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Sample records for acid-induced neuronal differentiation

  1. Transcriptional Elongation Factor Elongin A Regulates Retinoic Acid-Induced Gene Expression during Neuronal Differentiation

    Takashi Yasukawa

    2012-11-01

    Full Text Available Elongin A increases the rate of RNA polymerase II (pol II transcript elongation by suppressing transient pausing by the enzyme. Elongin A also acts as a component of a cullin-RING ligase that can target stalled pol II for ubiquitylation and proteasome-dependent degradation. It is not known whether these activities of Elongin A are functionally interdependent in vivo. Here, we demonstrate that Elongin A-deficient (Elongin A−/− embryos exhibit abnormalities in the formation of both cranial and spinal nerves and that Elongin A−/− embryonic stem cells (ESCs show a markedly decreased capacity to differentiate into neurons. Moreover, we identify Elongin A mutations that selectively inactivate one or the other of the aforementioned activities and show that mutants that retain the elongation stimulatory, but not pol II ubiquitylation, activity of Elongin A rescue neuronal differentiation and support retinoic acid-induced upregulation of a subset of neurogenesis-related genes in Elongin A−/− ESCs.

  2. Wnt signaling pathway participates in valproic acid-induced neuronal differentiation of neural stem cells

    Wang, Li; Liu, Yuan; Li, Sen; Zai-yun LONG; Wu, Ya-min

    2015-01-01

    Neural stem cells (NSCs) are multipotent cells that have the capacity for differentiation into the major cell types of the nervous system, i.e. neurons, astrocytes and oligodendrocytes. Valproic acid (VPA) is a widely prescribed drug for seizures and bipolar disorder in clinic. Previously, a number of researches have been shown that VPA has differential effects on growth, proliferation and differentiation in many types of cells. However, whether VPA can induce NSCs from embryonic cerebral cor...

  3. Retinoic Acid Induces Ubiquitination-Resistant RIP140/LSD1 Complex to Fine-Tune Pax6 Gene in Neuronal Differentiation.

    Wu, Cheng-Ying; Persaud, Shawna D; Wei, Li-Na

    2016-01-01

    Receptor-interacting protein 140 (RIP140) is a wide-spectrum coregulator for hormonal regulation of gene expression, but its activity in development/stem cell differentiation is unknown. Here, we identify RIP140 as an immediate retinoic acid (RA)-induced dual-function chaperone for LSD1 (lysine-specific demethylase 1). RIP140 protects LSD1's catalytic domain and antagonizes its Jade-2-mediated ubiquitination and degradation. In RA-induced neuronal differentiation, the increased RIP140/LSD1 complex is recruited by RA-elevated Pit-1 to specifically reduce H3K4me2 modification on the Pax6 promoter, thereby repressing RA-induction of Pax6. This study reveals a new RA-induced gene repressive mechanism that modulates the abundance, enzyme quality, and recruitment of histone modifier LSD1 to neuronal regulator Pax6, which provides a homeostatic control for RA induction of neuronal differentiation. PMID:26372689

  4. Properties of acid-induced currents in mouse dorsal root ganglia neurons.

    Ergonul, Zuhal; Yang, Lei; Palmer, Lawrence G

    2016-05-01

    Acid-sensing ion channels (ASICs) are cation channels that are activated by protons (H(+)). They are expressed in neurons throughout the nervous system and may play important roles in several neurologic disorders including inflammation, cerebral ischemia, seizures, neurodegeneration, anxiety, depression, and migraine. ASICs generally produce transient currents that desensitize in response to a decrease in extracellular pH Under certain conditions, the inactivation of ASICs can be incomplete and allow them to produce sustained currents. Here, we characterize the properties of both transient and sustained acid-induced currents in cultured mouse dorsal root ganglia (DRG) neurons. At pH levels between 7.3 and 7.1 they include "window currents" through ASICs. With stronger acid signals sustained currents are maintained in the absence of extracellular Na(+) or the presence of the ASIC blockers amiloride and Psalmotoxin-1(PcTx1). These sustained responses may have several different origins in these cells, including acid-induced stimulation of inward Cl(-) currents, block of outward K(+) currents, and augmentation of inward H(+) currents, properties that distinguish these novel sustained currents from the well-characterized transient currents. PMID:27173673

  5. Metabolic reprogramming during neuronal differentiation.

    Agostini, M; Romeo, F; Inoue, S; Niklison-Chirou, M V; Elia, A J; Dinsdale, D; Morone, N; Knight, R A; Mak, T W; Melino, G

    2016-09-01

    Newly generated neurons pass through a series of well-defined developmental stages, which allow them to integrate into existing neuronal circuits. After exit from the cell cycle, postmitotic neurons undergo neuronal migration, axonal elongation, axon pruning, dendrite morphogenesis and synaptic maturation and plasticity. Lack of a global metabolic analysis during early cortical neuronal development led us to explore the role of cellular metabolism and mitochondrial biology during ex vivo differentiation of primary cortical neurons. Unexpectedly, we observed a huge increase in mitochondrial biogenesis. Changes in mitochondrial mass, morphology and function were correlated with the upregulation of the master regulators of mitochondrial biogenesis, TFAM and PGC-1α. Concomitant with mitochondrial biogenesis, we observed an increase in glucose metabolism during neuronal differentiation, which was linked to an increase in glucose uptake and enhanced GLUT3 mRNA expression and platelet isoform of phosphofructokinase 1 (PFKp) protein expression. In addition, glutamate-glutamine metabolism was also increased during the differentiation of cortical neurons. We identified PI3K-Akt-mTOR signalling as a critical regulator role of energy metabolism in neurons. Selective pharmacological inhibition of these metabolic pathways indicate existence of metabolic checkpoint that need to be satisfied in order to allow neuronal differentiation. PMID:27058317

  6. STRUCTURAL REMODELING OF PROTEOGLYCANS UPON RETINOIC ACID-INDUCED DIFFERENTIATION OF NCCIT CELLS*

    Gasimli, Leyla; Stansfield, Hope E.; Nairn, Alison V.; Liu, Haiying; Paluh, Janet L.; Yang, Bo; Dordick, Jonathan S.; Moremen, Kelley W.; Linhardt, Robert J.

    2012-01-01

    Pluripotent and multipotent cells become increasingly lineage restricted through differentiation. Alterations to the cellular proteoglycan composition and structure should accompany these changes to influence cell proliferation, delineation of tissues and acquisition of cell migration capabilities. Retinoic acid plays an important role in pre-patterning of the early embryo. Retinoic acid can be used in vitro to induce differentiation, causing pluripotent and multipotent cells to become increasingly lineage restricted. We examined retinoic acid-induced changes in the cellular proteoglycan composition of the well-characterized teratocarcinoma line NCCIT. Our analysis revealed changes in the abundance of transcripts for genes encoding core proteins, enzymes that are responsible for early and late linkage region biosynthesis, as well as enzymes for GAG chain extension and modification. Transcript levels for genes encoding core proteins used as backbones for polysaccharide synthesis revealed highly significant increases in expression of lumican and decorin, 1500-fold and 2800-fold, respectively. Similarly, glypican 3, glypican 5, versican and glypican 6 showed increases between 5 and 70-fold. Significant decreases in biglycan, serglycin, glypican 4, aggrecan, neurocan, CD74 and glypican 1 were observed. Disaccharide analysis of the glycans in heparin/heparan sulfate and chondroitin/dermatan sulfate revealed retinoic acid-induced changes restricted to chondroitin/dermatan sulfate glycans. Our study provides the first detailed analysis of changes in the glycosaminoglycan profile of human pluripotent cells upon treatment with the retinoic acid morphogen. PMID:23053635

  7. Structural remodeling of proteoglycans upon retinoic acid-induced differentiation of NCCIT cells.

    Gasimli, Leyla; Stansfield, Hope E; Nairn, Alison V; Liu, Haiying; Paluh, Janet L; Yang, Bo; Dordick, Jonathan S; Moremen, Kelley W; Linhardt, Robert J

    2013-07-01

    Pluripotent and multipotent cells become increasingly lineage restricted through differentiation. Alterations to the cellular proteoglycan composition and structure should accompany these changes to influence cell proliferation, delineation of tissues and acquisition of cell migration capabilities. Retinoic acid plays an important role in pre-patterning of the early embryo. Retinoic acid can be used in vitro to induce differentiation, causing pluripotent and multipotent cells to become increasingly lineage restricted. We examined retinoic acid-induced changes in the cellular proteoglycan composition of the well-characterized teratocarcinoma line NCCIT. Our analysis revealed changes in the abundance of transcripts for genes encoding core proteins, enzymes that are responsible for early and late linkage region biosynthesis, as well as enzymes for GAG chain extension and modification. Transcript levels for genes encoding core proteins used as backbones for polysaccharide synthesis revealed highly significant increases in expression of lumican and decorin, 1,500-fold and 2,800-fold, respectively. Similarly, glypican 3, glypican 5, versican and glypican 6 showed increases between 5 and 70-fold. Significant decreases in biglycan, serglycin, glypican 4, aggrecan, neurocan, CD74 and glypican 1 were observed. Disaccharide analysis of the glycans in heparin/heparan sulfate and chondroitin/dermatan sulfate revealed retinoic acid-induced changes restricted to chondroitin/dermatan sulfate glycans. Our study provides the first detailed analysis of changes in the glycosaminoglycan profile of human pluripotent cells upon treatment with the retinoic acid morphogen. PMID:23053635

  8. Sprouty2 and -4 hypomorphism promotes neuronal survival and astrocytosis in a mouse model of kainic acid induced neuronal damage.

    Thongrong, Sitthisak; Hausott, Barbara; Marvaldi, Letizia; Agostinho, Alexandra S; Zangrandi, Luca; Burtscher, Johannes; Fogli, Barbara; Schwarzer, Christoph; Klimaschewski, Lars

    2016-05-01

    Sprouty (Spry) proteins play a key role as negative feedback inhibitors of the Ras/Raf/MAPK/ERK pathway downstream of various receptor tyrosine kinases. Among the four Sprouty isoforms, Spry2 and Spry4 are expressed in the hippocampus. In this study, possible effects of Spry2 and Spry4 hypomorphism on neurodegeneration and seizure thresholds in a mouse model of epileptogenesis was analyzed. The Spry2/4 hypomorphs exhibited stronger ERK activation which was limited to the CA3 pyramidal cell layer and to the hilar region. The seizure threshold of Spry2/4(+/-) mice was significantly reduced at naive state but no difference to wildtype mice was observed 1 month following KA treatment. Histomorphological analysis revealed that dentate granule cell dispersion (GCD) was diminished in Spry2/4(+/-) mice in the subchronic phase after KA injection. Neuronal degeneration was reduced in CA1 and CA3 principal neuron layers as well as in scattered neurons of the contralateral CA1 and hilar regions. Moreover, Spry2/4 reduction resulted in enhanced survival of somatostatin and neuropeptide Y expressing interneurons. GFAP staining intensity and number of reactive astrocytes markedly increased in lesioned areas of Spry2/4(+/-) mice as compared with wildtype mice. Taken together, although the seizure threshold is reduced in naive Spry2/4(+/-) mice, neurodegeneration and GCD is mitigated following KA induced hippocampal lesions, identifying Spry proteins as possible pharmacological targets in brain injuries resulting in neurodegeneration. The present data are consistent with the established functions of the ERK pathway in astrocyte proliferation as well as protection from neuronal cell death and suggest a novel role of Spry proteins in the migration of differentiated neurons. © 2015 The Authors Hippocampus Published by Wiley Periodicals, Inc. PMID:26540287

  9. Optimal condition of directional-differentiation of neurons from retinoic-acid induced MSESPU35 embryonic stem cell lines%维甲酸诱导MESPU35胚胎干细胞系定向分化神经细胞的最佳条件

    秦茂林; 蔡文琴; 姚忠祥

    2006-01-01

    BACKGROUND: Neural axon regeneration is one of the difficulties that must be overcome in treatment of injury of central nerve system. Significant therapeutic effects have been obtained in transplantation of neural stem cells (NSCs), embryonic stem cells (ESCs) and Schwann cells. But the bottleneck situation of insufficiency of cell provider has limited the development on it.OBJECTIVE: To observe directional-differentiation of retinoic-acid induced ESCs so as to find optimal condition for neuronal differentiation.DESIGN: Non-randomized controlled experiment was designed.SETTING: Teaching-Research Room of Histology and Embryology, Department of Basic Medicines. Third Military Medical University of Chinese PLAMATERIALS: The experiment was performed in Staff Room of Histology and Embryology, Third Military Medical University of Chinese PLA from January to May 2000. Eighteen Kunming mice in disoestrus were employed, of which. 12 mice were female and 6 mice male. They were placed in same cage at ratio of 2:1 for mating. The date of pregnancy was recorded. MESPU35 ESC line was prepared.METHODS: Removed head. internal organs and four limbs, feeder-layer Feeder-layer adherent culture was used to proliferate MESPU35 ESCs.Classic 4-/4+ method [The embryoid body (EB) grew naturally for 4 days,without retinoic acid added. In the coming 4 days, retinoic acid was added to induce neural EB of high proportion] was applied to induce the directional differentiation of the nerve. EB was cultured with serum of different concentrations. Phase contrast microscope was used to observe nerve-like EB in serum of different concentrations and to count numbers. ②Immunocytochemical technique was used to observe cellular morphological charac ters at various differentiating phase spots (5th. 9th, 14th days) and with retinoic acid at various concentrations. Flow cytometer (FCM) was used to count the proportion of differentiated neurons.MAIN OUTCOME MEASURES: ①Estimated measurement of the length

  10. PI3K/AKT and ERK regulate retinoic acid-induced neuroblastoma cellular differentiation

    Qiao, Jingbo [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Paul, Pritha; Lee, Sora [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Qiao, Lan; Josifi, Erlena; Tiao, Joshua R. [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Chung, Dai H., E-mail: dai.chung@vanderbilt.edu [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232 (United States)

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer Retinoic acid (RA) induces neuroblastoma cells differentiation, which is accompanied by G0/G1 cell cycle arrest. Black-Right-Pointing-Pointer RA resulted in neuroblastoma cell survival and inhibition of DNA fragmentation; this is regulated by PI3K pathway. Black-Right-Pointing-Pointer RA activates PI3K and ERK1/2 pathway; PI3K pathway mediates RA-induced neuroblastoma cell differentiation. Black-Right-Pointing-Pointer Upregulation of p21 is necessary for RA-induced neuroblastoma cell differentiation. -- Abstract: Neuroblastoma, the most common extra-cranial solid tumor in infants and children, is characterized by a high rate of spontaneous remissions in infancy. Retinoic acid (RA) has been known to induce neuroblastoma differentiation; however, the molecular mechanisms and signaling pathways that are responsible for RA-mediated neuroblastoma cell differentiation remain unclear. Here, we sought to determine the cell signaling processes involved in RA-induced cellular differentiation. Upon RA administration, human neuroblastoma cell lines, SK-N-SH and BE(2)-C, demonstrated neurite extensions, which is an indicator of neuronal cell differentiation. Moreover, cell cycle arrest occurred in G1/G0 phase. The protein levels of cyclin-dependent kinase inhibitors, p21 and p27{sup Kip}, which inhibit cell proliferation by blocking cell cycle progression at G1/S phase, increased after RA treatment. Interestingly, RA promoted cell survival during the differentiation process, hence suggesting a potential mechanism for neuroblastoma resistance to RA therapy. Importantly, we found that the PI3K/AKT pathway is required for RA-induced neuroblastoma cell differentiation. Our results elucidated the molecular mechanism of RA-induced neuroblastoma cellular differentiation, which may be important for developing novel therapeutic strategy against poorly differentiated neuroblastoma.

  11. Nerve growth factor protects cholinergic neurons against quinolinic acid-induced excitotoxicity in wistar rats

    Vasiljević Ivana D.; Jovanović Marina D.; Čolić Miodrag J.; Mićić D.; Ninković Milica; Maličević Živorad

    2004-01-01

    The etiology of neuronal death in neurodegenerative diseases, including Huntington's disease (HD) is still unknown. There could be a complex interplay between altered energy metabolism, excitotoxicity and oxidative stress. Excitotoxic striatal lesions induced by quinolinic acid (QA), were used to test for the neuroprotective actions of nerve growth factor (NGF) on striatal cholinergic and GABAergic neurons. QA is an endogenous excitotoxin acting on N-methyl-D-aspartate (NMDA) rec...

  12. Phenolic antioxidants attenuate hippocampal neuronal cell damage against kainic acid induced excitotoxicity

    M S Parihar; Taruna Hemnani

    2003-02-01

    Increasing evidence supports the role of excitotoxicity in neuronal cell injury. Thus, it is extremely important to explore methods to retard or reverse excitotoxic neuronal injury. In this regard, certain dietary compounds are begining to receive increased attention, in particular those involving phytochemicals found in medicinal plants in alleviating neuronal injury. In the present study, we examined whether medicinal plant extracts protect neurons against excitotoxic lesions induced by kainic acid (KA) in female Swiss albino mice. Mice were anesthetized with ketamine and xylazine (200 mg and 2 mg/kg body wt. respectively) and KA (0.25 g in a volume of 0.5 l) was administered to mice by intra hippocampal injections. The results showed an impairment of the hippocampus region of brain after KA injection. The lipid peroxidation and protein carbonyl content were significantly ( < 0.05) increased in comparison to controls. Glutathione peroxidase (GPx) activity (EC 1.11.1.9) and reduced glutathione (GSH) content declined after appearance of excitotoxic lesions. As GPx and GSH represent a major pathway in the cell for metabolizing hydrogen peroxide (H2O2), their depletion would be expected to allow H2O2 to accumulate to toxic levels. Dried ethanolic plant extracts of Withania somnifera (WS), Convolvulus pleuricauas (CP) and Aloe vera (AV) dissolved in distilled water were tested for their total antioxidant activity. The diet was prepared in terms of total antioxidant activity of plant extracts. The iron (Fe3+) reducing activity of plant extracts was also tested and it was found that WS and AV were potent reductants of Fe3+ at pH 5.5. CP had lower Fe3+ reducing activity in comparison to WS and AV. Plant extracts given singly and in combination 3 weeks prior to KA injections resulted in a decrease in neurotoxicity. Measures of lipid peroxidation and protein carbonyl declined. GPx activity and GSH content were elevated in hippocampus supplemented with WS and combination of

  13. Conditioned Medium Reconditions Hippocampal Neurons against Kainic Acid Induced Excitotoxicity: An In Vitro Study

    Bevinahal, Pradeep Kumar K.; Venugopal, Chaitra; Yencharla, Harish Chandra Prasad S.; Chandanala, Shashank; Trichur, Raju R.; Talakad, Sathyaprabha N.; Bhonde, Ramesh R.; Dhanushkodi, Anandh

    2014-01-01

    Stem cell therapy is gaining attention as a promising treatment option for neurodegenerative diseases. The functional efficacy of grafted cells is a matter of debate and the recent consensus is that the cellular and functional recoveries might be due to “by-stander” effects of grafted cells. In the present study, we investigated the neuroprotective effect of conditioned medium (CM) derived from human embryonic kidney (HEK) cells in a kainic acid (KA) induced hippocampal degeneration model system in in vitro condition. Hippocampal cell line was exposed to KA (200 µM) for 24 hrs (lesion group) whereas, in the treatment group, hippocampal cell line was exposed to KA in combination with HEK-CM (KA + HEK-CM). We observed that KA exposure to cells resulted in significant neuronal loss. Interestingly, HEK-CM cotreatment completely attenuated the excitotoxic effects of KA. In HEK-CM cotreatment group, the cell viability was ~85–95% as opposed to 47% in KA alone group. Further investigation demonstrated that treatment with HEK-CM stimulated the endogenous cell survival factors like brain derived neurotrophic factors (BDNF) and antiapoptotic factor Bcl-2, revealing the possible mechanism of neuroprotection. Our results suggest that HEK-CM protects hippocampal neurons against excitotoxicity by stimulating the host's endogenous cell survival mechanisms. PMID:25505907

  14. Conditioned Medium Reconditions Hippocampal Neurons against Kainic Acid Induced Excitotoxicity: An In Vitro Study

    Pradeep Kumar K. Bevinahal

    2014-01-01

    Full Text Available Stem cell therapy is gaining attention as a promising treatment option for neurodegenerative diseases. The functional efficacy of grafted cells is a matter of debate and the recent consensus is that the cellular and functional recoveries might be due to “by-stander” effects of grafted cells. In the present study, we investigated the neuroprotective effect of conditioned medium (CM derived from human embryonic kidney (HEK cells in a kainic acid (KA induced hippocampal degeneration model system in in vitro condition. Hippocampal cell line was exposed to KA (200 µM for 24 hrs (lesion group whereas, in the treatment group, hippocampal cell line was exposed to KA in combination with HEK-CM (KA + HEK-CM. We observed that KA exposure to cells resulted in significant neuronal loss. Interestingly, HEK-CM cotreatment completely attenuated the excitotoxic effects of KA. In HEK-CM cotreatment group, the cell viability was ~85–95% as opposed to 47% in KA alone group. Further investigation demonstrated that treatment with HEK-CM stimulated the endogenous cell survival factors like brain derived neurotrophic factors (BDNF and antiapoptotic factor Bcl-2, revealing the possible mechanism of neuroprotection. Our results suggest that HEK-CM protects hippocampal neurons against excitotoxicity by stimulating the host’s endogenous cell survival mechanisms.

  15. Modulation of the retinoic acid-induced cell apoptosis and differentiation by the human TR4 orphan nuclear receptor

    In our previous studies, the TR4 orphan nuclear receptor (TR4) has been demonstrated to suppress retinoic acid (RA)-induced transactivation via a negative feedback control mechanism and in situ analysis showed that TR4 is extensively expressed in mouse brain, especially in regions where the cells are proliferating. To further study the potential roles of TR4 during cell differentiation, a tetracycline-inducible system with anti-sense TR4 in teratocarcinoma P19 cell lines was generated to analyze the retinoic acid-induced differentiation of these cells. The results indicated that the expression of TR4 reduced by doxycycline anti-sense TR4 would alter the retinoic acid-induced differentiation pathway that results in the changes of cell morphology and cell cycle profile. Unexpectedly, our data further indicated that the RA-induced apoptosis, judging by DNA fragmentation, could also be altered by the induction of anti-sense TR4. Together, these findings provide the first in vivo evidence that an orphan nuclear receptor, such as TR4, may play major roles in the RA-mediated apoptosis or differentiation in P19 cells

  16. The BRPF2/BRD1-MOZ complex is involved in retinoic acid-induced differentiation of embryonic stem cells.

    Cho, Hye In; Kim, Min Seong; Jang, Yeun Kyu

    2016-08-01

    The scaffold protein BRPF2 (also called BRD1), a key component of histone acetyltransferase complexes, plays an important role in embryonic development, but its function in the differentiation of embryonic stem cells (ESCs) remains unknown. In the present study, we investigated whether BRPF2 is involved in mouse ESC differentiation. BRPF2 depletion resulted in abnormal formation of embryoid bodies, downregulation of differentiation-associated genes, and persistent maintenance of alkaline phosphatase activity even after retinoic acid-induced differentiation, indicating impaired differentiation of BRPF2-depleted ESCs. We also found reduced global acetylation of histone H3 lysine 14 (H3K14) in BRPF2-depleted ESCs, irrespective of differentiation status. Further, co-immunoprecipitation analysis revealed a physical association between BRPF2 and the histone acetyltransferase MOZ in differentiated ESCs, suggesting the role of BRPF2-MOZ complexes in ESC differentiation. Together, these results suggest that BRPF2-MOZ complexes play an important role in the differentiation of ESCs via H3K14 acetylation. PMID:27256846

  17. All-Trans Retinoic Acid Induces Expression of a Novel Intergenic Long Noncoding RNA in Adult rat Primary Hippocampal Neurons.

    Kour, Sukhleen; Rath, Pramod C

    2016-02-01

    Around 90% of the mammalian genome undergoes pervasive transcription into various types of small and long regulatory noncoding RNAs, whereas only ∼ 1.5% codes for proteins. Long noncoding RNAs (lncRNAs) constitute diverse classes of sense- and antisense transcripts that are abundantly expressed in the mammalian central nervous system (CNS) in cell type- and developmental stage-specific manners. They are implicated in brain development, differentiation, neuronal plasticity, and other cognitive functions. Mammalian brain requires the vitamin A metabolite all-trans retinoic acid (atRA) for its normal development, differentiation, and cell-fate determination. However, its role in adult brain function is less understood. Here, we report atRA-mediated transcriptional upregulation of endogenous expression of a novel long intergenic noncoding RNA-rat brain expressed (LINC-RBE) in cultured primary hippocampal neurons from adult rat. We have previously reported LINC-RBE as an intergenic, simple repeat sequence containing lncRNA highly expressed in the rat brain. This is a first-time report of involvement of atRA in transcriptional upregulation of lncRNA expression in rat hippocampal neurons. Therefore, it may be involved in regulation of brain function and disease. PMID:26572536

  18. STRUCTURAL REMODELING OF PROTEOGLYCANS UPON RETINOIC ACID-INDUCED DIFFERENTIATION OF NCCIT CELLS*

    Gasimli, Leyla; Stansfield, Hope E.; Nairn, Alison V.; Liu, Haiying; Janet L. Paluh; Yang, Bo; Dordick, Jonathan S.; Moremen, Kelley W.; Linhardt, Robert J.

    2012-01-01

    Pluripotent and multipotent cells become increasingly lineage restricted through differentiation. Alterations to the cellular proteoglycan composition and structure should accompany these changes to influence cell proliferation, delineation of tissues and acquisition of cell migration capabilities. Retinoic acid plays an important role in pre-patterning of the early embryo. Retinoic acid can be used in vitro to induce differentiation, causing pluripotent and multipotent cells to become increa...

  19. Retinoic acid induces nuclear accumulation of Raf1 during differentiation of HL-60 cells

    Smith, James; Bunaciu, Rodica P.; Reiterer, Gudrun [Department of Biomedical Sciences, T4-008 VRT, Cornell University, Ithaca, NY 14853 (United States); Coder, David; George, Thaddeus [Amnis Corporation, Seattle, Washington (United States); Asaly, Michael [Department of Biomedical Sciences, T4-008 VRT, Cornell University, Ithaca, NY 14853 (United States); Yen, Andrew, E-mail: ay13@cornell.edu [Department of Biomedical Sciences, T4-008 VRT, Cornell University, Ithaca, NY 14853 (United States)

    2009-08-01

    All trans-retinoic acid (RA) is a standard therapeutic agent used in differentiation induction therapy treatment of acute promyelocytic leukemia (APL). RA and its metabolites use a diverse set of signal transduction pathways during the differentiation program. In addition to the direct transcriptional targets of the nuclear RAR and RXR receptors, signals derived from membrane receptors and the Raf-MEK-ERK pathway are required. Raf1 phosphorylation and the prolonged activation of Raf1 persisting during the entire differentiation process are required for RA-dependent differentiation of HL-60 cells. Here we identify a nuclear redistribution of Raf1 during the RA-induced differentiation of HL-60 cells. In addition, the nuclear accumulation of Raf1 correlates with an increase in Raf1 phosphorylated at serine 621. The serine 621 phosphorylated Raf1 is predominantly localized in the nucleus. The RA-dependent nuclear accumulation of Raf1 suggests a novel nuclear role for Raf1 during the differentiation process.

  20. Differential microRNA expression in aristolochic acid-induced upper urothelial tract cancers ex vivo.

    Tao, Le; Zeng, Yigang; Wang, Jun; Liu, Zhihong; Shen, Bing; Ge, Jifu; Liu, Yong; Guo, Yifeng; Qiu, Jianxin

    2015-11-01

    Aristolochic acid (AA) is a carcinogenic, mutagenic and nephrotoxic compound commonly isolated from members of the plant family of Aristolochiaceae (such as Aristolochia and Asarum) and used in Chinese herbal medicine. Use of AA and AA‑containing plants causes chronic kidney disease (CKD) and upper urinary tract carcinoma (UUC); however, the underlying mechanism remains to be defined. miRNAs regulate a number of biological processes, including cell proliferation, differentiation and metabolism. This study explored differentially expressed miRNAs between AA‑induced upper urothelial tract cancer (AAN‑UUC) and non‑AAN‑UUC tissues. Patients with AAN‑UUC and non‑AAN‑UUC (n=20/group) were recruited in the present study. Five tissue samples from each group were used for miRNA microarray profiling and the rest of the tissue samples were subjected to reverse transcription-quantitative polymerase chain reaction analysis including seven selected miRNAs for confirmation. A total of 29 miRNAs were differentially expressed between AAN‑UUC and non‑AAN‑UUC tissues (Pontology analyses predicted the functions and targeted genes of these differentially expressed miRNAs, i.e. Akt3, FGFR3, PSEN1, VEGFa and AR. Subsequently, expression of the selected differentially expressed miRNAs (Hsa‑miR‑4795‑5p, Hsa‑miR‑488, Hsa‑miR‑4784, Hsa‑miR‑330, Hsa‑miR‑3916, Hsa‑miR‑4274 and Hsa‑miR‑181c) was validated in another set of tissue samples. A total of 29 miRNAs were identified to be differentially expressed between AAN‑UUC and non‑AAN‑UUC tissues and these miRNA target genes in FGFR3 and Akt pathways, which regulate cell growth and tumor progression, respectively. PMID:26397152

  1. Neuronal Differentiation of Human Mesenchymal Stem Cells Using Exosomes Derived from Differentiating Neuronal Cells

    Takeda, Yuji S.; Qiaobing Xu

    2015-01-01

    Exosomes deliver functional proteins and genetic materials to neighboring cells, and have potential applications for tissue regeneration. One possible mechanism of exosome-promoted tissue regeneration is through the delivery of microRNA (miRNA). In this study, we hypothesized that exosomes derived from neuronal progenitor cells contain miRNAs that promote neuronal differentiation. We treated mesenchymal stem cells (MSCs) daily with exosomes derived from PC12 cells, a neuronal cell line, for 1...

  2. Retinoic acid induces HL-60 cell differentiation via the upregulation of miR-663

    Zhuan Zhou

    2011-04-01

    Full Text Available Abstract Background Differentiation of the acute myeloid leukemia (AML cell line HL-60 can be induced by all trans-retinoic acid (ATRA; however, the mechanism regulating this process has not been fully characterized. Methods Using bioinformatics and in vitro experiments, we identified the microRNA gene expression profile of HL-60 cells during ATRA induced granulocytic differentiation. Results Six microRNAs were upregulated by ATRA treatment, miR-663, miR-494, miR-145, miR-22, miR-363* and miR-223; and three microRNAs were downregulated, miR-10a, miR-181 and miR-612. Additionally, miR-663 expression was regulated by ATRA. We used a lentivirus (LV backbone incorporating the spleen focus forming virus (SFFV-F promoter to drive miR-663 expression, as the CMV (Cytomegalovirus promoter is ineffective in some lymphocyte cells. Transfection of LV-miR-663 induced significant HL-60 cell differentiation in vitro. Conclusions Our results show miR-663 may play an important role in ATRA induced HL-60 cell differentiation. Lentivirus delivery of miR-663 could potentially be used directly as an anticancer treatment in hematological malignancies

  3. Influence of suppressor gene p16 on retinoic acid inducing cancer cell A549 differentiation

    2001-01-01

    Objective To investigate the role of suppressor gene p16 in the process of differential regulation of retinoic acid (RA) on the A549 lung cancer cells.Methods Tumor suppressor gene p16 was transferred into A549 cells and the cells were treated with all-trans retinoic acid (ATR) at the dosage of 5×10-6 mol/L for 4 d. After that, the proliferation and differentiation of A549 cells were examined by growth curve and cytometry analysis, the change of lung lineage-specific marker MUC1 was tested by immunohistochemical staining. Meanwhile, Western blot was used to observe the change of p16 protein expression in A549 cells treated with ATRA.Results ATRA could obviously inhibit the growth and induce the differentiation of A549 Cells that were transferred with p16 gene. There were more cells arrested in G1/G0 phase and the expression of MUG1 was markedly down-regulated than in control cells. The expression of p16 protein was up-regulated in A549 cells treated with ATRA.Conclusion Suppressor gene p16 could enhance the effects of RA and proliferated suppression and differential induction of A549 cells.

  4. Mechanisms of all-trans retinoic acid-induced differentiation of acute promyelocytic leukemia cells

    Ji-Wang Zhang; Jian Gu; Zhen-Yi Wang; Sai-Juan Chen; Zhu Chen

    2000-09-01

    Retinoic acids (RA) play a key role in myeloid differentiation through their agonistic nuclear receptors (RAR/RXR) to modulate the expression of target genes. In acute promyelocytic leukemia (APL) cells with rearrangement of retinoic acid receptor (RAR) (including: PML-RAR, PLZF-RAR, NPM-RAR, NuMA-RAR or STAT5b-RAR) as a result of chromosomal translocations, the RA signal pathway is disrupted and myeloid differentiation is arrested at the promyelocytic stage. Pharmacologic dosage of all-trans retinoic acid (ATRA) directly modulates PML-RAR and its interaction with the nuclear receptor co-repressor complex, which restores the wild-type RAR/RXR regulatory pathway and induces the transcriptional expression of downstream genes. Analysing gene expression profiles in APL cells before and after ATRA treatment represents a useful approach to identify genes whose functions are involved in this new cancer treatment. A chronologically well coordinated modulation of ATRA-regulated genes has thus been revealed which seems to constitute a balanced functional network underlying decreased cellular proliferation, initiation and progression of maturation, and maintenance of cell survival before terminal differentiation.

  5. MicroRNA gene expression during retinoic acid-induced differentiation of human acute promyelocytic leukemia.

    Garzon, R; Pichiorri, F; Palumbo, T; Visentini, M; Aqeilan, R; Cimmino, A; Wang, H; Sun, H; Volinia, S; Alder, H; Calin, G A; Liu, C-G; Andreeff, M; Croce, C M

    2007-06-14

    MicroRNAs (miRNAs) are small non-coding RNAs of 19-25 nucleotides that are involved in the regulation of critical cell processes such as apoptosis, cell proliferation and differentiation. However, little is known about the role of miRNAs in granulopoiesis. Here, we report the expression of miRNAs in acute promyelocytic leukemia patients and cell lines during all-trans-retinoic acid (ATRA) treatment by using a miRNA microarrays platform and quantitative real time-polymerase chain reaction (qRT-PCR). We found upregulation of miR-15a, miR-15b, miR-16-1, let-7a-3, let-7c, let-7d, miR-223, miR-342 and miR-107, whereas miR-181b was downregulated. Among the upregulated miRNAs, miR-107 is predicted to target NFI-A, a gene that has been involved in a regulatory loop involving miR-223 and C/EBPa during granulocytic differentiation. Indeed, we have confirmed that miR-107 targets NF1-A. To get insights about ATRA regulation of miRNAs, we searched for ATRA-modulated transcription factors binding sites in the upstream genomic region of the let-7a-3/let-7b cluster and identified several putative nuclear factor-kappa B (NF-kappaB) consensus elements. The use of reporter gene assays, chromatin immunoprecipitation and site-directed mutagenesis revealed that one proximal NF-kappaB binding site is essential for the transactivation of the let-7a-3/let-7b cluster. Finally, we show that ATRA downregulation of RAS and Bcl2 correlate with the activation of known miRNA regulators of those proteins, let-7a and miR-15a/miR-16-1, respectively. PMID:17260024

  6. Neuronal acid-induced [Zn²⁺]i elevations calibrated using the low-affinity ratiometric probe FuraZin-1.

    Kiedrowski, Lech

    2015-11-01

    The experiments were carried out on primary cultures of murine cortical neurons from cryopreserved preparations obtained from embryonic-day-16 fetuses. To calibrate acid-induced intracelluar [Zn(2+) ] ([Zn(2+) ]i ) elevations, a low affinity (Kd = 39 μM at pH 6.1) ratiometric Zn(2+) probe, FuraZin-1, was used. A pHi drop from 7.2 to 6.1 caused [Zn(2+) ]i elevations reaching 2 μM; when the thiol-reactive agent N-ethylmaleimide (NEM) was subsequently applied, [Zn(2+) ]i increased further to 5.6 μM; analogous acid- and NEM-induced [Zn(2+) ]i elevations could also be detected but not calibrated, using the high affinity Zn(2+) probe FluoZin-3. The data indicate that NEM causes Zn(2+) release from ligands that chelate Zn(2+) at pH 6.1. ATP could also chelate Zn(2+) at pH 6.1 because its pKa is about 6.8. Therefore, it was tested whether an ATP depletion affects the acid-induced [Zn(2+) ]i elevations. The ATP depletion was induced by inhibiting mitochondrial and glycolytic ATP production. Interestingly, an almost complete ATP depletion (confirmed using a luciferin/luciferase assay) failed to affect the acid-induced [Zn(2+) ]i increases. These data suggest that the total amount of Zn(2+) accumulated in intracellular ATP-dependent stores (Zn(2+) -ATP complexes and organelles that accumulate Zn(2+) in an ATP-dependent manner) is negligible compared to the amount of Zn(2+) accumulated in the acid-sensitive intracellular ligands. In vitro, upon acidification, Zn(2+) -cysteine complexes release Zn(2+) and ATP chelates the released Zn(2+) . However, in vivo (cultured neurons), an ATP depletion failed to enhance acid-induced [Zn(2+) ]i elevations. These [Zn(2+) ]i elevations were calibrated using a low affinity ratiometric probe FuraZin-1; they reached 2 µM levels and increased to 5 µM when a thiol-reactive agent, N-ethylmaleimide, compromised Zn(2+) binding by cysteines. PMID:26263185

  7. Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation.

    Simandi, Zoltan; Horvath, Attila; Nagy, Peter; Nagy, Laszlo

    2016-01-01

    Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to tissue and cell-type specific regulation of gene expression during the cellular differentiation. Thus, their identification and further investigation is important in order to understand how cell fate is determined. Integration of gene expression data (e.g., microarray or RNA-seq) and results of chromatin immunoprecipitation (ChIP)-based genome-wide studies (ChIP-seq) allows large-scale identification of these regulatory regions. However, functional validation of cell-type specific enhancers requires further in vitro and in vivo experimental procedures. Here we describe how active enhancers can be identified and validated experimentally. This protocol provides a step-by-step workflow that includes: 1) identification of regulatory regions by ChIP-seq data analysis, 2) cloning and experimental validation of putative regulatory potential of the identified genomic sequences in a reporter assay, and 3) determination of enhancer activity in vivo by measuring enhancer RNA transcript level. The presented protocol is detailed enough to help anyone to set up this workflow in the lab. Importantly, the protocol can be easily adapted to and used in any cellular model system. PMID:27403939

  8. Salicylic acid induces differential antioxidant response in spring maize under high temperature stress.

    Khanna, Palak; Kaur, Kamaljit; Gupta, Anil K

    2016-06-01

    High temperature is one of the important stress factors that affect crops in tropical countries. Plants do evolve or adopt different mechanisms to overcome such stress for survival. It is an interesting subject and has attracted many researchers to work upon. Here, we studied the effect of salicylic acid (SA) on seedling growth and antioxidative defense system in two spring maize (Zea mays L.) genotypes viz., CML-32 (relatively heat tolerant) and LM-11 (relatively heat susceptible), under high temperature stress. High temperature induced greater reduction in dry biomass of LM-1 1 seedlings as compared to those of CML-32. There was a parallel increase in ascorbate peroxidase and glutathione reductase activities in the roots of CML-32 seedlings. However, the activities of catalase and superoxide dismutase decreased and the contents of H202, proline and malonaldialdehyde (MDA) increased in seedlings of both the genotypes. Application of SA (400 µM) led to increased dry biomass in heat stressed CML-32 seedlings. It improved the efficiency of Halliwell-Asada pathway in roots of CML-32 seedlings as was evidenced by the enhanced ascorbate peroxidase and glutathione reductase activities. The activities of catalase and superoxide dismutase increased in both the tissues of LM-11 seedlings, whereas in CML-32, it was only in shoots, after SA application. Peroxidase activity increased in SA treated seedlings of both the genotypes, though the increase was comparatively higher in CML-32. The contents of H₂O₂ and MDA decreased and that of proline increased in SA treated seedlings of both the genotypes, under stress conditions. It may be concluded that SA induced differential antioxidant response by upregulating Halliwell-Asada pathway in roots and attaining high POX activity in both the tissues of CML-32 seedlings, under high temperature stress. PMID:27468465

  9. Cholinergic activation enhances retinoic acid-induced differentiation in the human NB-4 acute promyelocytic leukemia cell line.

    Chotirat, Sadudee; Suriyo, Tawit; Hokland, Marianne; Hokland, Peter; Satayavivad, Jutamaad; Auewarakul, Chirayu U

    2016-07-01

    The non-neuronal cholinergic system (NNCS) has been shown to play a role in regulating hematopoietic differentiation. We determined the expression of cholinergic components in leukemic cell lines by Western blotting and in normal leukocyte subsets by flow cytometry and found a heterogeneous expression of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), choline transporter (CHT), M3 muscarinic acetylcholine receptor (M3-mAChR) and α7 nicotinic acetylcholine receptor (α7-nAChR). We then evaluated NNCS role in differentiation of human NB-4 acute promyelocytic leukemia cell line and discovered a dramatic induction of M3-mAChR after all-trans retinoic acid (ATRA) treatment (p<0.0001). Adding carbachol which is a cholinergic agonist to the ATRA treatment resulted in an increase of a granulocytic differentiation marker (CD11b) as compared with ATRA treatment alone (p<0.05), indicating that cholinergic activation enhanced ATRA in inducing NB-4 maturation. The combination of carbachol and ATRA treatment for 72h also resulted in decreased viability and increased cleaved caspase-3 expression when compared with ATRA treatment alone (p<0.05). However, this combination did not cause poly (ADP-ribose) polymerase (PARP) cleavage. Overall, we have shown that NB-4 cells expressed M3-mAChR in a differentiation-dependent manner and cholinergic stimulation induced maturation and death of ATRA-induced differentiated NB-4 cells. PMID:27282572

  10. Auditory brain-stem evoked potentials in cat after kainic acid induced neuronal loss. I. Superior olivary complex.

    Zaaroor, M; Starr, A

    1991-01-01

    Auditory brain-stem potentials (ABRs) were studied in cats for up to 45 days after kainic acid had been injected unilaterally or bilaterally into the superior olivary complex (SOC) to produce neuronal destruction while sparing fibers of passage and the terminals of axons of extrinsic origin connecting to SOC neurons. The components of the ABR in cat were labeled by their polarity at the vertex (P, for positive) and their order of appearance (the arabic numerals 1, 2, etc.). Component P1 can be further subdivided into 2 subcomponents labeled P1a and P1b. The correspondences we have assumed between the ABR components in cat and man are indicated by providing a Roman numeral designation for the human component in parentheses following the feline notation, e.g., P4 (V). With bilateral SOC destruction, there was a significant and marked attenuation of waves P2 (III), P3 (IV), P4 (V), P5 (VI), and the sustained potential shift (SPS) amounting to as much as 80% of preoperative values. Following unilateral SOC destruction the attenuation of many of these same ABR components, in response to stimulation of either ear, was up to 50%. No component of the ABR was totally abolished even when the SOC was lesioned 100% bilaterally. In unilaterally lesioned cats with extensive neuronal loss (greater than 75%) the latencies of the components beginning at P3 (IV) were delayed to stimulation of the ear ipsilateral to the injection site but not to stimulation of the ear contralateral to the injection. Binaural interaction components of the ABR were affected in proportion to the attenuation of the ABR. These results are compatible with multiple brain regions contributing to the generation of the components of the ABR beginning with P2 (III) and that components P3 (IV), P4 (V), and P5 (VI) and the sustained potential shift depend particularly on the integrity of the neurons of the SOC bilaterally. The neurons of the lateral subdivision (LSO) and the medial nucleus of the trapezoid body

  11. Sonic hedgehog and retinoic Acid induce bone marrow-derived stem cells to differentiate into glutamatergic neural cells.

