Wnt/β-catenin signaling regulates neuronal differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) have been demonstrated to be able to differentiate into neuron-like cells, but the precise mechanisms controlling this process are unclear. Using neuron-specific enolase (NSE) and nestin as neuronal markers, we examined the role of Wnt/β-catenin signaling in MSC neuronal differentiation in present study. The results indicated that the expression of β-catenin increased markedly during the neuronal differentiation of MSCs. Blocking Wnt signaling by treating MSCs with β-catenin siRNA could decrease the differentiation of MSCs into neuron-like cells and up-regulation of Wnt signaling by treating MSCs with Wnt-3a could promote neuronal differentiation of MSCs. Above results suggest that Wnt/β-catenin signaling may play a pivotal role in neuronal differentiation of MSCs. Our data broaden the knowledge of molecular mechanisms involved in the neuronal differentiation of MSCs and provide a potential target for directing differentiation of MSCs for clinical application.     

川芎嗪体外诱导小鼠骨髓间充质干细胞分化为神经元样细胞的研究

为了探讨川芎嗪体外诱导小鼠骨髓间质干细胞（BMSCs）分化为神经元样细胞的作用,以小鼠骨髓间充质干细胞为研究对象,实验分为空白对照组、β-巯基乙醇（BME）阳性对照组和川芎嗪诱导组。采用荧光免疫化学和Western blot方法,分别检测神经干细胞巢蛋白（nestin）和经元特异性烯醇化酶（NSE）的表达;RT-PCR检测诱导不同时间对神经细胞相关基因Nestin、NSE、β-微管蛋白III（β-Tubulin III）和核受体相关因子-1（Nurr1）mRNA表达的影响。结果显示川芎嗪诱导间充质干细胞24 h后,细胞形态发生显著改变,细胞突起形成且数目不等,形成神经元样细胞。细胞死亡率低于β-巯基乙醇诱导组。免疫荧光化学法和western blot结果显示：川芎嗪诱导后的细胞nes-tin和NSE蛋白表达呈阳性,且表达丰度显著高于β-巯基乙醇诱导组。川芎嗪作用不同时间的BMSCs表达神经细胞相关基因Nestin、β-Tubulin III、NSE和Nurrl。结果表明川芎嗪能定向诱导小鼠骨髓间充质干细胞分化为神经元样细胞,是较理想的诱导剂。     

人骨髓间充质干细胞在成年大鼠脑内的迁移及分化

骨髓间充质干细胞 (mesenchymalstemcells,MSCs)是目前备受关注的一类具有多向分化潜能的组织干细胞 ,体外可以分化为骨、软骨、脂肪等多种细胞。因此 ,MSCs是细胞治疗和基因治疗的种子细胞之一。为了探索MSCs的迁移和分化趋势 ,为帕金森病 (Parkinsondisease,PD)的干细胞治疗提供理论和实验依据 ,本实验将体外扩增并转染增强型绿色荧光蛋白 (enhancedgreenfluorescentprotein ,EGFP)的人骨髓MSCs注入PD大鼠脑内纹状体 ,观察了人骨髓MSCs在大鼠脑内的存活、迁移、分化以及注射MSCs前后大鼠的行为变化。结果表明 ,人骨髓MSCs在大鼠脑内可存活较长时间 ( 10周以上 ) ;随着时间的延长 ,MSCs迁移范围扩大 ,分布于纹状体、胼胝体、皮质以及脑内血管壁 ;免疫组化法检测证实MSCs在大鼠脑内表达人神经丝蛋白 (neurofilament,NF)、神经元特异性烯醇化酶 (neuron specificeno lase,NSE)以及胶质原纤维酸性蛋白 ( glialfibrillaryacidprotein ,GFAP) ;PD大鼠的异常行为有所缓解 ,转圈数由 8 86±2 0 9r/min下降到 4 87± 2 0 6r/min ,统计学分析P <0 0 5为差异显著。以上观察结果表明 ,骨髓MSCs有望成为治疗PD的种子细胞     

