导前,处于G0/G1期的hMSCs占58．5％,S＋G2/M期的细胞占41．5％;诱导后,G0/G1期细胞比例升高,而S＋G2/M期细胞比例下降,NSE阳性细胞率达（77±0．35）％,细胞质中可见深蓝色的块状或颗粒状尼氏体。免疫荧光显示,诱导前后hMSCs内Notch1和JAG1均呈阳性表达,但RT-PCR检测发现诱导后Notch1、JAG1、PSl和HES1 mRNA表达量较诱导前明显降低（均P〈0．05）。结果表明,诱导hMSCs向神经细胞分化能抑制Notch信号分子表达,低水平的Notch信号激活可能有利于神经细胞的分化。     

Mesenchymal stem cells regulate the proliferation and differentiation of neural stem cells through Notch signaling

The effects of mesenchymal stem cells (MSCs) on proliferation and cell fate determination of neural stem cells (NSCs) have been investigated. NSCs were co-cultured with MSCs or NIH3T3 cells using an in vitro transwell system. After 4 days, immunofluorescence staining showed that the number of cells positive for the cell proliferation antigen, ki-67, in neurospheres in MSCs was greater than in NIH3T3 cells. In some experiments, the top-layers of MSCs and NIH3T3 cells were removed to induce NSCs differentiation. Seven days after initiating differentiation, the levels of the neuronal marker, NSE, were higher in NSCs in MSCs co-culture group, and those of glial fibrillary acidic protein (GFAP) were lower, compared with NIH3T3 cells co-culture group. These were confirmed by immunofluorescence. The role of the Notch signaling pathway analyzed with the specific inhibitor, DAPT, and by examining the expression of Notch-related genes using RT-PCR showed that after co-culturing with MSCs for 24 h, NSCs expressed much higher levels of ki-67, Notch1, and Hes1 than did NSCs co-cultured with NIH3T3 cells. Treatment with DAPT decreased ki-67, Notch1 and Hes1 expression in NCSs, and increased Mash1 expression. The data indicate that the interactions between MSCs and NSCs promote NSCs proliferation and are involved in specifying neuronal fate, mediated in part by Notch signaling.     

Deletion of RBPJK in Mesenchymal Stem Cells Enhances Osteogenic Activity by Up-Regulation of BMP Signaling

Recently we have demonstrated the importance of RBPjk-dependent Notch signaling in the regulation of mesenchymal stem cell (MSC) differentiation during skeletogenesis both in vivo and in vitro. Here we further performed RBPJK loss-of-function experiments to demonstrate for the first time that RBPJK deficient MSC shows enhanced differentiation and osteogenesis acts via up-regulation of the BMP signaling. In the present study, we first compared the spontaneous and osteogenic differentiation in normal and recombination signal binding protein for immunoglobulin kappa J region (RBPJK) deficient human bone marrow-derived mesenchymal stem cells (MSCs). It was found that RBPJK highly expressed in fresh isolated MSCs and its expression was progressing down-regulated during spontaneous differentiation and even greater in osteogenic media inducted differentiation. Deletion of RBPJK in MSCs not only enhances cell spontaneous differentiation, but also significantly accelerates condition media inducted osteogenic differentiation by showing enhanced alkaline phosphatase (ALP) activity, Alizarin red staining, gene expression of Runx2, Osteopontin (OPN), Type I collagen (COL1a1) in culture. Additionally, BMP signaling responsive reporter activity and phosphor-smad1/5/8 expression were also significantly increased upon removal of RBPJK in MSCs. These data proved that inhibition of Notch signaling in MSCs promotes cell osteogenic differentiation by up-regulation of BMP signaling, and RBPJK deficient MSC maybe a better cell population for cell-based bone tissue engineering.     

