Striatal PSA-NCAM(+) precursor cells from the newborn rat express functional glycine receptors

Immunocytochemical analysis showed that ionotropic glycine receptors are expressed in neurogenic progenitors purified from the newborn rat striatum and expressing the polysialylated form of the neural cell adhesion molecule, both in vitro and in situ. To ascertain whether glycine receptors were functional in vitro, whole-cell patch-clamp recordings demonstrated that glycine triggers inward strychnine-sensitive currents in the majority of these cells. Moreover, we found that glycine receptors expressed by these neurogenic progenitors display intermediate electrophysiological characteristics between those of glycine receptors expressed by neural stem cells and by mature interneurons from the rat striatum. Altogether, the present data show that functional strychnine-sensitive glycine receptors are expressed in neurogenic progenitors purified from the newborn rat striatum.     

Umbilical cord blood mesenchymal stromal cells are neuroprotective and promote regeneration in a rat optic tract model

Exploitation of the ability of stem cells to protect damaged neuronal tissue may be a more viable strategy than cell replacement for repair of the central nervous system (CNS). In this study we assessed the capacity of human umbilical cord blood (hUCB)-derived mesenchymal stromal cells (MSCs) to protect and promote regeneration of axotomised neurons within the rat optic system. The optic tract of neonatal rats was transected at the level of the lateral geniculate nucleus, and MSCs were introduced into the lesion site. MSCs survived well up to 2 weeks after grafting, and did not migrate significantly or differentiate. In the presence of MSC grafts, host axonal processes were found to be present in the lesion site, and there was stimulation of an endogenous neural precursor population. Four weeks after grafting, retrograde tracer experiments demonstrated that grafted MSCs, as well as cells of a human fibroblast line, exerted a neuroprotective effect, rescuing a significant percentage of axotomised retinal ganglion cells (RGCs). Further experiments with retrograde and anterograde tracers strongly indicated that MSCs could also promote re-growth of axotomised RGCs to their target, the superior colliculus (SC). Further analysis showed that hUCB-derived MSCs secreted several immunomodulatory and neurotrophic factors in vitro, including TGFβ1, CNTF, NT-3 and BDNF, which are likely to play a role in neuroprotection. Our data indicate that hUCB-derived MSCs may be an easily accessible, widely available source of cells that can contribute towards neural repair through rescue and regeneration of injured neurons.     

Rapid neural differentiation of human cord blood-derived mesenchymal stem cells

Human umbilical cord blood (UCB) contains hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs), both of which are regarded as valuable sources for cell transplantation and cell therapy. Adherent cells expressing MSCs-related antigens such as SH2, CD13, CD29, and ASMA, have been isolated from a mononuclear cell fraction of human UCB. Under proneurogenic conditions, these UCB-derived adherent cells rapidly assumed the morphology of multipolar neurons. Both immunofluorescence and RT-PCR analyses indicated that the expression of a number of neural markers including Tuj1, TrkA, GFAP and CNPases, was markedly elevated during this acute differentiation. The neurogenic potential of UCB-derived may facilitate stem cell therapeutic approaches to neurodegenerative diseases.     

Comparative analysis of neuroectodermal differentiation capacity of human bone marrow stromal cells using various conversion protocols

Human adult bone marrow-derived mesodermal stromal cells (hMSCs) are able to differentiate into multiple mesodermal tissues, including bone and cartilage. There is evidence that these cells are able to break germ layer commitment and differentiate into cells expressing neuroectodermal properties. There is still debate about whether this results from cell fusion, aberrant marker gene expression or real neuroectodermal differentiation. Here we extend our work on neuroectodermal conversion of adult hMSCs in vitro by evaluating various epigenetic conversion protocols using quantitative RT-PCR and immunocytochemistry. Undifferentiated hMSCs expressed high levels of fibronectin as well as several neuroectodermal genes commonly used to characterize neural cell types, such as nestin, beta-tubulin III, and GFAP, suggesting that hMSCs retain the ability to differentiate into neuroectodermal cell types. Protocols using a direct differentiation of hMSCs into a neural phenotype failed to induce significant changes in morphology and/or expression of markers of early and mature glial/neuronal cells types. In contrast, a multistep protocol with conversion of hMSCs into a neural stem cell-like population and subsequent terminal differentiation in mature glia and neurons generated relevant morphological changes as well as significant increase of expression levels of marker genes for early and late neural cell types, such as nestin, neurogenin2, MBP, and MAP2ab, accompanied by a loss of their mesenchymal properties. Our data provide an impetus for differentiating hMSCs in vitro into mature neuroectodermal cells. Neuroectodermally converted hMSCs may therefore ultimately help in treating acute and chronic neurodegenerative diseases. Analysis of marker gene expression for characterization of neural cells derived from MSCs has to take into account that several early and late neuroectodermal genes are already expressed in undifferentiated MSCs.     

