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Sample records for differentiated neuronal cells

  1. Schwann cells induce neuronal differentiation of bone marrow stromal cells.

    PubMed

    Zurita, Mercedes; Vaquero, Jesús; Oya, Santiago; Miguel, Miriam

    2005-04-04

    Bone marrow stromal cells are multipotent stem cells that have the potential to differentiate into bone, cartilage, fat and muscle. Recently, bone marrow stromal cells have been shown to have the capacity to differentiate into neurons under specific experimental conditions, using chemical factors. We now describe how bone marrow stromal cells can be induced to differentiate into neuron-like cells when they are co-cultured with Schwann cells. When compared with chemical differentiation, expression of neuronal differentiation markers begins later, but one week after beginning co-culture, most bone marrow stromal cells showed a typical neuronal morphology. Our present findings support the transdifferentiation of bone marrow stromal cells, and the potential utility of these cells for the treatment of degenerative and acquired disorders of the nervous system.

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

    PubMed Central

    Takeda, Yuji S.; Xu, Qiaobing

    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 week. After the treatment with PC12-derived exosomes, MSCs developed neuron-like morphology, and gene and protein expressions of neuronal markers were upregulated. Microarray analysis showed that the expression of miR-125b, which is known to play a role in neuronal differentiation of stem cells, was much higher in PC12-derived exosomes than in exosomes from B16-F10 melanoma cells. These results suggest that the delivery of miRNAs contained in PC12-derived exosomes is a possible mechanism explaining the neuronal differentiation of MSC. PMID:26248331

  3. Sambucus williamsii induced embryonic stem cells differentiated into neurons.

    PubMed

    Liu, Shih-Ping; Hsu, Chien-Yu; Fu, Ru-Huei; Huang, Yu-Chuen; Chen, Shih-Yin; Lin, Shinn-Zong; Shyu, Woei-Cherng

    2015-01-01

    The pluripotent stem cells, including embryonic stem cells (ESCs), are capable of self-renewal and differentiation into any cell type, thus making them the focus of many clinical application studies. However, the efficiency of ESCs differentiated into neurons needs to improve. In this study, we tried to increase efficiently to a neural fate in the presence of various transitional Chinese medicines through a three-step differentiation strategy. From extracts of 10 transitional Chinese medicine candidates, we determined that Sambucus williamsii (SW) extract triggers the up-regulation of Nestin and Tuj1 (neuron cells markers) gene expression levels. After determining the different concentrations of SW extract, the number of neurons in the 200 μg/ml SW extract group was higher than the control, 50, 100, and 400 μg/ml SW extract groups. In addition, the number of neurons in the 200 μg/ml SW extract group was higher and higher after each time passage (three times). We also detected the Oct4, Sox2 (stem cells markers), Tuj1, and Nestin genes expression levels by RT-PCR. In the differentiated process, Oct4 and Sox2 genes decreased while the Tuj1 and Nestin genes expression levels increased. In summary, we demonstrated that SW could induce pluripotent stem cells differentiated into neurons. Thus, SW might become a powerful material for neurons-differentiating strategies.

  4. Labeling of neuronal differentiation and neuron cells with biocompatible fluorescent nanodiamonds

    NASA Astrophysics Data System (ADS)

    Hsu, Tzu-Chia; Liu, Kuang-Kai; Chang, Huan-Cheng; Hwang, Eric; Chao, Jui-I.

    2014-05-01

    Nanodiamond is a promising carbon nanomaterial developed for biomedical applications. Here, we show fluorescent nanodiamond (FND) with the biocompatible properties that can be used for the labeling and tracking of neuronal differentiation and neuron cells derived from embryonal carcinoma stem (ECS) cells. The fluorescence intensities of FNDs were increased by treatment with FNDs in both the mouse P19 and human NT2/D1 ECS cells. FNDs were taken into ECS cells; however, FNDs did not alter the cellular morphology and growth ability. Moreover, FNDs did not change the protein expression of stem cell marker SSEA-1 of ECS cells. The neuronal differentiation of ECS cells could be induced by retinoic acid (RA). Interestingly, FNDs did not affect on the morphological alteration, cytotoxicity and apoptosis during the neuronal differentiation. Besides, FNDs did not alter the cell viability and the expression of neuron-specific marker β-III-tubulin in these differentiated neuron cells. The existence of FNDs in the neuron cells can be identified by confocal microscopy and flow cytometry. Together, FND is a biocompatible and readily detectable nanomaterial for the labeling and tracking of neuronal differentiation process and neuron cells from stem cells.

  5. Labeling of neuronal differentiation and neuron cells with biocompatible fluorescent nanodiamonds.

    PubMed

    Hsu, Tzu-Chia; Liu, Kuang-Kai; Chang, Huan-Cheng; Hwang, Eric; Chao, Jui-I

    2014-05-16

    Nanodiamond is a promising carbon nanomaterial developed for biomedical applications. Here, we show fluorescent nanodiamond (FND) with the biocompatible properties that can be used for the labeling and tracking of neuronal differentiation and neuron cells derived from embryonal carcinoma stem (ECS) cells. The fluorescence intensities of FNDs were increased by treatment with FNDs in both the mouse P19 and human NT2/D1 ECS cells. FNDs were taken into ECS cells; however, FNDs did not alter the cellular morphology and growth ability. Moreover, FNDs did not change the protein expression of stem cell marker SSEA-1 of ECS cells. The neuronal differentiation of ECS cells could be induced by retinoic acid (RA). Interestingly, FNDs did not affect on the morphological alteration, cytotoxicity and apoptosis during the neuronal differentiation. Besides, FNDs did not alter the cell viability and the expression of neuron-specific marker β-III-tubulin in these differentiated neuron cells. The existence of FNDs in the neuron cells can be identified by confocal microscopy and flow cytometry. Together, FND is a biocompatible and readily detectable nanomaterial for the labeling and tracking of neuronal differentiation process and neuron cells from stem cells.

  6. Integrating human stem cell expansion and neuronal differentiation in bioreactors

    PubMed Central

    Serra, Margarida; Brito, Catarina; Costa, Eunice M; Sousa, Marcos FQ; Alves, Paula M

    2009-01-01

    Background Human stem cells are cellular resources with outstanding potential for cell therapy. However, for the fulfillment of this application, major challenges remain to be met. Of paramount importance is the development of robust systems for in vitro stem cell expansion and differentiation. In this work, we successfully developed an efficient scalable bioprocess for the fast production of human neurons. Results The expansion of undifferentiated human embryonal carcinoma stem cells (NTera2/cl.D1 cell line) as 3D-aggregates was firstly optimized in spinner vessel. The media exchange operation mode with an inoculum concentration of 4 × 105 cell/mL was the most efficient strategy tested, with a 4.6-fold increase in cell concentration achieved in 5 days. These results were validated in a bioreactor where similar profile and metabolic performance were obtained. Furthermore, characterization of the expanded population by immunofluorescence microscopy and flow cytometry showed that NT2 cells maintained their stem cell characteristics along the bioreactor culture time. Finally, the neuronal differentiation step was integrated in the bioreactor process, by addition of retinoic acid when cells were in the middle of the exponential phase. Neurosphere composition was monitored and neuronal differentiation efficiency evaluated along the culture time. The results show that, for bioreactor cultures, we were able to increase significantly the neuronal differentiation efficiency by 10-fold while reducing drastically, by 30%, the time required for the differentiation process. Conclusion The culture systems developed herein are robust and represent one-step-forward towards the development of integrated bioprocesses, bridging stem cell expansion and differentiation in fully controlled bioreactors. PMID:19772662

  7. Human pluripotent stem cell differentiation into authentic striatal projection neurons.

    PubMed

    Delli Carri, Alessia; Onorati, Marco; Castiglioni, Valentina; Faedo, Andrea; Camnasio, Stefano; Toselli, Mauro; Biella, Gerardo; Cattaneo, Elena

    2013-08-01

    Here we present the principles and steps of a protocol that we have recently developed for the differentiation of hES/iPS cells into the authentic human striatal projection medium spiny neurons (MSNs) that die in Huntington's Disease (HD). Authenticity is judged by the convergence of multiple features within individual cells. Our procedure lasts 80 days and couples neural induction via BMP/TGF-β inhibition with exposure to the developmental factors sonic hedgehog (SHH) and dickkopf1 (DKK-1) to drive ventral telencephalic specification, followed by terminal differentiation [1]. Authenticity of the resulting neuronal population is monitored by the appearance of FOXG1(+)/GSX2(+) progenitor cells of the lateral ganglionic eminence (LGE) at day 15-25 of differentiation, followed by appearance of CTIP2-, FOXP1- and FOXP2-positive cells at day 45. These precursor cells then mature into MAP2(+)/GABA(+) neurons with 20 % of them ultimately co-expressing the DARPP-32 and CTIP2 diagnostic markers and carrying electrophysiological properties expected for fully functional MSNs.The protocol is characterized by its replicability in at least three human pluripotent cell lines. Altogether this protocol defines a useful platform for in vitro developmental neurobiology studies, drug screening, and regenerative medicine approaches.