Small molecule induction of neural-like cells from bone marrow-mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (MSCs) have been demonstrated to be able to differentiate into neuron-like cell, but the precise mechanisms controlling this process are unclear. We report here that LY294002, a small molecule inhibitor of PI3K/AKT signal pathway, can inhibit proliferation and promote neuronal differentiation of MSCs after MSCs incubated with LY294002 for 6 and 12 h. RT-PCR results indicated that mRNA expression of α5β1 integrin significantly increased in neuron-like cell from MSCs. Interestingly, neuron-like cells derived by this method adhere much more strongly than MSCs, which was related to the expression of α5β1 integrin and FAK phosphorylation. However, these effects could be attenuated by LiCL or GSK-3β-siRNA. Our results indicate that activation GSK-3β signaling may be involved in MSCs proliferation, differentiation, and adhesion. Furthermore, this study demonstrates that small molecule regulators of PI3K/AKT signaling may be valuable tools for stem cell research aimed at treatment of neurodegenerative disease.     

Jagged1 protein enhances the differentiation of mesenchymal stem cells into cardiomyocytes

BACKGROUND: Mesenchymal stem cells (MSCs) can differentiate into cardiomyocytes if an appropriate cellular environment is provided. Notch signals exchanged between neighboring cells through the Notch receptor can eventually dictate cell differentiation. In our study, we show that MSC differentiation into cardiomyocytes is dependent on the Notch signal. METHODS: We created a myocardial infarction model in rat by coronary ligation, administered direct intramyocardial injection of DAPI-labeled MSC immediately, and observed the differentiation of MSCs after 14 days by immunofluorescence staining against troponin T. We cultured MSCs and cardiomyocytes in four ways, respectively, in vitro. (1) MSCs cocultured with cardiomyocytes obtained from neonatal rat ventricles in a ratio of 1:10. (2) The two types of cells were cultured in two chambers separated by a semipermeable membrane as indirect coculture group. (3) Notch receptor-soluble jagged1 protein was added to indirect coculture group. (4) Both jagged1 protein and gamma-secretase inhibitor-DAPT were added to indirect coculture group. Two weeks later, we observed the differentiation percentage, respectively, by immunofluorescence staining. RESULTS: We found the differentiation of MSCs which were close to cardiomyocytes in vivo. The differentiation percentage of the four cell culture group was 30.13+/-2.16%, 12.52+/-1.18%, 26.33+/-2.20%, and 13.08+/-1.15%. CONCLUSIONS: MSCs can differentiate into cardiomyocytes in vitro and in vivo if a cardiomyocyte microenvironment is provided. 2. Cell-to-cell interaction is very important for the differentiation of MSCs into cardiomyocytes. 3. Jagged1 protein can activate Notch signal and enhance the differentiation of MSC into cardiomyocyte, while the effect can be inhibited by DAPT.     

The Notch Signaling Regulates CD105 Expression,Osteogenic Differentiation and Immunomodulation of Human Umbilical Cord Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a group of multipotent cells with key properties of multi-lineage differentiation, expressing a set of relatively specific surface markers and unique immunomodulatory functions. IDO1, a catabolic enzyme of tryptophan, represents a critical molecule mediating immunomodulatory functions of MSCs. However, the signaling pathways involved in regulating these key properties still remain elusive. To investigate the involvement of Notch signaling as well as other potential signaling pathway(s) in regulating these critical properties of MSCs, we treated human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) with γ-secreatase inhibitor I (GSI-I), which inhibits both Notch signaling and ubiquitin-proteasome activities. It was shown that the GSI-I treatment resulted in apoptosis, reduced expression of surface markers CD73, CD90 and CD105, reduced osteogenic differentiation, and reduction of the hUC-MSCs-mediated suppression of Th1 lymphocyte proliferation and the IFN-γ-induced IDO1 expression. Through distinguishing the effects of GSI-I between Notch inhibition and proteasome inhibition, it was further observed that, whereas both Notch inhibition and proteasome inhibition were attributable to the reduced CD105 expression and osteogenic differentiation, but not to the induced apoptosis. However, Notch inhibition, but not proteasome inhibition, only contributed to the significant effect of GSI-I on Th1 proliferation probably through reducing IDO1 promoter activity. In conclusion, the Notch signaling may represent a very important cell signaling capable of regulating multiple critical properties, especially the immunomodulatory functions of MSCs.     