    Yu, Zhenhai; Wu, Shixing; Liu, Zhen; Lin, Haiyan; Chen, Lei; Yuan, Xinli; Zhang, Zhiying; Liu, Fang; Zhang, Chuansen

    2015-01-01

    Studies have showed that transplanted stem cells in the inner ear won't regenerate to replace the damaged sensory hair cells. They can spontaneously differentiate into mesenchymal cells and fibrocytes in the damaged inner ear. Only mature sensory cells of MSCs-derived possess the great potency for cell transplantation in the treatment of sensorineural hearing loss. So, we try to establish an efficient generation of the glutamatergic sensory neural phenotype for the cell transplantation of the hearing loss. We isolated MSCs from femoral and tibial bones according to their adherence to culture dishes. After purification, proliferation, and passaged, cells became homogeneous in appearance, showing more uniformity and grew in a monolayer with a typical spindle-shape morphology. The cell surface markers were assessed using FACS to characterize the isolated cells. For neural induction to harvest the glutamatergic sensory neurons, passage 3 MSCs were incubated with preinduced medium for 24 hr, and neural-induced medium for an additional 14 days. The cells exhibit a typical neural shape. RT-PCR analysis indicated that the mRNA levels of the neural cell marker nestin, Tau, MAP-2, β-tubulin III, GluR-3, and GluR-4 were higher compared with primary MSCs. Immunohistochemistry and western-blotting proofed that nestin, MAP-2, β-tubulin III, and GluR-4 proteins indeed exhibit their expression difference in the induced cells compared to the MSCs. We show an efficient protocol by the combined applications of Sonic Hedgehog (Shh) and Retinoic Acid (RA) to induce MSCs to differentiate into the glutamatergic sensory neuron which were identified from the morphological, biochemical, and molecular characteristics. PMID:24547891

  12. YAP regulates neuronal differentiation through Sonic hedgehog signaling pathway

    Lin, Yi-Ting; Ding, Jing-Ya [Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei 112, Taiwan (China); Li, Ming-Yang [Department of Life Science, National Taiwan Normal University, Taipei 116, Taiwan (China); Yeh, Tien-Shun [Department of Anatomy and Cell Biology, National Yang-Ming University, Taipei 112, Taiwan (China); Wang, Tsu-Wei, E-mail: twwang@ntnu.edu.tw [Department of Life Science, National Taiwan Normal University, Taipei 116, Taiwan (China); Yu, Jenn-Yah, E-mail: jyyu@ym.edu.tw [Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei 112, Taiwan (China); Brain Research Center, National Yang-Ming University, Taipei 112, Taiwan (China)

    2012-09-10

    Tight regulation of cell numbers by controlling cell proliferation and apoptosis is important during development. Recently, the Hippo pathway has been shown to regulate tissue growth and organ size in Drosophila. In mammalian cells, it also affects cell proliferation and differentiation in various tissues, including the nervous system. Interplay of several signaling cascades, such as Notch, Wnt, and Sonic Hedgehog (Shh) pathways, control cell proliferation during neuronal differentiation. However, it remains unclear whether the Hippo pathway coordinates with other signaling cascades in regulating neuronal differentiation. Here, we used P19 cells, a mouse embryonic carcinoma cell line, as a model to study roles of YAP, a core component of the Hippo pathway, in neuronal differentiation. P19 cells can be induced to differentiate into neurons by expressing a neural bHLH transcription factor gene Ascl1. Our results showed that YAP promoted cell proliferation and inhibited neuronal differentiation. Expression of Yap activated Shh but not Wnt or Notch signaling activity during neuronal differentiation. Furthermore, expression of Yap increased the expression of Patched homolog 1 (Ptch1), a downstream target of the Shh signaling. Knockdown of Gli2, a transcription factor of the Shh pathway, promoted neuronal differentiation even when Yap was over-expressed. We further demonstrated that over-expression of Yap inhibited neuronal differentiation in primary mouse cortical progenitors and Gli2 knockdown rescued the differentiation defect in Yap over-expressing cells. In conclusion, our study reveals that Shh signaling acts downstream of YAP in regulating neuronal differentiation. -- Highlights: Black-Right-Pointing-Pointer YAP promotes cell proliferation and inhibits neuronal differentiation in P19 cells. Black-Right-Pointing-Pointer YAP promotes Sonic hedgehog signaling activity during neuronal differentiation. Black-Right-Pointing-Pointer Knockdown of Gli2 rescues the Yap

  13. Auditory brain-stem evoked potentials in cat after kainic acid induced neuronal loss. II. Cochlear nucleus.

    Zaaroor, M; Starr, A

    1991-01-01

    Auditory brain-stem potentials (ABRs) were studied in cats for up to 6 weeks after kainic acid had been injected unilaterally into the cochlear nucleus (CN) producing extensive neuronal destruction. The ABR components were labeled by the polarity at the vertex (P, for positive) and their order of appearance (the arabic numerals 1, 2, etc.). Component P1 can be further subdivided into 2 subcomponents, P1a and P1b. The assumed correspondence between the ABR components in cat and man is indicated by providing human Roman numeral designations in parentheses following the feline notation, e.g., P2 (III). To stimulation of the ear ipsilateral to the injection, the ABR changes consisted of a loss of components P2 (III) and P3 (IV), and an attenuation and prolongation of latency of components P4 (V) and P5 (VI). The sustained potential shift from which the components arose was not affected. Wave P1a (I) was also slightly but significantly attenuated compatible with changes of excitability of nerve VIII in the cochlea secondary to cochlear nucleus destruction. Unexpectedly, to stimulation of the ear contralateral to the injection side, waves P2 (III), P3 (IV), and P4 (V) were also attenuated and delayed in latency but to a lesser degree than to stimulation of the ear ipsilateral to the injection. Changes in binaural interaction of the ABR following cochlear nucleus lesions were similar to those produced in normal animals by introducing a temporal delay of the input to one ear. The results of the present set of studies using kainic acid to induce neuronal loss in auditory pathway when combined with prior lesion and recording experiments suggest that each of the components of the ABR requires the integrity of an anatomically diffuse system comprising a set of neurons, their axons, and the neurons on which they terminate. Disruption of any portion of the system will alter the amplitude and/or the latency of that component. PMID:1716569

  14. Phenazopyridine induces and synchronizes neuronal differentiation of embryonic stem cells.

    Suter, David M; Preynat-Seauve, Olivier; Tirefort, Diderik; Feki, Anis; Krause, Karl-Heinz

    2009-09-01

    Embryonic stem (ES) cells are powerful tools to understand mechanisms of neuronal differentiation and to engineer neurons for in vitro studies and cell therapy. We developed a screening approach to identify small organic molecules driving neuronal differentiation of ES cells. For this purpose, we used a lentivector carrying a dual luciferase reporter system to engineer an ES cell line which allowed us to screen for small organic molecules enhancing neuronal differentiation. One of them, phenazopyridine, was further analysed in human ES cells. Phenazopyridine: (i) enhanced neuronal differentiation, (ii) increased cell survival, (iii) decreased the amount of non-neuronal and undifferentiated cells and (iv) synchronized the cellular differentiation state. Phenazopyridine allowed the development of a differentiation protocol compatible with the generation of clinical grade neural precursors, which were able differentiate into different neuronal subtypes, astrocytes and oligodendrocytes. In summary, we describe a powerful approach to identify small molecules directing stem cell differentiation. This led to the establishment of a new application for an old drug and the development of a novel clinical grade protocol for neuronal differentiation of ES cells. PMID:20196783

  15. Alternative Splicing of G9a Regulates Neuronal Differentiation

    Ana Fiszbein; Luciana E. Giono; Ana Quaglino; Bruno G. Berardino; Lorena Sigaut; Catalina von Bilderling; Ignacio E. Schor; Juliana H. Enriqué Steinberg; Mario Rossi; Lía I. Pietrasanta; Julio J. Caramelo; Anabella Srebrow; Alberto R. Kornblihtt

    2016-01-01

    Chromatin modifications are critical for the establishment and maintenance of differentiation programs. G9a, the enzyme responsible for histone H3 lysine 9 dimethylation in mammalian euchromatin, exists as two isoforms with differential inclusion of exon 10 (E10) through alternative splicing. We find that the G9a methyltransferase is required for differentiation of the mouse neuronal cell line N2a and that E10 inclusion increases during neuronal differentiation of cultured cells, as well as i...

  16. Neuron differentiation and neuritogenesis stimulated by N-acetylcysteine(NAC)

    Hao-ran QIAN; Yi YANG

    2009-01-01

    Aim:To investigate the effect of N-acetylcysteine (NAC),a potent antioxidant,on neuron differentiation of cultured mouse embryonic stem cells (ESCs) induced by retinoic acid (RA) in vitro.Superior cervical ganglion (SCG) neurons were used to study the effect of NAC on neuritogenesis.Methods:Immunoblotting was performed to detect the expression of microtubule-associated protein 2 (MAP2).MTT assays were used to determine cell viability.Cell death was estimated with trypan blue exclusion and Hoechst 33342 staining.Immunocytochemical analysis was carried out to identify neurons.Results:We obtained a high percentage of MAP2-positive neurons derived from embryoid bodies (EBs) induced by RA by administering 1 mmol/L NAC at differentiation day O.On differentiation day 8,the expression of MAP2 protein was strongly upregulated in the presence of NAC.NAC promoted neuron differentiation of ES cells in a dose- and time-dependent manner.Notably,NAC suppressed cell death caused/Jy RA during neuron differentiation.In addition,neurite extension of SCG neurons was greatly stimulated in the presence of NAC.Conclusion:These results show that NAC enhanced both neuron differentiation and neuritogenesis,suggesting that it may be used in the development of novel therapeutic approaches targeting neuron loss and neurite dystrophy in neurodegenerative diseases.

  17. Differentiation renders susceptibility to excitotoxicity in HT22 neurons

    Minchao He; Jun Liu; Shaowu Cheng; Yigang Xing; William Z Suo

    2013-01-01

    HT22 is an immortalized mouse hippocampal neuronal cell line that does not express cholinergic and glutamate receptors like mature hippocampal neurons in vivo. This in part prevents its use as a model for mature hippocampal neurons in memory-related studies. We now report that HT22 cells were appropriately induced to differentiate and possess properties similar to those of mature hippocampal neurons in vivo, such as becoming more glutamate-receptive and excitatory. Results showed that sensitivity of HT22 cells to glutamate-induced toxicity changed dramatically when comparing undifferentiated with differentiated cells, with the half-effective concentration for differentiated cells reducing approximately two orders of magnitude. Moreover, glutamate-induced toxicity in differentiated cells, but not undifferentiated cells, was inhibited by the N-methyl-D- aspartate receptor antagonists MK-801 and memantine. Evidently, differentiated HT22 cells expressed N-methyl-D-aspartate receptors, while undifferentiated cells did not. Our experimental findings indicated that differentiation is important for immortalized cell lines to render post-mitotic neuronal properties, and that differentiated HT22 neurons represent a better model of hippocampal neurons than undifferentiated cells.

  18. ACQUISITION AND LOSS OF NEURONAL CA2+/CALMODULIN-DEPENDENT PROTEIN KINASE DURING NEURONAL DIFFERENTIATION

    Neurons display characteristic schedules by which they acquire and lose the neuron-specific Ca2+/calmodulin-dependent protein Kinase-Gr (CaM Kinase-Gr) during differentiation. uch schedules are exemplified by patterns of expression of this kinase in the developing cerebellum and ...

  19. Amyloid precursor protein expression and processing are differentially regulated during cortical neuron differentiation

    Bergström, Petra; Agholme, Lotta; Nazir, Faisal Hayat; Satir, Tugce Munise; Toombs, Jamie; Wellington, Henrietta; Strandberg, Joakim; Bontell, Thomas Olsson; Kvartsberg, Hlin; Holmström, Maria; Boreström, Cecilia; Simonsson, Stina; Kunath, Tilo; Lindahl, Anders; Blennow, Kaj; Hanse, Eric; Portelius, Erik; Wray, Selina; Zetterberg, Henrik

    2016-01-01

    Amyloid precursor protein (APP) and its cleavage product amyloid β (Aβ) have been thoroughly studied in Alzheimer’s disease. However, APP also appears to be important for neuronal development. Differentiation of induced pluripotent stem cells (iPSCs) towards cortical neurons enables in vitro mechanistic studies on human neuronal development. Here, we investigated expression and proteolytic processing of APP during differentiation of human iPSCs towards cortical neurons over a 100-day period. APP expression remained stable during neuronal differentiation, whereas APP processing changed. α-Cleaved soluble APP (sAPPα) was secreted early during differentiation, from neuronal progenitors, while β-cleaved soluble APP (sAPPβ) was first secreted after deep-layer neurons had formed. Short Aβ peptides, including Aβ1-15/16, peaked during the progenitor stage, while processing shifted towards longer peptides, such as Aβ1-40/42, when post-mitotic neurons appeared. This indicates that APP processing is regulated throughout differentiation of cortical neurons and that amyloidogenic APP processing, as reflected by Aβ1-40/42, is associated with mature neuronal phenotypes. PMID:27383650

  20. Divergent modulation of neuronal differentiation by caspase-2 and -9.

    Giuseppa Pistritto

    Full Text Available Human Ntera2/cl.D1 (NT2 cells treated with retinoic acid (RA differentiate towards a well characterized neuronal phenotype sharing many features with human fetal neurons. In view of the emerging role of caspases in murine stem cell/neural precursor differentiation, caspases activity was evaluated during RA differentiation. Caspase-2, -3 and -9 activity was transiently and selectively increased in differentiating and non-apoptotic NT2-cells. SiRNA-mediated selective silencing of either caspase-2 (si-Casp2 or -9 (si-Casp9 was implemented in order to dissect the role of distinct caspases. The RA-induced expression of neuronal markers, i.e. neural cell adhesion molecule (NCAM, microtubule associated protein-2 (MAP2 and tyrosine hydroxylase (TH mRNAs and proteins, was decreased in si-Casp9, but markedly increased in si-Casp2 cells. During RA-induced NT2 differentiation, the class III histone deacetylase Sirt1, a putative caspase substrate implicated in the regulation of the proneural bHLH MASH1 gene expression, was cleaved to a ∼100 kDa fragment. Sirt1 cleavage was markedly reduced in si-Casp9 cells, even though caspase-3 was normally activated, but was not affected (still cleaved in si-Casp2 cells, despite a marked reduction of caspase-3 activity. The expression of MASH1 mRNA was higher and occurred earlier in si-Casp2 cells, while was reduced at early time points during differentiation in si-Casp9 cells. Thus, caspase-2 and -9 may perform opposite functions during RA-induced NT2 neuronal differentiation. While caspase-9 activation is relevant for proper neuronal differentiation, likely through the fine tuning of Sirt1 function, caspase-2 activation appears to hinder the RA-induced neuronal differentiation of NT2 cells.

  1. Alternative Splicing of G9a Regulates Neuronal Differentiation.

    Fiszbein, Ana; Giono, Luciana E; Quaglino, Ana; Berardino, Bruno G; Sigaut, Lorena; von Bilderling, Catalina; Schor, Ignacio E; Steinberg, Juliana H Enriqué; Rossi, Mario; Pietrasanta, Lía I; Caramelo, Julio J; Srebrow, Anabella; Kornblihtt, Alberto R

    2016-03-29

    Chromatin modifications are critical for the establishment and maintenance of differentiation programs. G9a, the enzyme responsible for histone H3 lysine 9 dimethylation in mammalian euchromatin, exists as two isoforms with differential inclusion of exon 10 (E10) through alternative splicing. We find that the G9a methyltransferase is required for differentiation of the mouse neuronal cell line N2a and that E10 inclusion increases during neuronal differentiation of cultured cells, as well as in the developing mouse brain. Although E10 inclusion greatly stimulates overall H3K9me2 levels, it does not affect G9a catalytic activity. Instead, E10 increases G9a nuclear localization. We show that the G9a E10(+) isoform is necessary for neuron differentiation and regulates the alternative splicing pattern of its own pre-mRNA, enhancing E10 inclusion. Overall, our findings indicate that by regulating its own alternative splicing, G9a promotes neuron differentiation and creates a positive feedback loop that reinforces cellular commitment to differentiation. PMID:26997278

  2. Alternative Splicing of G9a Regulates Neuronal Differentiation

    Ana Fiszbein

    2016-03-01

    Full Text Available Chromatin modifications are critical for the establishment and maintenance of differentiation programs. G9a, the enzyme responsible for histone H3 lysine 9 dimethylation in mammalian euchromatin, exists as two isoforms with differential inclusion of exon 10 (E10 through alternative splicing. We find that the G9a methyltransferase is required for differentiation of the mouse neuronal cell line N2a and that E10 inclusion increases during neuronal differentiation of cultured cells, as well as in the developing mouse brain. Although E10 inclusion greatly stimulates overall H3K9me2 levels, it does not affect G9a catalytic activity. Instead, E10 increases G9a nuclear localization. We show that the G9a E10+ isoform is necessary for neuron differentiation and regulates the alternative splicing pattern of its own pre-mRNA, enhancing E10 inclusion. Overall, our findings indicate that by regulating its own alternative splicing, G9a promotes neuron differentiation and creates a positive feedback loop that reinforces cellular commitment to differentiation.

  3. Directed neuronal differentiation of human embryonic stem cells

    Noggle Scott A

    2003-10-01

    Full Text Available Abstract Background We have developed a culture system for the efficient and directed differentiation of human embryonic stem cells (HESCs to neural precursors and neurons. HESC were maintained by manual passaging and were differentiated to a morphologically distinct OCT-4+/SSEA-4- monolayer cell type prior to the derivation of embryoid bodies. Embryoid bodies were grown in suspension in serum free conditions, in the presence of 50% conditioned medium from the human hepatocarcinoma cell line HepG2 (MedII. Results A neural precursor population was observed within HESC derived serum free embryoid bodies cultured in MedII conditioned medium, around 7–10 days after derivation. The neural precursors were organized into rosettes comprised of a central cavity surrounded by ring of cells, 4 to 8 cells in width. The central cells within rosettes were proliferating, as indicated by the presence of condensed mitotic chromosomes and by phosphoHistone H3 immunostaining. When plated and maintained in adherent culture, the rosettes of neural precursors were surrounded by large interwoven networks of neurites. Immunostaining demonstrated the expression of nestin in rosettes and associated non-neuronal cell types, and a radial expression of Map-2 in rosettes. Differentiated neurons expressed the markers Map-2 and Neurofilament H, and a subpopulation of the neurons expressed tyrosine hydroxylase, a marker for dopaminergic neurons. Conclusion This novel directed differentiation approach led to the efficient derivation of neuronal cultures from HESCs, including the differentiation of tyrosine hydroxylase expressing neurons. HESC were morphologically differentiated to a monolayer OCT-4+ cell type, which was used to derive embryoid bodies directly into serum free conditions. Exposure to the MedII conditioned medium enhanced the derivation of neural precursors, the first example of the effect of this conditioned medium on HESC.

  4. A PU.1 suppressive target gene, metallothionein 1G, inhibits retinoic acid-induced NB4 cell differentiation.

    Naomi Hirako

    Full Text Available We recently revealed that myeloid master regulator SPI1/PU.1 directly represses metallothionein (MT 1G through its epigenetic activity of PU.1, but the functions of MT1G in myeloid differentiation remain unknown. To clarify this, we established MT1G-overexpressing acute promyelocytic leukemia NB4 (NB4MTOE cells, and investigated whether MT1G functionally contributes to all-trans retinoic acid (ATRA-induced NB4 cell differentiation. Real-time PCR analyses demonstrated that the inductions of CD11b and CD11c and reductions in myeloperoxidase and c-myc by ATRA were significantly attenuated in NB4MTOE cells. Morphological examination revealed that the percentages of differentiated cells induced by ATRA were reduced in NB4MTOE cells. Since G1 arrest is a hallmark of ATRA-induced NB4 cell differentiation, we observed a decrease in G1 accumulation, as well as decreases in p21WAF1/CIP1 and cyclin D1 inductions, by ATRA in NB4MTOE cells. Nitroblue tetrazolium (NBT reduction assays revealed that the proportions of NBT-positive cells were decreased in NB4MTOE cells in the presence of ATRA. Microarray analyses showed that the changes in expression of several myeloid differentiation-related genes (GATA2, azurocidin 1, pyrroline-5-carboxylate reductase 1, matrix metallopeptidase -8, S100 calcium-binding protein A12, neutrophil cytosolic factor 2 and oncostatin M induced by ATRA were disturbed in NB4MTOE cells. Collectively, overexpression of MT1G inhibits the proper differentiation of myeloid cells.

  5. Retinoic Acid Induces Embryonic Stem Cell Differentiation by Altering Both Encoding RNA and microRNA Expression.

    Jingcheng Zhang

    Full Text Available Retinoic acid (RA is a vitamin A metabolite that is essential for early embryonic development and promotes stem cell neural lineage specification; however, little is known regarding the impact of RA on mRNA transcription and microRNA levels on embryonic stem cell differentiation. Here, we present mRNA microarray and microRNA high-output sequencing to clarify how RA regulates gene expression. Using mRNA microarray analysis, we showed that RA repressed pluripotency-associated genes while activating ectoderm markers in mouse embryonic stem cells (mESCs. Moreover, RA modulated the DNA methylation of mESCs by altering the expression of epigenetic-associated genes such as Dnmt3b and Dnmt3l. Furthermore, H3K4me2, a pluripotent histone modification, was repressed by RA stimulation. From microRNA sequence data, we identified two downregulated microRNAs, namely, miR-200b and miR-200c, which regulated the pluripotency of stem cells. We found that miR-200b or miR-200c deficiency suppressed the expression of pluripotent genes, including Oct4 and Nanog, and activated the expression of the ectodermal marker gene Nestin. These results demonstrate that retinoid induces mESCs to differentiate by regulating miR-200b/200c. Our findings provide the landscapes of mRNA and microRNA gene networks and indicate the crucial role of miR-200b/200c in the RA-induced differentiation of mESCs.

  6. Retinoic Acid Induces Embryonic Stem Cell Differentiation by Altering Both Encoding RNA and microRNA Expression

    Yu, Mengying; Wu, Haibo; Ai, Zhiying; Wu, Yongyan; Liu, Hongliang; Du, Juan; Guo, Zekun; Zhang, Yong

    2015-01-01

    Retinoic acid (RA) is a vitamin A metabolite that is essential for early embryonic development and promotes stem cell neural lineage specification; however, little is known regarding the impact of RA on mRNA transcription and microRNA levels on embryonic stem cell differentiation. Here, we present mRNA microarray and microRNA high-output sequencing to clarify how RA regulates gene expression. Using mRNA microarray analysis, we showed that RA repressed pluripotency-associated genes while activating ectoderm markers in mouse embryonic stem cells (mESCs). Moreover, RA modulated the DNA methylation of mESCs by altering the expression of epigenetic-associated genes such as Dnmt3b and Dnmt3l. Furthermore, H3K4me2, a pluripotent histone modification, was repressed by RA stimulation. From microRNA sequence data, we identified two downregulated microRNAs, namely, miR-200b and miR-200c, which regulated the pluripotency of stem cells. We found that miR-200b or miR-200c deficiency suppressed the expression of pluripotent genes, including Oct4 and Nanog, and activated the expression of the ectodermal marker gene Nestin. These results demonstrate that retinoid induces mESCs to differentiate by regulating miR-200b/200c. Our findings provide the landscapes of mRNA and microRNA gene networks and indicate the crucial role of miR-200b/200c in the RA-induced differentiation of mESCs. PMID:26162091

  7. Retinoic Acid Induces Embryonic Stem Cell Differentiation by Altering Both Encoding RNA and microRNA Expression

    Jingcheng Zhang; Yang Gao; Mengying Yu; Haibo Wu; Zhiying Ai; Yongyan Wu; Hongliang Liu; Juan Du; Zekun Guo; Yong Zhang

    2015-01-01

    Retinoic acid (RA) is a vitamin A metabolite that is essential for early embryonic development and promotes stem cell neural lineage specification; however, little is known regarding the impact of RA on mRNA transcription and microRNA levels on embryonic stem cell differentiation. Here, we present mRNA microarray and microRNA high-output sequencing to clarify how RA regulates gene expression. Using mRNA microarray analysis, we showed that RA repressed pluripotency-associated genes while activ...

  8. Manganese inhibits the ability of astrocytes to promote neuronal differentiation

    Manganese (Mn) is a known neurotoxicant and developmental neurotoxicant. As Mn has been shown to accumulate in astrocytes, we sought to investigate whether Mn would alter astrocyte-neuronal interactions, specifically the ability of astrocytes to promote differentiation of neurons. We found that exposure of rat cortical astrocytes to Mn (50-500 μM) impaired their ability to promote axonal and neurite outgrowth in hippocampal neurons. This effect of Mn appeared to be mediated by oxidative stress, as it was reversed by antioxidants (melatonin and PBN) and by increasing glutathione levels, while it was potentiated by glutathione depletion in astrocytes. As the extracellular matrix protein fibronectin plays an important role in astrocyte-mediated neuronal neurite outgrowth, we also investigated the effect of Mn on fibronectin. Mn caused a concentration-dependent decrease of fibronectin protein and mRNA in astrocytes lysate and of fibronectin protein in astrocyte medium; these effects were also antagonized by antioxidants. Exposure of astrocytes to two oxidants, H2O2 and DMNQ, similarly impaired their neuritogenic action, and led to a decreased expression of fibronectin. Mn had no inhibitory effect on neurite outgrowth when applied directly onto hippocampal neurons, where it actually caused a small increase in neuritogenesis. These results indicate that Mn, by targeting astrocytes, affects their ability to promote neuronal differentiation by a mechanism which is likely to involve oxidative stress.

  9. Valproic acid induces differentiation and inhibition of proliferation in neural progenitor cells via the beta-catenin-Ras-ERK-p21Cip/WAF1 pathway

    Arenas Ernest

    2008-12-01

    Full Text Available Abstract Background Valproic acid (VPA, a commonly used mood stabilizer that promotes neuronal differentiation, regulates multiple signaling pathways involving extracellular signal-regulated kinase (ERK and glycogen synthase kinase3β (GSK3β. However, the mechanism by which VPA promotes differentiation is not understood. Results We report here that 1 mM VPA simultaneously induces differentiation and reduces proliferation of basic fibroblast growth factor (bFGF-treated embryonic day 14 (E14 rat cerebral cortex neural progenitor cells (NPCs. The effects of VPA on the regulation of differentiation and inhibition of proliferation occur via the ERK-p21Cip/WAF1 pathway. These effects, however, are not mediated by the pathway involving the epidermal growth factor receptor (EGFR but via the pathway which stabilizes Ras through β-catenin signaling. Stimulation of differentiation and inhibition of proliferation in NPCs by VPA occur independently and the β-catenin-Ras-ERK-p21Cip/WAF1 pathway is involved in both processes. The independent regulation of differentiation and proliferation in NPCs by VPA was also demonstrated in vivo in the cerebral cortex of developing rat embryos. Conclusion We propose that this mechanism of VPA action may contribute to an explanation of its anti-tumor and neuroprotective effects, as well as elucidate its role in the independent regulation of differentiation and inhibition of proliferation in the cerebral cortex of developing rat embryos.

  10. Effect of leukemia inhibitory factor on embryonic stem cell differentiation: implications for supporting neuronal differentiation

    Zhao HE; Jing-jing LI; Chang-hong ZHEN; Lin-ying FENG; Xiao-yan DING

    2006-01-01

    Aim: Leukemia inhibitory factor (LIF), a pleiotropic cytokine, has been used extensively in the maintenance of mouse embryonic stem cell pluripotency. In this current work, we examined the effect of the LIF signaling pathway in embryonic stem (ES) cell differentiation to a neural fate. Methods: In the presence of LIF (1000 U/mL), the production of neuronal cells derived from embryoid bodies (EB)was tested under various culture conditions. Inhibition of the LIF pathway was examined with specific inhibitors. The effects of cell apoptosis and proliferation on neural differentiation were examined. ES cell differentiation into three-gem layers was compared. Results: Under various culture conditions, neuronal differentiation was increased in the presence of LIF. Blocking the LIF-activated STAT3signaling pathway with specific inhibitors abolished the neuronal differentiation of ES cells, whereas inhibition of the LIF-activated MEK signaling pathway impaired the differentiation of ES cells toward a glial fate. LIF suppressed cell apoptosis and promoted cell proliferation during ES cell differentiation. LIF inhibited the differentiation of ES cells to both mesoderm and extraembryonic endoderm fates, but enhanced the determination of neural progenitors. Conclusion:These results suggest that LIF plays a positive role during the differentiation of ES cells into neuronal cells.

  11. Midline thalamic neurons are differentially engaged during hippocampus network oscillations.

    Lara-Vásquez, Ariel; Espinosa, Nelson; Durán, Ernesto; Stockle, Marcelo; Fuentealba, Pablo

    2016-01-01

    The midline thalamus is reciprocally connected with the medial temporal lobe, where neural circuitry essential for spatial navigation and memory formation resides. Yet, little information is available on the dynamic relationship between activity patterns in the midline thalamus and medial temporal lobe. Here, we report on the functional heterogeneity of anatomically-identified thalamic neurons and the differential modulation of their activity with respect to dorsal hippocampal rhythms in the anesthetized mouse. Midline thalamic neurons expressing the calcium-binding protein calretinin, irrespective of their selective co-expression of calbindin, discharged at overall low levels, did not increase their activity during hippocampal theta oscillations, and their firing rates were inhibited during hippocampal sharp wave-ripples. Conversely, thalamic neurons lacking calretinin discharged at higher rates, increased their activity during hippocampal theta waves, but remained unaffected during sharp wave-ripples. Our results indicate that the midline thalamic system comprises at least two different classes of thalamic projection neuron, which can be partly defined by their differential engagement by hippocampal pathways during specific network oscillations that accompany distinct behavioral contexts. Thus, different midline thalamic neuronal populations might be selectively recruited to support distinct stages of memory processing, consistent with the thalamus being pivotal in the dialogue of cortical circuits. PMID:27411890

  12. Optical Imaging for Stem Cell Differentiation to Neuronal Lineage

    Hwang, Do Won; Lee, Dong Soo [Seoul National Univ., Seoul (Korea, Republic of)

    2012-03-15

    In regenerative medicine, the prospect of stem cell therapy hold great promise for the recovery of injured tissues and effective treatment of intractable diseases. Tracking stem cell fate provides critical information to understand and evaluate the success of stem cell therapy. The recent emergence of in vivo noninvasive molecular imaging has enabled assessment of the behavior of grafted stem cells in living subjects. In this review, we provide an overview of current optical imaging strategies based on cell or tissue specific reporter gene expression and of in vivo methods to monitor stem cell differentiation into neuronal lineages. These methods use optical reporters either regulated by neuron-specific promoters or containing neuron-specific microRNA binding sites. Both systems revealed dramatic changes in optical reporter imaging signals in cells differentiating a yeast GAL4 amplification system or an engineering-enhanced luciferase reported gene. Furthermore, we propose an advanced imaging system to monitor neuronal differentiation during neurogenesis that uses in vivo multiplexed imaging techniques capable of detecting several targets simultaneously.

  13. Optical Imaging for Stem Cell Differentiation to Neuronal Lineage

    In regenerative medicine, the prospect of stem cell therapy hold great promise for the recovery of injured tissues and effective treatment of intractable diseases. Tracking stem cell fate provides critical information to understand and evaluate the success of stem cell therapy. The recent emergence of in vivo noninvasive molecular imaging has enabled assessment of the behavior of grafted stem cells in living subjects. In this review, we provide an overview of current optical imaging strategies based on cell or tissue specific reporter gene expression and of in vivo methods to monitor stem cell differentiation into neuronal lineages. These methods use optical reporters either regulated by neuron-specific promoters or containing neuron-specific microRNA binding sites. Both systems revealed dramatic changes in optical reporter imaging signals in cells differentiating a yeast GAL4 amplification system or an engineering-enhanced luciferase reported gene. Furthermore, we propose an advanced imaging system to monitor neuronal differentiation during neurogenesis that uses in vivo multiplexed imaging techniques capable of detecting several targets simultaneously

  14. Gold Nanoparticle-Decorated Scaffolds Promote Neuronal Differentiation and Maturation.

    Baranes, Koby; Shevach, Michal; Shefi, Orit; Dvir, Tal

    2016-05-11

    Engineered 3D neuronal networks are considered a promising approach for repairing the damaged spinal cord. However, the lack of a technological platform encouraging axonal elongation over branching may jeopardize the success of such treatment. To address this issue we have decorated gold nanoparticles on the surface of electrospun nanofiber scaffolds, characterized the composite material, and investigated their effect on the differentiation, maturation, and morphogenesis of primary neurons and on an immature neuronal cell line. We have shown that the nanocomposite scaffolds have encouraged a longer outgrowth of the neurites, as judged by the total length of the branching trees and the length and total distance of neurites. Moreover, neurons grown on the nanocomposite scaffolds had less neurites originating out of the soma and lower number of branches. Taken together, these results indicate that neurons cultivated on the gold nanoparticle scaffolds prefer axonal elongation over forming complex branching trees. We envision that such cellular constructs may be useful in the future as implantable cellular devices for repairing damaged neuronal tissues, such as the spinal cord. PMID:26674672

  15. Sexual differentiation of monoaminergic neurons--genetic or epigenetic?

    Reisert, I; Pilgrim, C

    1991-10-01

    It is currently believed that sexual differentiation of the brain is mediated entirely by the epigenetic action of gonadal steroids during a critical period of development. Ingrid Reisert and Christoph Pilgrim review sexual dimorphisms of monoaminergic systems, which also appear to be generated by sex steroids. However, there are a number of observations that are not explainable by the 'androgen theory of sexual differentiation'. Results obtained from cultures of embryonic rat brain tissue appear to indicate that dopaminergic neurons may develop morphological and functional sex differences in the absence of sex steroids. Hormone-independent and -dependent developmental processes may affect diencephalic and mesencephalic dopaminergic neurons in a regionally diverse fashion. Factors other than sex steroids need to be examined. It is possible that some sexual dimorphisms in the nervous system may develop under primary genetic control. PMID:1722367

  16. Differential Modulation of Excitatory and Inhibitory Neurons during Periodic Stimulation.

    Mahmud, Mufti; Vassanelli, Stefano

    2016-01-01

    Non-invasive transcranial neuronal stimulation, in addition to deep brain stimulation, is seen as a promising therapeutic and diagnostic approach for an increasing number of neurological diseases such as epilepsy, cluster headaches, depression, specific type of blindness, and other central nervous system disfunctions. Improving its effectiveness and widening its range of use may strongly rely on development of proper stimulation protocols that are tailored to specific brain circuits and that are based on a deep knowledge of different neuron types response to stimulation. To this aim, we have performed a simulation study on the behavior of excitatory and inhibitory neurons subject to sinusoidal stimulation. Due to the intrinsic difference in membrane conductance properties of excitatory and inhibitory neurons, we show that their firing is differentially modulated by the wave parameters. We analyzed the behavior of the two neuronal types for a broad range of stimulus frequency and amplitude and demonstrated that, within a small-world network prototype, parameters tuning allow for a selective enhancement or suppression of the excitation/inhibition ratio. PMID:26941602

  17. Targeted mass spektrometry based assay for monitoring neuronal differentiation

    Žižková, Martina; Suchá, Rita; Rákocyová, Michaela; Doležalová, D.; Červenka, Jakub; Kovářová, Hana

    2015-01-01

    Roč. 78, Suppl 2 (2015), s. 26-27. ISSN 1210-7859. [Conference on Animal Models for neurodegenerative Diseases /3./. 08.11.2015-10.11.2015, Liblice] R&D Projects: GA TA ČR(CZ) TA01011466; GA MŠk ED2.1.00/03.0124; GA MŠk(CZ) 7F14308 Institutional support: RVO:67985904 Keywords : pluripotent cells * neural differentiation * neurons

  18. "Color Timer" mice: visualization of neuronal differentiation with fluorescent proteins

    Kanki Hiroaki

    2010-02-01

    Full Text Available Abstract The molecular mechanisms governing the differentiation of neural stem cells (NSCs into neuronal progenitor cells and finally into neurons are gradually being revealed. The lack of convenient means for real-time determination of the stages of differentiation of individual neural cells, however, has been hindering progress in elucidating the mechanisms. In order to be able to easily identify the stages of differentiation of neural cells, we have been attempting to establish a mouse system that would allow progression of neuronal differentiation to be visualized based on transitions between fluorescence colors by using a combination of mouse genetics and the ever-expanding repertoire of fluorescent proteins. In this study we report the initial version of such a mouse system, which we call "Color Timer." We first generated transgenic (Tg; nestin/KOr Tg mice in which production of the fluorescent protein Kusabira-Orange (KOr is controlled by the gene regulatory elements within the 2nd intronic enhancer of the nestin gene, which is a good marker for NSCs, so that NSCs would emit orange fluorescence upon excitation. We then confirmed by immunohistochemical and immunocytochemical analyses that the KOr fluorescence closely reflected the presence of the Nestin protein. We also confirmed by a neurosphere formation assay that the intensity of the KOr fluorescence correlated with "stemness" of the cell and it was possible to readily identify NSCs in the two neurogenic regions, namely the dentate gyrus of the hippocampus and the subventricular zone of the lateral ventricle, in the brain of adult nestin/KOr Tg mice by the orange fluorescence they emitted. We then crossed nestin/KOr mice with doublecortin-enhanced Green Fluorescent Protein Tg mice, whose immature neurons emit green fluorescence upon excitation, and it was possible to visualize the progress of NSC-to-neuron differentiation by the transition between fluorescence colors from orange to

  19. Palmitic Acid-Induced Neuron Cell Cycle G2/M Arrest and Endoplasmic Reticular Stress through Protein Palmitoylation in SH-SY5Y Human Neuroblastoma Cells

    Yung-Hsuan Hsiao

    2014-11-01

    Full Text Available Obesity-related neurodegenerative diseases are associated with elevated saturated fatty acids (SFAs in the brain. An increase in SFAs, especially palmitic acid (PA, triggers neuron cell apoptosis, causing cognitive function to deteriorate. In the present study, we focused on the specific mechanism by which PA triggers SH-SY5Y neuron cell apoptosis. We found that PA induces significant neuron cell cycle arrest in the G2/M phase in SH-SY5Y cells. Our data further showed that G2/M arrest is involved in elevation of endoplasmic reticular (ER stress according to an increase in p-eukaryotic translation inhibition factor 2α, an ER stress marker. Chronic exposure to PA also accelerates beta-amyloid accumulation, a pathological characteristic of Alzheimer’s disease. Interestingly, SFA-induced ER stress, G2/M arrest and cell apoptosis were reversed by treatment with 2-bromopalmitate, a protein palmitoylation inhibitor. These findings suggest that protein palmitoylation plays a crucial role in SFA-induced neuron cell cycle G2/M arrest, ER stress and apoptosis; this provides a novel strategy for preventing SFA-induced neuron cell dysfunction.

  20. ADAM10 negatively regulates neuronal differentiation during spinal cord development.

    Xin Yan

    Full Text Available Members of the ADAM (a disintegrin and metalloprotease family are involved in embryogenesis and tissue formation via their proteolytic function, cell-cell and cell-matrix interactions. ADAM10 is expressed temporally and spatially in the developing chicken spinal cord, but its function remains elusive. In the present study, we address this question by electroporating ADAM10 specific morpholino antisense oligonucleotides (ADAM10-mo or dominant-negative ADAM10 (dn-ADAM10 plasmid into the developing chicken spinal cord as well as by in vitro cell culture investigation. Our results show that downregulation of ADAM10 drives precocious differentiation of neural progenitor cells and radial glial cells, resulting in an increase of neurons in the developing spinal cord, even in the prospective ventricular zone. Remarkably, overexpression of the dn-ADAM10 plasmid mutated in the metalloprotease domain (dn-ADAM10-me mimics the phenotype as found by the ADAM10-mo transfection. Furthermore, in vitro experiments on cultured cells demonstrate that downregulation of ADAM10 decreases the amount of the cleaved intracellular part of Notch1 receptor and its target, and increases the number of βIII-tubulin-positive cells during neural progenitor cell differentiation. Taken together, our data suggest that ADAM10 negatively regulates neuronal differentiation, possibly via its proteolytic effect on the Notch signaling during development of the spinal cord.

