Adipose tissue-derived stromal cells express neuronal phenotypes

Background Adipose tissue-derived stromal cell (ADSCs) can be greatly expanded in vitro, and induced to differentiate into multiple mesenchymal cell types, including osteogenic, chondrogenic, myogenic, and adipogenic cells. This study was designed to investigate the possibility of ADSCs differentiating into neurons. Methods Adipose tissue from rats was digested with collagenase, and adherent stromal cells were cultured. A medium containing a low concentration of fetal bovine serum was adopted to induce the cells to differentiate. ADSCs were identified by immunocytochemistry, and semi-quantitative RT-PCR was applied to detect mRNA expression of neurofilament 1 (NF1), nestin, and neuron-specific enolase (NSE). Results Nestin-positive cells were found occasionally among ADSCs. ADSCs were found to express NSE mRNA and nestin mRNA, but not NF1 mRNA. ADSCs could differentiate into neuron-like cells in a medium composed of a low concentration of fetal bovine serum, and these differentiated cells displayed complicated neuron-like morphologies. Conclusions The data support the hypothesis that adipose tissue contains stem cells capable of differentiating into neurons. These stem cells can overcome their mesenchymal commitment, and may represent an alternative autologous stem cell source for CNS cell transplantation.     

Targeted induction of differentiation of human bone mesenchymal stem cells into neuron-like cells

A systematic method of isolating and culturing human bone mesenchymal stem cells （hMSCs）, and inducing them to differentiate into neuron-like cells in vitro was established. The hMSCs were isolated from bone marrow with the lymphocyte-separating medium, cultured and expanded in vitro, and induced after addition of compound neuro-revulsants. The morphological changes of hMSCs were observed, and the expression of surface markers in induced hMSCs was immunocytochemically identified during induction period. The hMSCs could be separated, cultured and expanded in vitro. After induction by compound neuro-revulsants for 48 h, the changes of neuron-like cells, such as cellular shrinkage and neurite growth, were observed in some cells. The immunochemical staining revealed nestin （＋） or NF （＋）, and GFAP （-）. It was concluded that hMSCs were successfully cultured and induced to differentiate into neuron-like cells.     

Effect of IKVAV peptide nanofiber on proliferation,adhesion and differentiation into neurocytes of bone marrow stromal cells

This study examined the effect of IKVAV peptide nanofiber on proliferation, adhesion and differentiation into neurocytes of bone marrow stromal cells （BMSCs）. IKVAV Peptide-amphiphile was synthesized and purified. Then, hydrogen chloride was added to the diluted aqueous solutions of PA to induce spontaneous formation of nanofiber in vitro. The resultant samples was observed tmder transmission electron microscope. BMSCs were cultured with IKVAV peptide nanofiber. The effect of IKVAV nanofiber on the proliferation, adhesion and induction differentiation of BMSCs was observed by inverted microscopy, calcein-AM/PI staining, cell counting and immunofluorescence staining. The results demonstrated that IKVAV peptide-amphiphile could self-assemble to form nanofiber gel. BMSCs cultured in combination with IKVAV peptide nanofiber gel grew well and the percentage of live cells was over 90%. IKVAV peptide nanofiber gel exerted no influence on the proliferation of BMSCs and could promote the adhesion of BMSCs and raise the ra- tio of neurons when BMSCs were induced to differentiate into neurocytes. It is concluded that BMSCs could proliferate and adhere well and yield more neurons during when induced to differente into neurocytes on IKVAV peptide nanofiber gel.     

The experimental study on promoting vascularization by bone marrow stromal cells transplantation after local brain injury in rats

Objective To observe the effects of bone marrow stromal cells transplantation brain injury in rats, and to on vascularization after local investigate the mechanism of repairing by BMSC transplantation. Methods The animal model local brain injury in rat was established. The hBM- SCs were transplanted into the local brain. The immuno- histochemistry and reactance were used to observe endothelial cell and microcirculation of local brain. Results The hBMSCs were transplanted into the animal modd, the quantities and the expression of the FⅦ RAg positive cells increased and the microvascular structure was formed; the inside wall of the microvessel was smooth, the union between the ceils was firm; nucleuses were regularly organized, basement membranes were continued , and the vascular compression was relieved. Conclusion Local vascular endothelial cells were promoted to proliferate when hBMSCs were transplanted and the microvessel formed increaselly. Microcirculation of local brain was improved, and brain injury was repaired. 10 refs. 2 figs. 1 tab.     