Fluorescent labeling of rat auditory brainstem circuits for synaptic and electrophysiological studies

We visualized neurons with fluorescent agents, both retrogradely and anterogradely, to identify the input and output neuronal pathways in the rat auditory system. Output neurons of dorsal cochlear nucleus (DCN) were labeled retrogradely by injecting fluorescent microspheres into the inferior colliculus. Electrical recordings were made from the labeled fusiform cells of DCN with the whole-cell patch-clamp recording technique in slice preparations. DiI and Sindbis virus expressing membrane-targeted green fluorescent protein (GFP) were adopted for anterograde labeling. Auditory nerve fibers (ANFs) were labeled by injecting DiI into the cochlea, and the contralateral projection to the medial nucleus of the trapezoid body (MNTB) by injecting DiI and GFP into the ventral cochlear nucleus. A single ANF projecting to a DCN fusiform cell was electrically stimulated by a glass electrode and EPSCs were recorded using whole-cell patch-clamp recording methods. EPSCs were sensitive to the positioning of the electrode, and the size of EPSCs was constant irrespective of stimulus intensity, indicating that a single fiber was stimulated. Large EPSCs were generated from MNTB principal cells by stimulating the labeled fiber with a Calyx of Held terminal. The membrane excitability and EPSCs recorded after fluorescence labeling were similar to those previously reported. We confirm that the fluorescence labeling is effective to visualize neural networks and is useful to investigate the electrophysiological properties of neurons and synapses.     

Cytotoxicity of human umbilical cord blood-derived mesenchymal stem cells against human malignant glioma cells

Background Mesenchymal stem cells (MSCs) represent a potential useful source for cell-based glioma therapies because these cells evidence both orthodox and unorthodox plasticity and also show tropism for cancer. In this study, the authors attempted to access the cytotoxicity of human umbilical cord blood (hUCB)-derived MSCs, with or without cytokine activations against malignant glioma cells. Materials and methods hUCB-derived MSCs were activated by interleukin-2, interleukin-15, granulocyte macrophage colony-stimulating factor, and combinations. The hUCB-derived MSCs and activated hUCB-derived MSCs were effector cells. The cytotoxicity of the unactivated hUCB-derived MSCs and activated hUCB-derived MSCs against the target cells (human malignant glioma cells) was estimated via visual survival cell assays and transwell inserts. Phenotypic changes occurring in these hUCB-derived MSCs before and after cytokine activation were determined via flow cytometry. The secreted proteins from these effector cells were estimated via enzyme-linked immunosorbent assays. Results We noted a significant cytotoxicity of hUCB-derived MSCs against malignant glioma cells. In addition, the hUCB-derived MSCs activated with cytokines evidenced significantly higher cytotoxicity than that observed with unactivated hUCB-derived MSCs. Differentiated immune effectors cells from the hUCB-derived MSCs after cytokine activation were not shown to have increased in number. However, the activated hUCB-derived MSCs secreted more immune response-related proteins (interleukin 4, interferon-γ) than did the unactivated hUCB-derived MSCs. Conclusion The data collected herein confirm for the first time that hUCB-derived MSCs, with or without activation, evidence significant cytotoxicity against human malignant glioma cells, and the immune response-related proteins secreted in this process may perform relevant functions.     