Notch信号在盐酸法舒地尔诱导大鼠骨髓间充质干细胞向神经元分化过程中的作用

目的:探讨Notch信号通路在盐酸法舒地尔诱导大鼠骨髓间充质干细胞(MSCs)向神经元分化中的作用。方法:实验分为未转染组、转染组(转染Rn-Notch1-siRNA)、阳性对照组(转染Rn-MAPK-1 Control siRNA)及阴性对照组(转染Negative Control siRNA)等4组。采用盐酸法舒地尔诱导大鼠MSCs分化为神经元。倒置荧光显微镜下观察MSCs转染后荧光表达情况;RT-PCR检测Notch1、Hes1和MAPK1 mRNA的表达变化;免疫细胞化学法检测Notch1、神经元烯醇化酶(NSE)、神经微丝蛋白亚单位(NF-M)和胶质纤维酸性蛋白(GFAP)的表达变化;MTT方法检测细胞存活率。结果:①siRNA转染72h,MSCs荧光表达最强,转染率可达91.3%±4.2%;同时,转染组MSCs的Notch1和Hes1 mRNA转录下降(P0.05);MTT提示转染组细胞存活率也显著减少(P0.05)。②盐酸法舒地尔可以诱导MSCs向神经元分化,其中以转染组诱导效果最佳,NSE、NF-M的表达率显著的高于其它各组(P0.05)。结论:盐酸法舒地尔在诱导大鼠MSCs向神经元分化过程中,可能存在Notch信号通路与RhoA/Rho激酶通路信号的协同作用,共同促进MSCs向神经元分化。     

Down-regulation of nestin in mesenchymal stem cells derived from peripheral blood through blocking bone morphogenesis pathway

Different signaling pathways are implicated in proliferation and differentiation of stem cells. Bone Morphogenesis Pathway (BMP) signaling was known to display an important function in osteogenic and adipogenic differentiation of mesenchymal stem cells (MSCs). In the present study, the authors investigated whether blocking BMP signaling was associated with down regulation of Nestin expression as neural stem cell marker in peripheral blood derived mesenchymal stem cells (PB-MSCs). At first, MSCs were isolated from peripheral blood by plastic adherent ability and flow cytometry analysis. After reaching the confluence, the cells were treated with medium containing Noggin as antagonist of BMP signaling upon 8 days. Real time PCR analysis indicated that the expression of Nestin was diminished in PB-MSCs by attenuating BMP signaling. The obtained results suggested that BMP signaling might have a regulatory function on the Nestin expression in mesenchymal stem cells.     

Stromal derived factor-1 regulates bone morphogenetic protein 2-induced osteogenic differentiation of primary mesenchymal stem cells

Stromal derived factor-1 (SDF-1) is a chemokine signaling molecule that binds to its transmembrane receptor CXC chemokine receptor-4 (CXCR4). While we previously detected that SDF-1 was co-required with bone morphogenetic protein 2 (BMP2) for differentiating mesenchymal C2C12 cells into osteoblastic cells, it is unknown whether SDF-1 is similarly involved in the osteogenic differentiation of mesenchymal stem cells (MSCs). Therefore, here we examined the role of SDF-1 signaling during BMP2-induced osteogenic differentiation of primary MSCs that were derived from human and mouse bone marrow. Our data showed that blocking of the SDF-1/CXCR4 signal axis or adding SDF-1 protein to MSCs significantly affected BMP2-induced alkaline phosphatase (ALP) activity and osteocalcin (OCN) synthesis, markers of preosteoblasts and mature osteoblasts, respectively. Moreover, disrupting the SDF-1 signaling impaired bone nodule mineralization during terminal differentiation of MSCs. Furthermore, we detected that blocking of the SDF-1 signaling inhibited the BMP2-induced early expression of Runt-related factor-2 (Runx2) and osterix (Osx), two “master” regulators of osteogenesis, and the SDF-1 effect was mediated via intracellular Smad and Erk activation. In conclusion, our results demonstrated a regulatory role of SDF-1 in BMP2-induced osteogenic differentiation of MSCs, as perturbing the SDF-1 signaling affected the differentiation of MSCs towards osteoblastic cells in response to BMP2 stimulation. These data provide novel insights into molecular mechanisms underlying MSC osteogenesis, and will contribute to the development of MSC therapies for enhancing bone formation and regeneration in broad orthopaedic situations.