Pre‐activation of retinoid signaling facilitates neuronal differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) can differentiate into neurons in an appropriate cellular environment. Retinoid signaling pathway is required in neural development. However, the effect and mechanism through retinoid signaling regulates neuronal differentiation of MSCs are still poorly understood. Here, we report that all‐trans‐retinoic acid (ATRA) pre‐induction improved neuronal differentiation of rat MSCs. We found that, when MSCs were exposed to different concentrations of ATRA (0.01–100 μmol/L) for 24 h and then cultured with modified neuronal induction medium (MNM), 1 μmol/L ATRA pre‐induction significantly improved neuronal differentiation efficiency and neural‐cell survival. Compared with MNM alone induced neural‐like cells, ATRA/MNM induced cells expressed higher levels of Nestin, neuron specific enolase (NSE), microtubule‐associated protein‐2 (MAP‐2), but lower levels of CD68, glial fibrillary acidic protein (GFAP), and glial cell line‐derived neurotrophic factor(GDNF), also exhibited higher resting membrane potential and intracellular calcium concentration, supporting that ATRA pre‐induction promotes maturation and function of derived neurons but not neuroglia cells from MSCs. Endogenous retinoid X receptors (RXR) RXRα and RXRγ (and to a lesser extent, RXRβ) were weakly expressed in MSCs. But the expression of RARα and RARγ was readily detectable, whereas RARβ was undetectable. However, at 24 h after ATRA treatment, the expression of RARβ, not RARα or RARγ, increased significantly. We further found the subnuclear redistribution of RARβ in differentiated neurons, suggesting that RARβ may function as a major mediator of retinoid signaling during neuronal differentiation from MSCs. ATRA treatment upregulated the expression of Vimentin and Stra13, while it downregulated the expression of Brachyury in MSCs. Thus, our results demonstrate that pre‐activation of retinoid signaling by ATRA facilitates neuronal differentiation of MSCs.     

In vitro differentiation of human processed lipoaspirate cells into early neural progenitors

Human processed lipoaspirate (PLA) cells are multipotent stem cells, capable of differentiating into multiple mesenchymal lineages (bone, cartilage, fat, and muscle). To date, differentiation to nonmesodermal fates has not been reported. This study demonstrates that PLA cells can be induced to differentiate into early neural progenitors, which are of an ectodermal origin. Undifferentiated cultures of human PLA cells expressed markers characteristic of neural cells such as neuron-specific enolase (NSE), vimentin, and neuron-specific nuclear protein (NeuN). After 2 weeks of treatment of PLA cells with isobutylmethylxanthine, indomethacin, and insulin, about 20 to 25 percent of the cells differentiated into cells with typical neural morphologic characteristics, accompanied by increased expression of NSE, vimentin, and the nerve-growth factor receptor trk-A. However, induced PLA cells did not express the mature neuronal marker, MAP, or the mature astrocyte marker, GFAP. It was also found that neurally induced PLA cells displayed a delayed-rectifier type K+ current (an early developmental ion channel) concomitantly with morphologic changes and increased expression of neural-specific markers. The authors concluded that human PLA cells might have the potential to differentiate in vitro into cells that represent early progenitors of neurons and/or glia.     

miR-29a Modulates Neuronal Differentiation through Targeting REST in Mesenchymal Stem Cells

Objective

To investigate the modulation of microRNAs (miRNAs) upon the neuronal differentiation of mesenchymal stem cells (MSCs) through targeting RE-1 Silencing Factor (REST), a mature neuronal gene suppressor in neuronal and un-neuronal cells.

Methods

Rat bone marrow derived–MSCs were induced into neuron-like cells (MSC-NCs) by DMSO and BHA in vitro. The expression of neuron specific enolase (NSE), microtubule-associated protein tau (Tau), REST and its target genes, including synaptosomal-associated protein 25 (SNAP25) and L1 cell adhesion molecular (L1CAM), were detected in MSCs and MSC-NCs. miRNA array analysis was conducted to screen for the upregulated miRNAs after neuronal differentiation. TargetScan was used to predict the relationship between these miRNAs and REST gene, and dual luciferase reporter assay was applied to validate it. Gain and loss of function experiments were used to study the role of miR-29a upon neuronal differentiation of MSCs. The knockdown of REST was conducted to show that miR-29a affected this process through targeting REST.