  1. Neuroprotective Effect of Uncaria rhynchophylla in Kainic Acid-Induced Epileptic Seizures by Modulating Hippocampal Mossy Fiber Sprouting, Neuron Survival, Astrocyte Proliferation, and S100B Expression

    Chung-Hsiang Liu

    2012-01-01

    Full Text Available Uncaria rhynchophylla (UR, which is a traditional Chinese medicine, has anticonvulsive effect in our previous studies, and the cellular mechanisms behind this are still little known. Because of this, we wanted to determine the importance of the role of UR on kainic acid- (KA- induced epilepsy. Oral UR for 6 weeks can successfully attenuate the onset of epileptic seizure in animal tests. Hippocampal mossy fiber sprouting dramatically decreased, while neuronal survival increased with UR treatment in hippocampal CA1 and CA3 areas. Furthermore, oral UR for 6 weeks significantly attenuated the overexpression of astrocyte proliferation and S100B proteins but not γ-aminobutyric acid A (GABAA receptors. These results indicate that oral UR for 6 weeks can successfully attenuate mossy fiber sprouting, astrocyte proliferation, and S100B protein overexpression and increase neuronal survival in KA-induced epileptic rat hippocampus

  2. Expression of polysialylated neural cell adhesion molecules on adult stem cells after neuronal differentiation of inner ear spiral ganglion neurons

    Park, Kyoung Ho [Department of Otolaryngology Head and Neck Surgery, College of Medicine, Catholic University, Seoul (Korea, Republic of); Yeo, Sang Won, E-mail: swyeo@catholic.ac.kr [Department of Otolaryngology Head and Neck Surgery, College of Medicine, Catholic University, Seoul (Korea, Republic of); Troy, Frederic A., E-mail: fatroy@ucdavis.edu [Department of Biochemistry and Molecular Medicine, University of California, School of Medicine, Davis, CA 95616 (United States); Xiamen University, School of Medicine, Xiamen City (China)

    2014-10-17

    Highlights: • PolySia expressed on neurons primarily during early stages of neuronal development. • PolySia–NCAM is expressed on neural stem cells from adult guinea pig spiral ganglion. • PolySia is a biomarker that modulates neuronal differentiation in inner ear stem cells. - Abstract: During brain development, polysialylated (polySia) neural cell adhesion molecules (polySia–NCAMs) modulate cell–cell adhesive interactions involved in synaptogenesis, neural plasticity, myelination, and neural stem cell (NSC) proliferation and differentiation. Our findings show that polySia–NCAM is expressed on NSC isolated from adult guinea pig spiral ganglion (GPSG), and in neurons and Schwann cells after differentiation of the NSC with epidermal, glia, fibroblast growth factors (GFs) and neurotrophins. These differentiated cells were immunoreactive with mAb’s to polySia, NCAM, β-III tubulin, nestin, S-100 and stained with BrdU. NSC could regenerate and be differentiated into neurons and Schwann cells. We conclude: (1) polySia is expressed on NSC isolated from adult GPSG and on neurons and Schwann cells differentiated from these NSC; (2) polySia is expressed on neurons primarily during the early stage of neuronal development and is expressed on Schwann cells at points of cell–cell contact; (3) polySia is a functional biomarker that modulates neuronal differentiation in inner ear stem cells. These new findings suggest that replacement of defective cells in the inner ear of hearing impaired patients using adult spiral ganglion neurons may offer potential hope to improve the quality of life for patients with auditory dysfunction and impaired hearing disorders.

  3. Expression of polysialylated neural cell adhesion molecules on adult stem cells after neuronal differentiation of inner ear spiral ganglion neurons

    Highlights: • PolySia expressed on neurons primarily during early stages of neuronal development. • PolySia–NCAM is expressed on neural stem cells from adult guinea pig spiral ganglion. • PolySia is a biomarker that modulates neuronal differentiation in inner ear stem cells. - Abstract: During brain development, polysialylated (polySia) neural cell adhesion molecules (polySia–NCAMs) modulate cell–cell adhesive interactions involved in synaptogenesis, neural plasticity, myelination, and neural stem cell (NSC) proliferation and differentiation. Our findings show that polySia–NCAM is expressed on NSC isolated from adult guinea pig spiral ganglion (GPSG), and in neurons and Schwann cells after differentiation of the NSC with epidermal, glia, fibroblast growth factors (GFs) and neurotrophins. These differentiated cells were immunoreactive with mAb’s to polySia, NCAM, β-III tubulin, nestin, S-100 and stained with BrdU. NSC could regenerate and be differentiated into neurons and Schwann cells. We conclude: (1) polySia is expressed on NSC isolated from adult GPSG and on neurons and Schwann cells differentiated from these NSC; (2) polySia is expressed on neurons primarily during the early stage of neuronal development and is expressed on Schwann cells at points of cell–cell contact; (3) polySia is a functional biomarker that modulates neuronal differentiation in inner ear stem cells. These new findings suggest that replacement of defective cells in the inner ear of hearing impaired patients using adult spiral ganglion neurons may offer potential hope to improve the quality of life for patients with auditory dysfunction and impaired hearing disorders

  4. AUDITORY HAIR CELL EXPLANT CO-CULTURES PROMOTE THE DIFFERENTIATION OF STEM CELLS INTO BIPOLAR NEURONS

    Coleman, B.; Fallon, J. B.; Gillespie, L.N.; Silva, M.G.; Shepherd, R.K.

    2006-01-01

    Auditory neurons, the target neurons of the cochlear implant, degenerate following a sensorineural hearing loss. The goal of this research is to direct the differentiation of embryonic stem cells (SCs) into bipolar auditory neurons that can be used to replace degenerating neurons in the deafened mammalian cochlea. Successful replacement of auditory neurons is likely to result in improved clinical outcomes for cochlear implant recipients. We examined two post-natal auditory co-culture models w...

  5. Quercetin Protects against Okadaic Acid-Induced Injury via MAPK and PI3K/Akt/GSK3β Signaling Pathways in HT22 Hippocampal Neurons.

    Wei Jiang

    Full Text Available Increasing evidence shows that oxidative stress and the hyperphosphorylation of tau protein play essential roles in the progression of Alzheimer's disease (AD. Quercetin is a major flavonoid that has anti-oxidant, anti-cancer and anti-inflammatory properties. We investigated the neuroprotective effects of quercetin to HT22 cells (a cell line from mouse hippocampal neurons. We found that Okadaic acid (OA induced the hyperphosphorylation of tau protein at Ser199, Ser396, Thr205, and Thr231 and produced oxidative stress to the HT22 cells. The oxidative stress suppressed the cell viability and decreased the levels of lactate dehydrogenase (LDH, superoxide dismutase (SOD, mitochondria membrane potential (MMP and Glutathione peroxidase (GSH-Px. It up-regulated malondialdehyde (MDA production and intracellular reactive oxygen species (ROS. In addition, phosphoinositide 3 kinase/protein kinase B/Glycogen synthase kinase3β (PI3K/Akt/GSK3β and mitogen activated protein kinase (MAPK were also involved in this process. We found that pre-treatment with quercetin can inhibited OA-induced the hyperphosphorylation of tau protein and oxidative stress. Moreover, pre-treatment with quercetin not only inhibited OA-induced apoptosis via the reduction of Bax, and up-regulation of cleaved caspase 3, but also via the inhibition of PI3K/Akt/GSK3β, MAPKs and activation of NF-κB p65. Our findings suggest the therapeutic potential of quercetin to treat AD.

  6. Constitutive expression of the neuron-restrictive silencer factor (NRSF)/REST in differentiating neurons disrupts neuronal gene expression and causes axon pathfinding errors in vivo

    Paquette, Alice J.; Perez, Sharon E.; Anderson, David J.

    2000-01-01

    The neuron-restrictive silencer factor (NRSF; also known as REST for repressor element-1 silencing transcription factor) is a transcriptional repressor of multiple neuronal genes, but little is known about its function in vivo. NRSF is normally down-regulated upon neuronal differentiation. Constitutive expression of NRSF in the developing spinal cord of chicken embryos caused repression of two endogenous target genes, N-tubulin and Ng-CAM, but did not prevent overt...

  7. Carbon monoxide improves neuronal differentiation and yield by increasing the functioning and number of mitochondria.

    Almeida, Ana S; Sonnewald, Ursula; Alves, Paula M; Vieira, Helena L A

    2016-08-01

    The process of cell differentiation goes hand-in-hand with metabolic adaptations, which are needed to provide energy and new metabolites. Carbon monoxide (CO) is an endogenous cytoprotective molecule able to inhibit cell death and improve mitochondrial metabolism. Neuronal differentiation processes were studied using the NT2 cell line, which is derived from human testicular embryonic teratocarcinoma and differentiates into post-mitotic neurons upon retinoic acid treatment. CO-releasing molecule A1 (CORM-A1) was used do deliver CO into cell culture. CO treatment improved NT2 neuronal differentiation and yield, since there were more neurons and the total cell number increased following the differentiation process. CO supplementation enhanced the mitochondrial population in post-mitotic neurons derived from NT2 cells, as indicated by an increase in mitochondrial DNA. CO treatment during neuronal differentiation increased the extent of the classical metabolic change that occurs during neuronal differentiation, from glycolytic to more oxidative metabolism, by decreasing the ratio of lactate production and glucose consumption. The expression of pyruvate and lactate dehydrogenases was higher, indicating an augmented oxidative metabolism. Moreover, these findings were corroborated by an increased percentage of (13) C incorporation from [U-(13) C]glucose into the tricarboxylic acid cycle metabolites malate and citrate, and also glutamate and aspartate in CO-treated cells. Finally, under low levels of oxygen (5%), which enhances glycolytic metabolism, some of the enhancing effects of CO on mitochondria were not observed. In conclusion, our data show that CO improves neuronal and mitochondrial yield by stimulation of tricarboxylic acid cycle activity, and thus oxidative metabolism of NT2 cells during the process of neuronal differentiation. The process of cell differentiation is coupled with metabolic adaptations. Carbon monoxide (CO) is an endogenous cytoprotective

  8. Ultrastructure of neuronal-like cells differentiated from adult adipose-derived stromal cells

    Changqing Ye; Xiaodong Yuan; Hui Liu; Yanan Cai; Ya Ou

    2010-01-01

    β-mercaptoethanol induces in vitro adult adipose-derived stromal cells (ADSCs) to differentiate into neurons. However, the ultrastructural features of the differentiated neuronal-like cells remain unknown. In the present study, inverted phase contrast microscopy was utilized to observe β-mercaptcethanol-induced differentiation of neuronal-like cells from human ADSCs, and immunocytochemistry and real-time polymerase chain reaction were employed to detect expression of a neural stem cells marker (nestin), a neuronal marker (neuron-specific enolase), and a glial marker (glial fibrillary acidic protein). In addition, ultrastructure of neuronal-like cells was observed by transmission election microscopy. Results revealed highest expression rate of nestin and neuron-specific enolase at 3 and 5 hours following induced differentiation; cells in the 5-hour induction group exhibited a neuronal-specific structure, i.e., Nissl bodies. However, when induction solution was replaced by complete culture medium after 8-hour induction, the differentiated cells reverted to the fibroblast-like morphology from day 1. These results demonstrate that β-mercaptoethanol-induced ADSCs induced differentiation into neural stem cells, followed by morphology of neuronal-like cells. However, this differentiation state was not stable.

  9. FOXP2 drives neuronal differentiation by interacting with retinoic acid signaling pathways

    Jeroen Middelbeek

    2014-09-01

    Full Text Available FOXP2 was the first gene shown to cause a Mendelian form of speech and language disorder. Although developmentally expressed in many organs, loss of a single copy of FOXP2 leads to a phenotype that is largely restricted to orofacial impairment during articulation and linguistic processing deficits. Why perturbed FOXP2 function affects specific aspects of the developing brain remains elusive. We investigated the role of FOXP2 in neuronal differentiation and found that FOXP2 drives molecular changes consistent with neuronal differentiation in a human model system. We identified a network of FOXP2 regulated genes related to retinoic acid signaling and neuronal differentiation. FOXP2 also produced phenotypic changes associated with neuronal differentiation including increased neurite outgrowth and reduced migration. Crucially, cells expressing FOXP2 displayed increased sensitivity to retinoic acid exposure. This suggests a mechanism by which FOXP2 may be able to increase the cellular differentiation response to environmental retinoic acid cues for specific subsets of neurons in the brain. These data demonstrate that FOXP2 promotes neuronal differentiation by interacting with the retinoic acid signaling pathway and regulates key processes required for normal circuit formation such as neuronal migration and neurite outgrowth. In this way, FOXP2, which is found only in specific subpopulations of neurons in the brain, may drive precise neuronal differentiation patterns and/or control localization and connectivity of these FOXP2 positive cells.

  10. Differentiation patterns of mouse embryonic stem cells and induced pluripotent stem cells into neurons.

    Nakamura, Mai; Kamishibahara, Yu; Kitazawa, Ayako; Kawaguchi, Hideo; Shimizu, Norio

    2016-05-01

    Mouse embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have the ability to differentiate in vitro into various cell lineages including neurons. The differentiation of these cells into neurons has potential applications in regenerative medicine. Previously, we reported that a chick dorsal root ganglion (DRG)-conditioned medium (CM) promoted the differentiation of mouse ES and iPS cells into neurons. Here, we used real-time PCR to investigate the differentiation patterns of ES and iPS cells into neurons when DRG-CM was added. DRG-CM promoted the expression levels of βIII-tubulin gene (a marker of postmitotic neurons) in ES and iPS cells. ES cells differentiated into neurons faster than iPS cells, and the maximum peaks of gene expression involved in motor, sensory, and dopaminergic neurons were different. Rho kinase (ROCK) inhibitors could be very valuable at numerous stages in the production and use of stem cells in basic research and eventual cell-based therapies. Thus, we investigated whether the addition of a ROCK inhibitor Y-27632 and DRG-CM on the basis of the differentiation patterns promotes the neuronal differentiation of ES cells. When the ROCK inhibitor was added to the culture medium at the initial stages of cultivation, it stimulated the neuronal differentiation of ES cells more strongly than that stimulated by DRG-CM. Moreover, the combination of the ROCK inhibitor and DRG-CM promoted the neuronal differentiation of ES cells when the ROCK inhibitor was added to the culture medium at day 3. The ROCK inhibitor may be useful for promoting neuronal differentiation of ES cells. PMID:25354731

  11. TRIM32-dependent transcription in adult neural progenitor cells regulates neuronal differentiation

    Hillje, Anna-Lena; Pavlou, Maria Angeliki; Beckmann, Elisabeth; Worlitzer, Maik; Bahnassawy, Lamiaa; Lewejohann, Lars; Palm, Thomas; Schwamborn, Jens Christian

    2013-01-01

    In the adult mammalian brain, neural stem cells in the subventricular zone continuously generate new neurons for the olfactory bulb. Cell fate commitment in these adult neural stem cells is regulated by cell fate-determining proteins. Here, we show that the cell fate-determinant TRIM32 is upregulated during differentiation of adult neural stem cells into olfactory bulb neurons. We further demonstrate that TRIM32 is necessary for the correct induction of neuronal differentiation in these cells...

  12. Protoplasmic Astrocytes Enhance the Ability of Neural Stem Cells to Differentiate into Neurons In Vitro

    Yuan Liu; Li Wang; Zaiyun Long; Lin Zeng; Yamin Wu

    2012-01-01

    Protoplasmic astrocytes have been reported to exhibit neuroprotective effects on neurons, but there has been no direct evidence for a functional relationship between protoplasmic astrocytes and neural stem cells (NSCs). In this study, we examined neuronal differentiation of NSCs induced by protoplasmic astrocytes in a co-culture model. Protoplasmic astrocytes were isolated from new-born and NSCs from the E13-15 cortex of rats respectively. The differentiated cells labeled with neuron-specific...

  13. Synaptic network activity induces neuronal differentiation of adult hippocampal precursor cells through BDNF signaling

    HarishBabu

    2009-09-01

    Full Text Available Adult hippocampal neurogenesis is regulated by activity. But how do neural precursor cells in the hippocampus respond to surrounding network activity and translate increased neural activity into a developmental program? Here we show that long-term potential (LTP-like synaptic activity within a cellular network of mature hippocampal neurons promotes neuronal differentiation of newly generated cells. In co-cultures of precursor cells with primary hippocampal neurons, LTP-like synaptic plasticity induced by addition of glycine in Mg2+-free media for 5 min, produced synchronous network activity and subsequently increased synaptic strength between neurons. Furthermore, this synchronous network activity led to a significant increase in neuronal differentiation from the co-cultured neural precursor cells. When applied directly to precursor cells, glycine and Mg2+-free solution did not induce neuronal differentiation. Synaptic plasticity-induced neuronal differentiation of precursor cells was observed in the presence of GABAergic neurotransmission blockers but was dependent on NMDA-mediated Ca2+ influx. Most importantly, neuronal differentiation required the release of brain-derived neurotrophic factor (BDNF from the underlying substrate hippocampal neurons as well as TrkB receptor phosphorylation in precursor cells. This suggests that activity-dependent stem cell differentiation within the hippocampal network is mediated via synaptically evoked BDNF signaling.

  14. Human in vitro reporter model of neuronal development and early differentiation processes

    Bogdahn Ulrich

    2008-02-01

    Full Text Available Abstract Background During developmental and adult neurogenesis, doublecortin is an early neuronal marker expressed when neural stem cells assume a neuronal cell fate. To understand mechanisms involved in early processes of neuronal fate decision, we investigated cell lines for their capacity to induce expression of doublecortin upon neuronal differentiation and develop in vitro reporter models using doublecortin promoter sequences. Results Among various cell lines investigated, the human teratocarcinoma cell line NTERA-2 was found to fulfill our criteria. Following induction of differentiation using retinoic acid treatment, we observed a 16-fold increase in doublecortin mRNA expression, as well as strong induction of doublecortin polypeptide expression. The acquisition of a neuronal precursor phenotype was also substantiated by the establishment of a multipolar neuronal morphology and expression of additional neuronal markers, such as Map2, βIII-tubulin and neuron-specific enolase. Moreover, stable transfection in NTERA-2 cells of reporter constructs encoding fluorescent or luminescent genes under the control of the doublecortin promoter allowed us to directly detect induction of neuronal differentiation in cell culture, such as following retinoic acid treatment or mouse Ngn2 transient overexpression. Conclusion Induction of doublecortin expression in differentiating NTERA-2 cells suggests that these cells accurately recapitulate some of the very early events of neuronal determination. Hence, the use of reporter genes under the control of the doublecortin promoter in NTERA-2 cells will help us to investigate factors involved early in the course of neuronal differentiation processes. Moreover the ease to detect the induction of a neuronal program in this model will permit to perform high throughput screening for compounds acting on the early neuronal differentiation mechanisms.

  15. cGMP modulates stem cells differentiation to neurons in brain in vivo.

    Gómez-Pinedo, U; Rodrigo, R; Cauli, O; Herraiz, S; Garcia-Verdugo, J-M; Pellicer, B; Pellicer, A; Felipo, V

    2010-02-17

    During brain development neural stem cells may differentiate to neurons or to other cell types. The aim of this work was to assess the role of cGMP (cyclic GMP) in the modulation of differentiation of neural stem cells to neurons or non-neuronal cells. cGMP in brain of fetuses was reduced to 46% of controls by treating pregnant rats with nitroarginine-methylester (L-NAME) and was restored by co-treatment with sildenafil.Reducing cGMP during brain development leads to reduced differentiation of stem cells to neurons and increased differentiation to non-neuronal cells. The number of neurons in the prefrontal cortex originated from stem cells proliferating on gestational day 14 was 715+/-14/mm(2) in control rats and was reduced to 440+/-29/mm(2) (61% of control) in rats treated with L-NAME. In rats exposed to L-NAME plus sildenafil, differentiation to neurons was completely normalized, reaching 683+/-11 neurons/mm(2). In rats exposed to sildenafil alone the number of cells labelled with bromodeoxyuridine (BrdU) and NeuN was 841+/-16/mm(2). In prefrontal cortex of control rats 48% of the neural stem cells proliferating in gestational day 14 differentiate to neurons, but only 24% in rats exposed to L-NAME. This was corrected by sildenafil, 40% of cells differentiate to neurons. Similar results were obtained for neurons proliferating during all developmental period. Treatment with L-NAME did not reduce the total number of cells labelled with BrdU, further supporting that L-NAME reduces selectively the differentiation of stem cells to neurons. Similar results were obtained in hippocampus. Treatment with L-NAME reduced the differentiation of neural stem cells to neurons, although the effect was milder than in prefrontal cortex. These results support that cGMP modulates the fate of neural stem cells in brain in vivo and suggest that high cGMP levels promote its differentiation to neurons while reduced cGMP levels promote differentiation to non-neuronal cells. PMID:19958812

  16. Neuron-specific ELAV/Hu proteins suppress HuR mRNA during neuronal differentiation by alternative polyadenylation

    Mansfield, Kyle D.; Keene, Jack D.

    2011-01-01

    The ubiquitously expressed RNA-binding protein HuR increases the stability and translation of mRNAs encoding growth regulatory proteins that promote proliferation in a variety of cell types. However, the three neuron-specific ELAV/Hu proteins, HuB, HuC and HuD, while binding to the same types of mRNAs, are required instead for neuronal differentiation, and it becomes difficult to reconcile these contrary functions when all four Hu proteins are expressed in the same neuron. HuR mRNA exists as ...

  17. The role of metallothionein II in neuronal differentiation and survival

    Køhler, Lene B; Berezin, Vladimir; Bock, Elisabeth;

    2003-01-01

    -I+II can affect neurons directly. It is likely that MT isoforms could be beneficial also during neurodegenerative disorders. In this study, we have examined if MT-II affects survival and neurite extension of dopaminergic and hippocampal neurons. We show for the first time that MT-II treatment can...... significantly stimulate neurite extension from both dopaminergic and hippocampal neurons. Moreover, MT-II treatment significantly increases survival of dopaminergic neurons exposed to 6-hydroxydopamine (6-OHDA) and protects significantly hippocampal neurons from amyloid beta-peptide-induced neurotoxicity...

  18. Human stem cell neuronal differentiation on silk-carbon nanotube composite

    Chen, Chi-Shuo; Soni, Sushant; Le, Catherine; Biasca, Matthew; Farr, Erik; Chen, Eric Y.-T.; Chin, Wei-Chun

    2012-02-01

    Human embryonic stem cells [hESCs] are able to differentiate into specific lineages corresponding to regulated spatial and temporal signals. This unique attribute holds great promise for regenerative medicine and cell-based therapy for many human diseases such as spinal cord injury [SCI] and multiple sclerosis [MS]. Carbon nanotubes [CNTs] have been successfully used to promote neuronal differentiation, and silk has been widely applied in tissue engineering. This study aims to build silk-CNT composite scaffolds for improved neuron differentiation efficiency from hESCs. Two neuronal markers (β-III tubulin and nestin) were utilized to determine the hESC neuronal lineage differentiation. In addition, axonal lengths were measured to evaluate the progress of neuronal development. The results demonstrated that cells on silk-CNT scaffolds have a higher β-III tubulin and nestin expression, suggesting augmented neuronal differentiation. In addition, longer axons with higher density were found to associate with silk-CNT scaffolds. Our silk-CNT-based composite scaffolds can promote neuronal differentiation of hESCs. The silk-CNT composite scaffolds developed here can serve as efficient supporting matrices for stem cell-derived neuronal transplants, offering a promising opportunity for nerve repair treatments for SCI and MS patients.

  19. Trimethyltin chloride inhibits neuronal cell differentiation in zebrafish embryo neurodevelopment.

    Kim, Jin; Kim, C-Yoon; Song, Juha; Oh, Hanseul; Kim, Cheol-Hee; Park, Jae-Hak

    2016-01-01

    Trimethyltin chloride (TMT) is a neurotoxicant widely present in the aquatic environment, primarily from effluents of the plastic industry. It is known to cause acute neuronal death in the limbic-cerebellar system, particularly in the hippocampus. However, relatively few studies have estimated the effects of TMT toxicity on neurodevelopment. In this study, we confirmed the dose-dependent effects of TMT on neurodevelopmental stages through analysis of morphological changes and fluorescence assays using HuC-GFP and olig2-dsRed transgenic zebrafish embryos. In addition, we analyzed the expression of genes and proteins related to neurodevelopment. Exposure of embryos to TMT for 4days post fertilization (dpf) elicited a concentration-related decrease in body length and increase in axial malformation. TMT affected the fluorescent CNS structure by decreasing pattern of HuC-GFP and olig2-dsRed transgenic zebrafish. In addition, it significantly modulated the expression patterns of Sonic hedgehog a (Shha), Neurogenin1 (Ngn1), Embryonic lethal abnormal vision like protein 3 (Elavl3), and Glial fibrillary acidic protein (Gfap). The overexpression of Shha and Ngn1, and downregulation of Elavl3 and Gfap, indicate repression of proneural cell differentiation. Our study demonstrates that TMT inhibits specific neurodevelopmental stages in zebrafish embryos and suggests a possible mechanism for the toxicity of TMT in vertebrate neurodevelopment. PMID:26687135

  20. G9a inhibition promotes neuronal differentiation of human bone marrow mesenchymal stem cells through the transcriptional induction of RE-1 containing neuronal specific genes.

    Kim, Ho-Tae; Jeong, Sin-Gu; Cho, Goang-Won

    2016-06-01

    Recent studies have shown that epigenomic modifications are significantly associated with neuronal differentiation. Many neuronal specific genes contain the repressor element-1 (RE-1), which recruits epigenetic modulators, such as the histone methyltransferase G9a and interrupts the expression of neuronal genes in non-neuronal cells. This study investigated the functional role of G9a during neuronal differentiation of human bone marrow mesenchymal stem cells (BM-MSCs). Human BM-MSCs treated with the G9a inhibitor BIX01294 showed an increased expression of various neuronal-lineage genes. Using genomic sequence analysis, we identified RE-1 consensus sequences in the proximal region of several neuronal-specific genes. Chromatin immunoprecipitation (ChIP) assay results have showed that H3K9me2 (dimethylation of lysine 9 on histone 3) occupancy at RE-1-containing sequences from neuronal-specific genes was significantly decreased in BIX01294-MSCs. When BIX01294-MSCs were differentiated with neuronal induction medium, cells differentiated more effectively into neuron-like cells, complete with a cell body and dendrites. Expression of neuronal-specific genes containing the RE-1 sequences was significantly increased in differentiated BIX01294-MSCs, as confirmed by immunocytochemical staining and immunoblotting. Thus, this study shows that BIX01294 pretreated human BM-MSCs can be effectively differentiated into neuron-like cells by induced expression of neuronal-specific genes containing RE-1 sequences. PMID:26952575

  1. Differential sensitivity to nicotine among hypothalamic magnocellular neurons

    Mikkelsen, J D; Jacobsen, Julie; Kiss, Adrian Emil

    2012-01-01

    The magnocellular neurons in the hypothalamic paraventricular (PVN) and supraoptic nuclei (SON) either contain vasopressin or oxytocin. Even though both hormones are released after systemic administration of nicotine, the mechanism through which the two populations of neurons are activated is not...... known. This study was carried out in the rat to investigate the effect of increasing doses of nicotine on subsets of magnocellular neurons containing either oxytocin or vasopressin....

  2. Citalopram increases the differentiation efficacy of bone marrow mesenchymal stem cells into neuronal-like cells

    Verdi, Javad; Mortazavi-Tabatabaei, Seyed AbdolReza; Sharif, Shiva; Verdi, Hadi; Shoae-Hassani, Alireza

    2014-01-01

    Several studies have demonstrated that selective serotonin reuptake inhibitor antidepressants can promote neuronal cell proliferation and enhance neuroplasticity both in vitro and in vivo. It is hypothesized that citalopram, a selective serotonin reuptake inhibitor, can promote the neuronal differentiation of adult bone marrow mesenchymal stem cells. Citalopram strongly enhanced neuronal characteristics of the cells derived from bone marrow mesenchymal stem cells. The rate of cell death was d...

  3. Mitochondrial DNA mutations affect calcium handling in differentiated neurons.

    Trevelyan, A.J.; Kirby, D.M.; Smulders-Srinivasan, T.K.; Nooteboom, M.; Acin-Perez, R.; Enriquez, J.A.; Whittington, M.A.; Lightowlers, R.N.; Turnbull, D.M.

    2010-01-01

    Mutations in the mitochondrial genome are associated with a wide range of neurological symptoms, but many aspects of the basic neuronal pathology are not understood. One candidate mechanism, given the well-established role of mitochondria in calcium buffering, is a deficit in neuronal calcium homoeo

  4. Do Substantia Nigra Dopaminergic Neurons Differentiate Between Reward and Punishment?

    Michael J. Frank; D. James Surmeier

    2009-01-01

    The activity of dopaminergic neurons are thought to be increased by stimuli that predict reward and decreased by stimuli that predict aversive outcomes. Recent work by Matsumoto and Hikosaka challenges this model by asserting that stimuli associated with either rewarding or aversive outcomes increase the activity of dopaminergic neurons in the substantia nigra pars compacta.

  5. The Limited Utility of Multiunit Data in Differentiating Neuronal Population Activity.

    Corey J Keller

    Full Text Available To date, single neuron recordings remain the gold standard for monitoring the activity of neuronal populations. Since obtaining single neuron recordings is not always possible, high frequency or 'multiunit activity' (MUA is often used as a surrogate. Although MUA recordings allow one to monitor the activity of a large number of neurons, they do not allow identification of specific neuronal subtypes, the knowledge of which is often critical for understanding electrophysiological processes. Here, we explored whether prior knowledge of the single unit waveform of specific neuron types is sufficient to permit the use of MUA to monitor and distinguish differential activity of individual neuron types. We used an experimental and modeling approach to determine if components of the MUA can monitor medium spiny neurons (MSNs and fast-spiking interneurons (FSIs in the mouse dorsal striatum. We demonstrate that when well-isolated spikes are recorded, the MUA at frequencies greater than 100Hz is correlated with single unit spiking, highly dependent on the waveform of each neuron type, and accurately reflects the timing and spectral signature of each neuron. However, in the absence of well-isolated spikes (the norm in most MUA recordings, the MUA did not typically contain sufficient information to permit accurate prediction of the respective population activity of MSNs and FSIs. Thus, even under ideal conditions for the MUA to reliably predict the moment-to-moment activity of specific local neuronal ensembles, knowledge of the spike waveform of the underlying neuronal populations is necessary, but not sufficient.

  6. Tracking neuronal marker expression inside living differentiating cells using molecular beacons

    Ilieva, Mirolyuba; Della Vedova, Paolo; Hansen, Ole;

    2013-01-01

    Monitoring gene expression is an important tool for elucidating mechanisms of cellular function. In order to monitor gene expression during nerve cell development, molecular beacon (MB) probes targeting markers representing different stages of neuronal differentiation were designed and synthesized...

  7. CALBINDIN CONTENT AND DIFFERENTIAL VULNERABILITY OF MIDBRAIN EFFERENT DOPAMINERGIC NEURONS IN MACAQUES

    Iria G Dopeso-Reyes

    2014-12-01

    Full Text Available Calbindin (CB is a calcium binding protein reported to protect dopaminergic neurons from degeneration. Although a direct link between CB content and differential vulnerability of dopaminergic neurons has long been accepted, factors other than CB have also been suggested, particularly those related to the dopamine transporter. Indeed, several studies have reported that CB levels are not causally related to the differential vulnerability of dopaminergic neurons against neurotoxins. Here we have used dual stains for tyrosine hydroxylase (TH and CB in 3 control and 3 MPTP-treated monkeys to visualize dopaminergic neurons in the ventral tegmental area (VTA and in the dorsal and ventral tiers of the substantia nigra pars compacta (SNcd and SNcv co-expressing TH and CB. In control animals, the highest percentages of co-localization were found in VTA (58.2%, followed by neurons located in the SNcd (34.7%. As expected, SNcv neurons lacked CB expression. In MPTP-treated animals, the percentage of CB-ir/TH-ir neurons in the VTA was similar to control monkeys (62.1%, whereas most of the few surviving neurons in the SNcd were CB-ir/TH-ir (88.6%. Next, we have elucidated the presence of CB within identified nigrostriatal and nigroextrastriatal midbrain dopaminergic projection neurons. For this purpose, two control monkeys received one injection of Fluoro-Gold into the caudate nucleus and one injection of cholera toxin (CTB into the postcommissural putamen, whereas two more monkeys were injected with CTB into the internal division of the globus pallidus. As expected, all the nigrocaudate- and nigroputamen-projecting neurons were TH-ir, although surprisingly, all of these nigrostriatal-projecting neurons were negative for CB. Furthermore, all the nigropallidal-projecting neurons co-expressed both TH and CB. In summary, although CB-ir dopaminergic neurons seem to be less prone to MPTP-induced degeneration, our data clearly demonstrated that these neurons are not

  8. Acidic leucine-rich nuclear phosphoprotein 32 family member B (ANP32B) contributes to retinoic acid-induced differentiation of leukemic cells

    Yu, Yun; Shen, Shao-Ming; Zhang, Fei-Fei; Wu, Zhao-Xia; Han, Bin [Shanghai Universities E-Institute for Chemical Biology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Rui-Jin Hospital, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Wang, Li-Shun, E-mail: jywangls@shsmu.edu.cn [Shanghai Universities E-Institute for Chemical Biology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Rui-Jin Hospital, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China)

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer ANP32B was down-regulated during ATRA-induced leukemic cell differentiation. Black-Right-Pointing-Pointer Knockdown of ANP32B enhanced ATRA-induced leukemic cell differentiation. Black-Right-Pointing-Pointer Ectopic expression of ANP32B inhibited ATRA-induced leukemic cell differentiation. Black-Right-Pointing-Pointer ANP32B inhibited ATRA activated transcriptional activity of RAR{alpha}. -- Abstract: The acidic leucine-rich nuclear phosphoprotein 32B (ANP32B) is a member of a conserved superfamily of nuclear proteins whose functions are largely unknown. In our previous work, ANP32B was identified as a novel direct substrate for caspase-3 and acted as a negative regulator for leukemic cell apoptosis. In this work, we provided the first demonstration that ANP32B expression was down-regulated during differentiation induction of leukemic cells by all-trans retinoic acid (ATRA). Knockdown of ANP32B expression by specific shRNA enhanced ATRA-induced leukemic cell differentiation, while ectopic expression of ANP32B attenuated it, indicating an inhibitory role of ANP32B against leukemic cell differentiation. Furthermore, luciferase reporter assay revealed that ANP32B might exert this role through inhibiting the ATRA dependent transcriptional activity of retinoic acid receptor (RAR{alpha}). These data will shed new insights into understanding the biological functions of ANP32B protein.

  9. Acidic leucine-rich nuclear phosphoprotein 32 family member B (ANP32B) contributes to retinoic acid-induced differentiation of leukemic cells

    Highlights: ► ANP32B was down-regulated during ATRA-induced leukemic cell differentiation. ► Knockdown of ANP32B enhanced ATRA-induced leukemic cell differentiation. ► Ectopic expression of ANP32B inhibited ATRA-induced leukemic cell differentiation. ► ANP32B inhibited ATRA activated transcriptional activity of RARα. -- Abstract: The acidic leucine-rich nuclear phosphoprotein 32B (ANP32B) is a member of a conserved superfamily of nuclear proteins whose functions are largely unknown. In our previous work, ANP32B was identified as a novel direct substrate for caspase-3 and acted as a negative regulator for leukemic cell apoptosis. In this work, we provided the first demonstration that ANP32B expression was down-regulated during differentiation induction of leukemic cells by all-trans retinoic acid (ATRA). Knockdown of ANP32B expression by specific shRNA enhanced ATRA-induced leukemic cell differentiation, while ectopic expression of ANP32B attenuated it, indicating an inhibitory role of ANP32B against leukemic cell differentiation. Furthermore, luciferase reporter assay revealed that ANP32B might exert this role through inhibiting the ATRA dependent transcriptional activity of retinoic acid receptor (RARα). These data will shed new insights into understanding the biological functions of ANP32B protein.

  10. Quantification of dopaminergic neuron differentiation and neurotoxicity via a genetic reporter

    Jun Cui; Megan Rothstein; Theo Bennett; Pengbo Zhang; Ninuo Xia; Reijo Pera, Renee A.

    2016-01-01

    Human pluripotent stem cells provide a powerful human-genome based system for modeling human diseases in vitro and for potentially identifying novel treatments. Directed differentiation of pluripotent stem cells produces many specific cell types including dopaminergic neurons. Here, we generated a genetic reporter assay in pluripotent stem cells using newly-developed genome editing technologies in order to monitor differentiation efficiency and compare dopaminergic neuron survival under diffe...

  11. Omega-3 Polyunsaturated Fatty Acids Enhance Neuronal Differentiation in Cultured Rat Neural Stem Cells

    Masanori Katakura

    2013-01-01

    Full Text Available Polyunsaturated fatty acids (PUFAs can induce neurogenesis and recovery from brain diseases. However, the exact mechanisms of the beneficial effects of PUFAs have not been conclusively described. We recently reported that docosahexaenoic acid (DHA induced neuronal differentiation by decreasing Hes1 expression and increasing p27kip1 expression, which causes cell cycle arrest in neural stem cells (NSCs. In the present study, we examined the effect of eicosapentaenoic acid (EPA and arachidonic acid (AA on differentiation, expression of basic helix-loop-helix transcription factors (Hes1, Hes6, and NeuroD, and the cell cycle of cultured NSCs. EPA also increased mRNA levels of Hes1, an inhibitor of neuronal differentiation, Hes6, an inhibitor of Hes1, NeuroD, and Map2 mRNA and Tuj-1-positive cells (a neuronal marker, indicating that EPA induced neuronal differentiation. EPA increased the mRNA levels of p21cip1 and p27kip1, a cyclin-dependent kinase inhibitor, which indicated that EPA induced cell cycle arrest. Treatment with AA decreased Hes1 mRNA but did not affect NeuroD and Map2 mRNA levels. Furthermore, AA did not affect the number of Tuj-1-positive cells or cell cycle progression. These results indicated that EPA could be involved in neuronal differentiation by mechanisms alternative to those of DHA, whereas AA did not affect neuronal differentiation in NSCs.

  12. Enhancement of caffeic acid phenethyl ester on all-trans retinoic acid-induced differentiation in human leukemia HL-60 cells

    All-trans retinoic acid (ATRA) induces complete remission in a high proportion of patients with acute promyelocytic leukemia (APL); however, the response is sometimes very slow. Furthermore, relapse and resistance to treatment often occur despite continued treatment with ATRA. Thereafter, combination treatment strategies have been suggested to circumvent these problems. The present study demonstrates that caffeic acid phenethyl ester (CAPE), a major component of honeybee propolis, enhanced ATRA-induced granulocytic differentiation in HL-60, a human promyelocytic cell line. The differentiation was assessed by Wright-Giemsa stain, nitroblue tetrazolium reduction, and membrane differentiation marker CD11b. In addition, CAPE enhanced ATRA-induced cell cycle arrest at the G1 phase by decreasing the association of cdk2-cyclin E complex. Finally, it was demonstrated that CAPE promoted the ATRA-mediated nuclear transcription activation of RARα assessed by EMSA assay and enhanced the expression of target genes including RARα, C/EBPε, and p21 protein resulting in the differentiation development of leukemia. It is suggested that CAPE possesses the potential to enhance the efficiency of ATRA in the differentiation therapy of APL

  13. Synthetic Small Molecules Derived from Natural Vitamin K Homologues that Induce Selective Neuronal Differentiation of Neuronal Progenitor Cells.

    Suhara, Yoshitomo; Hirota, Yoshihisa; Hanada, Norika; Nishina, Shun; Eguchi, Sachiko; Sakane, Rie; Nakagawa, Kimie; Wada, Akimori; Takahashi, Kazuhiko; Tokiwa, Hiroaki; Okano, Toshio

    2015-09-10

    We synthesized new vitamin K2 analogues with ω-terminal modifications of the side chain and evaluated their selective differentiation of neuronal progenitor cells into neurons in vitro. The result of the assay showed that the menaquinone-3 analogue modified with the m-methylphenyl group had the most potent activity, which was twice as great as the control. This finding indicated that it is possible to obtain much more potent compounds with modification of the structure of vitamin K2. PMID:26305288

  14. Cis-vaccenic acid induces differentiation and up-regulates gamma globin synthesis in K562, JK1 and transgenic mice erythroid progenitor stem cells.

    Aimola, Idowu A; Inuwa, Hajiya M; Nok, Andrew J; Mamman, Aisha I; Bieker, James J

    2016-04-01

    Gamma globin induction remains a promising pharmacological therapeutic treatment mode for sickle cell anemia and beta thalassemia, however Hydroxyurea remains the only FDA approved drug which works via this mechanism. In this regard, we assayed the γ-globin inducing capacity of Cis-vaccenic acid (CVA). CVA induced differentiation of K562, JK1 and transgenic mice primary bone marrow hematopoietic progenitor stem cells. CVA also significantly up-regulated γ-globin gene expression in JK-1 and transgenic mice bone marrow erythroid progenitor stem cells (TMbmEPSCs) but not K562 cells without altering cell viability. Increased γ-globin expression was accompanied by KLF1 suppression in CVA induced JK-1 cells. Erythropoietin induced differentiation of JK-1 cells 24h before CVA induction did not significantly alter CVA induced differentiation and γ-globin expression in JK-1 cells. Inhibition of JK-1 and Transgenic mice bone marrow erythroid progenitor stem cells Fatty acid elongase 5 (Elovl5) and Δ(9) desaturase suppressed the γ-globin inductive effects of CVA. CVA treatment failed to rescue γ-globin expression in Elovl5 and Δ(9)-desaturase inhibited cells 48h post inhibition in JK-1 cells. The data suggests that CVA directly modulates differentiation of JK-1 and TMbmEPSCs, and indirectly modulates γ-globin gene expression in these cells. Our findings provide important clues for further evaluations of CVA as a potential fetal hemoglobin therapeutic inducer. PMID:26879870

  15. Citalopram increases the differentiation efifcacy of bone marrow mesenchymal stem cells into neuronal-like cells

    Javad Verdi; Seyed Abdolreza Mortazavi-Tabatabaei; Shiva Sharif; Hadi Verdi; Alireza Shoae-Hassani

    2014-01-01

    Several studies have demonstrated that selective serotonin reuptake inhibitor antidepressants can promote neuronal cell proliferation and enhance neuroplasticity both in vitro and in vivo. It is hypothesized that citalopram, a selective serotonin reuptake inhibitor, can promote the neuronal differentiation of adult bone marrow mesenchymal stem cells. Citalopram strongly enhanced neuronal characteristics of the cells derived from bone marrow mesenchymal stem cells. The rate of cell death was decreased in citalopram-treated bone marrow mesenchymal stem cells than in control cells in neurobasal medium. In addition, the cumulative population doubling level of the citalopram-treated cells was signiifcantly increased compared to that of control cells. Also BrdU incorporation was elevated in citalopram-treated cells. These ifndings suggest that citalopram can improve the neuronal-like cell differentiation of bone marrow mesenchymal stem cells by increasing cell proliferation and survival while maintaining their neuronal characteristics.