  8. Accelerated neuronal differentiation toward motor neuron lineage from human embryonic stem cell line (H9).

    PubMed

    Lu, David; Chen, Eric Y T; Lee, Philip; Wang, Yung-Chen; Ching, Wendy; Markey, Christopher; Gulstrom, Chase; Chen, Li-Ching; Nguyen, Thien; Chin, Wei-Chun

    2015-03-01

    Motor neurons loss plays a pivotal role in the pathoetiology of various debilitating diseases such as, but not limited to, amyotrophic lateral sclerosis, primary lateral sclerosis, progressive muscular atrophy, progressive bulbar palsy, pseudobulbar palsy, and spinal muscular atrophy. However, advancement in motor neuron replacement therapy has been significantly constrained by the difficulties in large-scale production at a cost-effective manner. Current methods to derive motor neuron heavily rely on biochemical stimulation, chemical biological screening, and complex physical cues. These existing methods are seriously challenged by extensive time requirements and poor yields. An innovative approach that overcomes prior hurdles and enhances the rate of successful motor neuron transplantation in patients is of critical demand. Iron, a trace element, is indispensable for the normal development and function of the central nervous system. Whether ferric ions promote neuronal differentiation and subsequently promote motor neuron lineage has never been considered. Here, we demonstrate that elevated iron concentration can drastically accelerate the differentiation of human embryonic stem cells (hESCs) toward motor neuron lineage potentially via a transferrin mediated pathway. HB9 expression in 500 nM iron-treated hESCs is approximately twofold higher than the control. Moreover, iron treatment generated more matured and functional motor neuron-like cells that are ∼1.5 times more sensitive to depolarization when compared to the control. Our methodology renders an expedited approach to harvest motor neuron-like cells for disease, traumatic injury regeneration, and drug screening.

  9. Differentiation of Spermatogonia Stem Cells into Functional Mature Neurons Characterized with Differential Gene Expression.

    PubMed

    Bojnordi, Maryam Nazm; Azizi, Hossein; Skutella, Thomas; Movahedin, Mansoureh; Pourabdolhossein, Fereshteh; Shojaei, Amir; Hamidabadi, Hatef Ghasemi

    2016-09-19

    Transplantation of embryonic stem cells (ESCs) is a promising therapeutic approach for the treatment of neurodegenerative diseases. However, ESCs are not usable clinically due to immunological and ethical limitations. The identification of an alternative safe cell source opens novel options via autologous transplantation in neuro-regeneration circumventing these problems. Here, we examined the neurogenic capacity of embryonic stem-like cells (ES-like cells) derived from the testis using neural growth factor inducers and utilized them to generate functional mature neurons. The neuronal differentiation of ES-like cells is induced in three stages. Stage 1 is related to embryoid body (EB) formation. To induce neuroprogenitor cells, EBs were cultured in the presence of retinoic acid, N2 supplement and fibroblast growth factor followed by culturing in a neurobasal medium containing B27, N2 supplements for additional 10 days, to allow the maturation and development of neuronal progenitor cells. The neurogenic differentiation was confirmed by immunostaining for markers of mature neurons. The differentiated neurons were positive for Tuj1 and Tau1. Real-time PCR dates indicated the expression of Nestin and Neuro D (neuroprogenitor markers) in induced cells at the second stage of the differentiation protocol. The differentiated mature neurons exhibited the specific neuron markers Map2 and β-tubulin. The functional maturity of neurons was confirmed by an electrophysiological analysis of passive and active neural membrane properties. These findings indicated a differentiation capacity of ES-like cells derived from the testis to functionally mature neurons, which proposes them as a novel cell source for neuroregenerative medicine.

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

    PubMed

    Park, Kyoung Ho; Yeo, Sang Won; Troy, Frederic A

    2014-10-17

    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.

  11. Statins induce differentiation and cell death in neurons and astroglia.

    PubMed

    März, Pia; Otten, Uwe; Miserez, André R

    2007-01-01

    Statins are potent inhibitors of the hydroxy-methyl-glutaryl-coenzyme A reductase, the rate limiting enzyme for cholesterol biosynthesis. Experimental and clinical studies with statins suggest that they have beneficial effects on neurodegenerative disorders. Thus, it was of interest to characterize the direct effects of statins on CNS neurons and glial cells. We have treated defined cultures of neurons and astrocytes of newborn rats with two lipophilic statins, atorvastatin and simvastatin, and analyzed their effects on morphology and survival. Treatment of astrocytes with statins induced a time- and dose-dependent stellation, followed by apoptosis. Similarly, statins elicited programmed cell death of cerebellar granule neurons but with a higher sensitivity. Analysis of different signaling cascades revealed that statins fail to influence classical pathways such as Akt or MAP kinases, known to be activated in CNS cells. In addition, astrocyte stellation triggered by statins resembled dibutryl-cyclic AMP (db-cAMP) induced morphological differentiation. However, in contrast to db-cAMP, statins induced upregulation of low-density lipoprotein receptors, without affecting GFAP expression, indicating separate underlying mechanisms. Analysis of the cholesterol biosynthetic pathway revealed that lack of mevalonate and of its downstream metabolites, mainly geranylgeranyl-pyrophosphate (GGPP), is responsible for the statin-induced apoptosis of neurons and astrocytes. Moreover, astrocytic stellation triggered by statins was inhibited by mevalonate and GGPP. Interestingly, neuronal cell death was significantly reduced in astrocyte/neuron co-cultures treated with statins. We postulate that under these conditions signals provided by astrocytes, e.g., isoprenoids play a key role in neuronal survival.

  12. Differentiation of dental pulp stem cells into neuron-like cells in serum-free medium.

    PubMed

    Zainal Ariffin, Shahrul Hisham; Kermani, Shabnam; Zainol Abidin, Intan Zarina; Megat Abdul Wahab, Rohaya; Yamamoto, Zulham; Senafi, Sahidan; Zainal Ariffin, Zaidah; Abdul Razak, Mohamad

    2013-01-01

    Dental pulp tissue contains dental pulp stem cells (DPSCs). Dental pulp cells (also known as dental pulp-derived mesenchymal stem cells) are capable of differentiating into multilineage cells including neuron-like cells. The aim of this study was to examine the capability of DPSCs to differentiate into neuron-like cells without using any reagents or growth factors. DPSCs were isolated from teeth extracted from 6- to 8-week-old mice and maintained in complete medium. The cells from the fourth passage were induced to differentiate by culturing in medium without serum or growth factors. RT-PCR molecular analysis showed characteristics of Cd146(+) , Cd166(+) , and Cd31(-) in DPSCs, indicating that these cells are mesenchymal stem cells rather than hematopoietic stem cells. After 5 days of neuronal differentiation, the cells showed neuron-like morphological changes and expressed MAP2 protein. The activation of Nestin was observed at low level prior to differentiation and increased after 5 days of culture in differentiation medium, whereas Tub3 was activated only after 5 days of neuronal differentiation. The proliferation of the differentiated cells decreased in comparison to that of the control cells. Dental pulp stem cells are induced to differentiate into neuron-like cells when cultured in serum- and growth factor-free medium.

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

    SciTech Connect

    Park, Kyoung Ho; Yeo, Sang Won; Troy, Frederic A.

    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.