Mesenchymal stem cells expressing neural antigens instruct a neurogenic cell fate on neural stem cells

The neurogenic response to injury in the postnatal brain is limited and insufficient for restoration of function. Recent evidence suggests that transplantation of mesenchymal stem cells (MSCs) into the injured brain is associated with improved functional recovery, mediated in part through amplification in the endogenous neurogenic response to injury. In the current study we investigate the interactions between bone marrow-derived MSCs and embryonic neural stem cells (NSCs) plus their differentiated progeny using an in vitro co-culture system. Two populations of MSCs were used, MSCs induced to express neural antigens (nestin+, Tuj-1+, GFAP+) and neural antigen negative MSCs. Following co-culture of induced MSCs with differentiating NSC/progenitor cells a significant increase in Tuj-1+ neurons was detected compared to co-cultures of non-induced MSCs in which an increase in astrocyte (GFAP+) differentiation was observed. The effect was mediated by soluble interactions between the two cell populations and was independent of any effect on cell death and proliferation. Induced and non-induced MSCs also promoted the survival of Tuj-1+ cell progeny in long-term cultures and both promoted axonal growth, an effect also seen in differentiating neuroblastoma cells. Therefore, MSCs provide instructive signals that are able to direct the differentiation of NSCs and promote axonal development in neuronal progeny. The data indicates that the nature of MSC derived signals is dependent not only on their microenvironment but on the developmental status of the MSCs. Pre-manipulation of MSCs prior to transplantation in vivo may be an effective means of enhancing the endogenous neurogenic response to injury.     

Improvement of neurological deficits by intracerebral transplantation of human adipose tissue-derived stromal cells after cerebral ischemia in rats

Mesenchymal stem cells can be expanded rapidly in vitro and differentiated into multiple mesodermal cell types. In addition, their differentiation into neuron-like cells expressing markers typical for mature neurons has been reported. We isolated human adipose tissue stromal cells (hATSCs) from human liposuction tissues and induced neural differentiation with azacytidine. Following neural induction, hATSCs changed toward neural morphology and displayed expression of MAP2 and GFAP. hATSCs, which were labeled with LacZ adenovirus, were injected into the lateral ventricle of the rat brain. Transplanted cells migrated to various parts of the brain, and ischemic brain injury by middle cerebral artery occlusion (MCAo) increased their migration to the injured cortex. Some of the transplanted cells expressed MAP2 and GFAP. Transplantation of hATSCs improved functional deficits in ischemic brain injury induced by MCAo. Intracerebral grafting of BDNF-transduced hATSCs significantly improved motor recovery of functional deficits in MCAo rats. These data indicate that transplanted hATSCs survive, migrate, and improve functional recovery after stroke and that genetically engineered hATSCs can express biologically active gene products and, therefore, can function as effective vehicles for therapeutic gene transfer to the brain.     

Differentiation assays of bone marrow-derived Multipotent Adult Progenitor Cell (MAPC)-like cells towards neural cells cannot depend on morphology and a limited set of neural markers

There are accumulating studies that report a neurogenic potential of bone marrow-derived cells both in vitro as well as in vivo. Most claims of neural "transdifferentiation" have based their conclusions on morphology and neural gene expression. Recently, doubts have been raised about the validity of both outcome parameters since non-neural cells can extend neurites and show aberrant neural gene expression as a response to stress inducing factors. In this study, we compared bone marrow-derived Multipotent Adult Progenitor Cell (MAPC)-like cells and neural stem cells (NSC) in their morphology and neural gene expression profile after neural differentiation using three differentiation protocols. We evaluated the expression of five neuroglial antigens [neurofilament 200 (NF200); beta III tubulin (beta3 tub); tau; Glial Fibrillary Acidic Protein (GFAP); Myelin Basic Protein (MBP) and RIP antigen] using real-time PCR (RT-PCR) and immunocytochemistry (ICC). MAPC-like cells adopted a neural-like morphology in one protocol but a fibroblast-like morphology in the two other protocols. RT-PCR and ICC show that MAPC-like cells already express the neural antigens beta III tubulin and NF200 at baseline, but no upregulation of these genes after exposure to three distinct differentiation protocols was seen. In contrast, NSC adopt neural and glial morphologies with a clear increase in expression of all neuroglial genes in all differentiation protocols used. In conclusion, our data demonstrate that neural-like morphology and expression of a limited set of neural marker genes by MAPC-like cells after differentiation are not absolute proof of neural transdifferentiation because MAPC-like cells only partially meet the criteria which are fulfilled by NSC after neural differentiation.     

