Correlation between receptor-interacting protein 140 expression and directed differentiation of human embr yonic stem cells into neural stem cells

Overexpression of receptor-interacting protein 140 (RIP140) promotes neuronal differentiation of N2a cells via extracellular regulated kinase 1/2 (ERK1/2) signaling. However, involvement of RIP140 in human neural differentiation remains unclear. We found both RIP140 and ERK1/2 expression increased during neural differentiation of H1 human embryonic stem cells. Moreover, RIP140 negatively correlat-ed with stem cell markers Oct4 and Sox2 during early stages of neural differentiation, and positively correlated with the neural stem cell marker Nestin during later stages. hTus, ERK1/2 signaling may provide the molecular mechanism by which RIP140 takes part in neural differentiation to eventually affect the number of neurons produced.     

Isolation and characterization of neural progenitor cells from post-mortem human cortex

Post-mortem human brain tissue represents a vast potential source of neural progenitor cells for use in basic research as well as therapeutic applications. Here we describe five human neural progenitor cell cultures derived from cortical tissue harvested from premature infants. Time-lapse videomicrography of the passaged cultures revealed them to be highly dynamic, with high motility and extensive, evanescent intercellular contacts. Karyotyping revealed normal chromosomal complements. Prior to differentiation, most of the cells were nestin, Sox2, vimentin, and/or GFAP positive, and a subpopulation was doublecortin positive. Multilineage potential of these cells was demonstrated after differentiation, with some subpopulations of cells expressing the neuronal markers beta-tubulin, MAP2ab, NeuN, FMRP, and Tau and others expressing the oligodendroglial marker O1. Still other cells expressed the classic glial marker glial fibrillary acidic protein (GFAP). RT-PCR confirmed nestin, SOX2, GFAP, and doublecortin expression and also showed epidermal growth factor receptor and nucleostemin expression during the expansion phase. Flow cytometry showed high levels of the neural stem cell markers CD133, CD44, CD81, CD184, CD90, and CD29. CD133 markedly decreased in high-passage, lineage-restricted cultures. Electrophysiological analysis after differentiation demonstrated that the majority of cells with neuronal morphology expressed voltage-gated sodium and potassium currents. These data suggest that post-mortem human brain tissue is an important source of neural progenitor cells that will be useful for analysis of neural differentiation and for transplantation studies.     

Establishment and characterization of a human primitive neuroectodermal tumor cell line from the cerebral hemisphere.

The primitive neuroectodermal tumors (PNET) comprise a class of malignant nervous system neoplasms that afflict children. These tumors consist of cells that are morphologically identical to the primitive neuroepithelial cells normally seen in early stages of neural embryogenesis, supporting the notion that PNET result from a disturbance in the process of normal neuronal or glial differentiation. In the central nervous system, PNET occur most commonly in the cerebellum (medulloblastomas), but only occasionally in the cerebral hemispheres. We report here the establishment and characterization of a new human cell line (PFSK) derived from a PNET from the cerebral hemisphere of a child. The growth characteristics of PFSK cells were typical of an immortalized, transformed cell line. Cytogenetic and molecular genetic studies showed that three different sublines were present. In one of these sublines, sequences from chromosome 17 had been lost during establishment in culture. Immunocytochemical studies showed that PFSK cells expressed nestin, an intermediate filament protein normally expressed by neuroepithelial stem cells during neurulation. The PFSK cells did not express antigens typically found in terminally differentiated neurons or glia, indicating that this tumor cell line might represent neuroepithelial stem cells prior to commitment to a neuronal or glial lineage.     

A human pluripotent carcinoma stem cell-based model for in vitro developmental neurotoxicity testing: Effects of methylmercury,lead and aluminum evaluated by gene expression studies

The major advantage of the neuronal cell culture models derived from human stem cells is their ability to replicate the crucial stages of neurodevelopment such as the commitment of human stem cells to the neuronal lineage and their subsequent stages of differentiation into neuronal and glial-like cell. In these studies we used mixed neuronal/glial culture derived from the NTERA-2 (NT-2) cell line, which has been established from human pluripotent testicular embryonal carcinoma cells. After characterization of the different stages of cell differentiation into neuronal- and glial-like phenotype toxicity studies were performed to evaluate whether this model would be suitable for developmental neurotoxicity studies. The cells were exposed during the differentiation process to non-cytotoxic concentrations of methylmercury chloride, lead chloride and aluminum nitrate for two weeks. The toxicity was then evaluated by measuring the mRNA levels of cell specific markers (neuronal and glial). The results obtained suggest that lead chloride and aluminum nitrate at low concentrations were toxic primarily to astrocytes and at the higher concentrations it also induced neurotoxicity. In contrast, MetHgCl was toxic for both cell types, neuronal and glial, as mRNA specific for astrocytes and neuronal markers were affected. The results obtained suggest that a neuronal mixed culture derived from human NT2 precursor cells is a suitable model for developmental neurotoxicity studies and gene expression could be used as a sensitive endpoint for initial screening of potential neurotoxic compounds.     