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

    PubMed Central

    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 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 significantly increased compared to that of control cells. Also BrdU incorporation was elevated in citalopram-treated cells. These findings 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. PMID:25206899

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

    PubMed

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

    2014-04-15

    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 significantly increased compared to that of control cells. Also BrdU incorporation was elevated in citalopram-treated cells. These findings 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. AUDITORY HAIR CELL EXPLANT CO-CULTURES PROMOTE THE DIFFERENTIATION OF STEM CELLS INTO BIPOLAR NEURONS

    PubMed Central

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

    2007-01-01

    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 with and without neurotrophic support, for their potential to direct the differentiation of mouse embryonic SCs into characteristic, bipolar, auditory neurons. The differentiation of SCs into neuron-like cells was facilitated by co-culture with auditory neurons or hair cell explants, isolated from post-natal day five rats. The most successful combination was the co-culture of hair cell explants with whole embryoid bodies, which resulted in significantly greater numbers of neurofilament positive, neuron-like cells. While further characterisation of these differentiated cells will be essential before transplantation studies commence, these data illustrate the effectiveness of post-natal explant co-culture, at providing valuable molecular cues for directed differentiation of SCs towards an auditory neuron lineage. PMID:17112512

  17. Differentiation of neuroepithelial stem cells into functional dopaminergic neurons in 3D microfluidic cell culture.

    PubMed

    Moreno, Edinson Lucumi; Hachi, Siham; Hemmer, Kathrin; Trietsch, Sebastiaan J; Baumuratov, Aidos S; Hankemeier, Thomas; Vulto, Paul; Schwamborn, Jens C; Fleming, Ronan M T

    2015-06-07

    A hallmark of Parkinson's disease is the progressive loss of nigrostriatal dopaminergic neurons. We derived human neuroepithelial cells from induced pluripotent stem cells and successfully differentiated them into dopaminergic neurons within phase-guided, three-dimensional microfluidic cell culture bioreactors. After 30 days of differentiation within the microfluidic bioreactors, in situ morphological, immunocytochemical and calcium imaging confirmed the presence of dopaminergic neurons that were spontaneously electrophysiologically active, a characteristic feature of nigrostriatal dopaminergic neurons in vivo. Differentiation was as efficient as in macroscopic culture, with up to 19% of differentiated neurons immunoreactive for tyrosine hydroxylase, the penultimate enzyme in the synthesis of dopamine. This new microfluidic cell culture model integrates the latest innovations in developmental biology and microfluidic cell culture to generate a biologically realistic and economically efficient route to personalised drug discovery for Parkinson's disease.

  18. Differential Localization of Acetylcholinesterase in Neuronal and Non-Neuronal Cells

    PubMed Central

    Thullbery, Matthew D.; Cox, Holly D.; Schule, Travis; Thompson, Charles M.; George, Kathleen M.

    2006-01-01

    Acetylcholinesterase (AChE) expression is regulated in cell types at the transcriptional and translational levels. In this study, we characterized and compared AChE catalytic activity, mRNA, protein expression, and protein localization in a variety of neuronal (SH-SY5Y neuroblastoma and primary cerebellar granule neurons (CGN)) and non-neuronal (LLC-MK2, HeLa, THP-1, and primary astrocytes) cell types. All cell lines expressed AChE catalytic activity; however the levels of AChE-specific activity were higher in neuronal cells than in the non-neuronal cell types. CGN expressed significantly more AChE activity than SH-SY5Y cells. All cell lines analyzed expressed AChE protein at equivalent levels, as well as mRNA splice variants. Localization of AChE was characterized by immunofluorescence and confocal microscopy. SH-SY5Y, CGN, and nerve-growth factor-differentiated PC-12 cells exhibited a pattern of AChE localization characterized as diffuse in the cytoplasm and punctate staining along neurites and on the plasma membrane. The localization in HeLa, LLC-MK2, fibroblasts, and undifferentiated PC-12 cells was significantly different than in neuronal cells—AChE was intensely localized in the perinuclear region, without staining near or on the plasma membrane. Based on the evidence presented here, we hypothesize that the presence of AChE protein doesn’t correlate with catalytic activity, and the diffuse cytoplasmic and plasma membrane localization of AChE is a property of neuronal cell types. PMID:16052514

  19. Immediate differentiation of neuronal cells from stem/progenitor-like cells in the avian iris tissues.

    PubMed

    Matsushita, Tamami; Fujihara, Ai; Royall, Lars; Kagiwada, Satoshi; Kosaka, Mitsuko; Araki, Masasuke

    2014-06-01

    A simple culture method that was recently developed in our laboratory was applied to the chick iris tissues to characterize neural stem/progenitor-like cells. Iris tissue is a non-neuronal tissue and does not contain any neuronal cells. In the present study we isolated iris tissues from chick embryos just prior to hatching. The isolated iris pigmented epithelium (IPE) or the stroma was embedded in Matrigel and cultured in Dulbecco's MEM supplemented with either fetal bovine serum or the synthetic serum replacement solution B27. Within 24 h of culture, elongated cells with long processes extended out from the explants of both tissues and were positively stained for various neuronal markers such as transitin, Tuj-1 and acetylated tubulin. After a longer culture period, cells positive for photoreceptor markers like rhodopsin, iodopsin and visinin were found, suggesting that the iris tissues contain retinal stem/progenitor-like cells. Several growth factors were examined to determine their effects on neuronal differentiation. EGF was shown to dramatically enhance neuronal cell differentiation, particularly the elongation of neuronal fibers. The addition of exogenous FGF2, however, did not show any positive effects on neuronal differentiation, although FGF signaling inhibitor, SU5402, suppressed neuronal differentiation. The results show that neuronal stem/progenitor-like cells can differentiate into neuronal cells immediately after they are transferred into an appropriate environment. This process did not require any exogenous factors, suggesting that neural stem/progenitor-like cells are simply suppressed from neuronal differentiation within the tissue, and isolation from the tissue releases the cells from the suppression mechanism.

  20. [Human Bone Marrow Mesenchymal Stem Cells Differentiate into Neuron-Like Cells In Vitro

    PubMed

    Guo, Zi-Kuan; Liu, Xiao-Dan; Hou, Chun-Mei; Li, Xiu-Sen; Mao, Ning

    2001-03-01

    Recent reports have clearly demonstrated that bone marrow cells can be differentiated into neurons, suggesting the existence of cells with the differentiation capacity in the bone marrow cell population. It is well known that hematopoietic stem cells as well as mesenchymal stem cells (MSCs) can be transplanted and therefore, alternative of them might contribute to the process. In the present study it was addressed whether marrow MSCs could be coaxed into neuron-specific antigen bearing cells and if so, whether the differentiated cells possess the cytochemical features seen in neurons. The report here showed that high concentration of 2-mercaptoethanol (2-ME) could induce some of the MSCs into neuron-like cells expressing neurofilament (NF) and neuron specific enolase (NSE). The neuron-like cells were alkaline phosphotase positive while the others MSCs were kept negative. Cells treated with 2-ME were positive for alpha-naphthylacetate esterase and glycogen and negative for acetylchonlinesterase, which were similar with the results seen in untreated cells. Furthermore, Nissel body was not observed in treated cells shown by toluidine blue staining. Therefore, it is likely that the cells described here seem not belong to the neuronal lineage. These findings, however, reveal that human MSCs could alter their committed fates under some circumstances.

  1. Neuronal expression of pathological tau accelerates oligodendrocyte progenitor cell differentiation

    PubMed Central

    Ossola, Bernardino; Zhao, Chao; Compston, Alastair; Pluchino, Stefano; Franklin, Robin J. M.

    2015-01-01

    Oligodendrocyte progenitor cell (OPC) differentiation is an important therapeutic target to promote remyelination in multiple sclerosis (MS). We previously reported hyperphosphorylated and aggregated microtubule‐associated protein tau in MS lesions, suggesting its involvement in axonal degeneration. However, the influence of pathological tau‐induced axonal damage on the potential for remyelination is unknown. Therefore, we investigated OPC differentiation in human P301S tau (P301S‐htau) transgenic mice, both in vitro and in vivo following focal demyelination. In 2‐month‐old P301S‐htau mice, which show hyperphosphorylated tau in neurons, we found atrophic axons in the spinal cord in the absence of prominent axonal degeneration. These signs of early axonal damage were associated with microgliosis and an upregulation of IL‐1β and TNFα. Following in vivo focal white matter demyelination we found that OPCs differentiated more efficiently in P301S‐htau mice than wild type (Wt) mice. We also found an increased level of myelin basic protein within the lesions, which however did not translate into increased remyelination due to higher susceptibility of P301S‐htau axons to demyelination‐induced degeneration compared to Wt axons. In vitro experiments confirmed higher differentiation capacity of OPCs from P301S‐htau mice compared with Wt mice‐derived OPCs. Because the OPCs from P301S‐htau mice do not ectopically express the transgene, and when isolated from newborn mice behave like Wt mice‐derived OPCs, we infer that their enhanced differentiation capacity must have been acquired through microenvironmental priming. Our data suggest the intriguing concept that damaged axons may signal to OPCs and promote their differentiation in the attempt at rescue by remyelination. GLIA 2016;64:457–471 PMID:26576485

  2. Immortalized human dorsal root ganglion cells differentiate into neurons with nociceptive properties.

    PubMed

    Raymon, H K; Thode, S; Zhou, J; Friedman, G C; Pardinas, J R; Barrere, C; Johnson, R M; Sah, D W

    1999-07-01

    A renewable source of human sensory neurons would greatly facilitate basic research and drug development. We had established previously conditionally immortalized human CNS cell lines that can differentiate into functional neurons (). We report here the development of an immortalized human dorsal root ganglion (DRG) clonal cell line, HD10.6, with a tetracycline-regulatable v-myc oncogene. In the proliferative condition, HD10.6 cells have a doubling time of 1.2 d and exhibit a neuronal precursor morphology. After differentiation of clone HD10.6 for 7 d in the presence of tetracycline, v-myc expression was suppressed, and >50% of the cells exhibited typical neuronal morphology, stained positively for neuronal cytoskeletal markers, and fired action potentials in response to current injection. Furthermore, this cell line was fate-restricted to a neuronal phenotype; even in culture conditions that promote Schwann cell or smooth muscle differentiation of neural crest stem cells, HD10.6 differentiated exclusively into neurons. Moreover, differentiated HD10.6 cells expressed sensory neuron-associated transcription factors and exhibited capsaicin sensitivity. Taken together, these data indicate that we have established an immortalized human DRG cell line that can differentiate into sensory neurons with nociceptive properties. The cell line HD10.6 represents the first example of a human sensory neuronal line and will be valuable for basic research, as well as for the discovery of novel drug targets and clinical candidates.