Results

MSCs were induced into neuron-like cells which presented neuronal cell shape and expressed NSE and Tau. The expression of REST declined and the expression of SNAP25 and L1CAM increased upon the neuronal differentiation of MSCs. Among 14 upregulated miRNAs, miR-29a was validated to target REST gene. During the neuronal differentiation of MSCs, miR-29a inhibition blocked the downregulation of REST, as well as the upregulation of SNAP25, L1CAM, NSE and Tau. REST knockdown rescued the effect of miR-29a inhibition on the expression of NSE and Tau. Meanwhile, miR-29a knockin significantly decreased the expression of REST and increased the expression of SNAP25 and L1CMA in MSCs, but did not significantly affect the expression of NSE and Tau.

Conclusion

miR-29a regulates neurogenic markers through targeting REST in mesenchymal stem cells, which provides advances in neuronal differentiation research and stem cell therapy for neurodegenerative diseases.     

Differentiation potential of bone marrow mesenchymal stem cells in duck

The bone marrow mesenchymal stem cells （MSCs） are multipotent stem cells which can differentiate into mesenchymal cells in vitro. In this study, MSCs in duck were isolated from bone marrow by density gradient centrifuge separation, purified and expanded in the me- dium. The primary MSCs were expanded for 11 passages. The different-passage MSCs were induced to differentiate into osteoblasts and neuron-like cells. Karyotype analysis indicated that MSCs kept diploid condition and the hereditary feature was stable. The different- passage MSCs expressed CD44, ICAM-1 and SSEA-4, but not CD34, CD45 and SSEA-1 when detected by immunofluorescence staining There was no significant difference among the positive rates of passages 2, 6 and 8 （P 〉 0.05）, but a significant difference existed among those of passages 2, 6, 8 and 11 （P 〈 0.05）. After the osteogenic inducement was added, the induced different-passage MSCs expressed high-level alkaline phosphatase （ALP）, and are positive for tetracycline staining, Alizarin Red staining and Von Kossa staining. After the neural inducement was added, about 70% cells exhibited typical neuron-like phenotype, the induced different-passage MSCs expressed Nestin, neuron-specific enolase （NSE） and glial fibrillary acidic protein （GFAP） when detected by immunofluorescence staining. There was no significant difference among the positive rates of passages 3, 4 and 6 （P〉0.05）, but a significant difference existed among those of passages 3, 4, 6 and 8 （P〈0.05）. These results suggest that MSCs in duck were capable of differentiating into osteoblasts and neuron-like cells in vitro.     

Differentiation potential of bone marrow mesenchymal stem cells into retina in normal and laser-injured rat eye

Bone marrow mesenchymal stem cells (MSCs) can develop into hematopoietic and mesenchymal lineages but have not been known to participate in the production of retina. Here we report that bone marrow mesenchymal stem cells, after being subretinally transplanted into normal or Nd: YAG laser-injured rat eye, can integrate into RPE layer, photoreceptor layer, bipolar cell layer and ganglion layer. DAPI-labeling detection was used to trace the origin of the repopulating cells. DAPI fluorescence was used to identify retina cells of bone marrow origin 10, 20, 35 and 50 days after transplantation. No formation of rosettes was found but some random cells were found at the end of the observation. MSCs-originated cells spread more widely in the injured retinas than in the normal ones. Immunohistochemical detection showed that though the cells could express neuronal nuclei (NeuN), neuron specific enolase (NSE), glial fibrillary acidic protein (GFAP) and cytokeratin (CK), the proteins expression in the injured transplantation group was abnormal in some region compared with that in the normal transplantation group. Electroretinogram (ERG) showed that ERG-b wave of the injured transplantation group is significantly higher than that of the two laser-injured control groups. These results suggest that a proportion of MSCs can differentiate into retina-like structure in vivo and the differentiation differs in normal and laser-injured retinas.     