In vitro Culture of Bone Marrow Mesenchymal Stem Cells in Rats and Differentiation into Retinal Neural-like Cells

In order to study the in vitro culture and expansion of bone marrow mesenchymal stem cells in rats(rMSCs) and the possibility of rMSCs differentiation into retinal neural cells,the bone marrow-derived cells in SD rats were isolated and cultured in vitro.The retinal neural cells in SD rats were cultured and the supernatants were collected to prepare conditioned medium.The cultured rMSCs were induced to differentiate by two steps.Immunofluorescence method and anti-nestin,anti-NeuN,anti-GFAP and anti-Thy1.1 antibodies were used to identify the cells derived from the rMSCs.The results showed that the in vitro cultured rMSCs grew well and expanded quickly.After induction with two conditioned media,rMSCs was induced to differentiate into neural progenitor cells,then into retinal neural-like cells which were positive for nestin,NeuN,GFAP and Thy1.1 detected by fluorescence method.The findings suggested that rMSCs could be culture and expanded in vitro,and induced to differentiate into retinal neural-like cells.     

Experimental Study of Cell Migration and Functional Differentiation of Transplanted Neural Stem Cells Co-labeled with Superparamagnetic Iron Oxide and Brdu in an Ischemic Rat Model

Objective To explore the migration of transplanted neural stem cells co-labeled with superparamagnetic iron oxide （SPIO） and bromodeoxyuridine （Brdu） using the 4.7T MR system and to study the cell differentiation with immuno-histochemical method in ischemic rats. Methods Rat neural stem cells （NSCs） co-labelled with SPIO mediated by poly-L-lysine and bromodeoxyuridine （BrdU） were transplanted into the unaffected side of rat brain with middle cerebral artery occlusion （MCAO）. At weeks 1, 2, 3, 4, 5, and 6 after MCAO, migration of the labelled cells was monitored by MRI. At week 6 the rats were killed and their brain tissue was cut according to the migration site of transplanted ceils indicated by MRI and subjected to Prussian blue staining and immunohistochemical staining to observe the migration and differentiation of the transplanted NSCs. Results Three weeks after transplantation, the linear hypointensity area derived from the migration of labelled NSCs was observed by MRI in the corpus callosum adjacent to the injection site. Six weeks after the transplantation, the linear hypointensity area was moved toward the midline along the corpus callosum. MRI findings were confirmed by Prussian blue staining and immunohistochemical staining of the specimen at week 6 after the transplantation. Flourescence co-labelled immunohistochemical methods demonstrated that the transplanted NSCs could differentiate into astrocytes and neurons. Conclusion MRI can monitor the migration of SPIO-labelled NSCs after transplantation in a dynamical and non-invasive manner. NSCs transplanted into ischemic rats can differentiate into astrocytes and neurons during the process of migration.     

An Improved Method for Directional Differentiation and Efficient Production of Neurons from Embryonic Stem Cells in vitro

To establish a method of directional differentiation and efficient production of neurons from embryonic stem cells (ES cells) in vitro, based on the 4-/4 protocol described by Bain, a new method was established to induce ES cells differentiating into neurons by means of three-step differentiation using all-trans retinoic acid (ATRA) combined with astrocyte-conditioned medium(ACM) in Vitro. The totipotency of ES cells was identified by observation of cells‘ morphology and formations of teratoma in immunocompromised mice. The cells‘ differentiation was evaluated continuously by the detection of the specific cellular markers of neural stem cells, neurons and astrocytes,including nestin, NSE and GFAP using immunohistochemistry assay. The NSE positive cells‘ ratio of the differentiated cells was determined by flow cytometry. It was found that the transparent circular clusters surrounding embryoid bodies induced with combining induction protocol formed just after 24 h and gradually enlarged later. This phenomenon could not be observed in EBs induced only by ATRA. The NSE positive cells‘ ratio in the ceils induced with ATRA and ACM was higher than that of the cells induced by ATRA at different time points of differentiation, and finally reached up to 73.5 % among the total differentiated population. It was concluded that ES cells could be induced into neurons with high purity and yield by means of inducing method combining with ATRAand ACM.     