  16. Cannabidiol Exposure During Neuronal Differentiation Sensitizes Cells Against Redox-Active Neurotoxins.

    Schönhofen, Patrícia; de Medeiros, Liana M; Bristot, Ivi Juliana; Lopes, Fernanda M; De Bastiani, Marco A; Kapczinski, Flávio; Crippa, José Alexandre S; Castro, Mauro Antônio A; Parsons, Richard B; Klamt, Fábio

    2015-08-01

    Cannabidiol (CBD), one of the most abundant Cannabis sativa-derived compounds, has been implicated with neuroprotective effect in several human pathologies. Until now, no undesired side effects have been associated with CBD. In this study, we evaluated CBD's neuroprotective effect in terminal differentiation (mature) and during neuronal differentiation (neuronal developmental toxicity model) of the human neuroblastoma SH-SY5Y cell line. A dose-response curve was performed to establish a sublethal dose of CBD with antioxidant activity (2.5 μM). In terminally differentiated SH-SY5Y cells, incubation with 2.5 μM CBD was unable to protect cells against the neurotoxic effect of glycolaldehyde, methylglyoxal, 6-hydroxydopamine, and hydrogen peroxide (H2O2). Moreover, no difference in antioxidant potential and neurite density was observed. When SH-SY5Y cells undergoing neuronal differentiation were exposed to CBD, no differences in antioxidant potential and neurite density were observed. However, CBD potentiated the neurotoxicity induced by all redox-active drugs tested. Our data indicate that 2.5 μM of CBD, the higher dose tolerated by differentiated SH-SY5Y neuronal cells, does not provide neuroprotection for terminally differentiated cells and shows, for the first time, that exposure of CBD during neuronal differentiation could sensitize immature cells to future challenges with neurotoxins. PMID:25108670

  17. Genome-wide target analysis of NEUROD2 provides new insights into regulation of cortical projection neuron migration and differentiation

    Bayam, Efil; Şahin, Gülcan Semra; Güzelsoy, Gizem; Güner, Gökhan; Kabakçıoğlu, Alkan; İnce-Dunn, Gülayşe

    2015-01-01

    Background: Cellular differentiation programs are controlled, to a large extent, by the combinatorial functioning of specific transcription factors. Corticalprojection neurons constitute the major excitatory neuron population within the cortex and mediate long distance communication between the cortex and other brain regions. Our understanding of effector transcription factors and their downstream transcriptional programs that direct the differentiation process ofcortical projection neurons i...

  18. Neuronal differentiation modulates the dystrophin Dp71d binding to the nuclear matrix

    The function of dystrophin Dp71 in neuronal cells remains unknown. To approach this issue, we have selected the PC12 neuronal cell line. These cells express both a Dp71f cytoplasmic variant and a Dp71d nuclear isoform. In this study, we demonstrated by electron and confocal microscopy analyses of in situ nuclear matrices and Western blotting evaluation of cell extracts that Dp71d associates with the nuclear matrix. Interestingly, this binding is modulated during NGF-induced neuronal differentiation of PC12 cells with a twofold increment in the differentiated cells, compared to control cells. Also, distribution of Dp71d along the periphery of the nuclear matrix observed in the undifferentiated cells is replaced by intense fluorescent foci localized in Center of the nucleoskeletal structure. In summary, we revealed that Dp71d is a dynamic component of nuclear matrix that might participate in the nuclear modeling occurring during neuronal differentiation

  19. Sox11 modulates neocortical development by regulating the proliferation and neuronal differentiation of cortical intermediate precursors

    Yongzhe Li; Qingsong Li; Jianjiao Wang; Yongri Zheng; Yan Zhao; Mian Guo; Yang Li; Qiuli Bao; Yu Zhang; Lizhuang Yang

    2012-01-01

    Neural precursor cells play important roles in the neocortical development,but the mechanisms of neural progenitor proliferation,neuronal differentiation,and migration,as well as patterning are still unclear.Sox11,one of SoxC family members,has been reported to be essential for embryonic and adult neurogenesis.But there is no report about the roles of Sox11 in corticogenesis.In order to investigate Sox11 function during cortical development,loss of function experiment was performed in this study.Knockdown of Sox11 by Sox11 siRNA constructs resulted in a diminished neuronal differentiation,but enhanced proliferation of intermediate progenitors.Accompanied with the high expression of Sox11 in the postmitotic neurons,but low expression of Sox11 in the dividing neural progenitors,all the observations indicate that Sox11 induces neuronal differentiation during the neocortical development.

  20. Sequential transcriptional waves direct the differentiation of newborn neurons in the mouse neocortex.

    Telley, Ludovic; Govindan, Subashika; Prados, Julien; Stevant, Isabelle; Nef, Serge; Dermitzakis, Emmanouil; Dayer, Alexandre; Jabaudon, Denis

    2016-03-25

    During corticogenesis, excitatory neurons are born from progenitors located in the ventricular zone (VZ), from where they migrate to assemble into circuits. How neuronal identity is dynamically specified upon progenitor division is unknown. Here, we study this process using a high-temporal-resolution technology allowing fluorescent tagging of isochronic cohorts of newborn VZ cells. By combining this in vivo approach with single-cell transcriptomics in mice, we identify and functionally characterize neuron-specific primordial transcriptional programs as they dynamically unfold. Our results reveal early transcriptional waves that instruct the sequence and pace of neuronal differentiation events, guiding newborn neurons toward their final fate, and contribute to a road map for the reverse engineering of specific classes of cortical neurons from undifferentiated cells. PMID:26940868

  1. Differentiation of neuronal stem cells into motor neurons using electrospun poly-L-lactic acid/gelatin scaffold.

    Binan, Loïc; Tendey, Charlène; De Crescenzo, Gregory; El Ayoubi, Rouwayda; Ajji, Abdellah; Jolicoeur, Mario

    2014-01-01

    Neural stem cells (NSCs) provide promising therapeutic potential for cell replacement therapy in spinal cord injury (SCI). However, high increases of cell viability and poor control of cell differentiation remain major obstacles. In this study, we have developed a non-woven material made of co-electrospun fibers of poly L-lactic acid and gelatin with a degradation rate and mechanical properties similar to peripheral nerve tissue and investigated their effect on cell survival and differentiation into motor neuronal lineages through the controlled release of retinoic acid (RA) and purmorphamine. Engineered Neural Stem-Like Cells (NSLCs) seeded on these fibers, with and without the instructive cues, differentiated into β-III-tubulin, HB-9, Islet-1, and choactase-positive motor neurons by immunostaining, in response to the release of the biomolecules. In addition, the bioactive material not only enhanced the differentiation into motor neuronal lineages but also promoted neurite outgrowth. This study elucidated that a combination of electrospun fiber scaffolds, neural stem cells, and controlled delivery of instructive cues could lead to the development of a better strategy for peripheral nerve injury repair. PMID:24161168

  2. Retinoic acid-induced differentiation of retrovirus-infected HL-60 cells is associated with enhanced transcription from the viral long terminal repeat.

    Collins, S J

    1988-01-01

    I infected different human leukemic cell lines with an amphotropic retrovirus vector (designated PA317/N2) which confers G418 resistance and contains the Moloney murine leukemia virus long terminal repeat. In retrovirus-infected G418-resistant HL-60 cells, induction of granulocyte differentiation by retinoic acid was invariably accompanied by a marked increase (5- to 10-fold) in the transcriptional activity of the integrated retroviral long terminal repeat.

  3. Differential Neuronal Plasticity of Dental Pulp Stem Cells From Exfoliated Deciduous and Permanent Teeth Towards Dopaminergic Neurons.

    Majumdar, Debanjana; Kanafi, Mohammad; Bhonde, Ramesh; Gupta, Pawan; Datta, Indrani

    2016-09-01

    Based on early occurrence in chronological age, stem-cells from human exfoliated deciduous teeth (SHED) has been reported to possess better differentiation-potential toward certain cell-lineage in comparison to stem-cells from adult teeth (DPSCs). Whether this same property between them extends for the yield of functional central nervous system neurons is still not evaluated. Hence, we aim to assess the neuronal plasticity of SHED in comparison to DPSCs toward dopaminergic-neurons and further, if the difference is reflected in a differential expression of sonic-hedgehog (SHH)-receptors and basal-expressions of tyrosine-hydroxylase [TH; through cAMP levels]. Human SHED and DPSCs were exposed to midbrain-cues [SHH, fibroblast growth-factor8, and basic fibroblast growth-factor], and their molecular, immunophenotypical, and functional characterization was performed at different time-points of induction. Though SHED and DPSCs spontaneously expressed early-neuronal and neural-crest marker in their naïve state, only SHED expressed a high basal-expression of TH. The upregulation of dopaminergic transcription-factors Nurr1, Engrailed1, and Pitx3 was more pronounced in DPSCs. The yield of TH-expressing cells decreased from 49.8% to 32.16% in SHED while it increased from 8.09% to 77.47% in DPSCs. Dopamine release and intracellular-Ca(2+) influx upon stimulation (KCl and ATP) was higher in induced DPSCs. Significantly lower-expression of SHH-receptors was noted in naïve SHED than DPSCs, which may explain the differential neuronal plasticity. In addition, unlike DPSCs, SHED showed a down-regulation of cyclic adenosine-monophosphate (cAMP) upon exposure to SHH; possibly another contributor to the lesser differentiation-potential. Our data clearly demonstrates for the first time that DPSCs possess superior neuronal plasticity toward dopaminergic-neurons than SHED; influenced by higher SHH-receptor and lower basal TH expression. J. Cell. Physiol. 231: 2048-2063, 2016. © 2016

  4. Suppression of KV7/KCNQ potassium channel enhances neuronal differentiation of PC12 cells.

    Zhou, Najing; Huang, Sha; Li, Li; Huang, Dongyang; Yan, Yunli; Du, Xiaona; Zhang, Hailin

    2016-10-01

    Membrane potential shift driven by electrical activity is critical in determining the cell fate of proliferation or differentiation. As such, the ion channels that underlie the membrane electrical activity play an important role in cell proliferation/differentiation. KV7/KCNQ potassium channels are critical in determining the resting membrane potentials in many neuronal cells. However, the role of these channels in cell differentiation is not well studied. In the present study, we used PC12 cells as well as primary cultured rat cortical neurons to study the role and mechanism of KV7/KCNQ in neuronal differentiation. NGF induced PC12 cell differentiation into neuron-like cells with growth of neurites showing typical growth cone-like extensions. The Kv7/KCNQ blocker XE991 promoted NGF-induced neurite outgrowth, whereas Kv7/KCNQ opener retigabine (RTG) inhibited outgrowth. M-type Kv7 channels are likely involved in regulating neurite growth because overexpression of KCNQ2/Q3 inhibited neurite growth whereas suppression of KCNQ2/Q3 with shRNA promoted neurite growth. Membrane depolarization possibly underpins enhanced neurite growth induced by the suppression of Kv7/KCNQ. Additionally, high extracellular K(+) likely induced membrane depolarization and also promoted neurite growth. Finally, T-type Ca(2+) channels may be involved in membrane-depolarization-induced neurite growth. This study provides a new perspective for understanding neuronal differentiation as well as KV7/KCNQ channel function. PMID:27450567

  5. Comparative Neuronal Differentiation of Self-Renewing Neural Progenitor Cell Lines Obtained from Human Induced Pluripotent Stem Cells

    Chiara Verpelli

    2013-10-01

    Full Text Available Most human neuronal disorders are associated with genetic alterations that cause defects in neuronal development and induce precocious neurodegeneration. In order to fully characterize the molecular mechanisms underlying the onset of these devastating diseases, it is important to establish in vitro models able to recapitulate the human pathology as closely as possible. Here we compared three different differentiation protocols for obtaining functional neurons from human induced pluripotent stem cells (hiPSCs: human neural progenitors (hNPs obtained from hiPSCs were differentiated by co-culturing them with rat primary neurons, glial cells or simply by culturing them on matrigel in neuronal differentiation medium, and the differentiation level was compared using immunofluorescence, biochemical and electrophysiological methods.We show that the differentiated neurons displayed distinct maturation properties depending on the protocol used and the faster morphological and functional maturation was obtained when hNPs were co-cultured with rat primary neurons.

  6. Differential glucocorticoid-induced closure of the blood-milk barrier during lipopolysaccharide- and lipoteichoic acid-induced mastitis in dairy cows.

    Wall, Samantha K; Hernández-Castellano, Lorenzo E; Ahmadpour, Amir; Bruckmaier, Rupert M; Wellnitz, Olga

    2016-09-01

    Bacteria invading the mammary gland can cause pathogen-dependent differences in the permeability of the blood-milk barrier leading to the differential paracellular transfer of blood and milk components. Glucocorticoids such as prednisolone (PRED) are known to increase the integrity of the blood-milk barrier and quickly restore the decreased milk quality associated with mastitis. The objective of this study was to examine the effect of intramammary PRED on the differential permeability of the blood-milk barrier during mastitis induced by lipopolysaccharide (LPS) from Escherichia coli or lipoteichoic acid (LTA) from Staphylococcus aureus. Thirty-one dairy cows, divided into 6 groups, were injected via a teat canal with LPS, LTA, LPS and PRED, LTA and PRED, saline (control), or PRED. Milk and blood samples were collected 0 to 8h after challenge and analyzed for somatic cell count, IgG, serum albumin, and lactate dehydrogenase in milk, or α-lactalbumin in plasma. Somatic cell count was similarly elevated in LPS- and LTA-challenged quarters and was reduced to control quarter levels only in LTA-challenged quarters with PRED administration. Lactate dehydrogenase activity was highly elevated in LPS quarters and only slightly elevated in LTA quarters, but decreased to control quarter levels with PRED administration. For serum albumin and IgG, only LPS quarters showed an elevation in concentration and PRED treatment reduced the concentration to control quarter level. We found no differences in α-lactalbumin concentrations in plasma in PRED-treated cows compared with cows that only received LPS or LTA. In conclusion, the pathogen-specific appearance of blood constituents in milk during mastitis demonstrates a differential activation of the blood-milk barrier that, in turn, can be manipulated by intramammary glucocorticoids. The results show that the administration of PRED during mastitis increases the blood-milk barrier integrity but has implications in reducing the

  7. Sexual differentiation of the brain requires perinatal kisspeptin-GnRH neuron signaling.

    Clarkson, Jenny; Busby, Ellen R; Kirilov, Milen; Schütz, Günther; Sherwood, Nancy M; Herbison, Allan E

    2014-11-12

    Sex differences in brain function underlie robust differences between males and females in both normal and disease states. Although alternative mechanisms exist, sexual differentiation of the male mammalian brain is initiated predominantly by testosterone secreted by the testes during the perinatal period. Despite considerable advances in understanding how testosterone and its metabolite estradiol sexually differentiate the brain, little is known about the mechanism that generates the male-specific perinatal testosterone surge. In mice, we show that a male-specific activation of GnRH neurons occurs 0-2 h following birth and that this correlates with the male-specific surge of testosterone occurring up to 5 h after birth. The necessity of GnRH signaling for the sexually differentiating effects of the perinatal testosterone surge was demonstrated by the persistence of female-like brain characteristics in adult male, GnRH receptor knock-out mice. Kisspeptin neurons have recently been identified to be potent, direct activators of GnRH neurons. We demonstrate that a population of kisspeptin neurons appears in the preoptic area of only the male between E19 and P1. The importance of kisspeptin inputs to GnRH neurons for the process of sexual differentiation was demonstrated by the lack of a normal neonatal testosterone surge, and disordered brain sexual differentiation of male mice in which the kisspeptin receptor was deleted selectively from GnRH neurons. These observations demonstrate the necessity of perinatal GnRH signaling for driving brain sexual differentiation and indicate that kisspeptin inputs to GnRH neurons are essential for this process to occur. PMID:25392497

  8. Morphine Regulates Dopaminergic Neuron Differentiation via miR-133b

    Sanchez-Simon, Fatima Macho; Zhang, Xiao Xiao; Loh, Horace H.; Law, Ping-Yee; Rodriguez, Raquel E.

    2010-01-01

    Morphine is one of the analgesics used most to treat chronic pain, although its long-term administration produces tolerance and dependence through neuronal plasticity. The ability of morphine to regulate neuron differentiation in vivo has been reported. However, the detailed mechanisms have not yet been elucidated because of the inability to separate maternal influences from embryonic events. Using zebrafish embryos as the model, we demonstrate that morphine decreases miR-133b expression, hen...

  9. Role of lipid rafts in neuronal differentiation of dental pulp-derived stem cells.

    Mattei, Vincenzo; Santacroce, Costantino; Tasciotti, Vincenzo; Martellucci, Stefano; Santilli, Francesca; Manganelli, Valeria; Piccoli, Luca; Misasi, Roberta; Sorice, Maurizio; Garofalo, Tina

    2015-12-10

    Human dental pulp-derived stem cells (hDPSCs) are characterized by a typical fibroblast-like morphology. They express specific markers for mesenchymal stem cells and are capable of differentiation into osteoblasts, adipoblasts and neurons in vitro. Previous studies showed that gangliosides are involved in the induction of early neuronal differentiation of hDPSCs. This study was undertaken to investigate the role of lipid rafts in this process. Lipid rafts are signaling microdomains enriched in glycosphingolipids, cholesterol, tyrosine kinase receptors, mono- or heterotrimeric G proteins and GPI-anchored proteins. We preliminary showed that established cells expressed multipotent mesenchymal stromal-specific surface antigens. Then, we analyzed the distribution of lipid rafts, revealing plasma membrane microdomains with GM2 and EGF-R enrichment. Following stimulation with EGF/bFGF, neuronal differentiation was observed. To analyze the functional role of lipid rafts in EGF/bFGF-induced hDPSCs differentiation, cells were preincubated with lipid raft affecting agents, i.e. [D]-PDMP or methyl-β-cyclodextrin. These compounds significantly prevented neuronal-specific antigen expression, as well as Akt and ERK 1/2 phosphorylation, induced by EGF/bFGF, indicating that lipid raft integrity is essential for EGF/bFGF-induced hDPSCs differentiation. These results suggest that lipid rafts may represent specific chambers, where multimolecular signaling complexes, including lipids (gangliosides, cholesterol) and proteins (EGF-R), play a role in hDPSCs differentiation. PMID:26586565

  10. NGF-mediated transcriptional targets of p53 in PC12 neuronal differentiation

    Labhart Paul

    2007-05-01

    Full Text Available Abstract Background p53 is recognized as a critical regulator of the cell cycle and apoptosis. Mounting evidence also suggests a role for p53 in differentiation of cells including neuronal precursors. We studied the transcriptional role of p53 during nerve growth factor-induced differentiation of the PC12 line into neuron-like cells. We hypothesized that p53 contributed to PC12 differentiation through the regulation of gene targets distinct from its known transcriptional targets for apoptosis or DNA repair. Results Using a genome-wide chromatin immunoprecipitation cloning technique, we identified and validated 14 novel p53-regulated genes following NGF treatment. The data show p53 protein was transcriptionally activated and contributed to NGF-mediated neurite outgrowth during differentiation of PC12 cells. Furthermore, we describe stimulus-specific regulation of a subset of these target genes by p53. The most salient differentiation-relevant target genes included wnt7b involved in dendritic extension and the tfcp2l4/grhl3 grainyhead homolog implicated in ectodermal development. Additional targets included brk, sdk2, sesn3, txnl2, dusp5, pon3, lect1, pkcbpb15 and other genes. Conclusion Within the PC12 neuronal context, putative p53-occupied genomic loci spanned the entire Rattus norvegicus genome upon NGF treatment. We conclude that receptor-mediated p53 transcriptional activity is involved in PC12 differentiation and may suggest a contributory role for p53 in neuronal development.

  11. Neuron-NG2 Cell Synapses: Novel Functions for Regulating NG2 Cell Proliferation and Differentiation

    Qian-Kun Yang

    2013-01-01

    Full Text Available NG2 cells are a population of CNS cells that are distinct from neurons, mature oligodendrocytes, astrocytes, and microglia. These cells can be identified by their NG2 proteoglycan expression. NG2 cells have a highly branched morphology, with abundant processes radiating from the cell body, and express a complex set of voltage-gated channels, AMPA/kainate, and GABA receptors. Neurons notably form classical and nonclassical synapses with NG2 cells, which have varied characteristics and functions. Neuron-NG2 cell synapses could fine-tune NG2 cell activities, including the NG2 cell cycle, differentiation, migration, and myelination, and may be a novel potential therapeutic target for NG2 cell-related diseases, such as hypoxia-ischemia injury and periventricular leukomalacia. Furthermore, neuron-NG2 cell synapses may be correlated with the plasticity of CNS in adulthood with the synaptic contacts passing onto their progenies during proliferation, and synaptic contacts decrease rapidly upon NG2 cell differentiation. In this review, we highlight the characteristics of classical and nonclassical neuron-NG2 cell synapses, the potential functions, and the fate of synaptic contacts during proliferation and differentiation, with the emphasis on the regulation of the NG2 cell cycle by neuron-NG2 cell synapses and their potential underlying mechanisms.

  12. Efficient Differentiation of Embryonic Stem Cells into Neurons in Glial Cell-conditioned Medium under Attaching Conditions

    Hai-Bin TIAN; Zeng-Liang BAI; Hong WANG; Jian-Quan CHEN; Guo-Xiang CHENG

    2005-01-01

    Embryonic stem (ES) cells can differentiate into neurons in vitro, which provides hope for the treatment of some neurodegenerative diseases through cell transplantation. However, it remains a challenge to efficiently induce ES cells to differentiate into neurons. Here, we show that murine ES cells can efficiently differentiate into neurons when cultured in glial cell- conditioned medium (GCM) under attaching conditions without the formation of embryoid bodies. In comparison with murine embryonic fibroblast-conditioned medium, we found that GCM has a positive effect on limiting the generation of non-neuronal cells, such as astrocytes. In addition, compared with suspension conditions, attaching conditions delay the differentiation process of ES cells.

  13. Differentiation of neurons from neural precursors generated in floating spheres from embryonic stem cells

    Forrester Jeff

    2009-09-01

    Full Text Available Abstract Background Neural differentiation of embryonic stem (ES cells is usually achieved by induction of ectoderm in embryoid bodies followed by the enrichment of neuronal progenitors using a variety of factors. Obtaining reproducible percentages of neural cells is difficult and the methods are time consuming. Results Neural progenitors were produced from murine ES cells by a combination of nonadherent conditions and serum starvation. Conversion to neural progenitors was accompanied by downregulation of Oct4 and NANOG and increased expression of nestin. ES cells containing a GFP gene under the control of the Sox1 regulatory regions became fluorescent upon differentiation to neural progenitors, and ES cells with a tau-GFP fusion protein became fluorescent upon further differentiation to neurons. Neurons produced from these cells upregulated mature neuronal markers, or differentiated to glial and oligodendrocyte fates. The neurons gave rise to action potentials that could be recorded after application of fixed currents. Conclusion Neural progenitors were produced from murine ES cells by a novel method that induced neuroectoderm cells by a combination of nonadherent conditions and serum starvation, in contrast to the embryoid body method in which neuroectoderm cells must be selected after formation of all three germ layers.

  14. Differentiation of Wharton's jelly mesenchymal stem cells into neurons in alginate scaffold

    Hosseini, Seyed Mojtaba; Vasaghi, Attiyeh; Nakhlparvar, Newsha; Roshanravan, Reza; Talaei-khozani, Tahereh; Razi, Zahra

    2015-01-01

    Alginate scaffold has been considered as an appropriate biomaterial for promoting the differentiation of embryonic stem cells toward neuronal cell lineage. We hypothesized that alginate scaffold is suitable for culturing Wharton's jelly mesenchymal stem cells (WJMSCs) and can promote the differentiation of WJMSCs into neuron-like cells. In this study, we cultured WJMSCs in a three-dimensional scaffold fabricated by 0.25% alginate and 50 mM CaCl2 in the presence of neurogenic medium containing 10 μM retinoic acid and 20 ng/mL basic fibroblast growth factor. These cells were also cultured in conventional two-dimensional culture condition in the presence of neurogenic medium as controls. After 10 days, immunofluorescence staining was performed for detecting β-tubulin (marker for WJMSCs-differentiated neuron) and CD271 (motor neuron marker). β-Tubulin and CD271 expression levels were significantly greater in the WJMSCs cultured in the three-dimensional alginate scaffold than in the conventional two-dimensional culture condition. These findings suggest that three-dimensional alginate scaffold cell culture system can induce neuronal differentiation of WJMSCs effectively. PMID:26487861

  15. Differentiation of Spiral Ganglion-Derived Neural Stem Cells into Functional Synaptogenetic Neurons.

    Li, Xiaoyang; Aleardi, Alicia; Wang, Jue; Zhou, Yang; Andrade, Rodrigo; Hu, Zhengqing

    2016-05-15

    Spiral ganglion neurons (SGNs) are usually damaged in sensorineural hearing loss. SGN-derived neural stem cells (NSCs) have been identified and proposed to differentiate into neurons to replace damaged SGNs. However, it remains obscure whether SGN-NSC-derived neurons (ScNs) are electrophysiologically functional and possess the capability to form neural connections. Here, we found that SGN-derived cells demonstrated NSC characteristics and differentiated into SGN-like glutamatergic neurons. Neurotrophins significantly increased neuronal differentiation and neurite length of ScNs. Patch clamp recording revealed that ScNs possessed SGN-like NaV and HCN channels, suggesting electrophysiological function. FM1-43 staining and synaptic protein immunofluorescence showed ScNs possess the ability to form neural connections. Astrocyte-conditioned medium was able to stimulate ScNs to express synaptic proteins. These data suggested that neurotrophins are able to stimulate postnatal SGN-NSCs to differentiate into functional glutamatergic ScNs with the capability to form synaptic connections in vitro. PMID:27021700

  16. Alternative Splicing of Neuronal Differentiation Factor TRF2 Regulated by HNRNPH1/H2

    Ioannis Grammatikakis

    2016-05-01

    Full Text Available During neuronal differentiation, use of an alternative splice site on the rat telomere repeat-binding factor 2 (TRF2 mRNA generates a short TRF2 protein isoform (TRF2-S capable of derepressing neuronal genes. However, the RNA-binding proteins (RBPs controlling this splicing event are unknown. Here, using affinity pull-down analysis, we identified heterogeneous nuclear ribonucleoproteins H1 and H2(HNRNPH as RBPs specifically capable of interacting with the spliced RNA segment (exon 7 of Trf2 pre-mRNA. HNRNPH proteins prevent the production of the short isoform of Trf2 mRNA, as HNRNPH silencing selectively elevates TRF2-S levels. Accordingly, HNRNPH levels decline while TRF2-S levels increase during neuronal differentiation. In addition, CRISPR/Cas9-mediated deletion of hnRNPH2 selectively accelerates the NGF-triggered differentiation of rat pheochromocytoma cells into neurons. In sum, HNRNPH is a splicing regulator of Trf2 pre-mRNA that prevents the expression of TRF2-S, a factor implicated in neuronal differentiation.

  17. Alternative Splicing of Neuronal Differentiation Factor TRF2 Regulated by HNRNPH1/H2.

    Grammatikakis, Ioannis; Zhang, Peisu; Panda, Amaresh C; Kim, Jiyoung; Maudsley, Stuart; Abdelmohsen, Kotb; Yang, Xiaoling; Martindale, Jennifer L; Motiño, Omar; Hutchison, Emmette R; Mattson, Mark P; Gorospe, Myriam

    2016-05-01

    During neuronal differentiation, use of an alternative splice site on the rat telomere repeat-binding factor 2 (TRF2) mRNA generates a short TRF2 protein isoform (TRF2-S) capable of derepressing neuronal genes. However, the RNA-binding proteins (RBPs) controlling this splicing event are unknown. Here, using affinity pull-down analysis, we identified heterogeneous nuclear ribonucleoproteins H1 and H2(HNRNPH) as RBPs specifically capable of interacting with the spliced RNA segment (exon 7) of Trf2 pre-mRNA. HNRNPH proteins prevent the production of the short isoform of Trf2 mRNA, as HNRNPH silencing selectively elevates TRF2-S levels. Accordingly, HNRNPH levels decline while TRF2-S levels increase during neuronal differentiation. In addition, CRISPR/Cas9-mediated deletion of hnRNPH2 selectively accelerates the NGF-triggered differentiation of rat pheochromocytoma cells into neurons. In sum, HNRNPH is a splicing regulator of Trf2 pre-mRNA that prevents the expression of TRF2-S, a factor implicated in neuronal differentiation. PMID:27117401

  18. Slug, mammalian homologue gene of Drosophila escargot, promotes neuronal-differentiation through suppression of HEB/daughterless.

    Yang, Dong-Jin; Chung, Ji-Yun; Lee, Su-Jin; Park, So-Young; Pyo, Jung-Hoon; Ha, Nam-Chul; Yoo, Mi-Ae; Park, Bum-Joon

    2010-07-15

    At the neuron developmental stage, neuron-precursor cells can be differentiated into neuron or glia cells. However, precise molecular mechanism to determine the cell fate has not been clearly demonstrated. In this study, we reveal that Drosophila esgarcot and its mammalian homologue genes, Snail and Slug, play a key role in neuronal differentiation. In Drosophila model system, overexpression of Esg, like as Wingless, suppresses the bristle formation. In contrast, elimination of Esg though RNAi promotes double bristle phenotype. We can also observe the similar phenotype in Snail-overexpression system. In mammalian system, overexpression of Slug or Snail can induce neuronal differentiation. Esg and its mammalian homologue gene Slug directly interact with Daughtherless and its mammalian homologue HEB and eliminate them through siah-1 mediated protein degradation. Thus, overexpression of siah-1 can promote neuron cell differentiation, whereas si-siah-1 blocks the Slug-induced HEB suppression. In fact, Drosophila SINA, Siah-1 homologue, has been also known to be involved in bristle formation and Neuronal differentiation. In addition, it has been revealed that CK1 is involved in Esg or Snail stability and Neuronal differentiation. However, Snail is regulated only by CK1 but not by Siah. Considering the fact that Slug mutations have been found in human genetic disease, waardenberg syndrome, major symptoms of which is loss of hearing neuron and odd eye, our result implies that slug/Snail system is required for proper neuronal differentiation, like as Esg in Drosophila. PMID:20647756

  19. The role of Tal2 and Tal1 in the differentiation of midbrain GABAergic neuron precursors

    Kaia Achim

    2013-08-01

    Midbrain- and hindbrain-derived GABAergic interneurons are critical for regulation of sleep, respiratory, sensory-motor and motivational processes, and they are implicated in human neurological disorders. However, the precise mechanisms that underlie generation of GABAergic neuron diversity in the midbrain–hindbrain region are poorly understood. Here, we show unique and overlapping requirements for the related bHLH proteins Tal1 and Tal2 in GABAergic neurogenesis in the midbrain. We show that Tal2 and Tal1 are specifically and sequentially activated during midbrain GABAergic neurogenesis. Similar to Gata2, a post-mitotic selector of the midbrain GABAergic neuron identity, Tal2 expression is activated very early during GABAergic neuron differentiation. Although the expression of Tal2 and Gata2 genes are independent of each other, Tal2 is important for normal midbrain GABAergic neurogenesis, possibly as a partner of Gata2. In the absence of Tal2, the majority of midbrain GABAergic neurons switch to a glutamatergic-like phenotype. In contrast, Tal1 expression is activated in a Gata2 and Tal2 dependent fashion in the more mature midbrain GABAergic neuron precursors, but Tal1 alone is not required for GABAergic neuron differentiation from the midbrain neuroepithelium. However, inactivation of both Tal2 and Tal1 in the developing midbrain suggests that the two factors co-operate to guide GABAergic neuron differentiation in a specific ventro-lateral midbrain domain. The observed similarities and differences between Tal1/Tal2 and Gata2 mutants suggest both co-operative and unique roles for these factors in determination of midbrain GABAergic neuron identities.

  20. Role of neurotrophin signalling in the differentiation of neurons from dorsal root ganglia and sympathetic ganglia.

    Ernsberger, Uwe

    2009-06-01

    Manipulation of neurotrophin (NT) signalling by administration or depletion of NTs, by transgenic overexpression or by deletion of genes coding for NTs and their receptors has demonstrated the importance of NT signalling for the survival and differentiation of neurons in sympathetic and dorsal root ganglia (DRG). Combination with mutation of the proapoptotic Bax gene allows the separation of survival and differentiation effects. These studies together with cell culture analysis suggest that NT signalling directly regulates the differentiation of neuron subpopulations and their integration into neural networks. The high-affinity NT receptors trkA, trkB and trkC are restricted to subpopulations of mature neurons, whereas their expression at early developmental stages largely overlaps. trkC is expressed throughout sympathetic ganglia and DRG early after ganglion formation but becomes restricted to small neuron subpopulations during embryogenesis when trkA is turned on. The temporal relationship between trkA and trkC expression is conserved between sympathetic ganglia and DRG. In DRG, NGF signalling is required not only for survival, but also for the differentiation of nociceptors. Expression of neuropeptides calcitonin gene-related peptide and substance P, which specify peptidergic nociceptors, depends on nerve growth factor (NGF) signalling. ret expression indicative of non-peptidergic nociceptors is also promoted by the NGF-signalling pathway. Regulation of TRP channels by NGF signalling might specify the temperature sensitivity of afferent neurons embryonically. The manipulation of NGF levels "tunes" heat sensitivity in nociceptors at postnatal and adult stages. Brain-derived neurotrophic factor signalling is required for subpopulations of DRG neurons that are not fully characterized; it affects mechanical sensitivity in slowly adapting, low-threshold mechanoreceptors and might involve the regulation of DEG/ENaC ion channels. NT3 signalling is required for the

  1. Transcription factor activating protein 2 beta (TFAP2B) mediates noradrenergic neuronal differentiation in neuroblastoma.

    Ikram, Fakhera; Ackermann, Sandra; Kahlert, Yvonne; Volland, Ruth; Roels, Frederik; Engesser, Anne; Hertwig, Falk; Kocak, Hayriye; Hero, Barbara; Dreidax, Daniel; Henrich, Kai-Oliver; Berthold, Frank; Nürnberg, Peter; Westermann, Frank; Fischer, Matthias

    2016-02-01

    Neuroblastoma is an embryonal pediatric tumor that originates from the developing sympathetic nervous system and shows a broad range of clinical behavior, ranging from fatal progression to differentiation into benign ganglioneuroma. In experimental neuroblastoma systems, retinoic acid (RA) effectively induces neuronal differentiation, and RA treatment has been therefore integrated in current therapies. However, the molecular mechanisms underlying differentiation are still poorly understood. We here investigated the role of transcription factor activating protein 2 beta (TFAP2B), a key factor in sympathetic nervous system development, in neuroblastoma pathogenesis and differentiation. Microarray analyses of primary neuroblastomas (n = 649) demonstrated that low TFAP2B expression was significantly associated with unfavorable prognostic markers as well as adverse patient outcome. We also found that low TFAP2B expression was strongly associated with CpG methylation of the TFAP2B locus in primary neuroblastomas (n = 105) and demethylation with 5-aza-2'-deoxycytidine resulted in induction of TFAP2B expression in vitro, suggesting that TFAP2B is silenced by genomic methylation. Tetracycline inducible re-expression of TFAP2B in IMR-32 and SH-EP neuroblastoma cells significantly impaired proliferation and cell cycle progression. In IMR-32 cells, TFAP2B induced neuronal differentiation, which was accompanied by up-regulation of the catecholamine biosynthesizing enzyme genes DBH and TH, and down-regulation of MYCN and REST, a master repressor of neuronal genes. By contrast, knockdown of TFAP2B by lentiviral transduction of shRNAs abrogated RA-induced neuronal differentiation of SH-SY5Y and SK-N-BE(2)c neuroblastoma cells almost completely. Taken together, our results suggest that TFAP2B is playing a vital role in retaining RA responsiveness and mediating noradrenergic neuronal differentiation in neuroblastoma. PMID:26598443

  2. Effect of matrix composition on differentiation of nestin-positive neural progenitors from circulation into neurons

    Jose, Anumol; Krishnan, Lissy K.

    2010-06-01

    The human peripheral blood mononuclear cell has a mixture of progenitor cells with potential to differentiate into a wide range of lineages. The ability of hematopoietic tissue-derived adult stem cells to differentiate into neural progenitor cells offers an alternative to embryonic stem cells as a viable source for cell transplantation therapies to cure neurodegenerative diseases. This approach could lead to the use of autologous progenitors from blood circulation; however, due to the limited numbers available, in vitro cell expansion may be indispensable. In addition, for successful transplantation there is the requirement of a delivery matrix on which cells can survive and differentiate. In this context we carried out this study to identify a suitable biodegradable matrix on which progenitor cells can home, multiply and differentiate. We designed different compositions of the biomimetic matrix containing fibrin, fibronectin, gelatin, growth factors, laminin and hyaluronic acid. The attached cells expressed proliferation markers in initial periods of culture and between days 6 and 9 in culture they differentiated into neurons and/or astrocytes. The differentiation of progenitors into neurons and asterocyte on the composed matrix was established by morphological and immunochemical analysis. Flow cytometric analysis of cells in culture was employed to track development of neurons which expressed an early marker β-tubulin3 and a terminal marker microtubule-associated protein-2 at a later culture period. In vitro experiments indicate that a highly specific niche consisting of various components of the extracellular matrix, including hyaluronic acid, promote cell homing, survival and differentiation.

  3. Differentiation of neuron-like cells from mouse parthenogenetic embryonic stem cells

    Xingrong Yan; Liwen Li; Fulin Chen; Yanhong Yang; Wei Liu; Wenxin Geng; Huichong Du; Jihong Cui; Xin Xie; Jinlian Hua; Shumin Yu

    2013-01-01

    Parthenogenetic embryonic stem cells have pluripotent differentiation potentials, akin to fertilized embryo-derived embryonic stem cells. The aim of this study was to compare the neuronal differentiation potential of parthenogenetic and fertilized embryo-derived embryonic stem cells. Before differentiation, karyotype analysis was performed, with normal karyotypes detected in both parthenogenetic and fertilized embryo-derived embryonic stem cells. Sex chromosomes were identified as XX. Immunocytochemistry and quantitative real-time PCR detected high expression of the pluripotent gene, Oct4, at both the mRNA and protein levels, indicating pluripotent differentiation potential of the two embryonic stem cell subtypes. Embryonic stem cells were induced with retinoic acid to form embryoid bodies, and then dispersed into single cells. Single cells were differentiated in N2 differentiation medium for 9 days. Immunocytochemistry showed parthenogenetic and fertilized embryo-derived embryonic stem cells both express the neuronal cell markers nestin, βIII-tubulin and myelin basic protein. Quantitative real-time PCR found expression of neurogenesis related genes (Sox-1, Nestin, GABA, Pax6, Zic5 and Pitx1) in both types of embryonic stem cells, and Oct4 expression was significantly decreased. Nestin and Pax6 expression in parthenogenetic embryonic stem cells was significantly higher than that in fertilized embryo-derived embryonic stem cells. Thus, our experimental findings indicate that parthenogenetic embryonic stem cells have stronger neuronal differentiation potential than fertilized embryo-derived embryonic stem cells.

  4. STAT3 modulation to enhance motor neuron differentiation in human neural stem cells.

    Rajalaxmi Natarajan

    Full Text Available Spinal cord injury or amyotrophic lateral sclerosis damages spinal motor neurons and forms a glial scar, which prevents neural regeneration. Signal transducer and activator of transcription 3 (STAT3 plays a critical role in astrogliogenesis and scar formation, and thus a fine modulation of STAT3 signaling may help to control the excessive gliogenic environment and enhance neural repair. The objective of this study was to determine the effect of STAT3 inhibition on human neural stem cells (hNSCs. In vitro hNSCs primed with fibroblast growth factor 2 (FGF2 exhibited a lower level of phosphorylated STAT3 than cells primed by epidermal growth factor (EGF, which correlated with a higher number of motor neurons differentiated from FGF2-primed hNSCs. Treatment with STAT3 inhibitors, Stattic and Niclosamide, enhanced motor neuron differentiation only in FGF2-primed hNSCs, as shown by increased homeobox gene Hb9 mRNA levels as well as HB9+ and microtubule-associated protein 2 (MAP2+ co-labeled cells. The increased motor neuron differentiation was accompanied by a decrease in the number of glial fibrillary acidic protein (GFAP-positive astrocytes. Interestingly, Stattic and Niclosamide did not affect the level of STAT3 phosphorylation; rather, they perturbed the nuclear translocation of phosphorylated STAT3. In summary, we demonstrate that FGF2 is required for motor neuron differentiation from hNSCs and that inhibition of STAT3 further increases motor neuron differentiation at the expense of astrogliogenesis. Our study thus suggests a potential benefit of targeting the STAT3 pathway for neurotrauma or neurodegenerative diseases.

  5. Quantitative glycomics monitoring of induced pluripotent- and embryonic stem cells during neuronal differentiation

    Michiyo Terashima

    2014-11-01

    Full Text Available Alterations in the structure of cell surface glycoforms occurring during the stages of stem cell differentiation remain unclear. We describe a rapid glycoblotting-based cellular glycomics method for quantitatively evaluating changes in glycoform expression and structure during neuronal differentiation of murine induced pluripotent stem cells (iPSCs and embryonic stem cells (ESCs. Our results show that changes in the expression of cellular N-glycans are comparable during the differentiation of iPSCs and ESCs. The expression of bisect-type N-glycans was significantly up-regulated in neurons that differentiated from both iPSCs and ESCs. From a glycobiological standpoint, iPSCs are an alternative neural cell source in addition to ESCs.

  6. D609 induces vascular endothelial cells and marrow stromal cells differentiation into neuron-like cells

    Nan WANG; Chun-qing DU; Shao-shan WANG; Kun XIE; Shang-li ZHANG; Jun-ying MIAO

    2004-01-01

    AIM: To investigate the effect of tricyclodecane-9-yl-xanthogenate (D609) on cell differentiation in vascular endothelial cells (VECs) and marrow stromal cells (MSCs). METHODS: Morphological changes were observed under phase contrast microscope. Electron microscope and immunostaining were used for VECs identification. The expressions of neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) were examined by immunohistochemistry. RESULTS: After 6 h of induction with D609, some VECs showed morphological changes characteristic of neurones. 9 h later, more VECs became neuron-like cells. About 30.8 % of VECs displayed positive NSE (P<0.01), while the expression of GFAP was negative. When MSCs were exposed to D609, the cells displayed neuronal morphologies, such as pyramidal cell bodies and processes formed extensive networks at 3 h. 6 h later, almost all of the cells exhibited a typical neuronal appearance, and 85.6 % of MSCs displayed intensive positive NSE, but GFAP did not express. CONCLUSION: D609 induces VECs and MSCs differentiation into neuron-like cells.

  7. Cholinergic neuronal differentiation of bone marrow mesenchymal stem cells in rhesus monkeys

    2010-01-01

    The purpose of the present study was to determine the best cholinergic neuronal differentiation method of rhesus monkey bone marrow mesenchymal stem cells(BMSCs).Four methods were used to induce differentiation,and the groups were assigned accordingly:basal inducing group(culture media,bFGF,and forskolin);SHH inducing group(SHH,inducing group);RA inducing group(RA,basal inducing group);and SHH+RA inducing group(SHH,RA,and basal inducing group).All groups displayed neuronal morphology and increased expression of nestin and neuron-specific enolase.The basal inducing group did not express synapsin,and cells from the SHH inducing group did not exhibit neuronal resting membrane potential.In contrast,results demonstrated that BMSCs from the RA and SHH+RA inducing groups exhibited neuronal resting membrane potential,and cells from the SHH+RA inducing group expressed higher levels of synapsin and acetylcholine.In conclusion,the induction of cholinergic differentiation through SHH+RA was determined to be superior to the other methods.