Molecular manipulation targeting regulation of dopaminergic differentiation and proliferation of neural stem cells or pluripotent stem cells

Parkinson's disease (PD) is a severe deliberating neurological disease caused by progressive degenerative death of dopaminergic neurons in the substantia nigra of midbrain. While cell replacement strategy by transplantation of neural stem cells and inducement of dopaminergic neurons is recommended for the treatment of PD, understanding the differentiation mechanism and controlled proliferation of grafted stem cells remain major concerns in their clinical application. Here we review recent studies on molecular signaling pathways in regulation of dopaminergic differentiation and proliferation of stem cells, particularly Wnt/beta-catenin signaling in stimulating formation of the dopaminergic phenotype, Notch signaling in inhibiting stem cell differentiation, and Sonic hedgehog functioning in neural stem cell proliferation and neuronal cell production. Activation of oncogenes involved in uncontrolled proliferation or tumorigenicity of stem cells is also discussed. It is proposed that a selective molecular manipulation targeting strategy will greatly benefit cell replacement therapy for PD by effectively promoting dopaminergic neuronal cell generation and reducing risk of tumorigenicity of in vivo stem cell applications.     

Differentiation of mesenchymal stem cells into neuronal cells on fetal bovine acellular dermal matrix as a tissue engineered nerve scaffold

The purpose of this study was to assess fetal bovine acellular dermal matrix as a scaffold for supporting the differentiation of bone marrow mesenchymal stem cells into neural cells following induction with neural differentiation medium.We performed long-term,continuous observation of cell morphology,growth,differentiation,and neuronal development using several microscopy techniques in conjunction with immunohistochemistry.We examined specific neuronal proteins and Nissl bodies involved in the differentiation process in order to determine the neuronal differentiation of bone marrow mesenchymal stem cells.The results show that bone marrow mesenchymal stem cells that differentiate on fetal bovine acellular dermal matrix display neuronal morphology with unipolar and bi/multipolar neurite elongations that express neuronal-specific proteins,including βIII tubulin.The bone marrow mesenchymal stem cells grown on fetal bovine acellular dermal matrix and induced for long periods of time with neural differentiation medium differentiated into a multilayered neural network-like structure with long nerve fibers that was composed of several parallel microfibers and neuronal cells,forming a complete neural circuit with dendrite-dendrite to axon-dendrite to dendrite-axon synapses.In addition,growth cones with filopodia were observed using scanning electron microscopy.Paraffin sectioning showed differentiated bone marrow mesenchymal stem cells with the typical features of neuronal phenotype,such as a large,round nucleus and a cytoplasm full of Nissl bodies.The data suggest that the biological scaffold fetal bovine acellular dermal matrix is capable of supporting human bone marrow mesenchymal stem cell differentiation into functional neurons and the subsequent formation of tissue engineered nerve.     

Pluripotent stem cell‐derived radial glia‐like cells as stable intermediate for efficient generation of human oligodendrocytes

Neural precursor cells (NPCs) derived from human pluripotent stem cells (hPSCs) represent an attractive tool for the in vitro generation of various neural cell types. However, the developmentally early NPCs emerging during hPSC differentiation typically show a strong propensity for neuronal differentiation, with more limited potential for generating astrocytes and, in particular, for generating oligodendrocytes. This phenomenon corresponds well to the consecutive and protracted generation of neurons and GLIA during normal human development. To obtain a more gliogenic NPC type, we combined growth factor‐mediated expansion with pre‐exposure to the differentiation‐inducing agent retinoic acid and subsequent immunoisolation of CD133‐positive cells. This protocol yields an adherent and self‐renewing population of hindbrain/spinal cord radial glia (RG)‐like neural precursor cells (RGL‐NPCs) expressing typical neural stem cell markers such as nestin, ASCL1, SOX2, and PAX6 as well as RG markers BLBP, GLAST, vimentin, and GFAP. While RGL‐NPCs maintain the ability for tripotential differentiation into neurons, astrocytes, and oligodendrocytes, they exhibit greatly enhanced propensity for oligodendrocyte generation. Under defined differentiation conditions promoting the expression of the major oligodendrocyte fate‐determinants OLIG1/2, NKX6.2, NKX2.2, and SOX10, RGL‐NPCs efficiently convert into NG2‐positive oligodendroglial progenitor cells (OPCs) and are subsequently capable of in vivo myelination. Representing a stable intermediate between PSCs and OPCs, RGL‐NPCs expedite the generation of PSC‐derived oligodendrocytes with O4‐, 4860‐, and myelin basic protein (MBP)‐positive cells that already appear within 7 weeks following growth factor withdrawal‐induced differentiation. Thus, RGL‐NPCs may serve as robust tool for time‐efficient generation of human oligodendrocytes from embryonic and induced pluripotent stem cells. GLIA 2015;63:2152–2167     