Mechanism of in Vitro Differentiation of Bone Marrow Stromal Cells into Neuron-like Cells~

In order to study whether marrow stromal cells (MSCs) can be induced into nerve-like cells in vitro, and the mechanism, the MSCs in Wistar rats were isolated and cultured, and then induced with DMSO and BHA in vitro. The expression of specific marking proteins in neurons, glia and neural stem cells were detected before preinduction, at 24 h of preinduction, at 6 h, 24 h, and 48 h of neuronal induction by using immunohistochemistry and Western blotting. The ultrastructural changes after the inducement were observed. The results showed that after the inducement, many MSCs turned into bipolar, multipolar and taper, and then intersected as network structure. At the same time, some MSCs had the typical neuron-like ultrastructure. Immunohistochemistry revealed that NeuN and Nestin expression was detectable after inducement, but there was no GFAP and CNP expression. Western blotting showed the expression of Nestin was strong at 6 h of neuronal induction, and decreased at 24 h, 48 h of the induction. NeuN was detectable at 6 h of neuronal induction, and increased at 24 h, 48 h of the induction. It was concluded MSCs were induced into neural stem cells, and then differentiated into neuron-like cells in vitro.     

Human adipose-derived mesenchymal stem cells: a better cell source for nervous system regeneration

Background In order to suggest an ideal source of adult stem cells for the treatment of nervous system diseases,MSCs from human adipose tissue and bone marrow were isolated and studied to explore the differences with regard to cell morphology,surface markers,neuronal differentiation capacity,especially the synapse structure formation and the secretion of neurotrophic factors.Methods The neuronal differentiation capacity of human mesenchymal stem cells from adipose tissue （hADSCs） and bone marrow （hBMSCs） was determined based on nissl body and synapse structure formation,and neural factor secretion function.hADSCs and hBMSCs were isolated and differentiated into neuron-like cells with rat brain-conditioned medium,a potentially rich source of neuronal differentiation promoting signals.Specific neuronal proteins and neural factors were detected by immunohistochemistry and enzyme-linked immunosorbent assay analysis,respectively.Results Flow cytometric analysis showed that both cell types had similar phenotypes.Cell growth curves showed that hADSCs proliferated more quickly than hBMSCs.Both kinds of cells were capable of osteogenic and adipogenic differentiation.The morphology of hADSCs and hBMSCs changed during neuronal differentiation and displayed neuronlike cell appearance after 14 days＇ differentiation.Both hADSCs and hBMSCs were able to differentiate into neuron-like cells based on their production of neuron specific proteins including β-tubulin-Ⅲ,neuron-specific enolase （NSE）,nissl bodies,and their ability to secrete brain derived neurotrophic factor （BDNF） and nerve growth factor （NGF）.Assessment of synaptop hysin and growth-associated protein-43 （GAP-43） suggested synapse structure formation in differentiated hADSCs and hBMSCs.Conclusions Our results demonstrate that hADSCs have neuronal differentiation potential similar to hBMSC,but with a higher proliferation capacity than hBMSC.Adipose tissue is abundant,easily available and would be a potential ideal source of adult stem cells for neural-related clinical research and application.     

人脐带来源的基质细胞分化为神经细胞

目的探索人脐带组织来源的基质细胞向神经细胞分化的可能性，为神经移植探寻新的细胞来源。方法采用组织块培养法培养人脐带基质细胞，酶消化法对细胞进行传代，应用免疫细胞化学的方法对细胞进行鉴定。结果从脐带组织成功培养出基质细胞，这种细胞可以随机分化为平滑肌细胞，表达平滑肌肌动蛋白；丹参注射液诱导基质细胞向神经细胞分化，分化的神经元样细胞具有典型的神经细胞的形态，早期表达巢蛋白、β-管蛋白和神经元特异性烯醇化酶，后期表达神经微丝200；在合适的诱导条件下，分化的神经元样细胞的比例在90％左右，分化的细胞中星型胶质细胞的比例在1％左右，无髓鞘基础蛋白的表达，说明无少突胶质细胞产生。结论脐带组织来源的基质细胞可以表达神经细胞的标志物，在体外能够分化为形态复杂的神经元样细胞，这种细胞有可能成为神经移植的种子细胞。     