  8. Co-culture of neuroepithelial stem cells with interstitial cells of Cajal results in neuron differentiation

    Zhao, Bin; Liu, Wei; Wu, Rongde

    2015-01-01

    Objective: The interstitial cells of Cajal (ICCs) interact morphologically and functionally with the elements of the enteric nervous system in the digestive tract. However, direct evidence that ICCs participate in the differentiation of the enteric nervous system is lacking. In this work, we examined in co-culture experiments whether ICCs could stimulate the differentiation of neuroepithelial stem cells (NESCs) to neurons. Methods: NESCs were harvested from the neural tube of embryonic (E11.5...

  9. Stem Cells from Human-Exfoliated Deciduous Teeth Can Differentiate into Dopaminergic Neuron-Like Cells

    Wang, Jinsong; Wang, Xuan; Sun, Zuoli; Wang, Xiaomin; Yang, Hui; Shi, Songtao; Wang, Songlin

    2010-01-01

    Stem cells from human exfoliated deciduous teeth (SHED) have been identified as a novel population of postnatal stem cells capable of differentiating into neural cells, odontogenic cells, and adipocytes. SHED were reported to differentiate into neural cells based on cellular morphology and the expression of early neuronal markers when cultured under neural inductive conditions. This study therefore investigated the therapeutic efficacy of SHED in alleviating Parkinson's disease (PD) in a rat ...

  10. Surface N-glycoproteome patterns reveal key proteins of neuronal differentiation

    Tylečková, Jiřina; Valeková, Ivona; Žižková, Martina; Rákocyová, Michaela; Maršala, S.; Maršala, M.; Gadher, S. J.; Kovářová, Hana

    2016-01-01

    Roč. 132, č. 1 (2016), s. 13-20. ISSN 1874-3919 R&D Projects: GA MŠk ED2.1.00/03.0124; GA TA ČR(CZ) TA01011466 Institutional support: RVO:67985904 Keywords : cell adhesion proteins * cell surface capture * neuronal differentiation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.888, year: 2014

  11. Neuronal glycosylation differentials in normal, injured and chondroitinase-treated environments

    Highlights: ► Carbohydrates are important in the CNS and ChABC has been used for spinal cord injury (SCI) treatment. ► Neuronal glycosylation in injury and after ChABC treatment is unknown. ► In silico mining verified that glyco-related genes were differentially regulated after SCI. ► In vitro model system revealed abnormal sialylation in an injured environment. ► The model indicated a return to normal neuronal glycosylation after ChABC treatment. -- Abstract: Glycosylation is found ubiquitously throughout the central nervous system (CNS). Chondroitin sulphate proteoglycans (CSPGs) are a group of molecules heavily substituted with glycosaminoglycans (GAGs) and are found in the extracellular matrix (ECM) and cell surfaces. Upon CNS injury, a glial scar is formed, which is inhibitory for axon regeneration. Several CSPGs are up-regulated within the glial scar, including NG2, and these CSPGs are key inhibitory molecules of axonal regeneration. Treatment with chondroitinase ABC (ChABC) can neutralise the inhibitory nature of NG2. A gene expression dataset was mined in silico to verify differentially regulated glycosylation-related genes in neurons after spinal cord injury and identify potential targets for further investigation. To establish the glycosylation differential of neurons that grow in a healthy, inhibitory and ChABC-treated environment, we established an indirect co-culture system where PC12 neurons were grown with primary astrocytes, Neu7 astrocytes (which overexpress NG2) and Neu7 astrocytes treated with ChABC. After 1, 4 and 8 days culture, lectin cytochemistry of the neurons was performed using five fluorescently-labelled lectins (ECA MAA, PNA, SNA-I and WFA). Usually α-(2,6)-linked sialylation scarcely occurs in the CNS but this motif was observed on the neurons in the injured environment only at day 8. Treatment with ChABC was successful in returning neuronal glycosylation to normal conditions at all timepoints for MAA, PNA and SNA-I staining

  12. Neuronal glycosylation differentials in normal, injured and chondroitinase-treated environments

    Kilcoyne, Michelle; Sharma, Shashank [Glycoscience Group, National Centre for Biomedical Engineering Science, National University of Ireland, Galway (Ireland); McDevitt, Niamh; O' Leary, Claire [Anatomy, School of Medicine, National University of Ireland, Galway (Ireland); Joshi, Lokesh [Glycoscience Group, National Centre for Biomedical Engineering Science, National University of Ireland, Galway (Ireland); McMahon, Siobhan S., E-mail: siobhan.mcmahon@nuigalway.ie [Anatomy, School of Medicine, National University of Ireland, Galway (Ireland)

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer Carbohydrates are important in the CNS and ChABC has been used for spinal cord injury (SCI) treatment. Black-Right-Pointing-Pointer Neuronal glycosylation in injury and after ChABC treatment is unknown. Black-Right-Pointing-Pointer In silico mining verified that glyco-related genes were differentially regulated after SCI. Black-Right-Pointing-Pointer In vitro model system revealed abnormal sialylation in an injured environment. Black-Right-Pointing-Pointer The model indicated a return to normal neuronal glycosylation after ChABC treatment. -- Abstract: Glycosylation is found ubiquitously throughout the central nervous system (CNS). Chondroitin sulphate proteoglycans (CSPGs) are a group of molecules heavily substituted with glycosaminoglycans (GAGs) and are found in the extracellular matrix (ECM) and cell surfaces. Upon CNS injury, a glial scar is formed, which is inhibitory for axon regeneration. Several CSPGs are up-regulated within the glial scar, including NG2, and these CSPGs are key inhibitory molecules of axonal regeneration. Treatment with chondroitinase ABC (ChABC) can neutralise the inhibitory nature of NG2. A gene expression dataset was mined in silico to verify differentially regulated glycosylation-related genes in neurons after spinal cord injury and identify potential targets for further investigation. To establish the glycosylation differential of neurons that grow in a healthy, inhibitory and ChABC-treated environment, we established an indirect co-culture system where PC12 neurons were grown with primary astrocytes, Neu7 astrocytes (which overexpress NG2) and Neu7 astrocytes treated with ChABC. After 1, 4 and 8 days culture, lectin cytochemistry of the neurons was performed using five fluorescently-labelled lectins (ECA MAA, PNA, SNA-I and WFA). Usually {alpha}-(2,6)-linked sialylation scarcely occurs in the CNS but this motif was observed on the neurons in the injured environment only at day 8. Treatment

  13. Differentiation of embryonic versus adult rat neural stem cells into dopaminergic neurons in vitro

    Chunlong Ke; Baili Chen; Shaolei Guo; Chao Yang

    2008-01-01

    BACKGROUND: It has been reported that the conversion of neural stem cells into dopaminergic neurons in vitro can be increased through specific cytokine combinations. Such neural stem cell-derived dopaminergic neurons could be used for the treatment of Parkinson's disease. However, little is known about the differences in dopaminergic differentiation between neural stem cells derived from adult and embryonic rats.OBJECTIVE: To study the ability of rat adult and embryonic-derived neural stem cells to differentiate into dopaminergic neurons in vitro.DESIGN: Randomized grouping design.SETTING: Department of Neurosurgery in the First Affiliated Hospital of Sun Yat-sen University.MATERIALS: This experiment was performed at the Surgical Laboratory in the First Affiliated Hospital of Sun Yat-scn University (Guangzhou, Guangdong, China) from June to December 2007. Eight, adult, male,Sprague Dawley rats and eight, pregnant, Sprague Dawley rats (embryonic day 14 or 15) were provided by the Experimental Animal Center of Sun Yat-sen University.METHODS: Neural stem cells derived from adult and embryonic rats were respectively cultivated in serum-free culture medium containing epidermal growth factor and basic fibroblast growth factor. After passaging, neural stem cells were differentiated in medium containing interleukin-1 ct, interleukin-11, human leukemia inhibition factor, and glial cell line-derived neurotrophic factor. Six days later, cells were analyzed by immunocytochemistry and flow cytometry.MAIN OUTCOME MEASURES: Alterations in cellular morphology after differentiation of neural stem cells derived from adult and embryonic rats; and percentage of tyrosine hydroxylase-positive neurons in the differentiated cells.RESULTS: Neural stem cells derived from adult and embryonic rats were cultivated in differentiation medium. Six days later, differentiated cells were immunoreactive for tyrosine hydroxylasc. The percentage of tyrosine hydroxylase positive neurons was (5.6 ± 2

  14. ALS/FTLD-linked TDP-43 regulates neurite morphology and cell survival in differentiated neurons

    Han, Jeong-Ho; Yu, Tae-Hoon; Ryu, Hyun-Hee; Jun, Mi-Hee; Ban, Byung-Kwan [Department of Biotechnology, College of Life Science and Nanotechnology, Hannam University, Dajeon 305-811 (Korea, Republic of); Jang, Deok-Jin [Department of Applied Biology, College of Ecology and Environment, Kyungpook National University, 386, Gajang-dong, Sangju-si, Kyungbuk 742-711 (Korea, Republic of); Lee, Jin-A, E-mail: leeja@hnu.kr [Department of Biotechnology, College of Life Science and Nanotechnology, Hannam University, Dajeon 305-811 (Korea, Republic of)

    2013-08-01

    Tar-DNA binding protein of 43 kDa (TDP-43) has been characterized as a major component of protein aggregates in brains with neurodegenerative diseases such as frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). However, physiological roles of TDP-43 and early cellular pathogenic effects caused by disease associated mutations in differentiated neurons are still largely unknown. Here, we investigated the physiological roles of TDP-43 and the effects of missense mutations associated with diseases in differentiated cortical neurons. The reduction of TDP-43 by siRNA increased abnormal neurites and decreased cell viability. ALS/FTLD-associated missense mutant proteins (A315T, Q331K, and M337V) were partially mislocalized to the cytosol and neurites when compared to wild-type and showed abnormal neurites similar to those observed in cases of loss of TDP-43. Interestingly, cytosolic expression of wild-type TDP-43 with mutated nuclear localization signals also induced abnormal neurtie morphology and reduction of cell viability. However, there was no significant difference in the effects of cytosolic expression in neuronal morphology and cell toxicity between wild-type and missense mutant proteins. Thus, our results suggest that mislocalization of missense mutant TDP-43 may contribute to loss of TDP-43 function and affect neuronal morphology, probably via dominant negative action before severe neurodegeneration in differentiated cortical neurons. Highlights: • The function of nuclear TDP-43 in neurite morphology in mature neurons. • Partial mislocalization of TDP-43 missense mutants into cytosol from nucleus. • Abnormal neurite morphology caused by missense mutants of TDP-43. • The effect of cytosolic expression of TDP-43 in neurite morphology and in cell survival.

  15. ALS/FTLD-linked TDP-43 regulates neurite morphology and cell survival in differentiated neurons

    Tar-DNA binding protein of 43 kDa (TDP-43) has been characterized as a major component of protein aggregates in brains with neurodegenerative diseases such as frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). However, physiological roles of TDP-43 and early cellular pathogenic effects caused by disease associated mutations in differentiated neurons are still largely unknown. Here, we investigated the physiological roles of TDP-43 and the effects of missense mutations associated with diseases in differentiated cortical neurons. The reduction of TDP-43 by siRNA increased abnormal neurites and decreased cell viability. ALS/FTLD-associated missense mutant proteins (A315T, Q331K, and M337V) were partially mislocalized to the cytosol and neurites when compared to wild-type and showed abnormal neurites similar to those observed in cases of loss of TDP-43. Interestingly, cytosolic expression of wild-type TDP-43 with mutated nuclear localization signals also induced abnormal neurtie morphology and reduction of cell viability. However, there was no significant difference in the effects of cytosolic expression in neuronal morphology and cell toxicity between wild-type and missense mutant proteins. Thus, our results suggest that mislocalization of missense mutant TDP-43 may contribute to loss of TDP-43 function and affect neuronal morphology, probably via dominant negative action before severe neurodegeneration in differentiated cortical neurons. Highlights: • The function of nuclear TDP-43 in neurite morphology in mature neurons. • Partial mislocalization of TDP-43 missense mutants into cytosol from nucleus. • Abnormal neurite morphology caused by missense mutants of TDP-43. • The effect of cytosolic expression of TDP-43 in neurite morphology and in cell survival

  16. Human neural progenitors differentiate into astrocytes and protect motor neurons in aging rats.

    Das, Melanie M; Avalos, Pablo; Suezaki, Patrick; Godoy, Marlesa; Garcia, Leslie; Chang, Christine D; Vit, Jean-Philippe; Shelley, Brandon; Gowing, Genevieve; Svendsen, Clive N

    2016-06-01

    Age-associated health decline presents a significant challenge to healthcare, although there are few animal models that can be used to test potential treatments. Here, we show that there is a significant reduction in both spinal cord motor neurons and motor function over time in the aging rat. One explanation for this motor neuron loss could be reduced support from surrounding aging astrocytes. Indeed, we have previously shown using in vitro models that aging rat astrocytes are less supportive to rat motor neuron function and survival over time. Here, we test whether rejuvenating the astrocyte niche can improve the survival of motor neurons in an aging spinal cord. We transplanted fetal-derived human neural progenitor cells (hNPCs) into the aging rat spinal cord and found that the cells survive and differentiate into astrocytes with a much higher efficiency than when transplanted into younger animals, suggesting that the aging environment stimulates astrocyte maturation. Importantly, the engrafted astrocytes were able to protect against motor neuron loss associated with aging, although this did not result in an increase in motor function based on behavioral assays. We also transplanted hNPCs genetically modified to secrete glial cell line-derived neurotrophic factor (GDNF) into the aging rat spinal cord, as this combination of cell and protein delivery can protect motor neurons in animal models of ALS. During aging, GDNF-expressing hNPCs protected motor neurons, though to the same extent as hNPCs alone, and again had no effect on motor function. We conclude that hNPCs can survive well in the aging spinal cord, protect motor neurons and mature faster into astrocytes when compared to transplantation into the young spinal cord. While there was no functional improvement, there were no functional deficits either, further supporting a good safety profile of hNPC transplantation even into the older patient population. PMID:27032721

  17. Differential effects of GABAA receptor antagonists in the control of respiratory neuronal discharge patterns.

    Dogas, Z; Krolo, M; Stuth, E A; Tonkovic-Capin, M; Hopp, F A; McCrimmon, D R; Zuperku, E J

    1998-11-01

    To ascertain the role of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in shaping and controlling the phasic discharge patterns of medullary respiratory premotor neurons, localized pressure applications of the competitive GABAA receptor antagonist bicuculline (BIC) and the noncompetitive GABAA receptor antagonist picrotoxin (PIC) were studied. Multibarrel micropipettes were used in halothane anesthetized, paralyzed, ventilated, vagotomized dogs to record single unit activity from inspiratory and expiratory neurons in the caudal ventral respiratory group and to picoeject GABAA receptor antagonists. The moving time average of phrenic nerve activity was used to determine respiratory phase durations and to synchronize cycle-triggered histograms of discharge patterns. Picoejection of BIC and PIC had qualitatively different effects on the discharge patterns of respiratory neurons. BIC caused an increase in the discharge rate during the neuron's active phase without inducing activity during the neuron's normally silent phase. The resulting discharge patterns were amplified replicas (x2-3) of the underlying preejection phasic patterns. In contrast, picoejection of PIC did not increase the peak discharge rate during the neuron's active phase but induced a tonic level of activity during the neuron's normally silent phase. The maximum effective BIC dose (15 +/- 1.8 pmol/min) was considerably smaller than that for PIC (280 +/- 53 pmol/min). These findings suggest that GABAA receptors with differential pharmacology mediate distinct functions within the same neuron, 1) gain modulation that is BIC sensitive but PIC insensitive and 2) silent-phase inhibition blocked by PIC. These studies also suggest that the choice of an antagonist is an important consideration in the determination of GABA receptor function within the respiratory motor control system. PMID:9819249

  18. Regulation of differentiation flux by Notch signalling influences the number of dopaminergic neurons in the adult brain

    Trujillo-Paredes, Niurka; Valencia, Concepción; Guerrero-Flores, Gilda; Arzate, Dulce-María; Baizabal, José-Manuel; Guerra-Crespo, Magdalena; Fuentes-Hernández, Ayari; Zea-Armenta, Iván; Covarrubias, Luis

    2016-01-01

    ABSTRACT Notch signalling is a well-established pathway that regulates neurogenesis. However, little is known about the role of Notch signalling in specific neuronal differentiation. Using Dll1 null mice, we found that Notch signalling has no function in the specification of mesencephalic dopaminergic neural precursor cells (NPCs), but plays an important role in regulating their expansion and differentiation into neurons. Premature neuronal differentiation was observed in mesencephalons of Dll1-deficient mice or after treatment with a Notch signalling inhibitor. Coupling between neurogenesis and dopaminergic differentiation was indicated from the coincident emergence of neuronal and dopaminergic markers. Early in differentiation, decreasing Notch signalling caused a reduction in NPCs and an increase in dopaminergic neurons in association with dynamic changes in the proportion of sequentially-linked dopaminergic NPCs (Msx1/2+, Ngn2+, Nurr1+). These effects in differentiation caused a significant reduction in the number of dopaminergic neurons produced. Accordingly, Dll1 haploinsufficient adult mice, in comparison with their wild-type littermates, have a consistent reduction in neuronal density that was particularly evident in the substantia nigra pars compacta. Our results are in agreement with a mathematical model based on a Dll1-mediated regulatory feedback loop between early progenitors and their dividing precursors that controls the emergence and number of dopaminergic neurons. PMID:26912775

  19. Human dental pulp stem cells express many pluripotency regulators and differentiate into neuronal cells

    Behnam Ebrahimi; Mohammad Mehdi Yaghoobi; Ali Mohammadi Kamal-abadi; Maryam Raoof

    2011-01-01

    Stem cells were isolated from human dental pulp using an optimized method, in which pulp pieces were digested by enzymes and immobilized to enhance cell outgrowth. Stem cell marker expression was detected by reverse transcription-PCR (RT-PCR), and differentiation markers were detected by real-time quantitative RT-PCR and immunocytochemistry. Results showed that dental pulp stem cells actively expressed nanog, oct4, nucleostemin slain-1, jmjd1a, jmjd2c, and cyclin D1. When stem cells were induced to differentiate into neurons, nucleostemin, nanog, and cyclin D1 expres-sion significantly decreased, whereas expression of neuronal markers, such as microtubule asso-ciated protein-2 and neurofilament-heavy, significantly increased. These results suggested that stem cells exited a pluripotent state and entered a neuronal differentiation pathway. In addition, results demonstrated that human dental pulp serves as a reservoir of stem cells that express defined stem cell markers; these cells were easily isolated and were induced to differentiate towards a desired cell lineage.

  20. Choline kinase alpha expression during RA-induced neuronal differentiation: role of C/EBPβ.

    Domizi, Pablo; Aoyama, Chieko; Banchio, Claudia

    2014-04-01

    Neuronal differentiation is a complex process characterized by a halt in proliferation and extension of neurites from the cell body. This process is accompanied by changes in gene expression that mediate the redirection leading to neurite formation and function. Acceleration of membrane phospholipids synthesis is associated with neurite elongation, and phosphatidylcholine (PtdCho) is the major membrane phospholipid in mammalian cells. The transcription of two genes in particular encoding key enzymes in the CDP-choline pathway for PtdCho biosynthesis are stimulated; the Chka gene for choline kinase (CK) alpha isoform and the Pcyt1a gene for the CTP:phosphocholine cytidylyltransferase (CCT) alpha isoform. We report that the stimulation of CKα expression during retinoic acid (RA) induced differentiation depends on a promoter region that contains two CCAAT/Enhancer-binding Protein-β (C/EBPβ) sites. We demonstrate that during neuronal differentiation of Neuro-2a cells, RA induces Chka expression by a mechanism that involves ERK1/2 activation which triggers C/EBPβ expression. Elevated levels of C/EBPβ bind to the Chka proximal promoter (Box1) inducing CKα expression. In addition we identified a downstream sequence named Box2 which together with Box1 is required for the promoter to reach the full induction. This is the first elucidation of the mechanism by which the expression of Chka is coordinately regulated during neuronal differentiation. PMID:24440820

  1. Electrospun polyurethane scaffolds for proliferation and neuronal differentiation of human embryonic stem cells

    Carlberg, Bjoern; Liu, Johan [BioNano Systems Laboratory, Department of Microtechnology and Nanoscience, Chalmers University of Technology, Goeteborg, SE-412 96 (Sweden); Axell, Mathilda Zetterstroem; Kuhn, H Georg [Center for Brain Repair and Rehabilitation, Institute of Neuroscience and Physiology, University of Gothenburg, Goeteborg, SE-413 45 (Sweden); Nannmark, Ulf, E-mail: bjorn.carlberg@chalmers.s, E-mail: mathilda.zetterstrom@neuro.gu.s, E-mail: georg.kuhn@neuro.gu.s, E-mail: ulf.nannmark@anatcell.gu.s, E-mail: jliu@chalmers.s [Department of Medical Chemistry and Cell Biology, Institute of Biomedicine, University of Gothenburg, Goeteborg, SE-405 30 (Sweden)

    2009-08-15

    Adult central nervous system (CNS) tissue has a limited capacity to recover after trauma or disease. Hence, tissue engineering scaffolds intended for CNS repair and rehabilitation have been subject to intense research effort. Electrospun porous scaffolds, mimicking the natural three-dimensional environment of the in vivo extracellular matrix (ECM) and providing physical support, have been identified as promising candidates for CNS tissue engineering. The present study demonstrates in vitro culturing and neuronal differentiation of human embryonic stem cells (hESCs) on electrospun fibrous polyurethane scaffolds. Electrospun scaffolds composed of biocompatible polyurethane resin (Desmopan 9370A, Bayer MaterialScience AG) were prepared with a vertical electrospinning setup. Resulting scaffolds, with a thickness of approximately 150{mu}m, exhibited high porosity (84%) and a bimodal pore size distribution with peaks at 5-6 and 1{mu}m. The mean fiber diameter was measured to approximately 360 nm with a standard deviation of 80 nm. The undifferentiated hESC line SA002 (Cellartis AB, Goeteborg, Sweden) was seeded and cultured on the produced scaffolds and allowed propagation and then differentiation for up to 47 days. Cultivation of hESC on electrospun fibrous scaffolds proved successful and neuronal differentiation was observed via standard immunocytochemistry. The results indicate that predominantly dopaminergic tyrosine hydroxylase (TH) positive neurons are derived in co-culture with fibrous scaffolds, in comparison to reference cultures under the same differentiation conditions displaying large proportions of GFAP positive cell types. Scanning electron micrographs confirm neurite outgrowth and connection to adjacent cells, as well as cell attachment to individual fibers of the fibrous scaffold. Consequently, electrospun polyurethane scaffolds have been proven feasible as a substrate for hESC propagation and neuronal differentiation. The physical interaction between

  2. Electrospun polyurethane scaffolds for proliferation and neuronal differentiation of human embryonic stem cells

    Adult central nervous system (CNS) tissue has a limited capacity to recover after trauma or disease. Hence, tissue engineering scaffolds intended for CNS repair and rehabilitation have been subject to intense research effort. Electrospun porous scaffolds, mimicking the natural three-dimensional environment of the in vivo extracellular matrix (ECM) and providing physical support, have been identified as promising candidates for CNS tissue engineering. The present study demonstrates in vitro culturing and neuronal differentiation of human embryonic stem cells (hESCs) on electrospun fibrous polyurethane scaffolds. Electrospun scaffolds composed of biocompatible polyurethane resin (Desmopan 9370A, Bayer MaterialScience AG) were prepared with a vertical electrospinning setup. Resulting scaffolds, with a thickness of approximately 150 μm, exhibited high porosity (84%) and a bimodal pore size distribution with peaks at 5-6 and 1 μm. The mean fiber diameter was measured to approximately 360 nm with a standard deviation of 80 nm. The undifferentiated hESC line SA002 (Cellartis AB, Goeteborg, Sweden) was seeded and cultured on the produced scaffolds and allowed propagation and then differentiation for up to 47 days. Cultivation of hESC on electrospun fibrous scaffolds proved successful and neuronal differentiation was observed via standard immunocytochemistry. The results indicate that predominantly dopaminergic tyrosine hydroxylase (TH) positive neurons are derived in co-culture with fibrous scaffolds, in comparison to reference cultures under the same differentiation conditions displaying large proportions of GFAP positive cell types. Scanning electron micrographs confirm neurite outgrowth and connection to adjacent cells, as well as cell attachment to individual fibers of the fibrous scaffold. Consequently, electrospun polyurethane scaffolds have been proven feasible as a substrate for hESC propagation and neuronal differentiation. The physical interaction between cells

  3. Functional differentiation of stem cell-derived neurons from different murine backgrounds

    Lydia eBarth

    2014-02-01

    Full Text Available Murine stem cell derived-neurons have been used to study a wide variety of neuropsychiatric diseases with a hereditary component, ranging from autism to Alzheimer’s. While a significant amount of data on their molecular biology has been generated, there is little data on the physiology of these cultures. Different mouse strains show clear differences in behavioural and other neurobiologically relevant readouts. We have studied the physiology of early differentiation and network formation in neuronal cultures derived from three different mouse embryonic stem cell lines. We have found largely overlapping patterns with some significant differences in the timing of the functional milestones. Neurons from R1 showed the fastest development of intrinsic excitability, while E14Tg2a and J1 were slower. This was also reflected in an earlier appearance of synaptic activity in R1 cultures, while E14Tg2a and J1 were delayed by up to two days. In conclusion, stem cells from all backgrounds could be successfully differentiated into functioning neural networks with similar developmental patterns. Differences in the timing of specific milestones, suggest that control cell lines and time-points should be carefully chosen when investigating genetic alterations that lead to subtle deficits in neuronal function.

  4. Differentiation of human adipose-derived stem cells into neuron-like cells by Radix Angelicae Sinensis

    Qiaozhi Wang; Lile Zhou; Yong Guo; Guangyi Liu; Jiyan Cheng; Hong Yu

    2013-01-01

    Human adipose tissues are an ideal source of stem cells. It is important to find inducers that can safely and effectively differentiate stem cells into functional neurons for clinical use. In this study, we investigate the use of Radix Angelicae Sinensis as an inducer of neuronal differentiation. Primary human adipose-derived stem cells were obtained from adult subcutaneous fatty tissue, then pre-induced with 10%Radix Angelicae Sinensis injection for 24 hours, and incubated in serum-free Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 containing 40% Radix Angelicae Si-nensis to induce its differentiation into neuron-like cells. Butylated hydroxyanisole, a common in-ducer for neuronal differentiation, was used as the control. After human adipose-derived stem cells differentiated into neuron-like cells under the induction of Radix Angelicae Sinensis for 24 hours, the positive expression of neuron-specific enolase was lower than that of the butylated hydroxyani-sole-induced group, and the expression of glial fibril ary acidic protein was negative. After they were induced for 48 hours, the positive expression of neuron specific enolase in human adipose-derived stem cells was significantly higher than that of the butylated hydroxyanisole-induced group. Our experimental findings indicate that Radix Angelicae Sinensis can induce human adipose-derived stem celldifferentiation into neuron-like cells and produce less cytotoxicity.

  5. Cell cultures derived from early zebrafish embryos differentiate in vitro into neurons and astrocytes

    Ghosh, Chandramallika; Liu, Yi; Ma, Chunguang; Collodi, Paul

    1997-01-01

    The zebrafish is a polular nonmammalian model for studies of neural development. We have derived cell cultures, initiated from blastula-stage zebrafish embryos, that differentiate in vitro into neurons and astrocytes. Cultures were initiated in basal nutrient medium supplemented with bovine insulin, trout serum, trout embryo extract and fetal bovine serum. After two weeks in culture the cells exhibited extensive neurite outgrowth and possessed elevated levels of acetylcholinesterase enzyme ac...

  6. Surface N-glycoproteome patterns reveal key proteins of neuronal differentiation

    Tylečková, Jiřina; Valeková, Ivona; Žižková, Martina; Rákocyová, Michaela; Maršala, S.; Maršala, M.; Gadher, S. J.; Kovářová, Hana

    Poznaň : PTP, 2015. s. 1-1. [Central and Eastern European Proteomic Conference (CEEPC) /9./. 15.06.2015-18.06.2015, Poznaň] R&D Projects: GA MŠk ED2.1.00/03.0124; GA TA ČR(CZ) TA01011466 Institutional support: RVO:67985904 Keywords : cell adhesion proteins * cell surface capture * neuronal differentiation Subject RIV: EB - Genetics ; Molecular Biology

  7. Transfection of the glial cell line-derived neurotrophic factor gene promotes neuronal differentiation

    Du, Jie; Gao, Xiaoqing; Deng, Li; Chang, Nengbin; Xiong, Huailin; Zheng, Yu

    2014-01-01

    Glial cell line-derived neurotrophic factor recombinant adenovirus vector-transfected bone marrow mesenchymal stem cells were induced to differentiate into neuron-like cells using inductive medium containing retinoic acid and epidermal growth factor. Cell viability, microtubule-associated protein 2-positive cell ratio, and the expression levels of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43 protein in the supernatant were significantly hig...

  8. Differentiation factors that influence neuronal markers expression in vitro from human amniotic epithelial cells

    H Niknejad

    2010-01-01

    Full Text Available The differentiation of neural cells from embryonic stem cells is influenced by several growth factors. Amniotic epithelial cells (AECs share many of the same characteristics as embryonic stem cells, and therefore those factors may similarly affect the derivation of neural cells from AECs. In this study, we examined the differentiation of neural cells in vitro from AECs following AECs treatment with retinoic acid (RA, basic fibroblast growth factor (bFGF as well as investigation of bFGF withdrawal on neuronal differentiation. We also studied whether blocking bone morphogenetic protein (BMP signaling using its antagonist, noggin, affects the derivation of neuronal cells from AECs. The effects of serum on the rate of neural markers expression were also examined. Analysis of AECs derived neurons was performed at some neural markers expression level by immunocytochemistry. All cultures treated with noggin showed the higher levels of neural markers expression than noggin free cultures. Combined treatment with bFGF and RA showed the highest level of neural markers in all treatment groups with or without noggin. bFGF withdrawal did not promote expression of neural markers, while its maintenance increased the expression of these markers. Serum-free condition decreased the viability of cells but increased the rate of neural markers expression. These results show the capability of AECs to express neural cell markers and this ability is affected by some factors including serum, noggin, bFGF and RA.

  9. Bone marrow mesenchymal stem cells stimulate proliferation and neuronal differentiation of retinal progenitor cells.

    Jing Xia

    Full Text Available During retina development, retinal progenitor cell (RPC proliferation and differentiation are regulated by complex inter- and intracellular interactions. Bone marrow mesenchymal stem cells (BMSCs are reported to express a variety of cytokines and neurotrophic factors, which have powerful trophic and protective functions for neural tissue-derived cells. Here, we show that the expanded RPC cultures treated with BMSC-derived conditioned medium (CM which was substantially enriched for bFGF and CNTF, expressed clearly increased levels of nuclear receptor TLX, an essential regulator of neural stem cell (NSC self-renewal, as well as betacellulin (BTC, an EGF-like protein described as supporting NSC expansion. The BMSC CM- or bFGF-treated RPCs also displayed an obviously enhanced proliferation capability, while BMSC CM-derived bFGF knocked down by anti-bFGF, the effect of BMSC CM on enhancing RPC proliferation was partly reversed. Under differentiation conditions, treatment with BMSC CM or CNTF markedly favoured RPC differentiation towards retinal neurons, including Brn3a-positive retinal ganglion cells (RGCs and rhodopsin-positive photoreceptors, and clearly diminished retinal glial cell differentiation. These findings demonstrate that BMSCs supported RPC proliferation and neuronal differentiation which may be partly mediated by BMSC CM-derived bFGF and CNTF, reveal potential limitations of RPC culture systems, and suggest a means for optimizing RPC cell fate determination in vitro.

  10. Preliminary Study on in Vitro Induced Differentiation of Embryonic Stem Cells into Neurons

    JianGe; ShunongLi; 等

    2002-01-01

    Purpose;To study preliminarily in vitro induced differentiation of embryonic stem cells into neurons for further investigation of an alternative for the treatment of glaumatous neuropathy.Materials and methods:Supernatant of cultured Buffalo rat liver cells (buffalorat liver cell-conditioned medium,BRL-CM)was used for culturing embryonic stem cells(ES-D3 cell line)..Morphological features of undifferentiated ES cells were studied by HE staining and electron microscopy.Based on the methods used by Bain et al,we modified the methods and used retinoic,acid(RA) as an inducer to differentiate ES-D3 cells and cytosine arabinoside(Ara-C) as inhibitor of proliferative cells.The growth of the cells was observed under phase contrast microscope.Reuslts:ES-D3 cells cultured by BRL-CM grew in aggregates and remained undifferentiated.Electromicroscopy showed large nucleus and a large amount of mitochondria in undifferentiated ES cells and many processes on the surfaces.In the first day after the adding of retinoic acid,some neuron-like cells with one,two or more processes were present.In the second day after adding RA and the first day after the plus of 10μm Ara-C,a large amount of neuron-like cells appeared,with the formation of neuron-like networks.Conclusions:Combined use of RA and Ara-C can induce ES cells to different into neuron-like cells.Our present preliminary study might provide insights into an alternative for the treatment of glaucomatous neuropathy by the transplantation of embryonic stem cells.Eye Science 2000;16:1-6.

  11. Requirement for zebrafish ataxin-7 in differentiation of photoreceptors and cerebellar neurons.

    Constantin Yanicostas

    Full Text Available The expansion of a polyglutamine (polyQ tract in the N-terminal region of ataxin-7 (atxn7 is the causative event in spinocerebellar ataxia type 7 (SCA7, an autosomal dominant neurodegenerative disorder mainly characterized by progressive, selective loss of rod-cone photoreceptors and cerebellar Purkinje and granule cells. The molecular and cellular processes underlying this restricted neuronal vulnerability, which contrasts with the broad expression pattern of atxn7, remains one of the most enigmatic features of SCA7, and more generally of all polyQ disorders. To gain insight into this specific neuronal vulnerability and achieve a better understanding of atxn7 function, we carried out a functional analysis of this protein in the teleost fish Danio rerio. We characterized the zebrafish atxn7 gene and its transcription pattern, and by making use of morpholino-oligonucleotide-mediated gene inactivation, we analysed the phenotypes induced following mild or severe zebrafish atxn7 depletion. Severe or nearly complete zebrafish atxn7 loss-of-function markedly impaired embryonic development, leading to both early embryonic lethality and severely deformed embryos. More importantly, in relation to SCA7, moderate depletion of the protein specifically, albeit partially, prevented the differentiation of both retina photoreceptors and cerebellar Purkinje and granule cells. In addition, [1-232] human atxn7 fragment rescued these phenotypes showing strong function conservation of this protein through evolution. The specific requirement for zebrafish atxn7 in the proper differentiation of cerebellar neurons provides, to our knowledge, the first in vivo evidence of a direct functional relationship between atxn7 and the differentiation of Purkinje and granule cells, the most crucial neurons affected in SCA7 and most other polyQ-mediated SCAs. These findings further suggest that altered protein function may play a role in the pathophysiology of the disease, an

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

    Yuping Feng; Jiao Wang; Shixin Ling; Zhuo Li; Mingsheng Li; Qiongyi Li; Zongren Ma; Sijiu Yu

    2014-01-01

    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 fol-lowing 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 speciifc neu-ronal 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 neuro-nal-speciifc proteins, includingβIII tubulin. The bone marrow mesenchymal stem cells grown on fetal bovine acellular dermal matrix and induced for long periods of time with neural differen-tiation medium differentiated into a multilayered neural network-like structure with long nerve ifbers that was composed of several parallel microifbers 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 sec-tioning 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.

  13. MicroRNA-9 promotes the neuronal differentiation of rat bone marrow mesenchymal stem cells by activating autophagy

    Guang-yu Zhang

    2015-01-01

    Full Text Available MicroRNA-9 (miR-9 has been shown to promote the differentiation of bone marrow mesenchymal stem cells into neuronal cells, but the precise mechanism is unclear. Our previous study confirmed that increased autophagic activity improved the efficiency of neuronal differentiation in bone marrow mesenchymal stem cells. Accumulating evidence reveals that miRNAs adjust the autophagic pathways. This study used miR-9-1 lentiviral vector and miR-9-1 inhibitor to modulate the expression level of miR-9. Autophagic activity and neuronal differentiation were measured by the number of light chain-3 (LC3-positive dots, the ratio of LC3-II/LC3, and the expression levels of the neuronal markers enolase and microtubule-associated protein 2. Results showed that LC3-positive dots, the ratio of LC3-II/LC3, and expression of neuron specific enolase and microtubule-associated protein 2 increased in the miR-9 + group. The above results suggest that autophagic activity increased and bone marrow mesenchymal stem cells were prone to differentiate into neuronal cells when miR-9 was overexpressed, demonstrating that miR-9 can promote neuronal differentiation by increasing autophagic activity.

  14. MicroRNA-9 promotes the neuronal differentiation of rat bone marrow mesenchymal stem cells by activating autophagy

    Guang-yu Zhang; Jun Wang; Yan-jie Jia; Rui Han; Ping Li; Deng-na Zhu

    2015-01-01

    MicroRNA-9 (miR-9) has been shown to promote the differentiation of bone marrow mesen-chymal stem cells into neuronal cells, but the precise mechanism is unclear. Our previous study conifrmed that increased autophagic activity improved the efifciency of neuronal differentiation in bone marrow mesenchymal stem cells. Accumulating evidence reveals that miRNAs adjust the autophagic pathways. This study used miR-9-1 lentiviral vector and miR-9-1 inhibitor to modulate the expression level of miR-9. Autophagic activity and neuronal differentiation were measured by the number of light chain-3 (LC3)-positive dots, the ratio of LC3-II/LC3, and the expression levels of the neuronal markers enolase and microtubule-associated protein 2. Re-sults showed that LC3-positive dots, the ratio of LC3-II/LC3, and expression of neuron speciifc enolase and microtubule-associated protein 2 increased in the miR-9+ group. The above results suggest that autophagic activity increased and bone marrow mesenchymal stem cells were prone to differentiate into neuronal cells when miR-9 was overexpressed, demonstrating that miR-9 can promote neuronal differentiation by increasing autophagic activity.

  15. Neuronal differentiation is associated with a redox-regulated increase of copper flow to the secretory pathway

    Hatori, Yuta; Yan, Ye; Schmidt, Katharina; Furukawa, Eri; Hasan, Nesrin M.; YANG, NAN; Liu, Chin-Nung; Sockanathan, Shanthini; Lutsenko, Svetlana

    2016-01-01

    Brain development requires a fine-tuned copper homoeostasis. Copper deficiency or excess results in severe neuro-pathologies. We demonstrate that upon neuronal differentiation, cellular demand for copper increases, especially within the secretory pathway. Copper flow to this compartment is facilitated through transcriptional and metabolic regulation. Quantitative real-time imaging revealed a gradual change in the oxidation state of cytosolic glutathione upon neuronal differentiation. Transiti...

  16. Identification of Centella asiatica’s Effective Ingredients for Inducing the Neuronal Differentiation

    Hui Jiang

    2016-01-01

    Full Text Available Centella asiatica, commonly known as Gotu kola, has been widely used as a traditional herb for decades. Yet, the study on which compounds or compound combinations actually lead to its brain benefits remains scarce. To study the neuroprotection effects of Centella asiatica, neuronal differentiation of PC12 cells was applied. In our pilot study, we isolated 45 Centella asiatica fractions and tested their abilities for inducing neuronal differentiation on PC12 cells. The most effective fraction showed robust induction in neurite outgrowth and neurofilament expression. LC-MS fingerprint analysis of this fraction revealed asiatic acid and madecassic acid as the dominant components. A further investigation on the pure combination of these two compounds indicated that the combination of these two compounds extensively promoted nerve differentiation in vitro. Application of PD98059, a protein MEK inhibitor, attenuated combination-induced neurofilament expression, indicating the combination-induced nerve differentiation through activation of MEK signaling pathway. Our results support the use of combination of asiatic acid and madecassic acid as an effective mean to intervene neurodegenerative diseases in which neurotrophin deficiency is involved.

  17. Microsphere-Incorporated Hybrid Thermogel for Neuronal Differentiation of Tonsil Derived Mesenchymal Stem Cells.

    Patel, Madhumita; Moon, Hyo Jung; Jung, Bo Kyung; Jeong, Byeongmoon

    2015-07-15

    Neuronal differentiation of tonsil-derived mesenchymal stem cells (TMSCs) is investigated in a 3D hybrid system. The hybrid system is prepared by increasing the temperature of poly(ethylene glycol)-poly(l-alanine) aqueous solution to 37 °C through the heat-induced sol-to-gel transition, in which TMSCs and growth factor releasing microspheres are suspended. The in situ formed gel exhibits a modulus of 800 Pa at 37 °C, similar to that of brain tissue, and it is robust enough to hold the microspheres and cells during the 3D culture of TMSCs. The neuronal growth factors are released over 12-18 d, and the TMSCs in a spherical shape initially undergo multipolar elongation during the 3D culture. Significantly higher expressions of the neuronal biomarkers such as nuclear receptor related protein (Nurr-1), neuron specific enolase, microtubule associated protein-2, neurofilament-M, and glial fibrillary acidic protein are observed in both mRNA level and protein level in the hybrid systems than in the control experiments. This study proves the significance of a controlled drug delivery concept in tissue engineering or regenerative medicine, and a 3D hybrid system with controlled release of growth factors from microspheres in a thermogel can be a very promising tool. PMID:26033880

  18. Differential regulation of the zebrafish orthopedia1 gene during fate determination of diencephalic neurons

    Tarallo Raffaella

    2006-10-01

    Full Text Available Abstract Background The homeodomain transcription factor Orthopedia (Otp is essential in restricting the fate of multiple classes of secreting neurons in the neuroendocrine hypothalamus of vertebrates. However, there is little information on the intercellular factors that regulate Otp expression during development. Results Here, we identified two otp orthologues in zebrafish (otp1 and otp2 and explored otp1 in the context of the morphogenetic pathways that specify neuroectodermal regions. During forebrain development, otp1 is expressed in anterior groups of diencephalic cells, positioned in the preoptic area (PO (anterior alar plate and the posterior tuberculum (PT (posterior basal plate. The latter structure is characterized by Tyrosine Hydroxylase (TH-positive cells, suggesting a role for otp1 in the lineage restriction of catecholaminergic (CA neurons. Disruptions of Hedgehog (HH and Fibroblast Growth Factor (FGF pathways point to the ability of SHH protein to trigger otp1 expression in PO presumptive neuroblasts, with the attenuating effect of Dzip1 and FGF8. In addition, our data disclose otp1 as a determinant of CA neurons in the PT, where otp1 activity is strictly dependent on Nodal signaling and it is not responsive to SHH and FGF. Conclusion In this study, we pinpoint the evolutionary importance of otp1 transcription factor in cell states of the diencephalon anlage and early neuronal progenitors. Furthermore, our data indicate that morphogenetic mechanisms differentially regulate otp1 expression in alar and basal plates.