Proteomic analysis of proteins in PC12 cells before and after treatment with nerve growth factor: increased levels of a 43-kDa chromogranin B-derived fragment during neuronal differentiation

Proteomic analysis is an important approach to characterizing the proteome and studying protein function in the post-genomic era. It is also a powerful screening method for detecting unexpected alterations in protein expression that may be missed by conventional biochemical techniques. The aim of this study was to perform a preliminary proteomic analysis of PC12 cells in order to investigate the effect of nerve growth factor (NGF) on protein expression in PC12 cells during neurite outgrowth. PC12 cell proteins were separated by two-dimensional electrophoresis (2DE) and visualized by silver staining, then certain proteins were identified by N-terminal amino acid microsequencing and a homology search of a protein sequence database. Over 400 proteins were detected, 10% of which showed a significant (greater than 30%) increase or decrease in expression during NGF-induced neuronal differentiation. Seven proteins in the 2DE map were identified; the levels of five of these were unaffected by NGF treatment, whereas the levels of the other two, beta-tubulin and a novel 43-kDa chromogranin B-derived fragment, were significantly increased by more than 30 and 200%, respectively. Our results suggest that chromogranin B processing is enhanced in PC12 cells during NGF-induced neuronal differentiation. In addition, since this increase in the levels of the chromogranin B-derived fragment was specifically blocked by PD98059, we suggest that the increased processing can be ascribed to activation of the MAP kinase pathway, and that the 43-kDa chromogranin B-derived fragment can serve as a new marker of neuronal differentiation for proteomic studies.     

Sources of neuronal material for implantation

The adult brain is an organ that does not have the natural ability to replace cells that have been lost through damage. Possible human interventions to rectify this situation include transplanting either developing neural tissue into the damaged host brain or transplantation of neural stem cells (cells that have the capacity to proliferate into neural cells and self‐replicate) into the damaged area. Fetal or embryonic stem cells can be extracted and differentiated in vitro into the specific desired progeny (e.g. neurons). The neuronal stem cells themselves can be extracted from fetuses and multiplied in culture and then transplanted into the damaged brain. There is the possibility of de‐differentiation, in which cells of one type can be converted into a different cell type; for example, a differentiated blood cell could be de‐differentiated back to its own hemopoietic stem cell and that stem cell could be converted into a neuronal stem cell which could then be differentiated into a neuron. It is probable that methods of generating large numbers of committed stem cells to treat conditions such as Alzheimer's disease will soon be increasingly common.     

建立人肾小管上皮细胞蛋白质组双向电泳的分离体系

背景：双向电泳分离技术是蛋白质组学研究的核心技术之一,但蛋白质样品的分离效果受各种实验条件的影响较大。因此,针对不同来源的蛋白样品进行实验条件的优化可获得具有较高分辨率的双向电泳图谱。 目的：拟建立优化的人肾小管上皮细胞株蛋白质组双向电泳分离体系。 方法：常规培养人肾小管上皮细胞株HK-2细胞并裂解提取全蛋白,按标准条件对蛋白质进行双向电泳分离,并对各个关键因素进行优化。等电聚焦采用缓慢升压模式,电泳参数根据Bio-Rad公司的预设方案进行调整。改良硝酸银法进行蛋白质斑点染色。采集电泳图谱并分析双向电泳图谱中蛋白斑点的数量、图像分辨率及背景条纹的变化。 结果与结论：通过对实验条件的筛选和优化,成功建立了具有较高的分辨率和重复性的人肾小管上皮细胞蛋白质组双向电泳分离体系。其中,优化后的裂解液配方成分为1% TBP,4%CHAPS,0.2% Bio-Lyte,40 mmol/L Tris,8 mol/L尿素,2 mol/L硫脲;采用pH 4~7的IPG胶条;上样方式选择被动的水化上样。等电聚焦过程中使用预设的缓慢升压模式,充分聚焦后选用合适的电压模式进行SDS-PAGE电泳,然后采用改良硝酸银法进行染色,最终获得了满意的蛋白质组双向电泳图谱。     