Surface protein patterns govern morphology, proliferation, and expression of cellular markers but have no effect on physiological properties of cortical precursor cells

The ability to differentiate and give rise to neurons, astrocytes, and oligodendrocytes is an inherent feature of neural stem cells, which raises hopes for cell-based therapies of neurodegenerative diseases. However, there are many hurdles to cross before such regimens can be applied clinically. A considerable challenge is to elucidate the factors that contribute to neural differentiation. In this study, we evaluated the possibility of steering neuronal maturation by growing cortical precursor cells on microscale surface patterns of extracellular matrix (ECM) proteins. When the cells were encouraged to extend processes along lines of ECM proteins, they displayed a much more mature morphology, less proliferation capacity, and greater expression of a neuronal marker in comparison with cells grown in clusters on ECM dots. This implied that the growth pattern alone could play a crucial role for neural differentiation. However, in spite of the strikingly different morphology, when performing whole-cell patch-clamp experiments, we never observed any differences in the functional properties between cells grown on the two patterns. These results clearly demonstrate that morphological appearances are not representative measures of the functional phenotype or grade of neuronal maturation, stressing the importance of complementary electrophysiological evidence. To develop successful transplantation therapies, increased cell survival is critical. Because process-bearing neurons are sensitive and break easily, it would be of clinical interest to explore further the differentiating capacity of the cells cultured on the ECM dot pattern, described in this article, which are devoid of processes but display the same functional properties as neurons with mature morphology.     

人脐带间充质干细胞体外扩增和向神经元样细胞定向诱导分化的研究

目的探讨人脐带间充质干细胞(MSCs)的体外分离、纯化、扩增和向神经元样细胞的定向诱导分化,以期为脐带MSCs的神经移植提供理论依据。方法无菌条件下收集剖宫产新生儿脐带,酶消化法获取MSCs,进行培养。用流式细胞仪检测MSCs的表面标志。取扩增3,5,10代的MSCs分别向神经元样细胞诱导,用免疫组化和RT-PCR法检测神经元样细胞特异性标志。结果脐带富含MSCs,且脐带MSCs(UCMSCs)强表达CD13、29、CD44、CD105,弱表达CD106,不表达CD34、CD11a、CD14、CD33、CD45。神经条件培养基诱导后的细胞平均有70%左右呈现典型的神经元样表型。免疫组化法检测发现不同代数的MSCs经诱导后均表达nestin,NSE,NeuN,NF-M,弱表达GFAP。RT-PCR显示诱导后NSEmRNA表达增加。结论MSCs存在于人脐带中,并且在体外有较强的增殖能力,特定条件下能够分化为神经元样细胞。     

Synaptic and morphologic properties in vitro of premotor rat nucleus tractus solitarius neurons labeled transneuronally from the stomach

Neurons in the rat nucleus tractus solitarius (NTS) possess morphologic characteristics that have been correlated with the type of synaptic information they receive. These features have been described for viscerosensory neurons but not for premotor NTS neurons. The morphologic and synaptic features of neurons in the rat caudal NTS were assessed using whole-cell patch-clamp recordings and biocytin labeling in brainstem slices. Gastric-related premotor NTS neurons were identified for recording after inoculation of the stomach wall with a transneuronal retrograde viral label that reports enhanced green fluorescent protein. Three morphologic groups of NTS neurons were identified based on quantitative aspects of soma area and proximal dendritic arborization, measures that were consistent across slice recordings. The most common type of cell (group I) had relatively small somata and one to three sparsely branching dendrites, whereas the other groups had larger somata and more than three dendrites, which branched predominantly close to (group II) or distant from (group III) the soma. Voltage-clamp recordings revealed spontaneous excitatory and inhibitory postsynaptic currents in all neurons, regardless of morphology. Gastric-related premotor NTS neurons composed two of the three morphologic types (i.e., groups I and II). Compared with unlabeled neurons, these cells were less likely to receive constant-latency synaptic input from the tractus solitarius. These results refute the hypothesis that general patterns of synaptic input to NTS neurons depend on morphology. Gastric premotor neurons comprise a subset of NTS morphologic types, the organization of the viscerosensory input to which has yet to be defined.     