  19. Differentiation of human gingival mesenchymal stem cells into neuronal lineages in 3D bioconjugated injectable protein hydrogel construct for the management of neuronal disorder.

    Rao, Suresh Ranga; Subbarayan, Rajasekaran; Dinesh, Murugan Girija; Arumugam, Gnanamani; Raja, Selvaraj Thirupathi Kumara

    2016-01-01

    The success of regeneration attempt is based on an ideal combination of stem cells, scaffolding and growth factors. Tissue constructs help to maintain stem cells in a required area for a desired time. There is a need for easily obtainable cells, potentially autologous stem cells and a biologically acceptable scaffold for use in humans in different difficult situations. This study aims to address these issues utilizing a unique combination of stem cells from gingiva and a hydrogel scaffold, based on a natural product for regenerative application. Human gingival mesenchymal stem cells (HGMSCs) were, with due induction, differentiated to neuronal lineages to overcome the problems associated with birth tissue-related stem cells. The differentiation potential of neuronal lineages was confirmed with suitable specific markers. The properties of mesenchymal stem cells in encapsulated form were observed to be similar to free cells. The encapsulated cells (3D) were then subjected to differentiation into neuronal lineages with suitable inducers, and the morphology and gene expression of transient cells were analyzed. HGMSCs was differentiated into neuronal lineages as both free and encapsulated forms without any significant differences. The presence of Nissl bodies and the neurite outgrowth confirm the differentiation. The advantages of this new combination appear to make it a promising tissue construct for translational application. PMID:26869025

  20. Differentiation of human gingival mesenchymal stem cells into neuronal lineages in 3D bioconjugated injectable protein hydrogel construct for the management of neuronal disorder

    Rao, Suresh Ranga; Subbarayan, Rajasekaran; Dinesh, Murugan Girija; Arumugam, Gnanamani; Raja, Selvaraj Thirupathi Kumara

    2016-01-01

    The success of regeneration attempt is based on an ideal combination of stem cells, scaffolding and growth factors. Tissue constructs help to maintain stem cells in a required area for a desired time. There is a need for easily obtainable cells, potentially autologous stem cells and a biologically acceptable scaffold for use in humans in different difficult situations. This study aims to address these issues utilizing a unique combination of stem cells from gingiva and a hydrogel scaffold, based on a natural product for regenerative application. Human gingival mesenchymal stem cells (HGMSCs) were, with due induction, differentiated to neuronal lineages to overcome the problems associated with birth tissue-related stem cells. The differentiation potential of neuronal lineages was confirmed with suitable specific markers. The properties of mesenchymal stem cells in encapsulated form were observed to be similar to free cells. The encapsulated cells (3D) were then subjected to differentiation into neuronal lineages with suitable inducers, and the morphology and gene expression of transient cells were analyzed. HGMSCs was differentiated into neuronal lineages as both free and encapsulated forms without any significant differences. The presence of Nissl bodies and the neurite outgrowth confirm the differentiation. The advantages of this new combination appear to make it a promising tissue construct for translational application. PMID:26869025

  1. Tryptanthrin induces growth inhibition and neuronal differentiation in the human neuroblastoma LA-N-1 cells.

    Liao, Xuemei; Leung, Kwok Nam

    2013-04-25

    Neuroblastoma is one of the most common extracranial solid cancers found in young children. The prognosis of neuroblastoma patients in advanced stages having N-myc amplification remains poor despite intensive multimodal therapy. Agents that trigger neuroblastoma cells to undergo cellular differentiation and thereby stop proliferation have attracted considerable interest as an alternative therapy. Tryptanthrin (12-dihydro-6,12-dioxoindolo-(2,1-b)-quinazoline) is a weakly basic alkaloid isolated from the dried roots of medicinal indigo plants known as Banlangen. It has been shown to possess various biological activities, such as anti-microbial, anti-inflammatory and anti-tumor activities. However, its effects and mechanism(s) of action on human neuroblastoma cells remain poorly understood. Therefore, the objective of this study is to investigate the effects of tryptanthrin on the growth and differentiation of human neuroblastoma LA-N-1 cells with N-myc amplification. Our results show that tryptanthrin inhibited the growth of the human neuroblastoma cells in a dose- and time-dependent manner. Mechanistic studies indicated that tryptanthrin induced cell cycle arrest of the human neuroblastoma LA-N-1 cells at the G0/G1 phase. Tryptanthrin also induced neuronal differentiation of LA-N-1 cells, as assessed by morphological criteria, enhancement of acetylcholine esterase activity and up-regulation of various differentiation markers. Moreover, tryptanthrin treatment led to the significant reduction of N-myc expression in LA-N-1 cells while siRNA directed against N-myc induced morphological differentiation of LA-N-1 cells. These results, when taken together, suggest that tryptanthrin suppressed the growth and induced neuronal differentiation in the human neuroblastoma LA-N-1 cells and might be exploited as a potential therapeutic candidate for the treatment of high-risk neuroblastomas with N-myc-amplification. PMID:23500671

  2. Differentiation of fetal pancreatic stem cells into neuron-like and islet-like cells in vitro ★

    Hua, Xiufeng; Wang, Yanwei; Lian, Peiwen; Zhang, Shouxin; Li, Jianyuan; Wang, Haiyan; Chen, Shulin; Gao, Wei

    2012-01-01

    Pancreatic stem cells were isolated and cultured from aborted human fetal pancreases of gestational age 14–20 weeks. They were seeded at a density of 1 × 104 in serum-free media for differentiation into neuron-like cells, expressing β-tubulin III and glial fibrillary acidic protein. These neuron-like cells displayed a synapse-like morphology and appeared to form a neuronal network. Pancreatic stem cells were also seeded at a density of 1 × 105 for differentiation into islet-like cells, expres...

  3. Differential cytotoxic effects of mono-(2-ethylhexyl) phthalate on blastomere-derived embryonic stem cells and differentiating neurons

    Potential applications of embryonic stem (ES) cells are not limited to regenerative medicine but can also include in vitro screening of various toxicants. In this study, we established mouse ES cell lines from isolated blastomeres of two-cell stage embryos and examined their potential use as an in vitro system for the study of developmental toxicity. Two ES cell lines were established from 69 blastomere-derived blastocysts (2.9%). The blastomere-derived ES (bm-ES) cells were treated with mono-(2-ethylhexyl) phthalate (MEHP) in an undifferentiated state or after directed differentiation into early neural cell types. We observed significantly decreased cell viability when undifferentiated bm-ES cells were exposed to a high dose of MEHP (1000 μM). The cytotoxic effects of MEHP were accompanied by increased DNA fragmentation, nuclear condensation, and activation of Caspase-3, which are biochemical and morphological features of apoptosis. Compared to undifferentiated bm-ES cells, considerably lower doses of MEHP (50 and 100 μM) were sufficient to induce cell death in early neurons differentiated from bm-ES cells. At the lower doses, the number of neural cells positive for the active form of Caspase-3 was greater than that for undifferentiated bm-ES cells. Thus, our data indicate that differentiating neurons are more sensitive to MEHP than undifferentiated ES cells, and that undifferentiated ES cells may have more efficient defense systems against cytotoxic stresses. These findings might contribute to the development of a new predictive screening method for assessment of hazards for developmental toxicity.

  4. HES6-1 and HES6-2 Function through Different Mechanisms during Neuronal Differentiation

    Vilas-Boas, Filipe; Henrique, Domingos

    2010-01-01

    Background Notch signalling plays a central role in the mechanisms regulating neuronal differentiation in the vertebrate nervous system. The transcriptional repressors encoded by Hes genes are the main effectors of this pathway, acting in neural progenitors during the lateral inhibition process to repress proneural genes and inhibit differentiation. However, Hes6 genes seem to behave differently: they are expressed in differentiating neurons and facilitate the activity of proneural genes in promoting neurogenesis. Still, the molecular mechanisms underlying this unique function of Hes6 genes are not yet understood. Methodology/Principal Findings Here, we identify two subgroups of Hes6 genes that seem conserved in most vertebrate species and characterize a novel Hes6 gene in chicken: cHes6-1. The embryonic expression pattern of cHes6-1 suggests roles for this gene in the formation of the pancreas, nervous system and in the generation of body asymmetry. We show that cHes6-1 is negatively regulated by Notch signalling in the developing embryonic spinal cord and in pancreatic progenitors, but requires Notch for the observed asymmetric expression at the lateral mesoderm. Functional studies by ectopic expression in the chick embryonic neural tube revealed that cHES6-1 up-regulates the expression of cDelta1 and cHes5 genes, in contrast with overexpression of cHES6-2, which represses the same genes. We show that this activity of cHES6-2 is dependent on its capacity to bind DNA and repress transcription, while cHES6-1 seems to function by sequestering other HES proteins and inhibit their activity as transcriptional repressors. Conclusions/Significance Our results indicate that the two chick HES6 proteins act at different phases of neuronal differentiation, contributing to the progression of neurogenesis by different mechanisms: while cHES6-2 represses the transcription of Hes genes, cHES6-1 acts later, sequestering HES proteins. Together, the two cHES6 proteins progressively

  5. IFN gamma regulates proliferation and neuronal differentiation by STAT1 in adult SVZ niche

    Leticia Pereira Gómez

    2015-07-01

    Full Text Available The adult subventricular zone (SVZ is the main neurogenic niche in normal adult brains of mice and rats. Interferon gamma (IFNγ has somewhat controversially been associated with SVZ progenitor proliferation and neurogenesis. The in vivo involvement of IFNγ in the physiology of the adult SVZ niche is not fully understood and its intracellular mediators are unknown. Here we show that IFNγ, through activation of its canonical STAT1 pathway, acts specifically on Nestin+ progenitors by decreasing both progenitor proliferation and the number of cycling cells. In addition, IFNγ increases the number of neuroblasts generated without shifting glial fate determination. The final result is deficient recruitment of newborn neurons to the olfactory bulb, indicating that IFNγ-induced stimulation of neuronal differentiation does not compensate for its antiproliferative effect. We conclude that IFNγ signaling via STAT1 in the SVZ acts dually as an antiproliferative and proneurogenic factor, and thereby regulates neurogenesis in normal adult brains.

  6. Differentiation of fetal pancreatic stem cells into neuron-like and islet-like cells in vitro

    Xiufeng Hua; Yanwei Wang; Peiwen Lian; Shouxin Zhang; Jianyuan Li; Haiyan Wang; Shulin Chen; Wei Gao

    2012-01-01

    Pancreatic stem cells were isolated and cultured from aborted human fetal pancreases of gestational age 14-20 weeks.They were seeded at a density of 1 × 104 in serum-free media for differentiation into neuron-like cells, expressing β-tubulin III and glial fibrillary acidic protein.These neuron-like cells displayed a synapse-like morphology and appeared to form a neuronal network.Pancreatic stem cells were also seeded at a density of 1 × 105 for differentiation into islet-like cells, expressing insulin and glucagon, with an islet-like morphology.These cells had glucose-stimulated secretion of human insulin and C-peptide.Results suggest that pancreatic stem cells can be differentiated into neuron-like and islet-like cells.

  7. GABA Receptors on Orexin and Melanin-Concentrating Hormone Neurons Are Differentially Homeostatically Regulated Following Sleep Deprivation123

    Toossi, Hanieh; del Cid-Pellitero, Esther

    2016-01-01

    Abstract Though overlapping in distribution through the hypothalamus, orexin (Orx) and melanin-concentrating hormone (MCH) neurons play opposite roles in the regulation of sleep–wake states. Orx neurons discharge during waking, whereas MCH neurons discharge during sleep. In the present study, we examined in mice whether GABAA and GABAB receptors (Rs) are present on Orx and MCH neurons and might undergo differential changes as a function of their different activities following sleep deprivation (SD) and sleep recovery (SR). Applying quantitative stereological image analysis to dual-immunofluorescent stained sections, we determined that the proportion of Orx neurons positively immunostained for GABAARs was significantly higher following SD (∼48%) compared with sleep control (SC; ∼24%) and SR (∼27%), and that the luminance of the GABAARs was significantly greater. In contrast, the average proportion of the MCH neurons immunostained for GABAARs was insignificantly lower following SD (∼43%) compared with SC (∼54%) and SR (56%), and the luminance of the GABAARs was significantly less. Although, GABABRs were observed in all Orx and MCH neurons (100%), the luminance of these receptors was differentially altered following SD. The intensity of GABABRs in the Orx neurons was significantly greater after SD than after SC and SR, whereas that in the MCH neurons was significantly less. The present results indicate that GABA receptors undergo dynamic and differential changes in the wake-active Orx neurons and the sleep-active MCH neurons as a function of and homeostatic adjustment to their preceding activity and sleep–wake state. PMID:27294196

  8. Valproic Acid Induced Hyperammonaemic Encephalopathy

    Objective: To observe clinical and laboratory features of valproic acid-induced hyperammonaemic encephalopathy in patients taking valproic acid. Methods: Observational study was conducted at the Neurology Department, Dow University of Health Sciences, Civil Hospital, Karachi, from February 26, 2010 to March 20, 2011. Ten patients on valproic acid therapy of any age group with idiopathic or secondary epilepsy, who presented with encephalopathic symptoms, were registered and followed up during the study. Serum ammonia level, serum valproic acid level, liver function test, cerebrospinal fluid examination, electroencephalogram and brain imaging of all the patients were done. Other causes of encephalopathy were excluded after clinical and appropriate laboratory investigations. Microsoft Excel 2007 was used for statistical analysis. Results: Hyperammonaemia was found in all patients with encephalopathic symptoms. Rise in serum ammonia was independent of dose and serum level of valproic acid. Liver function was also found to be normal in 80% (n=8) of the patients. Valproic acid was withdrawn in all patients. Three (30%) patients improved only after the withdrawal of valproic acid. Six (60%) patients improved after L-Carnitine replacement, one (10%) after sodium benzoate. On followup, serum ammonia had reduced to normal in five (50%) patients and to more than half of the baseline level in two (20%) patients. Three (30%) patients were lost to followup after complete clinical improvement. Conclusion: Within therapeutic dose and serum levels, valproic acid can cause symptomatic hyperammonaemia resulting in encephalopathy. All patients taking valproic acid presenting with encephalopathic symptoms must be monitored for the condition. (author)

  9. Mutant SOD1 accumulation in sensory neurons does not associate with endoplasmic reticulum stress features: Implications for differential vulnerability of sensory and motor neurons to SOD1 toxicity.

    Taiana, Michela; Sassone, Jenny; Lauria, Giuseppe

    2016-08-01

    Mutations in Cu/Zn-superoxide dismutase (SOD1) cause familial amyotrophic lateral sclerosis (ALS). Previous papers showed that mutant SOD1 accumulates and undergoes misfolding in motor neurons and that the specific interaction of mutant SOD1 with derlin-1 leads to endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR). Because evidence shows that mutant SOD1 expression also damages sensory neurons, we hypothesized that, similarly to motor neurons, the sensory neurons of ALS mouse model SOD1(G93A) accumulate mutant/misfolded SOD1 and suffer from ER stress and UPR activation. Our results reveal that SOD1(G93A) sensory neurons accumulate mutant/misfolded SOD1 but, surprisingly, do not suffer from ER stress and UPR activation. Moreover, the sensory neurons do not express detectable levels of the SOD1 interactor derlin-1. These results suggest a potential molecular mechanism underlying the differential vulnerability of motor and sensory neurons to mutant SOD1 toxicity. PMID:27241719

  10. Methamphetamine decreases dentate gyrus stem cell self-renewal and shifts the differentiation towards neuronal fate

    Sofia Baptista; Charlène Lasgi; Caroline Benstaali; Nuno Milhazes; Fernanda Borges; Carlos Fontes-Ribeiro; Fabienne Agasse; Ana Paula Silva

    2014-01-01

    Methamphetamine (METH) is a highly addictive psychostimulant drug of abuse that negatively interferes with neurogenesis. In fact, we have previously shown that METH triggers stem/progenitor cell death and decreases neuronal differentiation in the dentate gyrus (DG). Still, little is known regarding its effect on DG stem cell properties. Herein, we investigate the impact of METH on mice DG stem/progenitor cell self-renewal functions. METH (10 nM) decreased DG stem cell self-renewal, while 1 nM...

  11. Simulation of Networked Control System based on Smith Compensator and Single Neuron Incomplete Differential Forward PID

    Haitao Zhang

    2011-12-01

    Full Text Available In the networked control system with random time delay in forward and feedback channels, a kind of controller based on Smith compensator and signal neuron incomplete differential forward PID is presented. First, using root locus method and simulink simulation software, the influences of network’s time delay on the system stability and dynamic performance are analyzed. Then, combined with incomplete differential forward PID control algorithm, Smith compensation model is established. Compared with existing Smith compensator, the proposed control model is easy to be implemented, and can also get better control performance in the case of miss-matching compensator model. Finally, the simulation research on a DC motor is done, and the simulation results show the effectiveness of the proposed method.

  12. Extending neurites sense the depth of the underlying topography during neuronal differentiation and contact guidance.

    Chua, Jie Shi; Chng, Choon-Peng; Moe, Aung Aung Kywe; Tann, Jason Y; Goh, Eyleen L K; Chiam, Keng-Hwee; Yim, Evelyn K F

    2014-09-01

    The topography of the extracellular microenvironment influences cell morphology, provides conduct guidance and directs cell differentiation. Aspect ratio and dimension of topography have been shown to affect cell behaviours, but the ability and mechanism of depth-sensing is not clearly understood. We showed that murine neural progenitor cells (mNPCs) can sense the depth of the micro-gratings. Neurite elongation, alignment and neuronal differentiation were observed to increase with grating depth. We proposed a mechanism for depth-sensing by growing neurites: filopodial adhesion in the growth cones favour elongation but the bending rigidity of the neurite cytoskeleton resists it. Thus, perpendicular extension on deeper grooves is unfavourable as neurites need to bend over a larger angle. A quantitative model was developed and its prediction of neurite growth on gratings fit well with the experimental data. The results indicated that mNPC fate can be directed by appropriately designed patterned surfaces. PMID:24954734

  13. Differentiation of developing olfactory neurons analysed in terms of coupled epigenetic landscapes.

    Alsing, Anne Katrine; Sneppen, Kim

    2013-05-01

    The olfactory system integrates signals from receptors expressed in olfactory sensory neurons. Each sensory neuron expresses only one of many similar olfactory receptors (ORs). The choice of receptor is made stochastically early in the differentiation process and is maintained throughout the life of the neuron. The underlying mechanism of this stochastic commitment to one of multiple similar OR genes remains elusive. We present a theoretical analysis of a mechanism that invokes important epigenetic properties of the system. The proposed model combines nucleosomes and associated read-write enzymes as mediators of a cis-acting positive feedback with a trans-acting negative feedback, thereby coupling the local epigenetic landscape of the individual OR genes in a way that allow one and only one gene to be active at any time. The model pinpoint that singular gene selection does not require transient mechanisms, enhancer elements or transcription factors to separate choice from maintenance. In addition, our hypothesis allow us to combine all reported characteristics of singular OR gene selection, in particular that OR genes are silenced from OR transgenes. Intriguingly, it predicts that OR transgenes placed in close proximity should always be expressed simultaneously, though rarely. PMID:23519617

  14. Maternal immune activation differentially impacts mature and adult-born hippocampal neurons in male mice.

    Zhang, Zhi; van Praag, Henriette

    2015-03-01

    Schizophrenia is associated with deficits in the hippocampus, a brain area important for learning and memory. The dentate gyrus (DG) of the hippocampus develops both before and after birth. To study the relative contribution of mature and adult-born DG granule cells to disease etiology, we compared both cell populations in a mouse model of psychiatric illness resulting from maternal immune activation. Polyriboinosinic-polyribocytidilic acid (PolyIC, 5mg/kg) or saline was given on gestation day 15 to pregnant female C57Bl/6 mice. Male offspring (n=105), was administered systemic bromodeoxyuridine (BrdU, 50mg/kg) (n=52) or intracerebral retroviral injection into the DG (n=53), to label dividing cells at one month of age. Two months later behavioral tests were performed to evaluate disease phenotype. Immunohistochemistry and whole-cell patch clamping were used to assess morphological and physiological characteristics of DG cells. Three-month-old PolyIC exposed male offspring exhibited deficient pre-pulse inhibition, spatial maze performance and motor coordination, as well as increased depression-like behavior. Histological analysis showed reduced DG volume and parvalbumin positive interneuron number. Both mature and new hippocampal neurons showed modifications in intrinsic properties such as increased input resistance and lower current threshold, and decreased action potential number. Reduced GABAergic inhibitory transmission was observed only in mature DG neurons. Differential impairments in mature DG cells and adult-born new neurons may have implications for behavioral deficits associated with maternal immune activation. PMID:25449671

  15. Improving the neuronal differentiation efficiency of umbilical cord blood-derived mesenchymal stem cells cultivated under appropriate conditions

    Hassan Rafieemehr

    2015-11-01

    Full Text Available Objective(s: Umbilical cord blood-derived mesenchymal stromal cells (UCB-MSCs are ideally suited for use in various cell-based therapies. We investigated a novel induction protocol (NIP to improve the neuronal differentiation of human UCB-MSCs under appropriate conditions. Materials and Methods: This experimental study was performed in Iranian Blood Transfusion Organization (IBTO, Tehran, Iran. UCB-MSCs were cultured in DMEM medium supplemented with 10% FBS in a humidified incubator in equilibration with 5% CO2 at 37oC. For neuronal differentiation of UCB-MSCs, DMEM was removed and replaced with pre-induction medium containing RA, bFGF, EGF, and basal medium for two days. Then, NGF, IBMX, AsA, and Neurobasal medium were used for six days for this purpose. Real-time PCR was performed to analyze the neuronal differentiation of UCB-MSCs for the first time in Iran. Results: We found that the maximum and minimum levels of gene expression were related to GFAP and nestin, respectively. In addition, our study showed that compared to other neuronal inducers, RA might play the main role in neuronal differentiation and fate of MSCs compared to other neuronal inducers. Conclusion: Our data showed that the combination of chemical (RA, IBMX, AsA and growth factors (NGF, EGF, bFGF in NIP may improve the efficiency of neuronal differentiation of UCB-MSCs and may provide a new method for easy and quick application of UCB-MSCs in regenerative medicine in the future. However, the functionality of neuron-like cells must be carefully assessed in animal experiments prior to use in clinical applications.

  16. Endocannabinoids differentially modulate synaptic plasticity in rat hippocampal CA1 pyramidal neurons.

    Jian-Yi Xu

    Full Text Available BACKGROUND: Hippocampal CA1 pyramidal neurons receive two excitatory glutamatergic synaptic inputs: their most distal dendritic regions in the stratum lacunosum-moleculare (SLM are innervated by the perforant path (PP, originating from layer III of the entorhinal cortex, while their more proximal regions of the apical dendrites in the stratum radiatum (SR are innervated by the Schaffer-collaterals (SC, originating from hippocampal CA3 neurons. Endocannabinoids (eCBs are naturally occurring mediators capable of modulating both GABAergic and glutamatergic synaptic transmission and plasticity via the CB1 receptor. Previous work on eCB modulation of excitatory synapses in the CA1 region largely focuses on the SC pathway. However, little information is available on whether and how eCBs modulate glutamatergic synaptic transmission and plasticity at PP synapses. METHODOLOGY/PRINCIPAL FINDINGS: By employing somatic and dendritic patch-clamp recordings, Ca(2+ uncaging, and immunostaining, we demonstrate that there are significant differences in low-frequency stimulation (LFS- or DHPG-, an agonist of group I metabotropic glutamate receptors (mGluRs, induced long-term depression (LTD of excitatory synaptic transmission between SC and PP synapses in the same pyramidal neurons. These differences are eliminated by pharmacological inhibition with selective CB1 receptor antagonists or genetic deletion of the CB1 receptor, indicating that these differences likely result from differential modulation via a CB1 receptor-dependent mechanism. We also revealed that depolarization-induced suppression of excitation (DSE, a form of short-term synaptic plasticity, and photolysis of caged Ca(2+-induced suppression of Excitatory postsynaptic currents (EPSCs were less at the PP than that at the SC. In addition, application of WIN55212 (WIN induced a more pronounced inhibition of EPSCs at the SC when compared to that at the PP. CONCLUSIONS/SIGNIFICANCE: Our results suggest

  17. Induction of Human Embryonic Stem Cells into neuronal differentiation by increasing cyclic Adenosine Mono Phosphate

    Hossein Baharvand

    2006-04-01

    Full Text Available Introduction: To evaluate the cAMP -mediated IBMX (3-IsoButyle -1-Methyl Xanthin and db-cAMP (dibutyryl cAMP effects on differentiation of human Embryonic Stem Cells (hESCs into nerve cells were the objectives of this study. Methods: We have used Royan H1 hESC- derived embryoid bodies with four treatment groups: six days treatment with IBMX (5×10 -4M and db-cAMP (10 -9M (referred to as cAMP, retinoic acid (RA, 10 -6M, IBMX + db-cAMP + RA, and control (no treatment. Immunocytochemistry was carried out for neural specific antibodies including β-Tubulin III, Microtubule Associated Protein 2 (MAP-2, Neurofilament Protein-Heavy chain (NF-H, Glial Fibrilary Acidic Protein (GFAP and Synaptophysin as well as morphological studies. Semiquantitative RT-PCR was also used to evaluate gene expression involved in neurogenesis. Results: In the 4+6+4 days the neuronal process were apparently observed. Immunocytochemical studies using nerve specific antibodies for proteins such as β- Tubulin III, MAP-2, NF-H, GFAP and Synaptophysin showed the presence of these neuronal and astrocyte markers in differentiated cells by cAMP. Evaluation of expression of genes involved in neurogenesis showed that Hash1, Synaptophysin, β-Adrenergic Receptor and Acetylcholine Receptor- which were silent in embryoid bodies - switched on after treatment with cAMP and/or RA. Relative expression of nerve specific genes showed a significant enhancement in expression of Synaptophysin, NFM and β-Adrenergic Receptor during differentiation, which, with the enhancement in cAMP treated groups were more than those treated with RA and control (p < 0.05. Conclusion: In conclusion, this study showed that cAMP could be a neurogenic agent for human embryonic stem cells differentiation.

  18. Differentiation of Human Breast-Milk Stem Cells to Neural Stem Cells and Neurons

    Seyed Mojtaba Hosseini

    2014-01-01

    Full Text Available Objectives. Human breast milk contains a heterogeneous population of cells that have the potential to provide a noninvasive source of cells for cell therapy in many neurodegenerative diseases without any ethical concern. The objectives of this study were to differentiate the breast milk-derived stem cells (BMDSC toward neural stem cells and then into the neurons and neuroglia. Materials and Methods. To do this, the BMDSC were isolated from human breast milk and cultured in Dulbecco’s modified Eagle medium/F12 (DMEM/F12 containing fibroblast growth factor (bFGF. The cells were then characterized by evaluation of the embryonic and stem cell markers. Then, the cells were exposed to culture medium containing 1% B27 and 2% N2 for 7–10 days followed by medium supplemented with B27, N2, bFGF 10 µg/mL, and endothelial growth factor (EGF 20 µg/mL. Then, the sphere-forming assay was performed. The spheres were then differentiated into three neural lineages by withdrawing growth factor in the presence of 5% FBS (fetal bovine serum. The immunofluorescence was done for β-tubulin III, O4, and GFAP (glial fibrillary acidic protein. Results. The results indicated that the cells expressed both embryonic and mesenchymal stem cell (MSC markers. They also showed neurospheres formation that was nestin-positive. The cells were also differentiated into all three neural lineages. Conclusion. The BMDSC can behave in the same way with neural stem cells. They were differentiated into oligodendrocytes, and astrocytes as well as neurons.

  19. Role of gangliosides in the differentiation of human mesenchymal-derived stem cells into osteoblasts and neuronal cells

    Ghislain Moussavou

    2013-11-01

    Full Text Available Gangliosides are complex glycosphingolipids that are the majorcomponent of cytoplasmic cell membranes, and play a rolein the control of biological processes. Human mesenchymalstem cells (hMSCs have received considerable attention as alternativesources of adult stem cells because of their potentialto differentiate into multiple cell lineages. In this study, we focuson various functional roles of gangliosides in the differentiationof hMSCs into osteoblasts or neuronal cells. A relationshipbetween gangliosides and epidermal growth factor receptor(EGFR activation during osteoblastic differentiation ofhMSCs was observed, and the gangliosides may play a majorrole in the regulation of the differentiation. The roles of gangliosidesin osteoblast differentiation are dependent on the originof hMSCs. The reduction of ganglioside biosynthesis inhibitedthe neuronal differentiation of hMSCs during an earlystage of the differentiation process, and the ganglioside expressioncan be used as a marker for the identification of neuronaldifferentiation from hMSCs. [BMB Reports 2013; 46(11:527-532

  20. The ubiquitin ligase Praja1 reduces NRAGE expression and inhibits neuronal differentiation of PC12 cells.

    Teuber, Jan; Mueller, Bettina; Fukabori, Ryoji; Lang, Daniel; Albrecht, Anne; Stork, Oliver

    2013-01-01

    Evidence suggests that regulated ubiquitination of proteins plays a critical role in the development and plasticity of the central nervous system. We have previously identified the ubiquitin ligase Praja1 as a gene product induced during fear memory consolidation. However, the neuronal function of this enzyme still needs to be clarified. Here, we investigate its involvement in the nerve growth factor (NGF)-induced differentiation of rat pheochromocytoma (PC12) cells. Praja1 co-localizes with cytoskeleton components and the neurotrophin receptor interacting MAGE homologue (NRAGE). We observed an enhanced expression of Praja1 after 3 days of NGF treatment and a suppression of neurite formation upon Praja1 overexpression in stably transfected PC12 cell lines, which was associated with a proteasome-dependent reduction of NRAGE levels. Our data suggest that Praja1, through ubiquitination and degradation of NRAGE, inhibits neuronal differentiation. The two murine isoforms, Praja1.1 and Praja1.2, appear to be functionally homologous in this respect. PMID:23717400

  1. The ubiquitin ligase Praja1 reduces NRAGE expression and inhibits neuronal differentiation of PC12 cells.

    Jan Teuber

    Full Text Available Evidence suggests that regulated ubiquitination of proteins plays a critical role in the development and plasticity of the central nervous system. We have previously identified the ubiquitin ligase Praja1 as a gene product induced during fear memory consolidation. However, the neuronal function of this enzyme still needs to be clarified. Here, we investigate its involvement in the nerve growth factor (NGF-induced differentiation of rat pheochromocytoma (PC12 cells. Praja1 co-localizes with cytoskeleton components and the neurotrophin receptor interacting MAGE homologue (NRAGE. We observed an enhanced expression of Praja1 after 3 days of NGF treatment and a suppression of neurite formation upon Praja1 overexpression in stably transfected PC12 cell lines, which was associated with a proteasome-dependent reduction of NRAGE levels. Our data suggest that Praja1, through ubiquitination and degradation of NRAGE, inhibits neuronal differentiation. The two murine isoforms, Praja1.1 and Praja1.2, appear to be functionally homologous in this respect.

  2. Birth, survival and differentiation of neurons in an adult crustacean brain.

    Kim, Youngmi Faith; Sandeman, David C; Benton, Jeanne L; Beltz, Barbara S

    2014-06-01

    Life-long neurogenesis is a characteristic feature of many vertebrate and invertebrate species. In decapod crustaceans, new neurons are added throughout life to two cell clusters containing local (cluster 9) and projection (cluster 10) interneurons in the olfactory pathway. Adult-born neurons in clusters 9 and 10 in crayfish have the anatomical properties and chemistry of mature neurons by 6 months after birth. Here we use 5-bromo-2'-deoxyuridine (BrdU) incorporation to pulse label mitotically active cells in these cell clusters, followed by a survival time of up to 8 months, during which crayfish (Cherax destructor) were sacrificed at intervals and the numbers of BrdU-labeled cells quantified. We find a decrease in the numbers of BrdU-labeled cells in cell cluster 10 between the first and second weeks following BrdU exposure, suggesting a period of cell death shortly after proliferation. Additional delayed cell divisions in both cell clusters are indicated by increases in labeled cells long after the BrdU clearing time. The differentiation time of these cells into neurons was defined by detection of the first immunoreactivity for the transmitter SIFamide in cluster 10 BrdU-labeled cells, which begins at 4 weeks after BrdU labeling; the numbers of SIFamide-labeled cells continues to increase over the following month. Experiments testing whether proliferation and survival of Cluster 10 cells are influenced by locomotor activity provided no evidence of a correlation between activity levels and cell proliferation, but suggest a strong influence of locomotor activity on cell survival. PMID:24339155

  3. Isolation and Culture of Pig Spermatogonial Stem Cells and Their in Vitro Differentiation into Neuron-Like Cells and Adipocytes

    Xiaoyan Wang; Tingfeng Chen; Yani Zhang; Bichun Li; Qi Xu; Chengyi Song

    2015-01-01

    Spermatogonial stem cells (SSCs) renew themselves throughout the life of an organism and also differentiate into sperm in the adult. They are multipopent and therefore, can be induced to differentiate into many cells types in vitro. SSCs from pigs, considered an ideal animal model, are used in studies of male infertility, regenerative medicine, and preparation of transgenic animals. Here, we report on a culture system for porcine SSCs and the differentiation of these cells into neuron-like c...

  4. Human Breast Cancer Cell Lines Co-Express Neuronal, Epithelial, and Melanocytic Differentiation Markers In Vitro and In Vivo

    Qingbei Zhang; Hanli Fan; Jikun Shen; Hoffman, Robert M.; H Rosie Xing

    2010-01-01

    Differentiation programs are aberrant in cancer cells allowing them to express differentiation markers in addition to their tissue of origin. In the present study, we demonstrate the multi-lineage differentiation potential of breast cancer cell lines to express multiple neuronal/glial lineage-specific markers as well as mammary epithelial and melanocytic-specific markers. Multilineage expression was detected in luminal (MCF-7 and SKBR3) and basal (MDA-MB-231) types of human breast cancer cell...

  5. Coe genes are expressed in differentiating neurons in the central nervous system of protostomes.

    Adrien Demilly

    Full Text Available Genes of the coe (collier/olfactory/early B-cell factor family encode Helix-Loop-Helix transcription factors that are widely conserved in metazoans and involved in many developmental processes, neurogenesis in particular. Whereas their functions during vertebrate neural tube formation have been well documented, very little is known about their expression and role during central nervous system (CNS development in protostomes. Here we characterized the CNS expression of coe genes in the insect Drosophila melanogaster and the polychaete annelid Platynereis dumerilii, which belong to different subgroups of protostomes and show strikingly different modes of development. In the Drosophila ventral nerve cord, we found that the Collier-expressing cells form a subpopulation of interneurons with diverse molecular identities and neurotransmitter phenotypes. We also demonstrate that collier is required for the proper differentiation of some interneurons belonging to the Eve-Lateral cluster. In Platynereis dumerilii, we cloned a single coe gene, Pdu-coe, and found that it is exclusively expressed in post mitotic neural cells. Using an original technique of in silico 3D registration, we show that Pdu-coe is co-expressed with many different neuronal markers and therefore that, like in Drosophila, its expression defines a heterogeneous population of neurons with diverse molecular identities. Our detailed characterization and comparison of coe gene expression in the CNS of two distantly-related protostomes suggest conserved roles of coe genes in neuronal differentiation in this clade. As similar roles have also been observed in vertebrates, this function was probably already established in the last common ancestor of all bilaterians.

  6. A Flavonoid Compound Promotes Neuronal Differentiation of Embryonic Stem Cells via PPAR-β Modulating Mitochondrial Energy Metabolism

    Mei, Yu-qin; Pan, Zong-fu; Chen, Wen-teng; Xu, Min-hua; Zhu, Dan-yan; Yu, Yong-ping; Lou, Yi-jia

    2016-01-01

    Relatively little is known regarding mitochondrial metabolism in neuronal differentiation of embryonic stem (ES) cells. By using a small molecule, present research has investigated the pattern of cellular energy metabolism in neural progenitor cells derived from mouse ES cells. Flavonoid compound 4a faithfully facilitated ES cells to differentiate into neurons morphologically and functionally. The expression and localization of peroxisome proliferator-activated receptors (PPARs) were examined in neural progenitor cells. PPAR-β expression showed robust upregulation compared to solvent control. Treatment with PPAR-β agonist L165041 alone or together with compound 4a significantly promoted neuronal differentiation, while antagonist GSK0660 blocked the neurogenesis-promoting effect of compound 4a. Consistently, knockdown of PPAR-β in ES cells abolished compound 4a-induced neuronal differentiation. Interestingly, we found that mitochondrial fusion protein Mfn2 was also abolished by sh-PPAR-β, resulting in abnormal mitochondrial Ca2+ ([Ca2+]M) transients as well as impaired mitochondrial bioenergetics. In conclusion, we demonstrated that by modulating mitochondrial energy metabolism through Mfn2 and mitochondrial Ca2+, PPAR-β took an important role in neuronal differentiation induced by flavonoid compound 4a. PMID:27315062

  7. HIV Subtypes B and C gp120 and Methamphetamine Interaction: Dopaminergic System Implicates Differential Neuronal Toxicity.

    Samikkannu, Thangavel; Rao, Kurapati V K; Salam, Abdul Ajees Abdul; Atluri, Venkata S R; Kaftanovskaya, Elena M; Agudelo, Marisela; Perez, Suray; Yoo, Changwon; Raymond, Andrea D; Ding, Hong; Nair, Madhavan P N

    2015-01-01

    HIV subtypes or clades differentially induce HIV-associated neurocognitive disorders (HAND) and substance abuse is known to accelerate HIV disease progression. The HIV-1 envelope protein gp120 plays a major role in binding and budding in the central nervous system (CNS) and impacts dopaminergic functions. However, the mechanisms utilized by HIV-1 clades to exert differential effects and the methamphetamine (METH)-associated dopaminergic dysfunction are poorly understood. We hypothesized that clade B and C gp120 structural sequences, modeling based analysis, dopaminergic effect, and METH potentiate neuronal toxicity in astrocytes. We evaluated the effect of clade B and C gp120 and/or METH on the DRD-2, DAT, CaMKs and CREBP transcription. Both the structural sequence and modeling studies demonstrated that clade B gp120 in V1-V4, α -2 and N-glycosylated sites are distinct from clade C gp120. The distinct structure and sequence variation of clade B gp120 differentially impact DRD-2, DAT, CaMK II and CaMK IV mRNA, protein and intracellular expression compared to clade C gp120. However, CREB transcription is upregulated by both clade B and C gp120, and METH co-treatment potentiated these effects. In conclusion, distinct structural sequences of HIV-1 clade B and C gp120 differentially regulate the dopaminergic pathway and METH potentiates neurotoxicity. PMID:26057350

  8. Neurospheres induced from bone marrow stromal cells are multipotent for differentiation into neuron, astrocyte, and oligodendrocyte phenotypes

    Bone marrow stromal cells (MSCs) can be expanded rapidly in vitro and have the potential to be differentiated into neuronal, glial and endodermal cell types. However, induction for differentiation does not always have stable result. We present a new method for efficient induction and acquisition of neural progenitors, neuronal- and glial-like cells from MSCs. We demonstrate that rat MSCs can be induced to neurospheres and most cells are positive for nestin, which is an early marker of neuronal progenitors. In addition, we had success in proliferation of these neurospheres with undifferentiated characteristics and finally we could obtain large numbers of neuronal and glial phenotypes. Many of the cells expressed β-tubulin III when they were cultivated with our method. MSCs can become a valuable cell source as an autograft for clinical application involving regeneration of the central nervous system

  9. Intermediate Progenitor Cohorts Differentially Generate Cortical Layers and Require Tbr2 for Timely Acquisition of Neuronal Subtype Identity

    Anca B. Mihalas

    2016-06-01

    Full Text Available Intermediate progenitors (IPs amplify the production of pyramidal neurons, but their role in selective genesis of cortical layers or neuronal subtypes remains unclear. Using genetic lineage tracing in mice, we find that IPs destined to produce upper cortical layers first appear early in corticogenesis, by embryonic day 11.5. During later corticogenesis, IP laminar fates are progressively limited to upper layers. We examined the role of Tbr2, an IP-specific transcription factor, in laminar fate regulation using Tbr2 conditional mutant mice. Upon Tbr2 inactivation, fewer neurons were produced by immediate differentiation and laminar fates were shifted upward. Genesis of subventricular mitoses was, however, not reduced in the context of a Tbr2-null cortex. Instead, neuronal and laminar differentiation were disrupted and delayed. Our findings indicate that upper-layer genesis depends on IPs from many stages of corticogenesis and that Tbr2 regulates the tempo of laminar fate implementation for all cortical layers.

  10. Phenotypic differentiation of neonatal rat cochlear spiral ganglion neurons following trypsin dissociation and culture

    Dingjun Zha; Li Qiao; Lianjun Lu; Xue Gao; Tao Xue; Wenjuan Mi; Shunli Liu; Jianhua Qiu

    2008-01-01

    the cells adhered to the culture dish wall, DMEM/F12 supplemented with 10% fetal bovine serum was added. MAIN OUTCOME MEASURE: Using an inverted microscope, the adherent cultured cells were observed and neurite growth index was calculated. Immunocytochemistry was performed to identify the spiral ganglion neurons, and NeuN-positive cells were analyzed. Following immunofluorescence, cochlear spiral ganglion neurons were identified through a microscope. RESULTS: Observation of cellular morphology: after digestion, inoculated cells exhibited were spherical, well stacked, and had a transparent appearance. Six hours later, some cells adhered to the culture dish wall, and small neurites were detected in a small number of cells. Twelve hours later, the adherent cells developed into polarized cells. Eighteen hours after inoculation, the adherent cells presented an ellipsoidal appearance, clear cell membranes, homogeneous cytoplasm, good refraction, and a transparent cell body surrounded by a marked halation. Twenty-four hours later, most of the cochlear spiral ganglion neurons exhibited a bipolar neuronal morphology with neurite length ranging from 2-5 times the length of a cell body. Some cochlear spiral ganglion neurons exhibited a tripolar neuronal morphology with neurites that stretched in three directions; neurite length was several times greater than the transverse diameter. Forty-eight to seventy-two hours later, the cells further differentiated and exhibited interwoven neurites, with a length that was 7-8 times greater than the cell body length. Seven days later, cells began to degenerate and underwent apoptosis. Identification of cochlear spiral ganglion neurons: immunocytochemical staining revealed whole cochlear spiral ganglion neurons that were green-colored and exhibited an ellipsoidal cell body with clear neurites. Measurement of neurite growth index: neurite growth index was 0.52±0.13, 0.86±0.21, 1.22±0.33, and 1.05±0.26 for 24, 48, 72, and 120 hours after

  11. Serotonin differentially modulates excitatory and inhibitory synaptic inputs to putative sleep-promoting neurons of the ventrolateral preoptic nucleus.