脑源性神经营养因子体外诱导骨髓间充质干细胞分化为神经元样细胞

目的探讨脑源性神经营养因子(BDNF)体外诱导骨髓间充质干细胞(MSCs)分化成神经元样细胞的作用及其对诱导后的神经元样细胞保护作用，为治疗神经系统疾病提供更优良的细胞。方法体外培养MSCs至第5代后，分别用BDNF、β-巯基乙醇(β-ME)诱导MSCs，诱导后的第1、3和6小时计算两组神经元样细胞数，对各时间点两组神经元样细胞阳性率进行了比较；诱导后行神经细胞标记抗体的免疫细胞化学、RT-PCR及蛋白质印迹鉴定。结果两组诱导剂诱导后的细胞均呈神经元样细胞的形态；BDNF组诱导分化后细胞的存活时间远较β-ME组长；BDNF组在诱导后6h近于诱导高峰，而β-ME在诱导2h即达高峰，但随后神经元样细胞渐死亡。免疫细胞化学结果显示在两组诱导后细胞的巢蛋白(Nestin)、神经细胞特异性烯醇化酶(NSE)、微管相关蛋白-2(MAP-2)、神经丝蛋白(NF)表达均呈阳性，而胶质纤维酸性蛋白(GFAP)表达呈阴性；RT-PCR显示NSE、NFL，MAP-2的mRNA表达阳性，GFAP有较弱的表达；蛋白质印迹可见两组诱导剂诱导的细胞均表达神经元的特异性标记抗体NSE。结论BDNF能单独诱导MSCs分化形成神经元样细胞．而且分化后细胞的存活时间长．有望用于治疗脑缺血、神经变性等疾病。     

脂肪组织来源的基质细胞向神经元样细胞分化

目的：研究脂肪来源的基质细胞向神经元样细胞分化的可能性，为神经移植探索新的细胞来源。方法：采用β-巯基乙醇诱导脂肪来源的基质细胞向神经元样细胞分化，以免疫细胞化学方法对分化的和未分化的细胞进行鉴定。结果：从成年大鼠的脂肪组织中分离出基质细胞，在体外生长形态类似成纤维细胞，可以维持在未分化状态稳定增殖，体外扩增可超过10多代。β-巯基乙醇可诱导这种细胞向神经元样细胞分化，分化的细胞早期表达巢蛋白，后期则表达神经元的标志物——神经元特异性烯醇化酶和神经微丝，在最适合的诱导条件下约60％～85％的细胞表达这两种神经元的标志物。结论：脂肪组织中存在能分化为神经元样细胞的干细胞。     

大鼠骨髓基质干细胞的神经分化以及神经生长因子的表达

目的 探讨大鼠骨髓基质干细胞神经分化以及神经生长因子的表达.方法 体外培养大鼠骨髓基质干细胞(bone marrow stromal cell,BMSC),传代至第6代,进行神经诱导分化.先以bFGF (10 ng/ml)预诱导24 h,然后以胶质细胞源神经营养因子(glial cell line-derived neurotrophic factor,GDNF)10 ng/ml在胎肠培养基(fetal gut condition midium,FGCM)条件下诱导10 d.免疫荧光法检测神经元标志神经特异性烯醇化酶(NSE)和神经丝蛋白(NF)的表达.RT-PCR法检测神经营养因子GDNF和神经生长因子(nerve growth factor,NGF) mRNA的表达.结果 流式细胞检测BMSC高表达CD90(99.7%), 而CD45表达阴性.诱导10 d后,部分细胞在形态上表现出神经元样改变,免疫荧光染色神经元标志NSE和NF阳性,而胶质细胞标志GFAP阴性.RT-PCR检测显示,BMSC诱导前低表达神经营养因子NGF和GDNF mRNA,而诱导后表达水平显著增加.结论 GDNF不仅能在体外诱导BMSC分化为神经样细胞,而且能促进神经营养因子表达显著增加.     

