Bone marrow stromal cells express neural phenotypes in vitro and migrate in brain after transplantation in vivo

Objective To investigate the differentiation of bone marrow stromal cells （BMSC） into neuron-like cells and to explore their potential use for neural transplantation. Methods BMSC from rats and adult humans were cultured in serum-containing media. Salvia miltiorrhiza was used to induce human BMSC （hBMSC） to differentiate. BMSC were identified with immunocytochemistry. Semi-quantitative RT-PCR was used to examine mRNA expression of neurofilamentl （NF1）, nestin and neuron-specific enolase （NSE） in rat BMSC （rBMSC）. Rat BMSC labelled by Hoschst33258 were transplanted into striatum of rats to trace migration and distribution. Results rBMSC expressed NSE, NFI and nestin mRNA, and NF1 mRNA and expression was increased with induction of Salvia miltiorrhiza. A small number of hBMSC were stained by anti-nestin, anti-GFAP and anti-S 100. Salvia miltiorrhiza could induce hBMSC to differentiate into neuron-like cells. Some differentiated neuron-like cells, that expressed NSE, beta-tubulin and NF-200, showed typical neuron morphology, but some neuron-like cells also expressed alpha smooth muscle protein, making their neuron identification complicated, rBMSC could migrate and adapted in the host brains after being transplanted. Conclusion Bone marrow stromal cells could express phenotypes of neurons, and Salvia milliorrhiza could induce hBMSC to differentiate into neuron-like cells, If BMSC could be converted into neurons instead of mesenchymal derivatives, they would be an abundant and accessible cellular source to treat a variety of neurological diseases.     

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.     

Autologous serum can induce mesenchymal stem cells into hepatocyte-like cells

Objective： To investigate whether the rabbit serum after radiofrequency ablation to liver tumor can induce mesenchymal stem cells （MSCs） differentiating into hepatocyte-like cells in order to find a new source and culture process for repairing liver injury. Methods： A tumor piece of 1 mm× 1mm×1 mm was transplanted into a tunnel at right liver of rabbits. The model of liver tumor was established after 2-3 weeks. The serum was collected from rabbits 72 h after being subjected to radiofrequency ablation of the liver tumor. Mesenchymal stem cells were isolated from rabbit bone marrow and cultured in DMEM containing autologous rabbit serum. Three kinds of media （L-DMEM） were tested respectively： ① containing 10% fetal calf serum （FCS）; ② containing 30% rabbit autologous serum after radiofrequency ablation of the liver tumor （ASRF）; ③ containing 30% rabbit autologous serum （AS）. MSCs were cultured on 12-well plates until passage 2 and examined under the light and electron microscopy at indicted intervals. The expression of albumin and CKl8 was detected using immunofluorescence to identify the characteristics of differentiated cells. Results： MSCs performed differently in the presence of fetal calf serum, rabbit autologous serum and rabbit autologous serum after radiofrequency ablation of the liver tumor. Induced by the serum after radiofrequency ablation to liver tumor for 7 d, the spindle-shaped MSCs turned into round shaped and resembled hepatocyte-like cells. The reactions were not found in MSCs cultured in FCS and AS groups. After induction for 14 d, slender microvilli, cell-cell junction structure and cholangiole emerged, and the differentiated cells expressed albumin and CKl 8. All those could not been observed in 10% FCS and 30% autologous serum groups. Conclusion： Mesenchymal stem cells differentiate into hepatocyte-like cells in the serum after radiofrequency ablation of liver tumor, providing us a potential cell source and culture process for clinical application in liver     

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.     

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.     

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.     

Human Adipose-derived Mesenchymal Stem Cells: A Better Cell Source for Nervous System Diseases Application

In order to suggest an ideal source of adult stem cells for treatment of nervous system diseases, neuronal differentiation capacity of human mesenchymal stem cells from adipose tissue (hADSCs) and bone marrow (hBMSCs) were studied in terms of nissl body and synapse structure formation, neural factors secretion function for the first time. The hADSCs and hBMSCs were isolated and differentiated to the neuron-like cells with rat brain-conditioned medium which is potentially a rich source of signals promoting neuronal differentiation. The specific proteins of neurons and neural factors secreted by the cells were detected with immunohistochemistry and ELISA analysis, respectively. Flow cytometric analysis showed that these cells had similar phenotypes and 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 presented neuron-like cell appearance after 14 days differentiation. hADSCs and hBMSCs could differentiated into neuronal-like cells, which were identified by neuron specific proteins: β-Tubulin-Ш, neuron-specific enolase (NSE), and nissl body and their ability to secrete brain derived neurophic factor (BDNF) and nerve growth factor (NGF). Assessment of synaptophysin and growth-associated protein-43 (GAP-43) suggested synapse structure formation in differentiated hADSCs and hBMSCs. The present results demonstrated that hADSCs have a similar neuronal differentiation potential with hBMSCs but with higher proliferation capacity. Adipose tissue is an abundant and easily available, which would be a potential ideal source of adult stem cells for neural-related clinical research and application.     

Induced Differentiation of Human Cord Blood Mesenchymal Stem/Pro genitor Cells into Cardiomyocyte-like Cells In Vitro

Summary:The feasibility of usiug cord blood mesenchymal stem/progenitor cells(CB-MSPCs)to re-generate cardiomyocytes and the optimal inducing conditions were investigated.The CB mononuclearcells were cultured in low serum DMEM medium to produce an adherent layer.After expansion,theadherent cells were added into cardiomyocyte inducing medium supplemented with 5-azacytidine.Cardiogenic specific contractile protein troponin T staining was performed to identify the cardiomy-ocyte-like cells.The results showed that the frequency of CB-MSPCs clones in CB mononuclear cellswas 0.5×10~(-6) and about 1.3×10~7-fold expansion was achieved within 20 sub-cultivation.After car-diogenic induction,70％CB-MSPCs was differentiated into cardiomyocyte-like cells.It was indicat-ed that low serum culture could expand CB-MSPCs extensively and the expanded CB-MSPCs couldbe induced to differentiate into cardiomyocyte-like cells in high efficiency.     