    Sangare, Aude; Dubourget, Romain; Geoffroy, Hélène; Gallopin, Thierry; Rancillac, Armelle

    2016-10-01

    The role of serotonin (5-HT) in sleep-wake regulation has been a subject of intense debate and remains incompletely understood. In the ventrolateral preoptic nucleus (VLPO), the main structure that triggers non-rapid eye movement (NREM) sleep, putative sleep-promoting (PSP) neurons were shown ex vivo to be either inhibited (Type-1) or excited (Type-2) by 5-HT application. To determine the complex action of this neurotransmitter on PSP neurons, we recorded spontaneous and miniature excitatory and inhibitory postsynaptic currents (sEPSCs, sIPSCs, mEPSCs and mIPSCs) in response to bath application of 5-HT. We established in mouse acute VLPO slices that 5-HT reduces spontaneous and miniature EPSC and IPSC frequencies to Type-1 neurons, whereas 5-HT selectively increases sIPSC and mIPSC frequencies to Type-2 VLPO neurons. We further determined that Type-1 neurons display a lower action potential threshold and a smaller soma size than Type-2 neurons. Finally, single-cell RT-PCR designed to identify the 13 serotonergic receptor subtypes revealed the specific mRNA expression of the 5-HT1A,B,D,F receptors by Type-1 neurons. Furthermore, the 5-HT2A-C,4,7 receptors were found to be equivalently expressed by both neuronal types. Altogether, our results establish that the excitatory and inhibitory inputs to Type-1 and Type-2 VLPO PSP neurons are differentially regulated by 5-HT. Electrophysiological, morphological and molecular differences were also identified between these two neuronal types. Our results provide new insights regarding the orchestration of sleep regulation by 5-HT release, and strongly suggest that Type-2 neurons could play a permissive role, whereas Type-1 neurons could have an executive role in sleep induction and maintenance. PMID:27238836

  12. The CB1 cannabinoid receptor drives corticospinal motor neuron differentiation through the Ctip2/Satb2 transcriptional regulation axis

    Díaz-Alonso, Javier; Aguado, Tania; Wu, Chia-Shan; Palazuelos, Javier; Hofmann, Clementine; Garcez, Patricia; Guillemot, Francois; Lu, Hui-Chen; Lutz, Beat; Guzmán, Manuel; Galve-Roperh, Ismael

    2012-01-01

    The generation and specification of pyramidal neuron subpopulations during development relies on a complex network of transcription factors. The CB1 cannabinoid receptor is the major molecular target of endocannabinoids and marijuana active compounds. This receptor has been shown to influence neural progenitor proliferation and axonal growth, but its involvement in neuronal differentiation and the functional impact in the adulthood caused by altering its signaling during brain development are...

  13. VITAMIN C FACILITATES DOPAMINE NEURON DIFFERENTIATION IN FETAL MIDBRAIN THROUGH TET1- AND JMJD3-DEPENDENT EPIGENETIC CONTROL MANNER

    He, Xi-Biao; Kim, Mirang; Kim, Seon-Young; Yi, Sang-Hoon; Rhee, Yong-Hee; Kim, Taeho; Lee, Eun-Hye; Park, Chang-Hwan; Dixit, Shilpy; Harrison, Fiona E.; Lee, Sang-Hun

    2015-01-01

    Intracellular Vitamin C (VC) is maintained at high levels in the developing brain by the activity of sodium-dependent VC transporter 2 (Svct2), suggesting specific VC functions in brain development. A role of VC as a cofactor for Fe(II)-2-oxoglutarate-dependent dioxygenases has recently been suggested. We show that VC supplementation in neural stem cell (NSC) cultures derived from embryonic midbrains greatly enhanced differentiation towards midbrain-type DA (mDA) neurons, the neuronal subtype...

  14. Gene expression analysis of neuronal precursors from adult mouse brain and differential screen for neural stem cell markers

    Pennartz, Sandra

    2004-01-01

    In the adult mouse brain, neuronal precursor cells continuously emanate from neural stem cells (NSC) in the subventricular zone (SVZ) and migrate into the olfactory bulb (OB) where they differentiate to serve as replenishment for GABAergic interneurons. During the migration process, PSA-NCAM (Polysialic acid-Neural cell adhesion molecule) specifically marks the neuronal precursors (PSA+ cells). This phenomenon was exploited in the framework of this doctoral thesis to isolate a homogeneous cel...

  15. Mimicking the neurotrophic factor profile of embryonic spinal cord controls the differentiation potential of spinal progenitors into neuronal cells.

    Masaya Nakamura

    Full Text Available Recent studies have indicated that the choice of lineage of neural progenitor cells is determined, at least in part, by environmental factors, such as neurotrophic factors. Despite extensive studies using exogenous neurotrophic factors, the effect of endogenous neurotrophic factors on the differentiation of progenitor cells remains obscure. Here we show that embryonic spinal cord derived-progenitor cells express both ciliary neurotrophic factor (CNTF and brain-derived neurotrophic factor (BDNF mRNA before differentiation. BDNF gene expression significantly decreases with their differentiation into the specific lineage, whereas CNTF gene expression significantly increases. The temporal pattern of neurotrophic factor gene expression in progenitor cells is similar to that of the spinal cord during postnatal development. Approximately 50% of spinal progenitor cells differentiated into astrocytes. To determine the effect of endogenous CNTF on their differentiation, we neutralized endogenous CNTF by administration of its polyclonal antibody. Neutralization of endogenous CNTF inhibited the differentiation of progenitor cells into astrocytes, but did not affect the numbers of neurons or oligodendrocytes. Furthermore, to mimic the profile of neurotrophic factors in the spinal cord during embryonic development, we applied BDNF or neurotrophin (NT-3 exogenously in combination with the anti-CNTF antibody. The exogenous application of BDNF or NT-3 promoted the differentiation of these cells into neurons or oligodendrocytes, respectively. These findings suggest that endogenous CNTF and exogenous BDNF and NT-3 play roles in the differentiation of embryonic spinal cord derived progenitor cells into astrocytes, neurons and oligodendrocytes, respectively.

  16. Functionalizing Ascl1 with Novel Intracellular Protein Delivery Technology for Promoting Neuronal Differentiation of Human Induced Pluripotent Stem Cells.

    Robinson, Meghan; Chapani, Parv; Styan, Tara; Vaidyanathan, Ranjani; Willerth, Stephanie Michelle

    2016-08-01

    Pluripotent stem cells can become any cell type found in the body. Accordingly, one of the major challenges when working with pluripotent stem cells is producing a highly homogenous population of differentiated cells, which can then be used for downstream applications such as cell therapies or drug screening. The transcription factor Ascl1 plays a key role in neural development and previous work has shown that Ascl1 overexpression using viral vectors can reprogram fibroblasts directly into neurons. Here we report on how a recombinant version of the Ascl1 protein functionalized with intracellular protein delivery technology (Ascl1-IPTD) can be used to rapidly differentiate human induced pluripotent stem cells (hiPSCs) into neurons. We first evaluated a range of Ascl1-IPTD concentrations to determine the most effective amount for generating neurons from hiPSCs cultured in serum free media. Next, we looked at the frequency of Ascl1-IPTD supplementation in the media on differentiation and found that one time supplementation is sufficient enough to trigger the neural differentiation process. Ascl1-IPTD was efficiently taken up by the hiPSCs and enabled rapid differentiation into TUJ1-positive and NeuN-positive populations with neuronal morphology after 8 days. After 12 days of culture, hiPSC-derived neurons produced by Ascl1-IPTD treatment exhibited greater neurite length and higher numbers of branch points compared to neurons derived using a standard neural progenitor differentiation protocol. This work validates Ascl1-IPTD as a powerful tool for engineering neural tissue from pluripotent stem cells. PMID:27138845

  17. Spine formation pattern of adult-born neurons is differentially modulated by the induction timing and location of hippocampal plasticity.

    Noriaki Ohkawa

    Full Text Available In the adult hippocampus dentate gyrus (DG, newly born neurons are functionally integrated into existing circuits and play important roles in hippocampus-dependent memory. However, it remains unclear how neural plasticity regulates the integration pattern of new neurons into preexisting circuits. Because dendritic spines are major postsynaptic sites for excitatory inputs, spines of new neurons were visualized by retrovirus-mediated labeling to evaluate integration. Long-term potentiation (LTP was induced at 12, 16, or 21 days postinfection (dpi, at which time new neurons have no, few, or many spines, respectively. The spine expression patterns were investigated at one or two weeks after LTP induction. Induction at 12 dpi increased later spinogenesis, although the new neurons at 12 dpi didn't respond to the stimulus for LTP induction. Induction at 21 dpi transiently mediated spine enlargement. Surprisingly, LTP induction at 16 dpi reduced the spine density of new neurons. All LTP-mediated changes specifically appeared within the LTP-induced layer. Therefore, neural plasticity differentially regulates the integration of new neurons into the activated circuit, dependent on their developmental stage. Consequently, new neurons at different developmental stages may play distinct roles in processing the acquired information by modulating the connectivity of activated circuits via their integration.

  18. Differentiation of Embryonic Stem Cells into Neurons and Retina—like Structure in Nude Mice

    LiYP; GeJ

    1999-01-01

    Purpose:To investigate the intraocular growth and biological characteristics of mice embryonic stem cells in nude mice.Methods:Murine embryonic stem cells(D3 cell line)were cultured and maintained in an undifferentiated state in vitro,then transplanted into the anterior chamber of nude mice.Mophological and immunohistochemical examinations were implemented.Results:Two to three days after transplantation,yellow-white floating granules,sheets and masses were seen inside the anterior chamber and vitreous cavity,and enlarged gradually,14-20days later,the mice were executed.Morphological examination showed that there were undifferentiated cells and some round or polygonal differentiated cells in anterior chamber and vitreous cavity.The morphology of these differentiated cells were similar to that of the retina.The cells were highly positive in NSE staining.Conclusion:The tranplanted embryonic stem cells cold grow in the eyes of nude mice with tendency to differentiate into neurons and retina-like structure.

  19. Prenatal exposure to urban air nanoparticles in mice causes altered neuronal differentiation and depression-like responses.

    David A Davis

    Full Text Available Emerging evidence suggests that excessive exposure to traffic-derived air pollution during pregnancy may increase the vulnerability to neurodevelopmental alterations that underlie a broad array of neuropsychiatric disorders. We present a mouse model for prenatal exposure to urban freeway nanoparticulate matter (nPM. In prior studies, we developed a model for adult rodent exposure to re-aerosolized urban nPM which caused inflammatory brain responses with altered neuronal glutamatergic functions. nPMs are collected continuously for one month from a local freeway and stored as an aqueous suspension, prior to re-aerosolization for exposure of mice under controlled dose and duration. This paradigm was used for a pilot study of prenatal nPM impact on neonatal neurons and adult behaviors. Adult C57BL/6J female mice were exposed to re-aerosolized nPM (350 µg/m(3 or control filtered ambient air for 10 weeks (3×5 hour exposures per week, encompassing gestation and oocyte maturation prior to mating. Prenatal nPM did not alter litter size, pup weight, or postnatal growth. Neonatal cerebral cortex neurons at 24 hours in vitro showed impaired differentiation, with 50% reduction of stage 3 neurons with long neurites and correspondingly more undifferentiated neurons at Stages 0 and 1. Neuron number after 24 hours of culture was not altered by prenatal nPM exposure. Addition of exogenous nPM (2 µg/ml to the cultures impaired pyramidal neuron Stage 3 differentiation by 60%. Adult males showed increased depression-like responses in the tail-suspension test, but not anxiety-related behaviors. These pilot data suggest that prenatal exposure to nPM can alter neuronal differentiation with gender-specific behavioral sequelae that may be relevant to human prenatal exposure to urban vehicular aerosols.

  20. Habitat-Specific Shaping of Proliferation and Neuronal Differentiation in Adult Hippocampal Neurogenesis of Wild Rodents

    Nicole eCavegn

    2013-04-01

    Full Text Available Daily life of wild mammals is characterized by a multitude of attractive and aversive stimuli. The hippocampus processes complex polymodal information associated with such stimuli and mediates adequate behavioral responses. How newly generated hippocampal neurons in wild animals contribute to hippocampal function is still a subject of debate. Here, we test the relationship between adult hippocampal neurogenesis and habitat types. To this end, we compare wild Muridae species of southern Africa (Namaqua rock mouse (Micaelamys namaquensis, red veld rat (Aethomys chrysophilus, highveld gerbil (Tatera brantsii and spiny mouse (Acomys spinosissimus with data from wild European Muridae (long-tailed wood mice (Apodemus sylvaticus, pygmy field mice (Apodemus microps, yellow-necked wood mice (Apodemus flavicollis, and house mice (Mus musculus domesticus from previous studies. The pattern of neurogenesis, expressed in normalized numbers of Ki67- and DCX-positive cells to total granule cells, is similar for the species from a southern African habitat. However, we found low proliferation, but high neuronal differentiation in rodents from the southern African habitat compared to rodents from the European environment. Within the African rodents, we observe additional regulatory and morphological traits in the hippocampus. Namaqua rock mice with previous pregnancies showed lower adult hippocampal neurogenesis compared to males and nulliparous females. The phylogenetically closely related species (Namaqua rock mouse and red veld rat show a CA4, which is not usually observed in murine rodents. The specific features of the southern environment that may be associated with the high number of young neurons in African rodents still remain to be elucidated. This study provides the first evidence that a habitat can shape adult neurogenesis in rodents across phylogenetic groups.

  1. Ectopic expression of neurogenin 2 alone is sufficient to induce differentiation of embryonic stem cells into mature neurons.

    Eva C Thoma

    Full Text Available Recent studies show that combinations of defined key developmental transcription factors (TFs can reprogram somatic cells to pluripotency or induce cell conversion of one somatic cell type to another. However, it is not clear if single genes can define a cell̀s identity and if the cell fate defining potential of TFs is also operative in pluripotent stem cells in vitro. Here, we show that ectopic expression of the neural TF Neurogenin2 (Ngn2 is sufficient to induce rapid and efficient differentiation of embryonic stem cells (ESCs into mature glutamatergic neurons. Ngn2-induced neuronal differentiation did not require any additional external or internal factors and occurred even under pluripotency-promoting conditions. Differentiated cells displayed neuron-specific morphology, protein expression, and functional features, most importantly the generation of action potentials and contacts with hippocampal neurons. Gene expression analyses revealed that Ngn2-induced in vitro differentiation partially resembled neurogenesis in vivo, as it included specific activation of Ngn2 target genes and interaction partners. These findings demonstrate that a single gene is sufficient to determine cell fate decisions of uncommitted stem cells thus giving insights into the role of key developmental genes during lineage commitment. Furthermore, we present a promising tool to improve directed differentiation strategies for applications in both stem cell research and regenerative medicine.

  2. Characterization and evaluation of neuronal trans-differentiation with electrophysiological properties of mesenchymal stem cells isolated from porcine endometrium.

    Subbarao, Raghavendra Baregundi; Ullah, Imran; Kim, Eun-Jin; Jang, Si-Jung; Lee, Won-Jae; Jeon, Ryoung Hoon; Kang, Dawon; Lee, Sung-Lim; Park, Bong-Wook; Rho, Gyu-Jin

    2015-01-01

    Endometrial stromal cells (EMSCs) obtained from porcine uterus (n = 6) were positive for mesenchymal stem cell markers (CD29, CD44 and CD90), and negative for epithelial marker CD9 and hematopoietic markers CD34, CD45 analyzed by flow cytometry. Further the cells were positive for expression of mesenchymal markers, CD105, CD140b, and CD144 by PCR. Pluripotent markers OCT4, SOX2, and NANOG were positively expressed in EMSCs analyzed by Western blotting and PCR. Further, differentiation into adipocytes and osteocytes was confirmed by cytochemical staining and lineage specific gene expression by quantitative realtime-PCR. Adipocyte (FABP, LPL, AP2) and osteocyte specific genes (ON, BG, RUNX2) in differentiated EMSCs showed significant (p < 0.05) increase in expression compared to undifferentiated control cells. Neurogenic transdifferentiation of EMSCs exhibited distinctive dendritic morphology with axon projections and neuronal specific genes, NFM, NGF, MBP, NES, B3T and MAP2 and proteins, B3T, NFM, NGF, and TRKA were positively expressed in neuronal differentiated cells. Functional analysis of neuronal differentiated EMSCs displayed voltage-dependence and kinetics for transient outward K+ currents (Ito), at holding potential of -80 mV, Na+ currents and during current clamp, neuronal differentiated EMSCs was more negative than that of control EMSCs. Porcine EMSCs is a suitable model for studying molecular mechanism of transdifferentiation, assessment of electrophysiological properties and their efficiency during in vivo transplantation. PMID:26006231

  3. Characterization and Evaluation of Neuronal Trans-Differentiation with Electrophysiological Properties of Mesenchymal Stem Cells Isolated from Porcine Endometrium

    Raghavendra Baregundi Subbarao

    2015-05-01

    Full Text Available Endometrial stromal cells (EMSCs obtained from porcine uterus (n = 6 were positive for mesenchymal stem cell markers (CD29, CD44 and CD90, and negative for epithelial marker CD9 and hematopoietic markers CD34, CD45 analyzed by flow cytometry. Further the cells were positive for expression of mesenchymal markers, CD105, CD140b, and CD144 by PCR. Pluripotent markers OCT4, SOX2, and NANOG were positively expressed in EMSCs analyzed by Western blotting and PCR. Further, differentiation into adipocytes and osteocytes was confirmed by cytochemical staining and lineage specific gene expression by quantitative realtime-PCR. Adipocyte (FABP, LPL, AP2 and osteocyte specific genes (ON, BG, RUNX2 in differentiated EMSCs showed significant (p < 0.05 increase in expression compared to undifferentiated control cells. Neurogenic transdifferentiation of EMSCs exhibited distinctive dendritic morphology with axon projections and neuronal specific genes, NFM, NGF, MBP, NES, B3T and MAP2 and proteins, B3T, NFM, NGF, and TRKA were positively expressed in neuronal differentiated cells. Functional analysis of neuronal differentiated EMSCs displayed voltage-dependence and kinetics for transient outward K+ currents (Ito, at holding potential of −80 mV, Na+ currents and during current clamp, neuronal differentiated EMSCs was more negative than that of control EMSCs. Porcine EMSCs is a suitable model for studying molecular mechanism of transdifferentiation, assessment of electrophysiological properties and their efficiency during in vivo transplantation.

  4. Neuronal Differentiation of Induced Pluripotent Stem Cells on Surfactant Templated Chitosan Hydrogels.

    Worthington, Kristan S; Green, Brian J; Rethwisch, Mary; Wiley, Luke A; Tucker, Budd A; Guymon, C Allan; Salem, Aliasger K

    2016-05-01

    The development of effective tissue engineering materials requires careful consideration of several properties beyond biocompatibility, including permeability and mechanical stiffness. While surfactant templating has been used for over a decade to control the physical properties of photopolymer materials, the potential benefit of this technique with regard to biomaterials has yet to be fully explored. Herein we demonstrate that surfactant templating can be used to tune the water uptake and compressive modulus of photo-cross-linked chitosan hydrogels. Interestingly, templating with quaternary ammonium surfactants also hedges against property fluctuations that occur with changing pH. Further, we demonstrate that, after adequate surfactant removal, these materials are nontoxic, support the attachment of induced pluripotent stem cells and facilitate stem cell differentiation to neuronal phenotypes. These results demonstrate the utility of surfactant templating for optimizing the properties of biomaterials intended for a variety of applications, including retinal regeneration. PMID:27008004

  5. Stochastic differential equation models for ion channel noise in Hodgkin-Huxley neurons.

    Goldwyn, Joshua H; Imennov, Nikita S; Famulare, Michael; Shea-Brown, Eric

    2011-04-01

    The random transitions of ion channels between conducting and nonconducting states generate a source of internal fluctuations in a neuron, known as channel noise. The standard method for modeling the states of ion channels nonlinearly couples continuous-time Markov chains to a differential equation for voltage. Beginning with the work of R. F. Fox and Y.-N. Lu [Phys. Rev. E 49, 3421 (1994)], there have been attempts to generate simpler models that use stochastic differential equation (SDEs) to approximate the stochastic spiking activity produced by Markov chain models. Recent numerical investigations, however, have raised doubts that SDE models can capture the stochastic dynamics of Markov chain models.We analyze three SDE models that have been proposed as approximations to the Markov chain model: one that describes the states of the ion channels and two that describe the states of the ion channel subunits. We show that the former channel-based approach can capture the distribution of channel noise and its effects on spiking in a Hodgkin-Huxley neuron model to a degree not previously demonstrated, but the latter two subunit-based approaches cannot. Our analysis provides intuitive and mathematical explanations for why this is the case. The temporal correlation in the channel noise is determined by the combinatorics of bundling subunits into channels, but the subunit-based approaches do not correctly account for this structure. Our study confirms and elucidates the findings of previous numerical investigations of subunit-based SDE models. Moreover, it presents evidence that Markov chain models of the nonlinear, stochastic dynamics of neural membranes can be accurately approximated by SDEs. This finding opens a door to future modeling work using SDE techniques to further illuminate the effects of ion channel fluctuations on electrically active cells. PMID:21599202

  6. Methamphetamine decreases dentate gyrus stem cell self-renewal and shifts the differentiation towards neuronal fate

    Sofia Baptista

    2014-09-01

    Full Text Available Methamphetamine (METH is a highly addictive psychostimulant drug of abuse that negatively interferes with neurogenesis. In fact, we have previously shown that METH triggers stem/progenitor cell death and decreases neuronal differentiation in the dentate gyrus (DG. Still, little is known regarding its effect on DG stem cell properties. Herein, we investigate the impact of METH on mice DG stem/progenitor cell self-renewal functions. METH (10 nM decreased DG stem cell self-renewal, while 1 nM delayed cell cycle in the G0/G1-to-S phase transition and increased the number of quiescent cells (G0 phase, which correlated with a decrease in cyclin E, pEGFR and pERK1/2 protein levels. Importantly, both drug concentrations (1 or 10 nM did not induce cell death. In accordance with the impairment of self-renewal capacity, METH (10 nM decreased Sox2+/Sox2+ while increased Sox2−/Sox2− pairs of daughter cells. This effect relied on N-methyl-d-aspartate (NMDA signaling, which was prevented by the NMDA receptor antagonist, MK-801 (10 μM. Moreover, METH (10 nM increased doublecortin (DCX protein levels consistent with neuronal differentiation. In conclusion, METH alters DG stem cell properties by delaying cell cycle and decreasing self-renewal capacities, mechanisms that may contribute to DG neurogenesis impairment followed by cognitive deficits verified in METH consumers.

  7. Vitamin C facilitates dopamine neuron differentiation in fetal midbrain through TET1- and JMJD3-dependent epigenetic control manner.

    He, Xi-Biao; Kim, Mirang; Kim, Seon-Young; Yi, Sang-Hoon; Rhee, Yong-Hee; Kim, Taeho; Lee, Eun-Hye; Park, Chang-Hwan; Dixit, Shilpy; Harrison, Fiona E; Lee, Sang-Hun

    2015-04-01

    Intracellular Vitamin C (VC) is maintained at high levels in the developing brain by the activity of sodium-dependent VC transporter 2 (Svct2), suggesting specific VC functions in brain development. A role of VC as a cofactor for Fe(II)-2-oxoglutarate-dependent dioxygenases has recently been suggested. We show that VC supplementation in neural stem cell cultures derived from embryonic midbrains greatly enhanced differentiation toward midbrain-type dopamine (mDA) neurons, the neuronal subtype associated with Parkinson's disease. VC induced gain of 5-hydroxymethylcytosine (5hmC) and loss of H3K27m3 in DA phenotype gene promoters, which are catalyzed by Tet1 and Jmjd3, respectively. Consequently, VC enhanced DA phenotype gene transcriptions in the progenitors by Nurr1, a transcription factor critical for mDA neuron development, to be more accessible to the gene promoters. Further mechanism studies including Tet1 and Jmjd3 knockdown/inhibition experiments revealed that both the 5hmC and H3K27m3 changes, specifically in the progenitor cells, are indispensible for the VC-mediated mDA neuron differentiation. We finally show that in Svct2 knockout mouse embryos, mDA neuron formation in the developing midbrain decreased along with the 5hmC/H3k27m3 changes. These findings together indicate an epigenetic role of VC in midbrain DA neuron development. PMID:25535150

  8. Cell Signaling and Differential Protein Expression in Neuronal Differentiation of Bone Marrow Mesenchymal Stem Cells with Hypermethylated Salvador/Warts/Hippo (SWH Pathway Genes.

    Hui-Hung Tzeng

    Full Text Available Human mesenchymal stem cells (MSCs modified by targeting DNA hypermethylation of genes in the Salvador/Warts/Hippo pathway were induced to differentiate into neuronal cells in vitro. The differentiated cells secreted a significant level of brain-derived neurotrophy factor (BDNF and the expression of BDNF receptor tyrosine receptor kinase B (TrkB correlated well with the secretion of BDNF. In the differentiating cells, CREB was active after the binding of growth factors to induce phosphorylation of ERK in the MAPK/ERK pathway. Downstream of phosphorylated CREB led to the functional maturation of differentiated cells and secretion of BDNF, which contributed to the sustained expression of pERK and pCREB. In summary, both PI3K/Akt and MAPK/ERK signaling pathways play important roles in the neuronal differentiation of MSCs. The main function of the PI3K/Akt pathway is to maintain cell survival during neural differentiation; whereas the role of the MAPK/ERK pathway is probably to promote the maturation of differentiated MSCs. Further, cellular levels of protein kinase C epsilon type (PKC-ε and kinesin heavy chain (KIF5B increased with time of induction, whereas the level of NME/NM23 nucleoside diphosphate kinase 1 (Nm23-H1 decreased during the time course of differentiation. The correlation between PKC-ε and TrkB suggested that there is cross-talk between PKC-ε and the PI3K/Akt signaling pathway.

  9. Mechanosensory Neuron Aging: Differential Trajectories with Lifespan-Extending Alaskan Berry and Fungal Treatments in Caenorhabditis elegans.

    Scerbak, Courtney; Vayndorf, Elena M; Hernandez, Alicia; McGill, Colin; Taylor, Barbara E

    2016-01-01

    Many nutritional interventions that increase lifespan are also proposed to postpone age-related declines in motor and cognitive function. Potential sources of anti-aging compounds are the plants and fungi that have adapted to extreme environments. We studied the effects of four commonly consumed and culturally relevant Interior Alaska berry and fungus species (bog blueberry, lowbush cranberry, crowberry, and chaga) on the decline in overall health and neuron function and changes in touch receptor neuron morphology associated with aging. We observed increased wild-type Caenorhabditis elegans lifespan and improved markers of healthspan upon treatment with Alaskan blueberry, lowbush cranberry, and chaga extracts. Interestingly, although all three treatments increased lifespan, they differentially affected the development of aberrant morphologies in touch receptor neurons. Blueberry treatments decreased anterior mechanosensory neuron (ALM) aberrations (i.e., extended outgrowths and abnormal cell bodies) while lowbush cranberry treatment increased posterior mechanosensory neuron (PLM) aberrations, namely process branching. Chaga treatment both decreased ALM aberrations (i.e., extended outgrowths) and increased PLM aberrations (i.e., process branching and loops). These results support the large body of knowledge positing that there are multiple cellular strategies and mechanisms for promoting health with age. Importantly, these results also demonstrate that although an accumulation of abnormal neuron morphologies is associated with aging and decreased health, not all of these morphologies are detrimental to neuronal and organismal health. PMID:27486399

  10. Mechanosensory Neuron Aging: Differential Trajectories with Lifespan-Extending Alaskan Berry and Fungal Treatments in Caenorhabditis elegans

    Scerbak, Courtney; Vayndorf, Elena M.; Hernandez, Alicia; McGill, Colin; Taylor, Barbara E.

    2016-01-01

    Many nutritional interventions that increase lifespan are also proposed to postpone age-related declines in motor and cognitive function. Potential sources of anti-aging compounds are the plants and fungi that have adapted to extreme environments. We studied the effects of four commonly consumed and culturally relevant Interior Alaska berry and fungus species (bog blueberry, lowbush cranberry, crowberry, and chaga) on the decline in overall health and neuron function and changes in touch receptor neuron morphology associated with aging. We observed increased wild-type Caenorhabditis elegans lifespan and improved markers of healthspan upon treatment with Alaskan blueberry, lowbush cranberry, and chaga extracts. Interestingly, although all three treatments increased lifespan, they differentially affected the development of aberrant morphologies in touch receptor neurons. Blueberry treatments decreased anterior mechanosensory neuron (ALM) aberrations (i.e., extended outgrowths and abnormal cell bodies) while lowbush cranberry treatment increased posterior mechanosensory neuron (PLM) aberrations, namely process branching. Chaga treatment both decreased ALM aberrations (i.e., extended outgrowths) and increased PLM aberrations (i.e., process branching and loops). These results support the large body of knowledge positing that there are multiple cellular strategies and mechanisms for promoting health with age. Importantly, these results also demonstrate that although an accumulation of abnormal neuron morphologies is associated with aging and decreased health, not all of these morphologies are detrimental to neuronal and organismal health. PMID:27486399

  11. Negative regulation of microRNA-132 in expression of synaptic proteins in neuronal differentiation of embryonic neural stem cells.

    Yoshimura, Aya; Numakawa, Tadahiro; Odaka, Haruki; Adachi, Naoki; Tamai, Yoshitaka; Kunugi, Hiroshi

    2016-07-01

    MicroRNAs (miRs) play important roles in neuronal differentiation, maturation, and synaptic function in the central nervous system. They have also been suggested to be implicated in the pathogenesis of neurodegenerative and psychiatric diseases. Although miR-132 is one of the well-studied brain-specific miRs, which regulates synaptic structure and function in the postnatal brain, its function in the prenatal brain is still unclear. Here, we investigated miR-132 function during differentiation of rat embryonic neural stem cells (eNSCs). We found that miR-132 expression significantly increased during the fetal rat brain development and neural differentiation of eNSCs in vitro. Furthermore, miR-132 expression was increased during differentiation through MAPK/ERK1/2 pathway. Inhibition of ERK1/2 activation resulted in increased levels of synaptic proteins including PSD-95, GluR1 and synapsin I. Silencing of miR-132 also increased PSD-95 and GluR1. Considering that miR-132 increases synaptic proteins in differentiated cortical neurons, our result shows a novel function of miR-132 as a negative regulator for synaptic maturation in the neuronal differentiation of eNSCs. PMID:27131735

  12. Neural stem cells was induced to differentiate into cholinergic neurons in vitro

    The cholinergic-inducing effect of BMP4 on isolated and cultivated rat's cerebral neural stem cells (NSCs) was examined. NSCs which were isolated from two month's old rat's brain region like hippocampus and striatum were cultivated in a medium containing EGF and bFGF, and were identified with morphological character by microscope and nestin immunocytochemistry test. After 24 hours, half NSCs were cultivated with a BMP4-added medium as a experimental group instead of the primary medium, while the an other half NSCs being cultivated with the primary medium as a control group. After 8 days the expression of choline acetyltransferase (ChAT) of the cultivated cells was observated by indirect immunofluorescence test. Results showed that more positive cells were found in the experimental group, and the fluorescence intensity were stronger; while less positive cells were found in the control group, and the fluorescence intensity was weaker. The differentiational efficiency of the NSCs was examined by FITC-labelled Flow Cytometry. The results showed that about 16% cells of the experimental group appeared ChAT-positive, while that of control group only 7%. So BMP4 may have the function of inducing NSCs to differentiate into neurons with cholinergic characteristic. (authors)

  13. Peptide modified polymer poly (glycerol- dodecanedioate co-fumarate) for efficient control of motor neuron differentiation.

    Dai, Xizi; Huang, Yen-Chih; Leichner, Jared; Nair, Madhvan; Lin, Wei-Chiang; Li, Chen-Zhong

    2015-12-01

    Neural tissue engineering is one of the most promising approaches for healing nerve damage, which bypasses the limits of contemporary conventional treatments. In a previous study, we developed a fibrous scaffold via electrospinning poly (glycerol dodecanedioate) (PGD) and gelatin that mimics the structure of a native extracellular matrix (ECM) for soft tissue engineering application. In this study, fumaric acid (FA) was incorporated into the PGD synthesis process, which produced a PGD derivative referred to as poly (glycerol dodecanedioate co-fumarate) (PGDF). This introduced a new functional group, a double bond, into the polymer thus providing new modification possibilities. Arg-Gly-Asp-Cys (RGDC) and laminin peptides were chosen as biomolecules to modify the fiber and facilitate cell attachment and differentiation efficiency. The release of FA into the medium was quantified to investigate the bioreactivity of the derived scaffolds. In combination with UV crosslinking, the developed PGDF fiber mats were able to withstand degradation processes for up to 2 months, which ensures that neural tissue engineering applications are viable. Cell viability and motor neuron differentiation efficiency were demonstrated to be significantly improved with the addition of FA, RGDC and laminin peptides. PMID:26584592

  14. Factors inducing human umbilical cord blood-derived mesenchymal stem cells to differentiate into neuron-like cells

    Nawei Zhang; Fengqing Ji

    2006-01-01

    OBJECTIVE:Human umbilical cord blood-derived mesenchymal stem cells (HUCB-derived MSCs)can differentiate into neuron-like cells,which can be used to treat some central nervous system(CNS)diseases.To investigate the factors,which can induce HUCB-derived MSCs to differentiate into neuron-like cells,so as to find effective methods for future clinical application.DATA SOURCES:Using the key terms"human umbilical cord blood"combined with"mesenchymal stem cells,neuron-like cells,neural cells"respectively,the relevant articles in English published during the period from January 1999 to June 2006 were searched from the Medline database.Meanwhile,relevant Chinese articles published from January 1999 to June 2006 were searched Using the same key terms.STUDY SELECTION: All articles associated with the differentiation from human umbilical cord blood into neuron-like cells were selected firstly.Then the full texts were looked up by searchling Ovid medical Journals full-text database and Elsevier Electrical Journals Full-text Database.Articles with full expeiments,enrolled in inducible factors or involved inducible mechanism were retdeved.DATA EXTRACTION:Among 119 collected correlative articles,29 were involved and 90 were excluded.DATA SYNTHESIS:The inducible factors of HUCB-derived MSCs differentiatling into neuron-like cells included renal endothelial growth factors,fibroblasts,β-mercaptoethanol,dimethyl sulfoxide,butyl hydroxyl anisol,brain-derived neurotrophic factor,Danshen,retinoic acid,sodium ferulate and so on,but its mechanism was unclear.CONCLUSION:Human umbilical cord blood-derived MSCs can differentiate into neuron-like cells,with varied inductors.

  15. Induction of apoptotic death and retardation of neuronal differentiation of human neural stem cells by sodium arsenite treatment

    Ivanov, Vladimir N., E-mail: vni3@columbia.edu [Center for Radiological Research, Department of Radiation Oncology, College of Physicians and Surgeons, Columbia University, 630 West 168th Street, NY 10032 (United States); Hei, Tom K. [Center for Radiological Research, Department of Radiation Oncology, College of Physicians and Surgeons, Columbia University, 630 West 168th Street, NY 10032 (United States)

    2013-04-01

    Chronic arsenic toxicity is a global health problem that affects more than 100 million people worldwide. Long-term health effects of inorganic sodium arsenite in drinking water may result in skin, lung and liver cancers and in severe neurological abnormalities. We investigated in the present study whether sodium arsenite affects signaling pathways that control cell survival, proliferation and neuronal differentiation of human neural stem cells (NSC). We demonstrated that the critical signaling pathway, which was suppressed by sodium arsenite in NSC, was the protective PI3K–AKT pathway. Sodium arsenite (2–4 μM) also caused down-regulation of Nanog, one of the key transcription factors that control pluripotency and self-renewal of stem cells. Mitochondrial damage and cytochrome-c release induced by sodium arsenite exposure was followed by initiation of the mitochondrial apoptotic pathway in NSC. Beside caspase-9 and caspase-3 inhibitors, suppression of JNK activity decreased levels of arsenite-induced apoptosis in NSC. Neuronal differentiation of NSC was substantially inhibited by sodium arsenite exposure. Overactivation of JNK1 and ERK1/2 and down-regulation of PI3K–AKT activity induced by sodium arsenite were critical factors that strongly affected neuronal differentiation. In conclusion, sodium arsenite exposure of human NSC induces the mitochondrial apoptotic pathway, which is substantially accelerated due to the simultaneous suppression of PI3K–AKT. Sodium arsenite also negatively affects neuronal differentiation of NSC through overactivation of MEK–ERK and suppression of PI3K–AKT. - Highlights: ► Arsenite induces the mitochondrial apoptotic pathway in human neural stem cells. ► Arsenite-induced apoptosis is strongly upregulated by suppression of PI3K–AKT. ► Arsenite-induced apoptosis is strongly down-regulated by inhibition of JNK–cJun. ► Arsenite negatively affects neuronal differentiation by inhibition of PI3K–AKT.

  16. Induction of apoptotic death and retardation of neuronal differentiation of human neural stem cells by sodium arsenite treatment

    Chronic arsenic toxicity is a global health problem that affects more than 100 million people worldwide. Long-term health effects of inorganic sodium arsenite in drinking water may result in skin, lung and liver cancers and in severe neurological abnormalities. We investigated in the present study whether sodium arsenite affects signaling pathways that control cell survival, proliferation and neuronal differentiation of human neural stem cells (NSC). We demonstrated that the critical signaling pathway, which was suppressed by sodium arsenite in NSC, was the protective PI3K–AKT pathway. Sodium arsenite (2–4 μM) also caused down-regulation of Nanog, one of the key transcription factors that control pluripotency and self-renewal of stem cells. Mitochondrial damage and cytochrome-c release induced by sodium arsenite exposure was followed by initiation of the mitochondrial apoptotic pathway in NSC. Beside caspase-9 and caspase-3 inhibitors, suppression of JNK activity decreased levels of arsenite-induced apoptosis in NSC. Neuronal differentiation of NSC was substantially inhibited by sodium arsenite exposure. Overactivation of JNK1 and ERK1/2 and down-regulation of PI3K–AKT activity induced by sodium arsenite were critical factors that strongly affected neuronal differentiation. In conclusion, sodium arsenite exposure of human NSC induces the mitochondrial apoptotic pathway, which is substantially accelerated due to the simultaneous suppression of PI3K–AKT. Sodium arsenite also negatively affects neuronal differentiation of NSC through overactivation of MEK–ERK and suppression of PI3K–AKT. - Highlights: ► Arsenite induces the mitochondrial apoptotic pathway in human neural stem cells. ► Arsenite-induced apoptosis is strongly upregulated by suppression of PI3K–AKT. ► Arsenite-induced apoptosis is strongly down-regulated by inhibition of JNK–cJun. ► Arsenite negatively affects neuronal differentiation by inhibition of PI3K–AKT

  17. Changes in morphology and spatial position of coiled bodies during NGF-induced neuronal differentiation of PC12 cells.

    Janevski, J; Park, P C; De Boni, U

    1997-11-01

    Interphase nuclei are organized into structural and functional domains. The coiled body, a nuclear organelle of unknown function, exhibits cell type-specific changes in number and morphology. Its association with nucleoli and with small nuclear ribonucleo-proteins (snRNPs) indicates that it functions in RNA processing. In cycling cells, coiled bodies are round structures not associated with nucleoli. In contrast, in neurons, they frequently present as nucleolar "caps." To test the hypothesis that neuronal differentiation is accompanied by changes in the spatial association of coiled bodies with nucleoli and in their morphology, PC12 cells were differentiated into a neuronal phenotype with nerve growth factor (NGF) and coiled bodies detected by immunocytochemical localization of p80-coilin and snRNPs. The fraction of cells that showed coiled bodies as nucleolar caps increased from 1.6 +/- 0.9% (mean +/- SEM) in controls to 16.5 +/- 1.6% in NGF-differentiated cultures. The fraction of cells with ring-like coiled bodies increased from 17.2 +/- 5.0% in controls to 57.8 +/- 4.4% in differentiated cells. This was accompanied by a decrease, from 81.2 +/- 5.7% to 25.7 +/- 3.1%, in the fraction of cells with small, round coiled bodies. SnRNPs remained associated with typical coiled bodies and with ring-like coiled bodies during NGF-induced recruitment of snRNPs to the nuclear periphery. Together with the observation that coiled bodies are also present as nucleolar caps in sensory neurons, the results indicate that coiled bodies alter their morphology and increase their association with nucleoli during NGF-induced neuronal differentiation. PMID:9358854

  18. Induction of Neuronal Differentiation of Rat Muscle-Derived Stem Cells in Vitro Using Basic Fibroblast Growth Factor and Ethosuximide

    Jin Seon Kwon

    2013-03-01

    Full Text Available Several studies have demonstrated that basic fibroblast growth factor (bFGF can induce neural differentiation of mesenchymal stem cells. In this study, we investigated the neural differentiation of muscle-derived stem cells (MDSCs following treatment with bFGF and ethosuximide, a small molecule used as an anticonvulsant in humans. Stem cells isolated from rat skeletal muscle (rMDSCs were pre-induced by culturing with 25 ng/mL bFGF for 24 h and then were transferred to a medium supplemented with or without 4 mM ethosuximide. Neuronal differentiation was assessed by immunocytochemical and western blotting analyses of marker expression. Immunocytochemistry of rMDSCs treated with bFGF and ethosuximide identified abundant cells expressing neuronal markers (TuJ1, neuron-specific class III β-tubulin; NeuN, neuronal nuclear antigen; and NF-MH; neurofilament M and H. Olig2 (oligodendrocyte transcription factor 2-positive cells were also observed, indicating the presence of oligodendrocyte lineage cells. These findings were substantiated by western blotting analysis of marker proteins. In particular, the expression of NeuN and TuJ1 was significantly higher in rMDSCs treated with ethosuximide and bFGF than in cells stimulated with bFGF alone (NeuN, p < 0.05 and TuJ1, p < 0.001. Expression of the astrocyte marker GFAP (glial fibrillary acidic protein was not detected in this study. Collectively, the results showed that treatment with bFGF and ethosuximide induced effective transdifferentiation of rMDSCs into cells with a neural-like phenotype. Notably, rMDSCs treated with a combination of bFGF plus ethosuximide showed enhanced differentiation compared with cells treated with bFGF alone, implying that ethosuximide may stimulate neuronal differentiation.