Regulation of neuronal differentiation in human CNS stem cell progeny by leukemia inhibitory factor

The generation of diverse types of neural cells during development occurs through the progressive restriction of the fate potential of neuroepithelial progenitor cells. This process is controlled by factors intrinsic and extrinsic to the cell. While the effect of extrinsic cues on multipotent stem cells of the murine central nervous system (CNS) is becoming clearer, little is known of neural stem cells of human origin. We sought to establish the roles played by two cytokines, leukemia inhibitory (LIF) and ciliary neurotrophic factor (CNTF), and by nerve growth factor (NGF) and platelet-derived growth factor (PDGF) in regulating neuronal and astroglial differentiation in cultured embryonic diencephalic human stem cells. While NGF did not influence either neuronal or glial formation, PDGF surprisingly decreased the percentage of stem cell-generated neurons, an effect opposite to that observed in murine progenitors. Furthermore, while we confirmed the known ability of LIF and CNTF to support astroglial differentiation, we also observed that, in contrast with their murine counterparts, the fraction of CNS stem cell-generated neurons in human cultures was enhanced twofold in the presence of both cytokines. These findings highlight important differences between humans and rodents in regard to the way epigenetic cues regulate the function of neural stem cells.     

Differentiation of radial glia-like cells from embryonic stem cells

Radial glial cells play important roles in neural development. They provide support and guidance for neuronal migration and give rise to neurons and glia. In vitro, neurons, astrocytes, and oligodendrocytes can be generated from neural and embryonic stem cells, but the generation of radial glial cells from these stem cells has not yet been reported. Since the differentiation of radial glial cells is indispensable during brain development, we hypothesize that stem cells also generate radial glial cells during in vitro neural differentiation. To test this hypothesis, we utilized five different clones of mouse embryonic (ES) and embryonal carcinoma (EC) stem cell lines to investigate the differentiation of radial glial cells during in vitro neural differentiation. Here, we demonstrate that radial glia-like cells can be generated from ES/EC cell lines. These ES/EC cell-derived radial glia-like cells are similar in morphology to radial glial cells in vivo, i.e., they are bipolar with an unbranched long process and a short process. They also express several cytoskeletal markers, such as nestin, RC2, and/or GFAP, that are characteristics of radial glial cells in vivo. The processes of these in vitro generated radial glia-like cells are organized into parallel arrays that resemble the radial glial scaffolds in neocortical development. Since radial glia-like cells were observed in all five clones of ES/EC cells tested, we suggest that the differentiation of radial glial cells may be a common pathway during in vitro neural differentiation of ES cells. This novel in vitro model system should facilitate the investigation of regulation of radial glial cell differentiation and its biological function.     

Neurons derived from human-induced pluripotent stem cells express mu and kappa opioid receptors

Neuroprotection studies have shown that induced pluripotent stem(iPS) cells have the possibility to transform neuroprotection research. In the present study, iPS cells were generated from human renal epithelial cells and were then differentiated into neurons. Cells in the iPScell group were maintained in stem cell medium. In contrast, cells in the iPS-neuron group were first maintained in neural induction medium and expansion medium containing ROCK inhibitors, and then cultivated in neuronal differentiation medium and neuronal maturation medium to induce the neural stem cells to differentiate into neurons. The expression of relevant markers was compared at different stages of differentiation. Immunofluorescence staining revealed that cells in the iPS-neuron group expressed the neural stem cell markers SOX1 and nestin on day 11 of induction, and neuronal markers TUBB3 and NeuN on day 21 of induction. Polymerase chain reaction results demonstrated that, compared with the iPS-cell group, TUBB3 gene expression in the iPS-neuron group was increased 15.6-fold. Further research revealed that, compared with the iPS-cell group, the gene expression and immunoreactivity of mu opioid receptor in the iPS-neuron group were significantly increased(38.3-fold and 5.7-fold, respectively), but those of kappa opioid receptor had only a slight change(1.33-fold and 1.57-fold increases, respectively). Together, these data indicate that human iPS cells can be induced into mu opioid receptor-and kappa opioid receptor-expressing neurons, and that they may be useful to simulate human opioid receptor function in vitro and explore the underlying mechanisms of human conditions.     