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。     

人脐血单个核细胞诱导分化为神经元样细胞

目的 探讨人脐血单个核细胞体外向神经元样细胞定向诱导分化的条件。方法 无菌条件下采集正常人脐血，分离单个核细胞。用DMEM/F12培养基进行纯化和扩增培养。取扩增5代的间充质干细胞(MSC)，分别用B-巯基乙醇(B—ME)、二甲基亚砜(DMSO)、神经条件培养液诱导脐血MSC向神经元样细胞分化。免疫组化法检测脐血MSC的Nestin表达和神经元样细胞特异性标志。结果 脐血原代和2、5代MSC的Nestin表达阳性率分别为6．7％、12．4％和20．8％。神经条件培养液诱导神经元样细胞阳性率最高达36％，B—ME、DMSO分别为33％和25％。结论脐血MSC具有神经干／祖细胞的特性，在一定条件下能向神经元样细胞分化。     

Differentiation of mesenchymal stem cells into dopaminergic neuron-like cells in vitro

INTRODUCTION The therapy of Parkinson’s disease is difficult, sopeople have been exploring more effective methods.Drugs can only treat symptoms of the diseases, andbrain tissue transplantation has been the most prosp-ective way, but the sources of transplantation cells needto be explored. The people have a new viewpoint ofthe central nervous system (CNS) regeneration andthe therapy of CNS diseases[1,2] because of the recentdiscovery of stem cell populations in CNS. But adultneura…     

人脐血单个核细胞诱导分化为神经元样细胞

目的 探讨人脐血单个核细胞体外向神经元样细胞定向诱导分化的条件。方法 无菌条件下采集正常人脐血,分离单个核细胞。用DMEM/F12培养基进行纯化和扩增培养。取扩增5代的间充质干细胞(MSC),分别用β-巯基乙醇(β-ME)、二甲基亚砜(DMSO)、神经条件培养液诱导脐血MSC向神经元样细胞分化。免疫组化法检测脐血MSC的Nestin表达和神经元样细胞特异性标志。结果 脐血原代和2、5代MSC的Nestin表达阳性率分别为6.7%、12.4%和20.8%。神经条件培养液诱导神经元样细胞阳性率最高达36%,β-ME、DMSO分别为33%和25%。结论 脐血MSC具有神经干/祖细胞的特性,在一定条件下能向神经元样细胞分化。     

透明质酸水凝胶体外促进骨髓间充质干细胞神经分化的研究

目的观察以透明质酸(hyaluronic acid,HA)水凝胶作细胞支架,骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells,BMSCs)在HA水凝胶中的生长、增殖和分化情况,探讨HA水凝胶支持、促进BMSCs分化为神经元细胞的作用。方法采用Percoll法从SD雄鼠的骨髓中分离纯化BMSCs,传代培养,倒置显微镜观察BMSCs的生长情况和形态变化。各组以不同的方法诱导BMSCs分化。采用免疫细胞化学法检测各组BMSCs在相应时间点神经元特异性烯醇化酶(NSE)和神经丝蛋白(NF)及巢蛋白(nestin)的阳性细胞数。RT-PCR检测各组BMSCs相应时间点的NSE mRNA、NF mRNA和nestin mRNA表达。结果传代后BMSCs形似纤维细胞。诱导后,HA水凝胶组(A组)和脑组织提取液组(B组)BMSCs分化为神经元样细胞:A组细胞黏附在HA水凝胶表面,长出较多突起和分支;B组细胞出现突起及少量分支。诱导后A组和B组NSE、NF阳性细胞增多(P<0.05)。诱导2d后及7d后A组和B组nestin阳性细胞均增多:2d后,A组高于B组(P<0.05);7d后,A组低于B组(P<0.05)。诱导7d后A组和B组的NSE mRNA、NF mRNA表达均增加,A组增加明显(P<0.05)。诱导2d后A组和B组的nestin mRNA表达增加,A组更显著(P<0.05);7d后A组和B组的nestin mRNA表达则降低,A组更明显(P<0.05)。结论 HA水凝胶有利于BMSCs的黏附,为BMSCs生长、增殖和分化提供了结构支持和适宜的微环境,促使BMSCs向神经元细胞分化、增殖并表达神经元特异性蛋白。     

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。     

大鼠骨髓间充质干细胞体外分化为神经元样细胞的研究

目的：研究大鼠骨髓间充质干细胞(rMSCs)体外扩增不同诱导剂向神经元样细胞诱导分化的潜能。方法：采用贴壁筛选法分离Wistar大鼠股骨rMSCs，体外扩增5-10代后，分别用2-巯基乙醇、硫代甘油等无血清的DMEM培养液诱导hMSCs分化为神经元。用流式细胞仪鉴定rMSCs特异抗原表达，免疫组化鉴定诱导后神经元样细胞的神经元烯醇化酶(NSH)、神经丝蛋白(NF)、胶质纤维酸性蛋白(GFAP)。结果：大鼠MSCs体外扩增传至5代后，流式细胞仪显示rMSCsCD27、CD44、CD90呈阳性表达；将rMSC加入2-巯基乙醇和硫代甘油等诱导剂3h后，rMSC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表达阳性，GFAP阴性。结论：大鼠骨髓间充质干细胞在体外-定条件下可以分化为神经元样细胞。     

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。