  3. Neurogenic differentiation of dental pulp stem cells to neuron-like cells in dopaminergic and motor neuronal inductive media.

    PubMed

    Chang, Chia-Chieh; Chang, Kai-Chun; Tsai, Shang-Jye; Chang, Hao-Hueng; Lin, Chun-Pin

    2014-12-01

    Dental pulp stem cells (DPSCs) have been proposed as a promising source of stem cells in nerve regeneration due to their close embryonic origin and ease of harvest. The aim of this study was to evaluate the efficacy of dopaminergic and motor neuronal inductive media on transdifferentiation of human DPSCs (hDPSCs) into neuron-like cells. Isolation, cultivation, and identification of hDPSCs were performed with morphological analyses and flow cytometry. The proliferation potential of DPSCs was evaluated with an XTT [(2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide)] assay. Media for the induction of dopaminergic and spinal motor neuronal differentiation were prepared. The efficacy of neural induction was evaluated by detecting the expression of neuron cell-specific cell markers in DPSCs by immunocytochemistry and quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). In the XTT assay, there was a 2.6- or 2-fold decrease in DPSCs cultured in dopaminergic or motor neuronal inductive media, respectively. The proportions of βIII-tubulin (βIII-tub), glial fibrillary acidic protein (GFAP), and oligodendrocyte (O1)-positive cells were significantly higher in DPSCs cultured in both neuronal inductive media compared with those cultured in control media. Furthermore, hDPSC-derived dopaminergic and spinal motor neuron cells after induction expressed a higher density of neuron cell markers than those before induction. These findings suggest that in response to the neuronal inductive stimuli, a greater proportion of DPSCs stop proliferation and acquire a phenotype resembling mature neurons. Such neural crest-derived adult DPSCs may provide an alternative stem cell source for therapy-based treatments of neuronal disorders and injury. Copyright © 2014. Published by Elsevier B.V.

  4. Thrombospondin-4 Promotes Neuronal Differentiation of NG2 Cells via the ERK/MAPK Pathway.

    PubMed

    Yang, Hai Jie; Ma, Shuang Ping; Ju, Fei; Zhang, Ya Ping; Li, Zhi Chao; Zhang, Bin Bin; Lian, Jun Jiang; Wang, Lei; Cheng, Bin Feng; Wang, Mian; Feng, Zhi Wei

    2016-12-01

    NG2-expressing neural progenitors can produce neurons in the central nervous system, providing a potential cell resource of therapy for neurological disorders. However, the mechanism underlying neuronal differentiation of NG2 cells remains largely unknown. In this report, we found that a thrombospondin (TSP) family member, TSP4, is involved in the neuronal differentiation of NG2 cells. When TSP4 was overexpressed, NG2 cells underwent spontaneous neuronal differentiation, as demonstrated by the induction of various neuronal differentiation markers such as NeuN, Tuj1, and NF200, at the messenger RNA and protein levels. In contrast, TSP4 silencing had an opposite effect on the expression of neuronal differentiation markers in NG2 cells. Next, the signaling pathway responsible for TSP4-mediated NG2 cell differentiation was investigated. We found that ERK but not p38 and AKT signaling was affected by TSP4 overexpression. Furthermore, when ERK signaling was blocked by the inhibitor U0126, the neuronal marker expression of NG2 cells was substantially increased. Together, these findings suggested that TSP4 promoted neuronal differentiation of NG2 cells by inhibiting ERK/MAPK signaling, revealing a novel role of TSP4 in cell fate specification of NG2 cells.

  5. Effect of taurine on human fetal neuron cells: proliferation and differentiation.

    PubMed

    Chen, X C; Pan, Z L; Liu, D S; Han, X

    1998-01-01

    The purpose of this study was to investigate the effect of taurine on human fetal brain neuron cell proliferation and differentiation using a glial-free, pure cerebral neuronal culture grown in a serum-free environment. We found that taurine was necessary for neuronal survival and neurite extension. Taurine, on the other hand, has a trophic effect on the human fetal brain cell, promoting both proliferation and differentiation. Results showed that DNA synthesis of the neurons was increased in a dose-dependent manner when neurons were cultured in the medium containing taurine (100-6400 microM). The protein content of neuronal cells was also significantly increased in the neurons treated with taurine as compared to the control. At day 15, the expression of neuron-specific enolase (NSE) was only detected in the neurons cultured in the medium containing taurine. These results establish taurine as a putative human fetal brain neurontrophic factor in the process of human brain development.

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

    PubMed

    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.

  7. Astroglial cells regulate the developmental timeline of human neurons differentiated from induced pluripotent stem cells.

    PubMed

    Tang, Xin; Zhou, Li; Wagner, Alecia M; Marchetto, Maria C N; Muotri, Alysson R; Gage, Fred H; Chen, Gong

    2013-09-01

    Neurons derived from human induced-pluripotent stem cells (hiPSCs) have been used to model a variety of neurological disorders. Different protocols have been used to differentiate hiPSCs into neurons, but their functional maturation process has varied greatly among different studies. Here, we demonstrate that laminin, a commonly used substrate for iPSC cultures, was inefficient to promote fully functional maturation of hiPSC-derived neurons. In contrast, astroglial substrate greatly accelerated neurodevelopmental processes of hiPSC-derived neurons. We have monitored the neural differentiation and maturation process for up to two months after plating hiPSC-derived neuroprogenitor cells (hNPCs) on laminin or astrocytes. We found that one week after plating hNPCs, there were 21-fold more newly differentiated neurons on astrocytes than on laminin. Two weeks after plating hNPCs, there were 12-fold more dendritic branches in neurons cultured on astrocytes than on laminin. Six weeks after plating hNPCs, the Na(+) and K(+) currents, as well as glutamate and GABA receptor currents, were 3-fold larger in neurons cultured on astrocytes than on laminin. And two months after plating hNPCs, the spontaneous synaptic events were 8-fold more in neurons cultured on astrocytes than on laminin. These results highlight a critical role of astrocytes in promoting neural differentiation and functional maturation of human neurons derived from hiPSCs. Moreover, our data presents a thorough developmental timeline of hiPSC-derived neurons in culture, providing important benchmarks for future studies on disease modeling and drug screening.

  8. Astroglial cells regulate the developmental timeline of human neurons differentiated from induced pluripotent stem cells

    PubMed Central

    Tang, Xin; Zhou, Li; Wagner, Alecia M.; Marchetto, Maria C.N.; Muotri, Alysson R.; Gage, Fred H.; Chen, Gong

    2014-01-01

    Neurons derived from human induced-pluripotent stem cells (hiPSCs) have been used to model a variety of neurological disorders. Different protocols have been used to differentiate hiPSCs into neurons, but their functional maturation process has varied greatly among different studies. Here, we demonstrate that laminin, a commonly used substrate for iPSC cultures, was inefficient to promote fully functional maturation of hiPSC-derived neurons. In contrast, astroglial substrate greatly accelerated neurodevelopmental processes of hiPSC-derived neurons. We have monitored the neural differentiation and maturation process for up to two months after plating hiPSC-derived neuroprogenitor cells (hNPCs) on laminin or astrocytes. We found that one week after plating hNPCs, there were 21-fold more newly differentiated neurons on astrocytes than on laminin. Two weeks after plating hNPCs, there were 12-fold more dendritic branches in neurons cultured on astrocytes than on laminin. Six weeks after plating hNPCs, the Na+ and K+ currents, as well as glutamate and GABA receptor currents, were 3-fold larger in neurons cultured on astrocytes than on laminin. And two months after plating hNPCs, the spontaneous synaptic events were 8-fold more in neurons cultured on astrocytes than on laminin. These results highlight a critical role of astrocytes in promoting neural differentiation and functional maturation of human neurons derived from hiPSCs. Moreover, our data presents a thorough developmental timeline of hiPSC-derived neurons in culture, providing important benchmarks for future studies on disease modeling and drug screening. PMID:23759711

  9. Morphological and electrophysiological features of mature neurons in differentiated skin-derived precursor cells.

    PubMed

    Liebmann, L; Beetz, C; Thorwarth, M; Deufel, T; Hübner, Ca

    2012-01-01

    In vitro modelling of neuronal pathologies is, in particular, demanding and a lot of efforts have been undertaken to differentiate skin derived precursor cells into neuronal cells. However, so far all attempts did not result in cells with functional features of neurons like the ability to generate action potentials or synaptic activity. Here, we report that simple modifications of the protocols result in neuronal cells that display action potentials and synaptic activity. We think that our observation is an important step to model individual neuronal pathologies in vitro.