Mitogen activated protein kinase signaling pathways participate in the active principle region of Buyang Huanwu decoction-induced differentiation of bone marrow mesenchymal stem cells

Our preliminary studies confirmed that an active principle region of Buyang Huanwu decoction, comprising alkaloid, polysaccharide, aglycon, glucoside and volatile oil, can induce bone marrow mesenchymal stem cell differentiation into neurons. Mitogen-activated protein kinase signaling was identified as one of the key pathways underlying this differentiation process. The present study shows phosphorylated extracellular signal-regulated protein kinase and phosphorylated p38 protein expression was increased after differentiation. Cellular signaling pathway blocking agents, PD98059 and SB203580, inhibited extracellular signal-regulated protein kinase and p38 in mitogen-activated protein kinase signaling pathways respectively. mRNA and protein expression of the neuronal marker, neuron specific enolase, and neural stem cell marker, nestin, were decreased in bone marrow mesenchymal stem cells after treatment with the active principle region of Buyang Huanwu decoction. Experimental findings indicate that, extracellular signal-regulated protein kinase and p38 in mitogen-activated protein kinase signaling pathways participate in bone marrow mesenchymal stem cell differentiation into neuron-like cells, induced by the active principle region of Buyang Huanwu decoction.     

Wnt-3a protein promote neuronal differentiation of neural stem cells derived from adult mouse spinal cord

BACKGROUND: Wnt proteins as growth factor have multiple functions in neural development, and especially serve key roles in differentiation and development. Wnt-3a is an intercellular signaling molecule that is involved in a variety of morphogenetic events. The purpose of this study was to investigate the effects of Wnt-3a signal protein on proliferation and differentiation of neural stem cells derived from adult mouse spinal cord. METHODS: Adult mouse neural stem cells were cultured with serum free incubation. The recombined plasmid pSecTag2/Hygro B-Wnt3a for eukaryotic expression transfected adult neural stem cell, then the expression protein was detected by Western blot. The differentiation of adult neural stem cells was identified by the immunocytochemical technique. RESULTS: The inducing differentiated rates of neurons were improved greatly by Wnt-3a protein compared with control (p<0.05). CONCLUSION: Wnt-3a has obvious influence on the neuronal differentiation of adult neural stem cell.     

Oct-3/4 repression accelerates differentiation of neural progenitor cells in vitro and in vivo

Oct-3/4 (Oct-3/Oct-4/POU5F1) is a critical regulator of embryonic stem (ES) cell differentiation, though its role in tissue stem cells that persist in differentiated tissues has not been shown. Here, we show that Oct-3/4 is expressed in neurospheres (NS) composed of neural stem cells and neural progenitor cells and that up- or down-regulation of Oct-3/4 by using adenovirus vectors influences cell fate. Oct-3/4 down-regulation accelerates neuronal differentiation of progenitor cells while its sustained expression prevents neuronal differentiation. Transplantation of neurospheres into the adult rat brain shows that down-regulation of Oct-3/4 promotes differentiation of NS cells in vivo. Our findings indicate that Oct-3/4 is an essential regulator of NS cell differentiation and suggest that the modulation of Oct-3/4 could be a useful tool in clinical application of NS cells.     

Behavior of human neural progenitor cells transplanted to rat brain

Human neural stem/progenitor cells provide a useful tool for studies of neural development and differentiation, as well as a potential means for neuroreplacement therapeutic needs in the human CNS. Stem cells isolated from developing human central nervous system of 8-12-week fetuses were transplanted to the forebrain and cerebellum of young and adult rats after 14 days of in vitro expansion. Cells were labeled by bisbenzimide prior to transplantation without immunosuppression. Recipient brains were examined 10 and 20 days after transplantation. Labeled stem cells were found in the neocortex, lateral ventricle and caudate nucleus in the forebrain, and in the molecular layer, Purkinje cell layer, and granular layer of the cerebellum. Mitotically dividing stem cells were observed in graft core, confirming their proliferative potential in new microenvironment. Engrafted cells migrate through the parenchyme of striatum, along the ventricular ependymal layer and callosal fibers, some of them reaching the opposite hemisphere. Some cells migrating along the capillaries express glial acid fibrillary protein, demonstrating their differentiation into astrocytes. Grafted cells expressing calbindin were found in the Purkinje cell layer, suggesting their differentiation into the Purkinje cells. At the same time, some grafted cells were undifferentiated and expressed vimentin. Our results demonstrate that cultured human neural stem/progenitor cells migrate and differentiate into both neurons and astrocytes after transplantation to the rat forebrain or cerebellum of young and adult rats.