碱性成纤维细胞生长因子预诱导对骨髓基质干细胞向多巴胺能神经元分化的影响

目的探讨碱性成纤维细胞生长因子（bFGF）预诱导对骨髓基质干细胞（MSCs）向多巴胺（DA）能神经元分化的影响。方法取雄性Wistar大鼠股骨和胫骨骨髓，进行MSCs的体外培养、传代扩增及纯化。bFGF预诱导24h后，依据加入的神经营养因子不同分为单唾液酸四己糖神经节苷脂（GMl）组、胶质源性神经营养因子（GDNF）组和GDNF＋GMl组，以及对照组。倒置显微镜下观察细胞形态变化，分别在预诱导第3d、7d进行神经元特异性烯醇化酶（NSE）、神经胶质酸性蛋白（GFAP）、酪氨酸羟化酶（TH）免疫细胞化学检测。计数NSE和TH阳性细胞数，并计算阳性细胞百分比。结果对照组见少量NSE阳性细胞。实验组于诱导第3d、7d见较多数量的NSE、TH阳性细胞，GFAP阴性。bFGF预诱导各组中GDNF＋GMl组NSE、TH阳性细胞率最高，GDNF组次之，GMl组最低，组间比较差异有统计学意义（均P〈0．01）。结论bF—GF预诱导不仅可明显促进GDNF、GMl诱导MSCs向神经元样细胞分化，表达神经元细胞标志物——NSE；还可促进MSCs向DA能神经元分化，表达DA能神经元标志物——TH。     

星形胶质细胞源性因子对神经干细胞分化的实验研究

目的探讨星形胶质细胞源性因子对神经干细胞分化的影响。方法分离和培养新生大鼠脑组织的神经干细胞;采用差速贴壁法和振荡法分离纯化星形胶质细胞,用免疫细胞化学染色法,胶质纤维酸性蛋白(GFAP)标记星形胶质细胞,进行细胞的纯度鉴定;将星形胶质细胞和神经干细胞在互不接触的情况下进行共培养,免疫荧光法观察神经干细胞分化后神经元特异性烯醇化酶(NSE)、GFAP和酪氨酸羟化酶(TH)的表达。结果纯化的星形胶质细胞GFAP抗体标记阳性,细胞纯度达98%;星形胶质细胞与神经干细胞共培养时,神经干细胞贴壁分化加快,NSE阳性细胞及TH阳性细胞明显多于对照组(P<0·05)。结论星形胶质细胞源性因子可快速诱导神经干细胞向神经元细胞、包括多巴胺神经元细胞分化,提示星形胶质细胞支持神经元发生。     

Loss of RE-1 silencing factor in mesenchymal stem cell-derived dopamine progenitors induces functional maturity

Stem cell-derived dopamine (DA) neurons hold great promise for Parkinson's disease (PD). Mesenchymal stem cells (MSCs) have great potential for clinical applications. The generation of DA cells from MSCs using sonic hedgehog (SHH) and fibroblast growth factors (FGF8 and bFGF) has been reported. However, the DA cells showed weak electrical properties, representing DA neuron progenitors. Since RE-1 Silencing Factor (REST), suppresses mature neuronal genes in neuronal progenitors, we studied its role in the maturation of MSC-derived DA cells. REST expression did not change during the induction process, thus we knocked down REST and subjected MSCs to the same neural induction cocktail. We observed increases in the protein level of the Na(+) voltage-gated channel and tyrosine hydroxylase (TH). Electrophysiological analyses showed spontaneous firings and spontaneous postsynaptic currents, similar to native DA neurons. Taken together, these results show REST as the limiting gene in the generation of functional mature neurons from MSCs.     