Differentiation potential of bone marrow mesenchymal stem cells into retina in normal and laser-injured rat eye

Withinthebonemarrowstromathereexistsasubsetofnonhematopoieticcellsreferredtoasmes-enchymalstemormesenchymalprogenitorcells.Mesenchymalstemcells(MSCs)areapopulationofpluripotentcellswithinthehuman,birdorrodentbonemarrowmicroenvironmentdefinedbytheirability,eitherinvitroorinvivo,todifferentiateintocellsoftheosteogenic,chondrogenic,tendonogenic,adipo-genic,neuralcellsandmyogeniclineages[1].Themethodologiestoisolateandculture-expandMSCsfromhumanbonemarrowforestablishingthecellularortissuediffere…     

Multilineage potential of stable human mesenchymal stem cell line derived from fetal marrow

Human bone marrow contains two major cell types, hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). MSCs possess self-renewal capacity and pluripotency defined by their ability to differentiate into osteoblasts, chondrocytes, adipocytes and muscle cells. MSCs are also known to differentiate into neurons and glial cells in vitro, and in vivo following transplantation into the brain of animal models of neurological disorders including ischemia and intracerebral hemorrhage (ICH) stroke. In order to obtain sufficient number and homogeneous population of human MSCs, we have clonally isolated permanent and stable human MSC lines by transfecting primary cell cultures of fetal human bone marrow MSCs with a retroviral vector encoding v-myc gene. One of the cell lines, HM3.B10 (B10), was found to differentiate into neural cell types including neural stem cells, neurons, astrocytes and oligodendrocytes in vitro as shown by expression of genetic markers for neural stem cells (nestin and Musashi1), neurons (neurofilament protein, synapsin and MAP2), astrocytes (glial fibrillary acidic protein, GFAP) and oligodendrocytes (myelin basic protein, MBP) as determined by RT-PCR assay. In addition, B10 cells were found to differentiate into neural cell types as shown by immunocytochical demonstration of nestin (for neural stem cells), neurofilament protein and beta-tubulin III (neurons) GFAP (astrocytes), and galactocerebroside (oligodendrocytes). Following brain transplantation in mouse ICH stroke model, B10 human MSCs integrate into host brain, survive, differentiate into neurons and astrocytes and induce behavioral improvement in the ICH animals. B10 human MSC cell line is not only a useful tool for the studies of organogenesis and specifically for the neurogenesis, but also provides a valuable source of cells for cell therapy studies in animal models of stroke and other neurological disorders.     

Differentiation of single cell derived human mesenchymal stem cells into cells with a neuronal phenotype: RNA and microRNA expression profile

The adult bone marrow contains a subset of non-haematopoietic cells referred to as bone marrow mesenchymal stem cells (BMSCs). Mesenchymal stem cells (MSCs) have attracted immense research interest in the field of regenerative medicine due to their ability to be cultured for successive passages and multi-lineage differentiation. The molecular mechanisms governing the self-renewal and differentiation of MSCs remain largely unknown. In a previous paper we demonstrated the ability to induce human clonal MSCs to differentiate into cells with a neuronal phenotype (DMSCs). In the present study we evaluated gene expression profiles by Sequential Analysis of Gene Expression (SAGE) and microRNA expression profiles before and after the neuronal differentiation process. Various tissue-specific genes were weakly expressed in MSCs, including those of non-mesodermal origin, suggesting multiple potential tissue-specific differentiation, as well as stemness markers. Expression of OCT4, KLF4 and c-Myc cell reprogramming factors, which are modulated during the differentiation process, was also observed. Many peculiar nervous tissue genes were expressed at a high level in DMSCs, along with genes related to apoptosis. MicroRNA profiling and correlation with mRNA expression profiles allowed us to identify putative important genes and microRNAs involved in the differentiation of MSCs into neuronal-like cells. The profound difference in gene and microRNA expression patterns between MSCs and DMSCs indicates a real functional change during differentiation from MSCs to DMSCs.     