  10. Brn4 and TH synergistically promote the differentiation of neural stem cells into dopaminergic neurons.

    PubMed

    Tan, Xuefeng; Zhang, Lei; Zhu, Huixia; Qin, Jianbing; Tian, Meiling; Dong, Chuanming; Li, Haoming; Jin, Guohua

    2014-06-13

    Neural stem cells (NSCs) are pluripotent cells capable of differentiation into dopaminergic (DA) neurons, which are the major cell types damaged in Parkinson's disease (PD). Therefore, NSCs are considered the most promising cell source for cell replacement therapy of PD. However, the poor differentiation and maturation of DA neurons and decreased cell survival after transplantation are a challenge. We have previously demonstrated that Brn4, a member of the POU domain family of transcription factors, induced the differentiation of NSCs into neurons and promoted their maturation. In this study, we directly transduced tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine biosynthesis, into NSCs to induce DA neuronal differentiation. However, these DA neurons were morphologically immature and seldom expressed dopamine transporter (DAT), a late marker of mature DA neurons. In contrast, TH co-transfected with Brn4 generated increased number of mature DA neurons. Furthermore, Brn4 significantly induced the expression of glial cell line-derived neurotrophic factor (GDNF) with its receptors GFRα-1 and Ret, which may contribute to the maturation and survival of differentiated DA neurons. Our findings may be of future importance for the use of NSCs in cell replacement therapy of PD. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  11. Differentiation of mouse induced pluripotent stem cells into neurons using conditioned medium of dorsal root ganglia.

    PubMed

    Kitazawa, Ayako; Shimizu, Norio

    2011-07-01

    Mouse induced pluripotent stem (iPS) cells are known to have the ability to differentiate into various cell lineages including neurons in vitro. We have reported that chick dorsal root ganglion (DRG)-conditioned medium (CM) promoted the differentiation of mouse embryonic stem (ES) cells into motor neurons. We investigated the formation of undifferentiated iPS cell colonies and the differentiation of iPS cells into neurons using DRG-CM. When iPS cells were cultured in DMEM containing leukemia inhibitory factor (LIF), the iPS cells appeared to be maintained in an undifferentiated state for 19 passages. The number of iPS cell colonies (200 μm in diameter) was maximal at six days of cultivation and the colonies were maintained in an undifferentiated state, but the iPS cell colonies at ten days of cultivation had hollows inside the colonies and were differentiated. By contrast, the number of ES cell colonies (200 μm in diameter) was maximal at ten days of cultivation. The iPS cells were able to proliferate and differentiate easily into various cell lineages, compared to ES cells. When iPS cell colonies were cultured in a manner similar to ES cells with DMEM/F-12K medium supplemented with DRG-CM, the iPS cells mainly differentiated into motor and sensory neurons. These results suggested that the differentiation properties of iPS cells differ from those of ES cells.

  12. Method of derivation and differentiation of mouse embryonic stem cells generating synchronous neuronal networks.

    PubMed

    Gazina, Elena V; Morrisroe, Emma; Mendis, Gunarathna D C; Michalska, Anna E; Chen, Joseph; Nefzger, Christian M; Rollo, Benjamin N; Reid, Christopher A; Pera, Martin F; Petrou, Steven

    2017-08-18

    Stem cells-derived neuronal cultures hold great promise for in vitro disease modelling and drug screening. However, currently stem cells-derived neuronal cultures do not recapitulate the functional properties of primary neurons, such as network properties. Cultured primary murine neurons develop networks which are synchronised over large fractions of the culture, whereas neurons derived from mouse embryonic stem cells (ESCs) display only partly synchronised network activity and human pluripotent stem cells-derived neurons have mostly asynchronous network properties. Therefore, strategies to improve correspondence of derived neuronal cultures with primary neurons need to be developed to validate the use of stem cell-derived neuronal cultures as in vitro models. By combining serum-free derivation of ESCs from mouse blastocysts with neuronal differentiation of ESCs in morphogen-free adherent culture we generated neuronal networks with properties recapitulating those of mature primary cortical cultures. After 35days of differentiation ESC-derived neurons developed network activity very similar to that of mature primary cortical neurons. Importantly, ESC plating density was critical for network development. Compared to the previously published methods this protocol generated more synchronous neuronal networks, with high similarity to the networks formed in mature primary cortical culture. We have demonstrated that ESC-derived neuronal networks recapitulating key properties of mature primary cortical networks can be generated by optimising both stem cell derivation and differentiation. This validates the approach of using ESC-derived neuronal cultures for disease modelling and in vitro drug screening. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Effect of Cuscuta chinensis glycoside on the neuronal differentiation of rat pheochromocytoma PC12 cells.

    PubMed

    Jian-Hui, Liu; Bo, Jiang; Yong-Ming, Bao; Li-Jia, An

    2003-08-01

    Exposure of rat pheochromocytoma PC12 cells to Cuscuta chinensis glycoside induced neuronal differentiation with resulting outgrowth of neurites and increase of acetylcholinesterase activity. A specific inhibitor of mitogen-activated protein kinase (MAPK) kinase, PD98059, prevented this effect of C. chinensis on PC12 cells. These results suggested that C. chinensis glycoside induced neuronal differentiation in PC12 cells linked to the mitogen-activated protein kinase signaling cascade.

  14. Differentiation of human dental pulp stem cells into neuronal by resveratrol.

    PubMed

    Geng, Ya-Wei; Zhang, Zhen; Liu, Ming-Yue; Hu, Wei-Ping

    2017-08-07

    Dental pulp stem cells (DPSCs) have been proposed as a promising source of stem cells in nerve regeneration due to their close embryonic origin and ease of harvest. Resveratrol (RSV) is a natural polyphenolic and possesses many biological functions such as anti-inflammatory activity and protection against atherosclerosis and neuroprotective activities. There is increasing evidence showing that RSV plays a pivotal role in neuron protection and neuronal differentiation. In this study, we isolated DPSCs from impacted third molars and investigated whether RSV induces neuronal differentiation of DPSCs. To avoid loss of DPSCs multipotency, all the experiments were conducted on cells at early passages. RT-PCR results showed that RSV-treated DPSCs (RSV-DPSCs) significantly increased the expression of the neuroprogenitor marker Nestin. When RSV-DPSCs were differentiated with neuronal induction media (RSV-dDPSCs), they showed a cell morphology similar to neurons. The expression of neuronal-specific marker genes Nestin, Musashi, and NF-M in RSV-dDPSCs was significantly increased. Immunocytochemical staining and Western blot analysis showed that the expression of neuronal marker proteins, Nestin, and NF-M, was significantly increased in RSV-dDPSCs. Therefore, we have shown that RSV treatment, along with the use of neuronal induction media, effectively promotes neuronal cell differentiation of DPSCs. © 2017 International Federation for Cell Biology.

  15. Cellular Zinc Homeostasis Contributes to Neuronal Differentiation in Human Induced Pluripotent Stem Cells

    PubMed Central

    Pfaender, Stefanie; Föhr, Karl; Lutz, Anne-Kathrin; Putz, Stefan; Achberger, Kevin; Linta, Leonhard; Liebau, Stefan; Boeckers, Tobias M.; Grabrucker, Andreas M.