  19. Differential adeno-associated virus mediated gene transfer to sensory neurons following intrathecal delivery by direct lumbar puncture

    Kitto Kelley F

    2010-05-01

    Full Text Available Abstract Background Neuronal transduction by adeno-associated viral (AAV vectors has been demonstrated in cortex, brainstem, cerebellum, and sensory ganglia. Intrathecal delivery of AAV serotypes that transduce neurons in dorsal root ganglia (DRG and spinal cord offers substantial opportunities to 1 further study mechanisms underlying chronic pain, and 2 develop novel gene-based therapies for the treatment and management of chronic pain using a non-invasive delivery route with established safety margins. In this study we have compared expression patterns of AAV serotype 5 (AAV5- and AAV serotype 8 (AAV8-mediated gene transfer to sensory neurons following intrathecal delivery by direct lumbar puncture. Results Intravenous mannitol pre-treatment significantly enhanced transduction of primary sensory neurons after direct lumbar puncture injection of AAV5 (rAAV5-GFP or AAV8 (rAAV8-GFP carrying the green fluorescent protein (GFP gene. The presence of GFP in DRG neurons was consistent with the following evidence for primary afferent origin of the majority of GFP-positive fibers in spinal cord: 1 GFP-positive axons were evident in both dorsal roots and dorsal columns; and 2 dorsal rhizotomy, which severs the primary afferent input to spinal cord, abolished the majority of GFP labeling in dorsal horn. We found that both rAAV5-GFP and rAAV8-GFP appear to preferentially target large-diameter DRG neurons, while excluding the isolectin-B4 (IB4 -binding population of small diameter neurons. In addition, a larger proportion of CGRP-positive cells was transduced by rAAV5-GFP, compared to rAAV8-GFP. Conclusions The present study demonstrates the feasibility of minimally invasive gene transfer to sensory neurons using direct lumbar puncture and provides evidence for differential targeting of subtypes of DRG neurons by AAV vectors.

  20. Neuromelanin, neurotransmitter status and brainstem location determine the differential vulnerability of catecholaminergic neurons to mitochondrial DNA deletions

    Elstner Matthias

    2011-12-01

    Full Text Available Abstract Background Deletions of the mitochondrial DNA (mtDNA accumulate to high levels in dopaminergic neurons of the substantia nigra pars compacta (SNc in normal aging and in patients with Parkinson's disease (PD. Human nigral neurons characteristically contain the pigment neuromelanin (NM, which is believed to alter the cellular redox-status. The impact of neuronal pigmentation, neurotransmitter status and brainstem location on the susceptibility to mtDNA damage remains unclear. We quantified mtDNA deletions (ΔmtDNA in single pigmented and non-pigmented catecholaminergic, as well as non-catecholaminergic neurons of the human SNc, the ventral tegmental area (VTA and the locus coeruleus (LC, using laser capture microdissection and single-cell real-time PCR. Results In healthy aged individuals, ΔmtDNA levels were highest in pigmented catecholaminergic neurons (25.2 ± 14.9%, followed by non-pigmented catecholamergic (18.0 ± 11.2% and non-catecholaminergic neurons (12.3 ± 12.3%; p Conclusions Catecholaminergic brainstem neurons are differentially susceptible to mtDNA damage. Pigmented dopaminergic neurons of the SNc show the highest ΔmtDNA levels, possibly explaining the exceptional vulnerability of the nigro-striatal system in PD and aging. Although loss of pigmented noradrenergic LC neurons also is an early feature of PD pathology, mtDNA levels are not elevated in this nucleus in healthy controls. Thus, ΔmtDNA are neither an inevitable consequence of catecholamine metabolism nor a universal explanation for the regional vulnerability seen in PD.

  1. Electrophysiological study on differentiation of rat bone marrow stromal stem cells into neuron-like cells in vitro by edaravone

    ZENG Rong; HU Zi-bing; GUO Wei-tao; LIN Hao; SUN Xin; WEI Jin-song; WU Shao-ke

    2009-01-01

    To explore the electrophysiological proper-ties of differentiation of rat bone marrow-derived stromal stem cells (rBMSCs) to neuron-like cells in vitro by edaravone, a new type of free radical scavenger. Methods: Stromal stem cells were separated from rat bone marrow with Ficoll-Paque reagent and expanded in different culture medium in vitro, rBMSCs were induced by edaravone containing serum-free L-DMEM. Morphologic observation and Western blot analysis including the ex-pression of Nav1.6, Kv1.2, Kv1.3, Cav1.2 were performed, and whole patch-clamp technique was used. Results: Cyton contraction and long processes were shown in differentiated stromal stem cells. Nav1.6, Kv1.2, Kv1.3 and Cav1.2 were expressed in both differentiated and undifferentiated cells. However, the expression of channel proteins in differentiated cells was up-regulated. Consistently, their resting potential and outward currents were also enhanced in the differentiated cells, which was especially significant in the outward rectifier potassium current. Conclusion: In vitro, neuron-like cells derived from rBMSCs, induced by edaravone, possess electrophysiologi-cal properties of neurons.

  2. Poly(Dimethylsiloxane) (PDMS) Affects Gene Expression in PC12 Cells Differentiating into Neuronal-Like Cells

    Lopacinska, Joanna M.; Emnéus, Jenny; Dufva, Martin

    2013-01-01

    accumulated research. In contrast, the experience base is limited for materials used in microfludics chip fabrication. Methods: The effect of different materials (PS, PMMA and perforated PMMA with a piece of PDMS underneath) on the growth and differentiation of PC12 (adrenal phaeochromocytoma) cells into...... contrast, 41 genes showed different expression for PC12 cells differentiating on PMMA as compared to on PS. In contrast, 677 genes showed different expression on PMMA with PDMS underneath as compared with PC12 cells on PS. The differentially expressed genes are involved in neuronal cell development and...

  3. Carbon Monoxide Releasing Molecule-A1 (CORM-A1) Improves Neurogenesis: Increase of Neuronal Differentiation Yield by Preventing Cell Death

    Almeida, Ana S.; Soares, Nuno L.; Vieira, Melissa; Gramsbergen, Jan Bert

    2016-01-01

    Cerebral ischemia and neurodegenerative diseases lead to impairment or death of neurons in the central nervous system. Stem cell based therapies are promising strategies currently under investigation. Carbon monoxide (CO) is an endogenous product of heme degradation by heme oxygenase (HO) activity. Administration of CO at low concentrations produces several beneficial effects in distinct tissues, namely anti-apoptotic and anti-inflammatory. Herein the CO role on modulation of neuronal differentiation was assessed. Three different models with increasing complexity were used: human neuroblastoma SH-S5Y5 cell line, human teratocarcinoma NT2 cell line and organotypic hippocampal slice cultures (OHSC). Cell lines were differentiated into post-mitotic neurons by treatment with retinoic acid (RA) supplemented with CO-releasing molecule A1 (CORM-A1). CORM-A1 positively modulated neuronal differentiation, since it increased final neuronal production and enhanced the expression of specific neuronal genes: Nestin, Tuj1 and MAP2. Furthermore, during neuronal differentiation process, there was an increase in proliferative cell number (ki67 mRNA expressing cells) and a decrease in cell death (lower propidium iodide (PI) uptake, limitation of caspase-3 activation and higher Bcl-2 expressing cells). CO supplementation did not increase the expression of RA receptors. In the case of SH-S5Y5 model, small amounts of reactive oxygen species (ROS) generation emerges as important signaling molecules during CO-promoted neuronal differentiation. CO’s improvement of neuronal differentiation yield was validated using OHSC as ex vivo model. CORM-A1 treatment of OHSC promoted higher levels of cells expressing the neuronal marker Tuj1. Still, CORM-A1 increased cell proliferation assessed by ki67 expression and also prevented cell death, which was followed by increased Bcl-2 expression, decreased levels of active caspase-3 and PI uptake. Likewise, ROS signaling emerged as key factors in CO

  4. The HER2 inhibitor TAK165 Sensitizes Human Acute Myeloid Leukemia Cells to Retinoic Acid-Induced Myeloid Differentiation by activating MEK/ERK mediated RARα/STAT1 axis.

    Shao, Xuejing; Liu, Yujia; Li, Yangling; Xian, Miao; Zhou, Qian; Yang, Bo; Ying, Meidan; He, Qiaojun

    2016-01-01

    The success of all-trans retinoic acid (ATRA) in differentiation therapy for patients with acute promyelocytic leukemia (APL) highly encourages researches to apply this therapy to other types of acute myeloid leukemia (AML). However, AML, with the exception of APL, fails to respond to differentiation therapy. Therefore, research strategies to further sensitize cells to retinoids and to extend the range of AMLs that respond to retinoids beyond APLs are urgently needed. In this study, we showed that TAK165, a HER2 inhibitor, exhibited a strong synergy with ATRA to promote AML cell differentiation. We observed that TAK165 sensitized the AML cells to ATRA-induced cell growth inhibition, G0/G1 phase arrest, CD11b expression, mature morphologic changes, NBT reduction and myeloid regulator expression. Unexpectedly, HER2 pathway might not be essential for TAK165-enhanced differentiation when combined with ATRA, while the enhanced differentiation was dependent on the activation of the RARα/STAT1 axis. Furthermore, the MEK/ERK cascade regulated the activation of STAT1. Taken together, our study is the first to evaluate the synergy of TAK165 and ATRA in AML cell differentiation and to assess new opportunities for the combination of TAK165 and ATRA as a promising approach for future differentiation therapy. PMID:27074819

  5. Differential regulation of microtubule severing by APC underlies distinct patterns of projection neuron and interneuron migration

    Eom, Tae-Yeon; Stanco, Amelia; Guo, Jiami; Wilkins, Gary; Deslauriers, Danielle; Yan, Jessica; Monckton, Chase; Blair, Josh; Oon, Eesim; Perez, Abby; Salas, Eduardo; Oh, Adrianna; Ghukasyan, Vladimir; Snider, William D; John L R Rubenstein

    2014-01-01

    Coordinated migration of distinct classes of neurons to appropriate positions leads to the formation of functional neuronal circuitry in the cerebral cortex. Two major classes of cortical neurons, interneurons and projection neurons, utilize distinctly different modes (radial vs. tangential) and routes of migration to arrive at their final positions in the cerebral cortex. Here, we show that adenomatous polyposis coli (APC) modulates microtubule (MT) severing in interneurons to facilitate tan...

  6. Isolation and Culture of Pig Spermatogonial Stem Cells and Their in Vitro Differentiation into Neuron-Like Cells and Adipocytes.

    Wang, Xiaoyan; Chen, Tingfeng; Zhang, Yani; Li, Bichun; Xu, Qi; Song, Chengyi

    2015-01-01

    Spermatogonial stem cells (SSCs) renew themselves throughout the life of an organism and also differentiate into sperm in the adult. They are multipopent and therefore, can be induced to differentiate into many cells types in vitro. SSCs from pigs, considered an ideal animal model, are used in studies of male infertility, regenerative medicine, and preparation of transgenic animals. Here, we report on a culture system for porcine SSCs and the differentiation of these cells into neuron-like cells and adipocytes. SSCs and Sertoli cells were isolated from neonatal piglet testis by differential adhesion and SSCs were cultured on a feeder layer of Sertoli cells. Third-generation SSCs were induced to differentiate into neuron-like cells by addition of retinoic acid, β-mercaptoethanol, and 3-isobutyl-1-methylxanthine (IBMX) to the induction media and into adipocytes by the addition of hexadecadrol, insulin, and IBMX to the induction media. The differentiated cells were characterized by biochemical staining, qRT-PCR, and immunocytochemistry. The cells were positive for SSC markers, including alkaline phosphatase and SSC-specific genes, consistent with the cells being undifferentiated. The isolated SSCs survived on the Sertoli cells for 15 generations. Karyotyping confirmed that the chromosomal number of the SSCs were normal for pig (2n = 38, n = 19). Pig SSCs were successfully induced into neuron-like cells eight days after induction and into adipocytes 22 days after induction as determined by biochemical and immunocytochemical staining. qPCR results also support this conclusion. The nervous tissue markers genes, Nestin and β-tubulin, were expressed in the neuron-like cells and the adipocyte marker genes, PPARγ and C/EBPα, were expressed in the adipocytes. PMID:26556335

  7. Isolation and Culture of Pig Spermatogonial Stem Cells and Their in Vitro Differentiation into Neuron-Like Cells and Adipocytes

    Xiaoyan Wang

    2015-11-01

    Full Text Available Spermatogonial stem cells (SSCs renew themselves throughout the life of an organism and also differentiate into sperm in the adult. They are multipopent and therefore, can be induced to differentiate into many cells types in vitro. SSCs from pigs, considered an ideal animal model, are used in studies of male infertility, regenerative medicine, and preparation of transgenic animals. Here, we report on a culture system for porcine SSCs and the differentiation of these cells into neuron-like cells and adipocytes. SSCs and Sertoli cells were isolated from neonatal piglet testis by differential adhesion and SSCs were cultured on a feeder layer of Sertoli cells. Third-generation SSCs were induced to differentiate into neuron-like cells by addition of retinoic acid, β-mercaptoethanol, and 3-isobutyl-1-methylxanthine (IBMX to the induction media and into adipocytes by the addition of hexadecadrol, insulin, and IBMX to the induction media. The differentiated cells were characterized by biochemical staining, qRT-PCR, and immunocytochemistry. The cells were positive for SSC markers, including alkaline phosphatase and SSC-specific genes, consistent with the cells being undifferentiated. The isolated SSCs survived on the Sertoli cells for 15 generations. Karyotyping confirmed that the chromosomal number of the SSCs were normal for pig (2n = 38, n = 19. Pig SSCs were successfully induced into neuron-like cells eight days after induction and into adipocytes 22 days after induction as determined by biochemical and immunocytochemical staining. qPCR results also support this conclusion. The nervous tissue markers genes, Nestin and β-tubulin, were expressed in the neuron-like cells and the adipocyte marker genes, PPARγ and C/EBPα, were expressed in the adipocytes.

  8. Analytical Study of Fractional-Order Multiple Chaotic FitzHugh-Nagumo Neurons Model Using Multistep Generalized Differential Transform Method

    Shaher Momani

    2014-01-01

    Full Text Available The multistep generalized differential transform method is applied to solve the fractional-order multiple chaotic FitzHugh-Nagumo (FHN neurons model. The algorithm is illustrated by studying the dynamics of three coupled chaotic FHN neurons equations with different gap junctions under external electrical stimulation. The fractional derivatives are described in the Caputo sense. Furthermore, we present figurative comparisons between the proposed scheme and the classical fourth-order Runge-Kutta method to demonstrate the accuracy and applicability of this method. The graphical results reveal that only few terms are required to deduce the approximate solutions which are found to be accurate and efficient.

  9. In vitro effects of fetal rat cerebrospinal fluid on viability and neuronal differentiation of PC12 cells

    Nabiuni Mohammad

    2012-06-01

    Full Text Available Abstract Background Fetal cerebrospinal fluid (CSF contains many neurotrophic and growth factors and has been shown to be capable of supporting viability, proliferation and differentiation of primary cortical progenitor cells. Rat pheochromocytoma PC12 cells have been widely used as an in vitro model of neuronal differentiation since they differentiate into sympathetic neuron-like cells in response to growth factors. This study aimed to establish whether PC12 cells were responsive to fetal CSF and therefore whether they might be used to investigate CSF physiology in a stable cell line lacking the time-specific response patterns of primary cells previously described. Methods In vitro assays of viability, proliferation and differentiation were carried out after incubation of PC12 cells in media with and without addition of fetal rat CSF. An MTT tetrazolium assay was used to assess cell viability and/or cell proliferation. Expression of neural differentiation markers (MAP-2 and β-III tubulin was determined by immunocytochemistry. Formation and growth of neurites was measured by image analysis. Results PC12 cells differentiate into neuronal cell types when exposed to bFGF. Viability and cell proliferation of PC12 cells cultured in CSF-supplemented medium from E18 rat fetuses were significantly elevated relative to the control group. Neuronal-like outgrowths from cells appeared following the application of bFGF or CSF from E17 and E19 fetuses but not E18 or E20 CSF. Beta-III tubulin was expressed in PC12 cells cultured in any media except that supplemented with E18 CSF. MAP-2 expression was found in control cultures and in those with E17 and E19 CSF. MAP2 was located in neurites except in E17 CSF when the whole cell was positive. Conclusions Fetal rat CSF supports viability and stimulates proliferation and neurogenic differentiation of PC12 cells in an age-dependent way, suggesting that CSF composition changes with age. This feature may be important

  10. Chronic treatment with the GLP1 analogue liraglutide increases cell proliferation and differentiation into neurons in an AD mouse model.

    Vadivel Parthsarathy

    Full Text Available Neurogenesis is a life long process, but the rate of cell proliferation and differentiation decreases with age. In Alzheimer's patients, along with age, the presence of Aβ in the brain inhibits this process by reducing stem cell proliferation and cell differentiation. GLP-1 is a growth factor that has neuroprotective properties. GLP1 receptors are present on neuronal progenitor cells, and the GLP-1 analogue liraglutide has been shown to increase cell proliferation in an Alzheimer's disease (AD mouse model. Here we investigated acute and chronic effects of liraglutide on progenitor cell proliferation, neuroblast differentiation and their subsequent differentiation into neurons in wild type and APP/PS-1 mice at different ages. APP/PS1 and their littermate controls, aged 3, 6, 12, 15 months were injected acutely or chronically with 25 nmol/kg liraglutide. Acute treatment with liraglutide showed an increase in cell proliferation in APP/PS1 mice, but not in controls whereas chronic treatment increased cell proliferation at all ages (BrdU and Ki67 markers. Moreover, numbers of immature neurons (DCX were increased in both acute and chronic treated animals at all ages. Most newly generated cells differentiated into mature neurons (NeuN marker. A significant increase was observed with chronically treated 6, 12, 15 month APP/PS1 and WT groups. These results demonstrate that liraglutide, which is currently on the market as a treatment for type 2 diabetes (Victoza(TM, increases neurogenesis, which may have beneficial effects in neurodegenerative disorders like AD.

  11. S6K Promotes Dopaminergic Neuronal Differentiation Through PI3K/Akt/mTOR-Dependent Signaling Pathways in Human Neural Stem Cells.

    Lee, Jeong Eun; Lim, Mi Sun; Park, Jae Hyun; Park, Chang Hwan; Koh, Hyun Chul

    2016-08-01

    It has recently been reported that the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway regulates neuronal differentiation of neural stem cells (NSCs) derived from rats or mice and is essential for the self-renewal of human embryonic stem cells (hESCs). However, the roles of PI3K/Akt/mTOR signaling pathways during proliferation and dopaminergic neuronal differentiation of human neural stem cells (hNSCs) are poorly understood. In this study, we examined the effect of regulation of these intracellular signaling pathways in hNSCs on the potential to maintain proliferation and induce dopaminergic neuronal differentiation. Dopaminergic neuronal differentiation depended on the concentration of insulin in our culture system. Inhibition of PI3K/Akt with LY294002 reduced proliferation and inhibited dopaminergic neuronal differentiation of these cells. We also found that rapamycin, a specific inhibitor of mTOR, significantly reduced neuronal differentiation without affecting proliferation. Inhibition of the Akt/mTOR signaling pathway led to inhibition of p70 ribosomal S6 kinase (S6K) signaling, which reduced dopaminergic neuronal differentiation in hNSCs. Inhibition of S6K by a specific chemical inhibitor, PF-4708671 inhibited dopaminergic neuronal differentiation of hNSCs. As expected, transduction with a dominant negative S6K1 (S6K1-DN) construct impaired dopaminergic neuronal differentiation of hNSCs. Conversely, overexpression of constitutively active S6K1 (S6K1-CA) promoted dopaminergic neuronal differentiation of these cells. In a survival study, 4 weeks after transplantation, no or very few donor cells were viable in striata grafted with S6K1-DN-transduced hNSCs. In contrast, S6K1-CA-transduced hNSCs survived, integrated into striata to generate tubular masses of grafts and differentiated toward TH-positive cells. Taken together, these data demonstrated that insulin promotes dopaminergic neuronal differentiation through a PI

  12. Overexpression of microRNA-124 promotes the neuronal differentiation of bone marrow-derived mesenchymal stem cells

    Defeng Zou; Yi Chen; Yaxin Han; Chen Lv; Guanjun Tu

    2014-01-01

    microRNAs (miRNAs) play an important regulatory role in the self-renewal and differentiation of stem cells. In this study, we examined the effects of miRNA-124 (miR-124) overexpression in bone marrow-derived mesenchymal stem cells. In particular, we focused on the effect of overexpression on the differentiation of bone marrow-derived mesenchymal stem cells into neurons. First, we used GeneChip technology to analyze the expression of miRNAs inbone marrow-derived mesen-chymal stem cells, neural stem cells and neurons. miR-124 expression was substantially reduced inbone marrow-derived mesenchymal stem cells compared with the other cell types. We con-structed a lentiviral vector overexpressing miR-124 and transfected it intobone marrow-derived mesenchymal stem cells. Intracellular expression levels of the neuronal early markersβ-III tu-bulin and microtubule-associated protein-2 were signiifcantly increased, and apoptosis induced by oxygen and glucose deprivation was reduced in transfected cells. After miR-124-transfected bone marrow-derived mesenchymal stem cells were transplanted into the injured rat spinal cord, a large number of cells positive for the neuronal marker neurofilament-200 were observed in the transplanted region. The Basso-Beattie-Bresnahan locomotion scores showed that the motor function of the hind limb of rats with spinal cord injury was substantially improved. These re-sults suggest that miR-124 plays an important role in the differentiation ofbone marrow-derived mesenchymal stem cells into neurons. Our ifndings should facilitate the development of novel strategies for enhancing the therapeutic efifcacy ofbone marrow-derived mesenchymal stem cell transplantation for spinal cord injury.

  13. Nanotopography induced contact guidance of the F11 cell line during neuronal differentiation: a neuronal model cell line for tissue scaffold development

    Wieringa, Paul; Tonazzini, Ilaria; Micera, Silvestro; Cecchini, Marco

    2012-07-01

    The F11 hybridoma, a dorsal root ganglion-derived cell line, was used to investigate the response of nociceptive sensory neurons to nanotopographical guidance cues. This established this cell line as a model of peripheral sensory neuron growth for tissue scaffold design. Cells were seeded on substrates of cyclic olefin copolymer (COC) films imprinted via nanoimprint lithography (NIL) with a grating pattern of nano-scale grooves and ridges. Different ridge widths were employed to alter the focal adhesion formation, thereby changing the cell/substrate interaction. Differentiation was stimulated with forskolin in culture medium consisting of either 1 or 10% fetal bovine serum (FBS). Per medium condition, similar neurite alignment was achieved over the four day period, with the 1% serum condition exhibiting longer, more aligned neurites. Immunostaining for focal adhesions found the 1% FBS condition to also have fewer, less developed focal adhesions. The robust response of the F11 to guidance cues further builds on the utility of this cell line as a sensory neuron model, representing a useful tool to explore the design of regenerative guidance tissue scaffolds.

  14. Hyaluronan and Fibrin Biomaterial as Scaffolds for Neuronal Differentiation of Adult Stem Cells Derived from Adipose Tissue and Skin

    Chiara Gardin

    2011-10-01

    Full Text Available Recently, we have described a simple protocol to obtain an enriched culture of adult stem cells organized in neurospheres from two post-natal tissues: skin and adipose tissue. Due to their possible application in neuronal tissue regeneration, here we tested two kinds of scaffold well known in tissue engineering application: hyaluronan based membranes and fibrin-glue meshes. Neurospheres from skin and adipose tissue were seeded onto two scaffold types: hyaluronan based membrane and fibrin-glue meshes. Neurospheres were then induced to acquire a glial and neuronal-like phenotype. Gene expression, morphological feature and chromosomal imbalance (kariotype were analyzed and compared. Adipose and skin derived neurospheres are able to grow well and to differentiate into glial/neuron cells without any chromosomal imbalance in both scaffolds. Adult cells are able to express typical cell surface markers such as S100; GFAP; nestin; βIII tubulin; CNPase. In summary, we have demonstrated that neurospheres isolated from skin and adipose tissues are able to differentiate in glial/neuron-like cells, without any chromosomal imbalance in two scaffold types, useful for tissue engineering application: hyaluronan based membrane and fibrin-glue meshes.

  15. Differentiation and molecular heterogeneity of inhibitory and excitatory neurons associated with midbrain dopaminergic nuclei.

    Lahti, Laura; Haugas, Maarja; Tikker, Laura; Airavaara, Mikko; Voutilainen, Merja H; Anttila, Jenni; Kumar, Suman; Inkinen, Caisa; Salminen, Marjo; Partanen, Juha

    2016-02-01

    Local inhibitory GABAergic and excitatory glutamatergic neurons are important for midbrain dopaminergic and hindbrain serotonergic pathways controlling motivation, mood, and voluntary movements. Such neurons reside both within the dopaminergic nuclei, and in adjacent brain structures, including the rostromedial and laterodorsal tegmental nuclei. Compared with the monoaminergic neurons, the development, heterogeneity, and molecular characteristics of these regulatory neurons are poorly understood. We show here that different GABAergic and glutamatergic subgroups associated with the monoaminergic nuclei express specific transcription factors. These neurons share common origins in the ventrolateral rhombomere 1, where the postmitotic selector genes Tal1, Gata2 and Gata3 control the balance between the generation of inhibitory and excitatory neurons. In the absence of Tal1, or both Gata2 and Gata3, the GABAergic precursors adopt glutamatergic fates and populate the glutamatergic nuclei in excessive numbers. Together, our results uncover developmental regulatory mechanisms, molecular characteristics, and heterogeneity of central regulators of monoaminergic circuits. PMID:26718003

  16. Nanoparticle-mediated transcriptional modification enhances neuronal differentiation of human neural stem cells following transplantation in rat brain.

    Li, Xiaowei; Tzeng, Stephany Y; Liu, Xiaoyan; Tammia, Markus; Cheng, Yu-Hao; Rolfe, Andrew; Sun, Dong; Zhang, Ning; Green, Jordan J; Wen, Xuejun; Mao, Hai-Quan

    2016-04-01

    Strategies to enhance survival and direct the differentiation of stem cells in vivo following transplantation in tissue repair site are critical to realizing the potential of stem cell-based therapies. Here we demonstrated an effective approach to promote neuronal differentiation and maturation of human fetal tissue-derived neural stem cells (hNSCs) in a brain lesion site of a rat traumatic brain injury model using biodegradable nanoparticle-mediated transfection method to deliver key transcriptional factor neurogenin-2 to hNSCs when transplanted with a tailored hyaluronic acid (HA) hydrogel, generating larger number of more mature neurons engrafted to the host brain tissue than non-transfected cells. The nanoparticle-mediated transcription activation method together with an HA hydrogel delivery matrix provides a translatable approach for stem cell-based regenerative therapy. PMID:26828681

  17. Multidimensional Characterization and Differentiation of Neurons in the Anteroventral Cochlear Nucleus

    Marei Typlt; Bernhard Englitz; Mandy Sonntag; Susanne Dehmel; Cornelia Kopp-Scheinpflug; Rudolf Ruebsamen

    2012-01-01

    Multiple parallel auditory pathways ascend from the cochlear nucleus. It is generally accepted that the origin of these pathways are distinct groups of neurons differing in their anatomical and physiological properties. In extracellular in vivo recordings these neurons are typically classified on the basis of their peri-stimulus time histogram. In the present study we reconsider the question of classification of neurons in the anteroventral cochlear nucleus (AVCN) by taking a wider range of r...

  18. CEND1 and NEUROGENIN2 Reprogram Mouse Astrocytes and Embryonic Fibroblasts to Induced Neural Precursors and Differentiated Neurons

    Katerina Aravantinou-Fatorou

    2015-09-01

    Full Text Available Recent studies demonstrate that astroglia from non-neurogenic brain regions can be reprogrammed into functional neurons through forced expression of neurogenic factors. Here we explored the effect of CEND1 and NEUROG2 on reprogramming of mouse cortical astrocytes and embryonic fibroblasts. Forced expression of CEND1, NEUROG2, or both resulted in acquisition of induced neuronal cells expressing subtype-specific markers, while long-term live-cell imaging highlighted the existence of two different modes of neuronal trans-differentiation. Of note, a subpopulation of CEND1 and NEUROG2 double-transduced astrocytes formed spheres exhibiting neural stem cell properties. mRNA and protein expression studies revealed a reciprocal feedback loop existing between the two molecules, while knockdown of endogenous CEND1 demonstrated that it is a key mediator of NEUROG2-driven neuronal reprogramming. Our data suggest that common reprogramming mechanisms exist driving the conversion of lineage-distant somatic cell types to neurons and reveal a critical role for CEND1 in NEUROG2-driven astrocytic reprogramming.

  19. Differential regulation of Aβ42-induced neuronal C1q synthesis and microglial activation

    Tenner Andrea J

    2005-01-01

    Full Text Available Abstract Expression of C1q, an early component of the classical complement pathway, has been shown to be induced in neurons in hippocampal slices, following accumulation of exogenous Aβ42. Microglial activation was also detected by surface marker expression and cytokine production. To determine whether C1q induction was correlated with intraneuronal Aβ and/or microglial activation, D-(--2-amino-5-phosphonovaleric acid (APV, an NMDA receptor antagonist and glycine-arginine-glycine-aspartic acid-serine-proline peptide (RGD, an integrin receptor antagonist, which blocks and enhances Aβ42 uptake, respectively, were assessed for their effect on neuronal C1q synthesis and microglial activation. APV inhibited, and RGD enhanced, microglial activation and neuronal C1q expression. However, addition of Aβ10–20 to slice cultures significantly reduced Aβ42 uptake and microglial activation, but did not alter the Aβ42-induced neuronal C1q expression. Furthermore, Aβ10–20 alone triggered C1q production in neurons, demonstrating that neither neuronal Aβ42 accumulation, nor microglial activation is required for neuronal C1q upregulation. These data are compatible with the hypothesis that multiple receptors are involved in Aβ injury and signaling in neurons. Some lead to neuronal C1q induction, whereas other(s lead to intraneuronal accumulation of Aβ and/or stimulation of microglia.

  20. Neural progenitor differentiation patterns of surface and secreted proteins for cell-replacement therapies of neuronal disorders

    Tylečková, Jiřina; Valeková, Ivona; Žižková, Martina; Rákocyová, Michaela; Maršala, S.; Maršala, M.; Gadher, S. J.; Kovářová, Hana

    Split : Mediterranean Institute for life Sciences, 2015. s. 1-1. [OMICs in Biomedical Research. 08.06.2015-12.06.2015, Split] R&D Projects: GA MŠk ED2.1.00/03.0124; GA TA ČR(CZ) TA01011466 Institutional support: RVO:67985904 Keywords : neuronal differentiation * surface N-glycoproteome * cell adhesion proteins Subject RIV: EB - Genetics ; Molecular Biology

  1. Induction of apoptotic death and retardation of neuronal differentiation of human neural stem cells by sodium arsenite treatment

    Ivanov, Vladimir N.; Hei, Tom K.

    2012-01-01

    Chronic arsenic toxicity is a global health problem that affects more than 100 million people worldwide. Long-term health effects of inorganic sodium arsenite in drinking water may result in skin, lung and liver cancer and severe neurological abnormalities. We investigated in the present study whether sodium arsenite affects signaling pathways that control cell survival, proliferation and neuronal differentiation of human neural stem cells (NSC). We demonstrated that the critical signaling pa...

  2. Overexpression of microRNA-124 promotes the neuronal differentiation of bone marrow-derived mesenchymal stem cells

    Zou, Defeng; Chen, Yi; Han, Yaxin; Lv, Chen; Tu, Guanjun

    2014-01-01

    microRNAs (miRNAs) play an important regulatory role in the self-renewal and differentiation of stem cells. In this study, we examined the effects of miRNA-124 (miR-124) overexpression in bone marrow-derived mesenchymal stem cells. In particular, we focused on the effect of overexpression on the differentiation of bone marrow-derived mesenchymal stem cells into neurons. First, we used GeneChip technology to analyze the expression of miRNAs in bone marrow-derived mesenchymal stem cells, neural...

  3. BDNF Increases Survival and Neuronal Differentiation of Human Neural Precursor Cells Cotransplanted with a Nanofiber Gel to the Auditory Nerve in a Rat Model of Neuronal Damage

    Yu Jiao

    2014-01-01

    Full Text Available Objectives. To study possible nerve regeneration of a damaged auditory nerve by the use of stem cell transplantation. Methods. We transplanted HNPCs to the rat AN trunk by the internal auditory meatus (IAM. Furthermore, we studied if addition of BDNF affects survival and phenotypic differentiation of the grafted HNPCs. A bioactive nanofiber gel (PA gel, in selected groups mixed with BDNF, was applied close to the implanted cells. Before transplantation, all rats had been deafened by a round window niche application of β-bungarotoxin. This neurotoxin causes a selective toxic destruction of the AN while keeping the hair cells intact. Results. Overall, HNPCs survived well for up to six weeks in all groups. However, transplants receiving the BDNF-containing PA gel demonstrated significantly higher numbers of HNPCs and neuronal differentiation. At six weeks, a majority of the HNPCs had migrated into the brain stem and differentiated. Differentiated human cells as well as neurites were observed in the vicinity of the cochlear nucleus. Conclusion. Our results indicate that human neural precursor cells (HNPC integration with host tissue benefits from additional brain derived neurotrophic factor (BDNF treatment and that these cells appear to be good candidates for further regenerative studies on the auditory nerve (AN.

  4. The role of the PDK1/PKB kinases in regulating neuronal survival and differentiation: characterization of the PDK1 K465E knock-in mice

    Zurashvili, Tinatin

    2015-01-01

    Neuronal cell death programmes are counteracted by survival signals during development in order to maintain the tissue homeostasis. Neuronal differentiation is a mechanism generating functionally integrated neuronal cells from their progenitors. These processes appear to be mediated via activation of the Ras/Raf/MAPK and the PI3K/PDK1/PKB signaling pathways and are associated with a selective increase in protein translation. Protein kinase B (PKB/Akt) is a serine/threonine prot...

  5. Differential neuronal vulnerability identifies IGF-2 as a protective factor in ALS.

    Allodi, Ilary; Comley, Laura; Nichterwitz, Susanne; Nizzardo, Monica; Simone, Chiara; Benitez, Julio Aguila; Cao, Ming; Corti, Stefania; Hedlund, Eva

    2016-01-01

    The fatal disease amyotrophic lateral sclerosis (ALS) is characterized by the loss of somatic motor neurons leading to muscle wasting and paralysis. However, motor neurons in the oculomotor nucleus, controlling eye movement, are for unknown reasons spared. We found that insulin-like growth factor 2 (IGF-2) was maintained in oculomotor neurons in ALS and thus could play a role in oculomotor resistance in this disease. We also showed that IGF-1 receptor (IGF-1R), which mediates survival pathways upon IGF binding, was highly expressed in oculomotor neurons and on extraocular muscle endplate. The addition of IGF-2 induced Akt phosphorylation, glycogen synthase kinase-3β phosphorylation and β-catenin levels while protecting ALS patient motor neurons. IGF-2 also rescued motor neurons derived from spinal muscular atrophy (SMA) patients from degeneration. Finally, AAV9::IGF-2 delivery to muscles of SOD1(G93A) ALS mice extended life-span by 10%, while preserving motor neurons and inducing motor axon regeneration. Thus, our studies demonstrate that oculomotor-specific expression can be utilized to identify candidates that protect vulnerable motor neurons from degeneration. PMID:27180807

  6. Immortalized mesenchymal stem cells: an alternative to primary mesenchymal stem cells in neuronal differentiation and neuroregeneration associated studies

    Gong Min

    2011-11-01

    Full Text Available Abstract Background Mesenchymal stem cells (MSCs can be induced to differentiate into neuronal cells under appropriate cellular conditions and transplanted in brain injury and neurodegenerative diseases animal models for neuroregeneration studies. In contrast to the embryonic stem cells (ESCs, MSCs are easily subject to aging and senescence because of their finite ability of self-renewal. MSCs senescence seriously affected theirs application prospects as a promising tool for cell-based regenerative medicine and tissue engineering. In the present study, we established a reversible immortalized mesenchymal stem cells (IMSCs line by using SSR#69 retrovirus expressing simian virus 40 large T (SV40T antigen as an alternative to primary MSCs. Methods The retroviral vector SSR#69 expressing simian virus 40 large T (SV40T antigen was used to construct IMSCs. IMSCs were identified by flow cytometry to detect cell surface makers. To investigate proliferation and differentiation potential of IMSCs, cell growth curve determination and mesodermal trilineage differentiation tests were performed. Neuronal differentiation characteristics of IMSCs were detected in vitro. Before IMSCs transplantation, we excluded its tumorigenicity in nude mice firstly. The Morris water maze tests and shuttle box tests were performed five weeks after HIBD models received cells transplantation therapy. Results In this study, reversible IMSCs were constructed successfully and had the similar morphology and cell surface makers as primary MSCs. IMSCs possessed better ability of proliferation and anti-senescence compared with primary MSCs, while maintained multilineage differentiation capacity. Neural-like cells derived from IMSCs had similar expressions of neural-specific genes, protein expression patterns and resting membrane potential (RMP compared with their counterparts derived from primary MSCs. There was no bump formation in nude mice subcutaneously injected with IMSCs. IMSCs

  7. Dual Inhibition of Activin/Nodal/TGF-β and BMP Signaling Pathways by SB431542 and Dorsomorphin Induces Neuronal Differentiation of Human Adipose Derived Stem Cells

    Vedavathi Madhu

    2016-01-01

    Full Text Available Damage to the nervous system can cause devastating diseases or musculoskeletal dysfunctions and transplantation of progenitor stem cells can be an excellent treatment option in this regard. Preclinical studies demonstrate that untreated stem cells, unlike stem cells activated to differentiate into neuronal lineage, do not survive in the neuronal tissues. Conventional methods of inducing neuronal differentiation of stem cells are complex and expensive. We therefore sought to determine if a simple, one-step, and cost effective method, previously reported to induce neuronal differentiation of embryonic stem cells and induced-pluripotent stem cells, can be applied to adult stem cells. Indeed, dual inhibition of activin/nodal/TGF-β and BMP pathways using SB431542 and dorsomorphin, respectively, induced neuronal differentiation of human adipose derived stem cells (hADSCs as evidenced by formation of neurite extensions, protein expression of neuron-specific gamma enolase, and mRNA expression of neuron-specific transcription factors Sox1 and Pax6 and matured neuronal marker NF200. This process correlated with enhanced phosphorylation of p38, Erk1/2, PI3K, and Akt1/3. Additionally, in vitro subcutaneous implants of SB431542 and dorsomorphin treated hADSCs displayed significantly higher expression of active-axonal-growth-specific marker GAP43. Our data offers novel insights into cell-based therapies for the nervous system repair.

  8. Brain-derived neurotrophic factor differentially modulates excitability of two classes of hippocampal output neurons.

    Graves, A R; Moore, S J; Spruston, N; Tryba, A K; Kaczorowski, C C

    2016-08-01

    Brain-derived neurotrophic factor (BDNF) plays an important role in hippocampus-dependent learning and memory. Canonically, this has been ascribed to an enhancing effect on neuronal excitability and synaptic plasticity in the CA1 region. However, it is the pyramidal neurons in the subiculum that form the primary efferent pathways conveying hippocampal information to other areas of the brain, and yet the effect of BDNF on these neurons has remained unexplored. We present new data that BDNF regulates neuronal excitability and cellular plasticity in a much more complex manner than previously suggested. Subicular pyramidal neurons can be divided into two major classes, which have different electrophysiological and morphological properties, different requirements for the induction of plasticity, and different extrahippocampal projections. We found that BDNF increases excitability in one class of subicular pyramidal neurons yet decreases excitability in the other class. Furthermore, while endogenous BDNF was necessary for the induction of synaptic plasticity in both cell types, BDNF enhanced intrinsic plasticity in one class of pyramidal neurons yet suppressed intrinsic plasticity in the other. Taken together, these data suggest a novel role for BDNF signaling, as it appears to dynamically and bidirectionally regulate the output of hippocampal information to different regions of the brain. PMID:27146982

  9. VASCULAR ENDOTHELIAL GROWTH FACTOR MEDIATES ATORVASTATIN-INDUCED MAMMALIAN ACHAETE-SCUTE HOMOLOGUE-1 GENE EXPRESSION AND NEURONAL DIFFERENTIATION AFTER STROKE IN RETIRED BREEDER RATS

    Chen, J.; ZACHAREK, A.; Li, A; Zhang, C; Ding, J; Roberts, C.; Lu, M.; KAPKE, A.; CHOPP, M.

    2006-01-01

    Neurogenesis declines with advancing age. The mammalian achaete-scute homologue-1 encodes a basic helix–loop– helix transcription factor, which controls neuronal differentiation. In this study, we first tested whether atorvastatin treatment enhances neurological functional outcome and neuronal differentiation after stroke in retired breeder 12 month rats. Rats were subjected to middle cerebral artery occlusion and treated with or without atorvastatin (3 mg/kg) for 7 days. Atorvastatin signifi...

  10. In vitro induction and differentiation of umbilical cord mesenchymal stem cells into neuron-like cells by alltrans retinoic acid

    Wei; Jin; Yao-Peng; Xu; An-Huai; Yang; Yi-Qiao; Xing

    2015-01-01

    AIM: To determine the optimal concentration for inducing the differentiation of human umbilical cord-derived mesenchymal stem cells(h UC-MSCs) into neuron-like cells, although it is understood that all-trans retinoic acid(ATRA) regulates cell proliferation in the nervous system by modulating the balance between mitosis and apoptosis.METHODS: The abilities of ATRA to promote apoptosis as well as neural differentiation were assessed in cultured h UC-MSCs by morphological observation, MTT assay, annexin V-FITC/PI flow cytometry and immunocytochemistry.RESULTS: The data showed that low concentrations of ATRA(0.5 μmol, 0.25 μmol) had no effect on the number of cells. However, treatment with 1.0 μmol or 2.0 μmol ATRA induced a 24.16% and 52.67% reduction in cell number, respectively, compared with vehicle-treated cultures. Further, 4.0 μmol ATRA had a potent effect on cell number, with almost no adherent cells recovered after 24 h. We further showed that 0.5 μmol ATRA caused these cells to express characteristic markers of neuronal progenitor cells.CONCLUSION: Taken together, we conclude that ATRA has a dose-dependent influence on the neural differentiation and apoptosis of h UC-MSCs. These findings have implications on the use of ATRA-differentiated h UC-MSCs for the study of neural degeneration diseases.