In vitro differentiation of human marrow stromal cells into early progenitors of neural cells by conditions that increase intracellular cyclic AMP

Human marrow stromal cells (hMSCs) are multipotential stem cells that can be differentiated into bone, cartilage, fat, and muscle. In the experiments here, we found that undifferentiated cultures of hMSCs express some markers characteristic of neural cells such as microtubule-associated protein 1B (MAP1B), neuron-specific tubulin (TuJ-1), neuron-specific enolase (NSE), and vimentin. By treating hMSCs with 0.5 mM isobutylmethylxanthine (IBMX)/1 mM dibutyryl cyclic AMP (dbcAMP) for 6 days, about 25% of the hMSCs differentiated into cells with a typical neural cell morphology and with increased levels of both NSE and vimentin. The data suggested that the hMSCs may have been differentiated into early progenitors of neural cells in vitro under conditions that increase the intracellular level of cAMP.     

Calcium: A novel and efficient inducer of differentiation of adipose‐derived stem cells into neuron‐like cells

This study comparatively investigated the effectiveness of calcium and other well‐known inducers such as isobutylmethylxanthine (IBMX) and insulin in differentiating human adipose‐derived stem cells (ADSCs) into neuronal‐like cells. ADSCs were immunophenotyped and differentiated into neuron‐like cells with different combinations of calcium, IBMX, and insulin. Calcium mobilization across the membrane was determined. Differentiated cells were characterized by cell cycle profiling, staining of Nissl bodies, detecting the gene expression level of markers such as neuronal nuclear antigen (NeuN), microtubule associated protein 2 (MAP2), neuron‐specific enolase (NSE), doublecortin, synapsin I, glial fibrillary acidic protein (GFAP), and myelin basic protein (MBP) by quantitative real‐time polymerase chain reaction (quantitative real‐time polymerase chain reaction (qRT‐PCR) and protein level by the immunofluorescence technique. Treatment with Ca + IBMX + Ins induced neuronal appearance and projection of neurite‐like processes in the cells, accompanied with inhibition of proliferation and halt in the cell cycle. A significantly higher expression of MBP, GFAP, NeuN, NSE, synapsin 1, doublecortin, and MAP2 was detected in differentiated cells, confirming the advantages of Ca + IBMX + Ins to the other combinations of inducers. Here, we showed an efficient protocol for neuronal differentiation of ADSCs, and calcium fostered differentiation by augmenting the number of neuron‐like cells and instantaneous increase in the expression of neuronal markers.     

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。     

The immunobiological development of human bone marrow mesenchymal stem cells in the course of neuronal differentiation

The central nervous system (CNS) has been referred to as the "immunological privileged site". However, it is now clear that the privileged status of the CNS is a result of a balance between immune privilege and effective response. In vitro, human bone marrow mesenchymal stem cells (MSCs) have the ability to differentiate into neurons. Based on this biological attribute we gain the possibility by means of using MSCs as the donors to develop a future cell therapy in clinical application. But using MSCs as donor cells inevitably raises the question as to whether these donor cells would be immunogenic, and if so, would they be rejected after transplantation. To investigate this, human MSCs were cultured in vitro and induced to differentiate along neuronal lineage. The expression of human leukocyte antigen (HLA) class I and class II molecules and the co-stimulatory protein CD80 were increased on the surface of MSCs in the course of neuronal differentiation. But neither of the co-stimulatory proteins, CD40 or CD86, was expressed. After IFN-gamma exposure, the expression of the HLA molecules was further enhanced, but the co-stimulatory proteins were unaffected. MSCs that had been differentiated along neuronal lineage were not capable of inducing the proliferation of peripheral blood lymphocytes (PBLs). Even after IFN-gamma exposure, PBLs remained unresponsive. Furthermore, MSCs differentiated along neuronal lineage suppressed the proliferation of PBLs induced by allogeneic PBLs and mitogens. The mechanisms involved in the immunosuppression may be related to the effect of soluble factors and cell-cell interactions of neuronal differentiated MSCs and PBLs. From the above data we suggested that the low immunogenicity and immunomodulatory function of MSCs in the course of neuronal differentiation in vitro, which will be helpful to further investigation in order to establish the new way for future medical application.