    2016-01-01

    Disturbances in neuronal differentiation and function are an underlying factor of many brain disorders. Zinc homeostasis and signaling are important mediators for a normal brain development and function, given that zinc deficiency was shown to result in cognitive and emotional deficits in animal models that might be associated with neurodevelopmental disorders. One underlying mechanism of the observed detrimental effects of zinc deficiency on the brain might be impaired proliferation and differentiation of stem cells participating in neurogenesis. Thus, to examine the molecular mechanisms regulating zinc metabolism and signaling in differentiating neurons, using a protocol for motor neuron differentiation, we characterized the expression of zinc homeostasis genes during neurogenesis using human induced pluripotent stem cells (hiPSCs) and evaluated the influence of altered zinc levels on the expression of zinc homeostasis genes, cell survival, cell fate, and neuronal function. Our results show that zinc transporters are highly regulated genes during neuronal differentiation and that low zinc levels are associated with decreased cell survival, altered neuronal differentiation, and, in particular, synaptic function. We conclude that zinc deficiency in a critical time window during brain development might influence brain function by modulating neuronal differentiation. PMID:27247802

  16. [Neuronal differentiation of human small cell lung cancer cell line PC-6 by Solcoseryl].

    PubMed

    Shimizu, T

    1997-11-01

    Solcoseryl is composed of extracts from calf blood, and is a drug known to activate tissue respiration. In the present study, I demonstrated the cell biological effects of Solcoseryl on a human small cell lung cancer cell line, PC-6, by analyzing cell morphology, cell growth, expression of neuronal differentiation markers, and the ras proto-oncogene product(ras p21). Exposure of PC-6 cells to Solcoseryl at the concentration of 200 microliters/ml induced (1) cell morphological changes, including neurodendrite-like projections from the cell surface, and (2) complete inhibition of cell growth, that was shown by the loss of Ki-67 expression. Solcoseryl also induced the expression of neurofilament protein and acetylcholinesterase, both of which are markers of neuronal differentiation. Moreover, it upregulated the expression of the ras proto-oncogene product, ras p21. Taken together, these data suggest that Solcoseryl is composed of component(s) which can induce neuronal differentiation of the human small cell lung cancer cell line, PC-6.

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

    NASA Astrophysics Data System (ADS)

    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.

  18. Differentiation of Human Dental Pulp Stem Cells into Dopaminergic Neuron-like Cells in Vitro.

    PubMed

    Chun, So Young; Soker, Shay; Jang, Yu-Jin; Kwon, Tae Gyun; Yoo, Eun Sang

    2016-02-01

    We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.

  19. The role of miR-9 during neuron differentiation of mouse retinal stem cells.

    PubMed

    Qi, Xin

    2016-12-01

    Retinal stem cells (RSCs) have been defined as neural cells with the potential to self-renew and to generate all the different cell types of the nervous system following differentiation, which are an ideal engraft in retinal regeneration. In this research, mouse RSCs were isolated from retina, induced differentiation into neuron cells in vitro after over-expression of miR-9. The results showed that the RSCs could induce differentiation into neuron cells under the special medium, but when the miR-9 was over-expressed, the differentiated efficiency of neuron cells from RSCs could be promoted. This reason was demonstrated that polypyrimidine tract-binding protein 1 (PTBP1) was a repressor for polypyrimidine tract-binding protein 2 (PTBP2), during neuronal differentiation, miR-9 reduced PTBP1 levels, leading to the accumulation of correctly spliced PTBP2 mRNA and a dramatic increase in PTBP2 protein. And then miR-9 promoted neuron cells from RSCs were successful colonized into injured spinal cord for participation in tissue-repair. In conclusion, our research showed that the miR-9 promoted the differentiation of neuronal cells from RSCs, and this mechanism was miR-9 reduced the expression of PTBP1, increased the expression of PTBP2.

  20. Maintenance and Neuronal Differentiation of Chicken Induced Pluripotent Stem-Like Cells

    PubMed Central

    Rossello, Ricardo; Chen, Chun-chun; Kessler, Joeran; Davison, Ian; Jarvis, Erich D.

    2014-01-01

    Pluripotent stem cells have the potential to become any cell in the adult body, including neurons and glia. Avian stem cells could be used to study questions, like vocal learning, that would be difficult to examine with traditional mouse models. Induced pluripotent stem cells (iPSCs) are differentiated cells that have been reprogrammed to a pluripotent stem cell state, usually using inducing genes or other molecules. We recently succeeded in generating avian iPSC-like cells using mammalian genes, overcoming a limitation in the generation and use of iPSCs in nonmammalian species (Rosselló et al., 2013). However, there were no established optimal cell culture conditions for avian iPSCs to establish long-term cell lines and thus to study neuronal differentiation in vitro. Here we present an efficient method of maintaining chicken iPSC-like cells and for differentiating them into action potential generating neurons. PMID:25610469

  1. Maintenance and neuronal differentiation of chicken induced pluripotent stem-like cells.

    PubMed

    Dai, Rui; Rossello, Ricardo; Chen, Chun-Chun; Kessler, Joeran; Davison, Ian; Hochgeschwender, Ute; Jarvis, Erich D

    2014-01-01

    Pluripotent stem cells have the potential to become any cell in the adult body, including neurons and glia. Avian stem cells could be used to study questions, like vocal learning, that would be difficult to examine with traditional mouse models. Induced pluripotent stem cells (iPSCs) are differentiated cells that have been reprogrammed to a pluripotent stem cell state, usually using inducing genes or other molecules. We recently succeeded in generating avian iPSC-like cells using mammalian genes, overcoming a limitation in the generation and use of iPSCs in nonmammalian species (Rosselló et al., 2013). However, there were no established optimal cell culture conditions for avian iPSCs to establish long-term cell lines and thus to study neuronal differentiation in vitro. Here we present an efficient method of maintaining chicken iPSC-like cells and for differentiating them into action potential generating neurons.

  2. Human periapical cyst-mesenchymal stem cells differentiate into neuronal cells.

    PubMed

    Marrelli, M; Paduano, F; Tatullo, M

    2015-06-01

    It was recently reported that human periapical cysts (hPCys), a commonly occurring odontogenic cystic lesion of inflammatory origin, contain mesenchymal stem cells (MSCs) with the capacity for self-renewal and multilineage differentiation. In this study, periapical inflammatory cysts were compared with dental pulp to determine whether this tissue may be an alternative accessible tissue source of MSCs that retain the potential for neurogenic differentiation. Flow cytometry and immunofluorescence analysis indicated that hPCy-MSCs and dental pulp stem cells spontaneously expressed the neuron-specific protein β-III tubulin and the neural stem-/astrocyte-specific protein glial fibrillary acidic protein (GFAP) in their basal state before differentiation occurs. Furthermore, undifferentiated hPCy-MSCs showed a higher expression of transcripts for neuronal markers (β-III tubulin, NF-M, MAP2) and neural-related transcription factors (MSX-1, Foxa2, En-1) as compared with dental pulp stem cells. After exposure to neurogenic differentiation conditions (neural media containing epidermal growth factor [EGF], basic fibroblast growth factor [bFGF], and retinoic acid), the hPCy-MSCs showed enhanced expression of β-III tubulin and GFAP proteins, as well as increased expression of neurofilaments medium, neurofilaments heavy, and neuron-specific enolase at the transcript level. In addition, neurally differentiated hPCy-MSCs showed upregulated expression of the neural transcription factors Pitx3, Foxa2, Nurr1, and the dopamine-related genes tyrosine hydroxylase and dopamine transporter. The present study demonstrated for the first time that hPCy-MSCs have a predisposition toward the neural phenotype that is increased when exposed to neural differentiation cues, based on upregulation of a comprehensive set of proteins and genes that define neuronal cells. In conclusion, these results provide evidence that hPCy-MSCs might be another optimal source of neural/glial cells for cell

  3. Comparative neuronal differentiation of self-renewing neural progenitor cell lines obtained from human induced pluripotent stem cells

    PubMed Central

    Verpelli, Chiara; Carlessi, Luigi; Bechi, Giulia; Fusar Poli, Elena; Orellana, Daniel; Heise, Christopher; Franceschetti, Silvana; Mantegazza, Renato; Mantegazza, Massimo; Delia, Domenico; Sala, Carlo

    2013-01-01

    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. PMID:24109433

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

    PubMed Central

    Katakura, Masanori; Hashimoto, Michio; Okui, Toshiyuki; Shahdat, Hossain Md; Matsuzaki, Kentaro; Shido, Osamu

    2013-01-01

    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. PMID:23365582

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

    PubMed

    Wang, Li; Liu, Yuan; Li, Sen; Long, Zai-Yun; 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 cortex differentiate into neurons and its possible molecular mechanism is also not clear. Wnt signaling is implicated in the control of cell growth and differentiation during CNS development in animal model, but its action at the cellular level has been poorly understood. In this experiment, we examined neuronal differentiation of NSCs induced by VPA culture media using vitro immunochemistry assay. The neuronal differentiation of NSCs was examined after treated with 0.75 mM VPA for three, seven and ten days. RT-PCR assay was employed to examine the level of Wnt-3α and β-catenin. The results indicated that there were more β-tublin III positive cells in NSCs treated with VPA medium compared to the control group. The expression of Wnt-3α and β-catenin in NSCs treated with VPA medium was significantly greater compared to that of control media. In conclusion, these findings indicated that VPA could induce neuronal differentiation of NSCs by activating Wnt signal pathway.