猫骨髓源性神经干细胞诱导分化的实验研究

目的探讨猫骨髓分离培养、诱导分化神经干细胞的可行性。方法无菌条件下行骨穿,梯度密度离心获取猫骨髓基质细胞,以“神经干细胞培养基”培养,用分化诱导因子进行体外培养和诱导分化。结果猫骨髓基质细胞在相应培养条件下能在体外培养中增殖、分化,克隆形成细胞球(或称“神经球”),这些细胞球能表达神经干细胞特异性抗原nestin,而且能进一步诱导分化出胶质样细胞和神经元样细胞,免疫细胞化学检测可见有胶质源性纤维酸性蛋白抗体(GFAP)和神经元特异性烯醇化酶(NSE)抗原表达。结论猫骨髓基质细胞在一定条件诱导下可分化成神经胶质样和神经元样细胞。     

Modification of the brain-derived neurotrophic factor gene: a portal to transform mesenchymal stem cells into advantageous engineering cells for neuroregeneration and neuroprotection

Multipotential mesenchymal stem cells (MSCs) are ideal seed cells for recruiting the loss of neural cells due to their strong proliferative capacity, easy acquisition, and considerable tolerance of genetic modifications. After transduction of brain-derived neurotrophic factor (BDNF) gene via recombinant retroviral vectors into the human MSCs, nearly 100% of cells expressed BDNF (which were therefore transformed into BNDF-MSCs) as detected by immunocytochemistry, and the quantity of BDNF in the culture medium was increased by approximately 20,000-fold. In spite of the genomic integration of an exogenous gene, BDNF-MSCs did not present any structural aberration in the chromosomes. All-trans-retinoic acid (RA) induction caused the BDNF-MSCs to differentiate into neural cells with significantly increased expressions of such neural-specific proteins as nestin, NeuN, O4, and glial fibrillary acidic protein (GFAP). The voltage-dependent K+/Ca2+ currents were recorded from the induced BDNF-MSCs using patch-clamp technique. Compared with the MSCs induced by both RA and BDNF, BDNF-MSCs survived in significantly greater number in the induction medium, and also more cells were induced into neuron-like cells (NeuN, P < 0.01) and oligodendrocyte-like cells (O4, P < 0.05). We suppose that, once engrafted into human central nervous system, the BDNF-MSCs would not only recruit the neuronal losses, but also provide, by way of paracrine, large quantities of BDNF that effectively perform the functions of neuroprotection and neuroregeneration, promoting the activation of endogenous neural stem/progenitor cells and their chemotactic migration. On the other hand, the BDNF-MSCs that can survive in the host environment and differentiate subsequently into functional mature cells may also serve as specifically targeting vectors for ex vivo gene therapy.     

大鼠骨髓间充质干细胞体外培养及分化为神经干细胞的实验研究

目的研究大鼠骨髓间充质干细胞(bone marrow mesenchymal stemcells,MSCs)体外分化为神经干细胞(neural stemcells,NSCs)的可能性。方法取4～6周SD大鼠双侧股骨和胫骨骨髓细胞进行体外培养,通过传代得到纯化的MSCs后换用分化液诱导,通过形态学观察诱导后细胞形态变化,并用免疫荧光检测不同天数细胞nestin的表达率;NSCs分化实验对所诱导的NSC的分化潜能进行检测。结果MSCs诱导后的细胞nestin表达率随天数增加逐渐升高,1周后形成高表达nestin的神经干细胞球形细胞团;在含血清培养基中可自然分化为神经元和神经胶质样细胞。结论MSCs能于体外分化出神经干细胞,且具有进一步的分化能力,骨髓来源的神经干细胞将可能作为种子细胞用于神经系统疾病的治疗。     

骨髓间充质干细胞体外诱导分化为神经元样细胞的实验研究

目的　探索骨髓间充质干细胞 (MSCs)能否在体外诱导分化成神经样细胞 ,并初步探讨其分化机制。方法　培养大鼠MSCs,用二甲亚砜 (DMSO)和丁羟茴醚 (BHA)诱导分化 ,鉴定诱导分化前后的细胞是否表达神经细胞及神经干细胞的特异性标记蛋白 ,并研究其超微结构的变化。结果　诱导分化后 ,大部分MSCs变成双极、多极和锥形 ,并相互交织成网络结构 ,出现神经元特异性核蛋白 (NeuN)和巢蛋白 (Nestin)表达 ,无胶质纤维酸性蛋白(GFAP)和 2 3 环核苷酸磷酸二脂酶 (CNP)表达。部分MSCs转变为典型的神经元超微结构。结论　MSCs可以在体外诱导分化为神经元样细胞。