成人骨髓间质干细胞诱导分化为神经元样细胞及其与细胞分裂的关系

目的探讨成人骨髓间质干细胞(hMSC)向神经元样细胞分化过程中分化与细胞分裂的关系。方法从成人骨髓中分离骨髓间质干细胞(MSC),培养扩增。用参芪液诱导MSC分化为神经元样细胞,经免疫组化鉴定其神经元烯醇化酶(NSE)、神经丝蛋白(NF)、胶质纤维酸性蛋白(GFAP)的表达。诱导后进行神经元样细胞计数,掺入Brdu测定,计算标记系数。采用双标记间接免疫荧光法检测神经元样细胞特异性表面标记(NSE、NF)与细胞分裂的标记Brdu的关系。抑制DNA合成,计算Brdu、NSE和NF阳性细胞数。结果MSC经参芪液诱导后,可见神经元样细胞。免疫组化显示诱导出的神经元样细胞NSE、NF阳性,GFAP阴性。MSC诱导分化后的细胞总数增加,诱导分化后12h内细胞增殖较快。30%以上分化的神经元样细胞在表达NSE和NF的同时可见Brdu标记,而另一部分分化的神经元则无Brdu标记。抑制DNA合成,并不会阻止神经元的分化,仍有70%的分化神经元表达神经元特异性标记蛋白NSE。结论MSC诱导分化的部分神经元样细胞可进行细胞分裂,但抑制细胞分裂不影响神经元分化。     

丹参注射液诱导间质干细胞分化为神经元样细胞

[目的]丹参注射液体外定向诱导成人骨髓间质干细胞(MSC)分化为神经元样细胞.[方法]采用Ficoll-Paque液离心分离成人MSC,体外扩增,流式细胞仪检测MSC表面抗原表达,分别采用含丹参注射液或硫代甘油等试剂的无血清达乐伯克改良必需基本培养基(DMEM)诱导MSC分化为神经元,免疫组化鉴定神经元特异性烯醇化酶(NSE)、神经丝蛋白(NF-M)、胶质纤维酸性蛋白(GFAP)、巢蛋白(nestin)的表达.[结果]成人骨髓间质干细胞在体外扩增原代可获得0.5×106,15代可获得(2～3)×1012个细胞,细胞流式细胞仪检测结果显示CD29、CD44、CD90、CD105、CD166表达阳性,CD11a、CD14、CD34、CD38、CD45、CD80、CD86为阴性.加入丹参或硫代甘油诱导后,MSC胞体收缩,突起伸出;免疫组化显示诱导出的神经元样细胞NSE、NF-M、nestin表达阳性,GFAP阴性.[结论]丹参可以在体外诱导成人骨髓间质干细胞分化为神经元样细胞.     

神经受体在人骨髓源性神经细胞上的表达

目的:检测神经受体在人骨髓源性神经细胞上的表达,探讨人骨髓间充质干细胞( hBMSC)体外诱导分化为神经细胞的方法及人骨髓源性神经细胞的功能特征.方法:分离纯化hBMSC,流式细胞仪检测CD34、CD45、CD44、CD117在hBMSC上的表达;应用DMSO/BHA/FSK/BDNF联合诱导hBMSC向神经细胞分化;...     

睾酮定向诱导MSC分化的实验研究

目的：激素体外定向诱导大鼠骨髓间质干细胞(MSC)分化为神经元样细胞。方法：通过贴壁法分离大鼠MSC，体外扩增培养，睾酮定向诱导MSC分化为类神经元样细胞。光镜下观察细胞形态，免疫细胞化学检测神经细胞特异性抗原标志。结果：大鼠骨髓间质干细胞可通过贴壁法成功分离并可在体外大量扩增。睾酮诱导6h后大部分MSC转变为神经元样细胞，出现胞体和突起，免疫细胞化学染色NSE、Nestin呈阳性、GFAP阴性。结论：大鼠骨髓间质干细胞可在体外经睾酮诱导分化为神经元样细胞。