  6. The embryonic midbrain directs neuronal specification of embryonic stem cells at early stages of differentiation.

    PubMed

    Baizabal, José-Manuel; Covarrubias, Luis

    2009-01-01

    Specific neuronal differentiation of Embryonic Stem Cells (ESCs) depends on their capacity to interpret environmental cues. At present, it is not clear at which stage of differentiation ESCs become competent to produce multiple neuronal lineages in response to the niche of the embryonic brain. To unfold the developmental potential of ESC-derived precursors, we transplanted these cells into the embryonic midbrain explants, where neurogenesis occurs as in normal midbrain development. Using this experimental design, we show that the transition from ESCs to Embryoid Body (EB) precursors is necessary to differentiate into Lmx1a(+)/Ptx3(+)/TH(+) dopaminergic neurons around the ventral midline of the midbrain. In addition, EB cells placed at other dorsal-ventral levels of the midbrain give rise to Nkx6.1(+) red nucleus (RN) neurons, Nkx2.2(+) ventral interneurons and Pax7(+) dorsal neurons at the correct positions. Notably, differentiation of ESCs into Neural Precursor Cells (NPCs) prior to transplantation markedly reduces specification at the Lmx1a, Nkx6.1 and Pax7 expression domains, without affecting neuronal differentiation. Finally, exposure to Fgf8 and Shh in vitro promotes commitment of some ESC-derived NPCs to differentiate into putative Lmx1a(+) dopaminergic neurons in the midbrain. Our data demonstrate intrinsic developmental potential differences among ESC-derived precursor populations.

  7. Differentiation of Neural Precursors and Dopaminergic Neurons from Human Embryonic Stem Cells

    PubMed Central

    Zhang, Xiao-Qing; Zhang, Su-Chun

    2010-01-01

    Directed differentiation of human embryonic stem cells (hESCs) to a functional cell type, including neurons, is the foundation for application of hESCs. We describe here a reproducible, chemically-defined protocol that allows directed differentiation of hESCs to nearly pure neuroectodermal cells and neurons. First, hESC colonies are detached from mouse fibroblast feeder layers and form aggregates to initiate the differentiation procedure. Second, after 4 days of suspension culture, the ESC growth medium is replaced with neural induction medium to guide neuroectodermal specification. Third, the differentiating hESC aggregates are attached onto the culture surface at day 6-7, where columnar neural epithelial cells appear and organize into rosettes. Fourth, the neural rosettes are enriched by detaching rosettes and leaving the peripheral flat cells attached, and expanded as neuroepithelial aggregates in the same medium. Finally, the neuroepithelial aggregates are dissociated and differentiated to nearly pure neurons. This stepwise differentiation protocol results in the generation of primitive neuroepithelia at day 8-10, neural progenitors at the 2nd and 3rd week, and postmitotic neurons at the 4th week, which mirrors the early phase of neural development in a human embryo. Identification of the primitive neuroepithelial cells permits efficient patterning of region-specific progenitors and neuronal subtypes such as midbrain dopaminergic neurons. PMID:19907987

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

    PubMed

    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.

  9. Human mesenchymal stem cells express neuronal markers after osteogenic and adipogenic differentiation.

    PubMed

    Foudah, Dana; Redondo, Juliana; Caldara, Cristina; Carini, Fabrizio; Tredici, Giovanni; Miloso, Mariarosaria

    2013-06-01

    Mesenchymal stem cells (MSCs) are multipotent cells that are able to differentiate into mesodermal lineages (osteogenic, adipogenic, chondrogenic), but also towards non-mesodermal derivatives (e.g. neural cells). Recent in vitro studies revealed that, in the absence of any kind of differentiation stimuli, undifferentiated MSCs express neural differentiation markers, but the literature data do not all concur. Considering their promising therapeutic potential for neurodegenerative diseases, it is very important to expand our knowledge about this particular biological property of MSCs. In this study, we confirmed the spontaneous expression of neural markers (neuronal, glial and progenitor markers) by undifferentiated human MSCs (hMSCs) and in particular, we demonstrated that the neuronal markers βIII-tubulin and NeuN are expressed by a very high percentage of hMSCs, regardless of the number of culture passages and the culture conditions. Moreover, the neuronal markers βIII-tubulin and NeuN are still expressed by hMSCs after in vitro osteogenic and adipogenic differentiation. On the other hand, chondrogenically differentiated hMSCs are negative for these markers. Our findings suggest that the expression of neuronal markers could be common to a wide range of cellular types and not exclusive for neuronal lineages. Therefore, the expression of neuronal markers alone is not sufficient to demonstrate the differentiation of MSCs towards the neuronal phenotype. Functional properties analysis is also required.

  10. Accumulation of neurons differentiated from mouse embryonic stem cells in particular areas of culture plate surface.

    PubMed

    Kitazawa, Ayako; Naka, Yukie; Yamaguchi, Hiroko; Shimizu, Norio

    2010-08-01

    Nanoscale magnetic beads coated with nerve growth factor (NGF) allow us to accumulate neurons differentiated from mouse ES cells in a selected area of the culture plate surface using a magnet. Neurons with neurite outgrowths within a particular area expressed TrkA and incorporated beads in the soma.

  11. Optimizing neuronal differentiation from induced pluripotent stem cells to model ASD

    PubMed Central

    Kim, Dae-Sung; Ross, P. Joel; Zaslavsky, Kirill; Ellis, James

    2014-01-01

    Autism spectrum disorder (ASD) is an early-onset neurodevelopmental disorder characterized by deficits in social communication, and restricted and repetitive patterns of behavior. Despite its high prevalence, discovery of pathophysiological mechanisms underlying ASD has lagged due to a lack of appropriate model systems. Recent advances in induced pluripotent stem cell (iPSC) technology and neural differentiation techniques allow for detailed functional analyses of neurons generated from living individuals with ASD. Refinement of cortical neuron differentiation methods from iPSCs will enable mechanistic studies of specific neuronal subpopulations that may be preferentially impaired in ASD. In this review, we summarize recent accomplishments in differentiation of cortical neurons from human pluripotent stems cells and efforts to establish in vitro model systems to study ASD using personalized neurons. PMID:24782713

  12. A not cytotoxic nickel concentration alters the expression of neuronal differentiation markers in NT2 cells.

    PubMed

    Ceci, Claudia; Barbaccia, Maria Luisa; Pistritto, Giuseppa

    2015-03-01

    Nickel, a known occupational/environmental hazard, may cross the placenta and reach appreciable concentrations in various fetal organs, including the brain. The aim of this study was to investigate whether nickel interferes with the process of neuronal differentiation. Following a 4 week treatment with retinoic acid (10μM), the human teratocarcinoma-derived NTera2/D1 cell line (NT2 cells) terminally differentiate into neurons which recapitulate many features of human fetal neurons. The continuous exposure of the differentiating NT2 cells to a not cytotoxic nickel concentration (10μM) increased the expression of specific neuronal differentiation markers such as neural cell adhesion molecule (NCAM) and microtubule associated protein 2 (MAP2). Furthermore, nickel exposure increased the expression of hypoxia-inducible-factor-1α (HIF-1α) and induced the activation of the AKT/PKB kinase pathway, as shown by the increase of P(Ser-9)-GSK-3β, the inactive form of glycogen synthase kinase-3β (GSK-3β). Intriguingly, by the end of the fourth week the expression of tyrosine hydroxylase (TH) protein, a marker of dopaminergic neurons, was lower in nickel-treated than in control cultures. Thus, likely by partially mimicking hypoxic conditions, a not-cytotoxic nickel concentration appears to alter the process of neuronal differentiation and hinder the expression of the dopaminergic neuronal phenotype. Taken together, these results suggest that nickel, by altering normal brain development, may increase susceptibility to neuro-psychopathology later in life.

  13. Neurotrophic property of geniposide for inducing the neuronal differentiation of PC12 cells.

    PubMed

    Liu, Jianhui; Zheng, Xuxu; Yin, Fei; Hu, Yinhe; Guo, Lixia; Deng, Xiaohong; Chen, Gang; Jiajia, Jing; Zhang, Heng

    2006-11-01

    The emerging data show that the insulinotrophic hormone glucagon-like peptide-1(GLP-1) and its agonist extendin-4 have neurotrophic function to inducing neuronal differentiation of PC12 cells and prevent neurons damage challenged by oxidative stress. Here, with the model of high throughput screen for GLP-1 receptor agonists, we screen and identify that geniposide is a novel agonist for GLP-1 receptor. Furthermore, geniposide induces the neuronal differentiation of PC12 cells with resulting neurites outgrowth; we also observe an increase in expression of growth-associated protein-43. U0126, a selective MEK inhibitor, prevents neurites out growth and phosphorylation of mitogen-activated kinase proteins in PC12 cells induced by geniposide. All these results show that activation of GLP-1 receptor by geniposide to induce the neuronal differentiation of PC12 cells involves in MAPK signaling cascade.

  14. Bach2 is involved in neuronal differentiation of N1E-115 neuroblastoma cells

    SciTech Connect

    Shim, Ki Shuk; Rosner, Margit; Freilinger, Angelika; Lubec, Gert . E-mail: gert.lubec@meduniwien.ac.at; Hengstschlaeger, Markus

    2006-07-15

    Bach1 and Bach2 are evolutionarily related members of the BTB-basic region leucine zipper transcription factor family. We found that Bach2 downregulates cell proliferation of N1E-115 cells and negatively affects their potential to differentiate. Nuclear localization of the cyclin-dependent kinase inhibitor p21 is known to arrest cell cycle progression, and cytoplasmic p21 has been shown to promote neuronal differentiation of N1E-115 cells. We found that ectopic Bach2 causes upregulation of p21 expression in the nucleus and in the cytoplasm in undifferentiated N1E-115 cells. In differentiated cells, Bach2 specifically triggers upregulation of cytoplasmic p21. Our data suggest that Bach2 expression could represent a switch during the process of neuronal differentiation. Bach2 is not expressed in neuronal precursor cells. It would have negative effects on proliferation and differentiation of these cells. In differentiated neuronal cells Bach2 expression is upregulated, which could allow Bach2 to function as a gatekeeper of the differentiated status.

  15. Gut-neuron interaction via Hh signaling regulates intestinal progenitor cell differentiation in Drosophila.

    PubMed

    Han, Hui; Pan, Chenyu; Liu, Chunying; Lv, Xiangdong; Yang, Xiaofeng; Xiong, Yue; Lu, Yi; Wu, Wenqing; Han, Junhai; Zhou, Zhaocai; Jiang, Hai; Zhang, Lei; Zhao, Yun

    2015-01-01

    Intestinal homeostasis is maintained by intestinal stem cells (ISCs) and their progenies. A complex autonomic nervous system spreads over posterior intestine. However, whether and how neurons regulate posterior intestinal homeostasis is largely unknown. Here we report that neurons regulate Drosophila posterior intestinal homeostasis. Specifically, downregulation of neuronal Hedgehog (Hh) signaling inhibits the differentiation of ISCs toward enterocytes (ECs), whereas upregulated neuronal Hh signaling promotes such process. We demonstrate that, among multiple sources of Hh ligand, those secreted by ECs induces similar phenotypes as does neuronal Hh. In addition, intestinal JAK/STAT signaling responds to activated neuronal Hh signaling, suggesting that JAK/STAT signaling acts downstream of neuronal Hh signaling in intestine. Collectively, our results indicate that neuronal Hh signaling is essential for the determination of ISC fate.

  16. Gut–neuron interaction via Hh signaling regulates intestinal progenitor cell differentiation in Drosophila

    PubMed Central

    Han, Hui; Pan, Chenyu; Liu, Chunying; Lv, Xiangdong; Yang, Xiaofeng; Xiong, Yue; Lu, Yi; Wu, Wenqing; Han, Junhai; Zhou, Zhaocai; Jiang, Hai; Zhang, Lei; Zhao, Yun

    2015-01-01

    Intestinal homeostasis is maintained by intestinal stem cells (ISCs) and their progenies. A complex autonomic nervous system spreads over posterior intestine. However, whether and how neurons regulate posterior intestinal homeostasis is largely unknown. Here we report that neurons regulate Drosophila posterior intestinal homeostasis. Specifically, downregulation of neuronal Hedgehog (Hh) signaling inhibits the differentiation of ISCs toward enterocytes (ECs), whereas upregulated neuronal Hh signaling promotes such process. We demonstrate that, among multiple sources of Hh ligand, those secreted by ECs induces similar phenotypes as does neuronal Hh. In addition, intestinal JAK/STAT signaling responds to activated neuronal Hh signaling, suggesting that JAK/STAT signaling acts downstream of neuronal Hh signaling in intestine. Collectively, our results indicate that neuronal Hh signaling is essential for the determination of ISC fate. PMID:27462407

  17. Chromatin remodeling during in vivo neural stem cells differentiating to neurons in early Drosophila embryos

    PubMed Central

    Ye, Youqiong; Li, Min; Gu, Liang; Chen, Xiaolong; Shi, Jiejun; Zhang, Xiaobai; Jiang, Cizhong

    2017-01-01

    Neurons are a key component of the nervous system and differentiate from multipotent neural stem cells (NSCs). Chromatin remodeling has a critical role in the differentiation process. However, its in vivo epigenetic regulatory role remains unknown. We show here that nucleosome depletion regions (NDRs) form in both proximal promoters and distal enhancers during NSCs differentiating into neurons in the early Drosophila embryonic development. NDR formation in the regulatory regions involves nucleosome shift and eviction. Nucleosome occupancy in promoter NDRs is inversely proportional to the gene activity. Genes with promoter NDR formation during differentiation are enriched for functions related to neuron development and maturation. Active histone-modification signals (H3K4me3 and H3K9ac) in promoters are gained in neurons in two modes: de novo establishment to high levels or increase from the existing levels in NSCs. The gene sets corresponding to the two modes have different neuron-related functions. Dynamic changes of H3K27ac and H3K9ac signals in enhancers and promoters synergistically repress genes associated with neural stem or progenitor cell-related pluripotency and upregulate genes associated with neuron projection morphogenesis, neuron differentiation, and so on. Our results offer new insights into chromatin remodeling during in vivo neuron development and lay a foundation for its epigenetic regulatory mechanism study of other lineage specification. PMID:27858939

  18. Histamine is required during neural stem cell proliferation to increase neuron differentiation.

    PubMed

    Rodríguez-Martínez, G; Velasco, I; García-López, G; Solís, K H; Flores-Herrera, H; Díaz, N F; Molina-Hernández, A

    2012-08-02

    Histamine in the adult central nervous system (CNS) acts as a neurotransmitter. This amine is one of the first neurotransmitters to appear during development reaching its maximum concentration simultaneously with neuron differentiation peak. This suggests that HA plays an important role in neurogenesis. We have previously shown that HA is able to increase neuronal differentiation of neural stem cells (NSCs) in vitro, by activating the histamine type 1 receptor. However the mechanism(s) by which HA has a neurogenic effect on NSCs has not been explored. Here we explore how HA is able to increase neuron phenotype. Cortex neuroepithelium progenitors were cultured and at passage two treatments with 100 μM HA were given during cell proliferation and differentiation or only during differentiation. Immunocytochemistry was performed on differentiated cultures to detect mature neurons. To explore the expression of certain important transcriptional factors involved on asymmetric cell division and commitment, RT-PCR and qRT-PCR were performed. Results indicate that HA is required during cell proliferation in order to increase neuron differentiation and suggest that this amine increases neuron commitment during the proliferative phase probably by rising prospero1 and neurogenin1 expression.

  19. In Vitro Differentiation of Human Wharton's Jelly-Derived Mesenchymal Stem Cells into Auditory Hair Cells and Neurons.

    PubMed

    Kil, Kicheol; Choi, Mi Young; Park, Kyoung Ho

    2016-04-01

    We attempted to induce mesenchymal stem cells (MSCs) from human Wharton's jelly (WJ) to differentiate into neuronal progenitor cells, neurons, and auditory hair cells. MSCs were isolated from WJ from human umbilical cords and cultured in medium containing epidermal growth factor and basic fibroblast growth factor. Differentiation into hair cells and neurons was induced using a neurobasal medium containing glial cell-derived neurotrophic factor, brain-derived neurotrophic factor, and neurotrophic factor 3. Fluorescence-activated cell sorting (FACS), immunocytochemistry, and reverse transcription polymerase chain reaction were performed to characterize the differentiated auditory hair cells and neurons. MSCs isolated from human WJ were confirmed by FACS. Double immunocytochemistry confirmed the expression of the hair cell markers myosin VIIA and TRPA1 and the functional marker C-terminal binding protein 2. Differentiation into neurons was revealed using neurofilament and βIII-tubulin markers. Gene expression of neuronal lineage-specific markers confirmed the neuronal differentiation state. MSCs from human WJ can be successfully induced to differentiate into auditory hair cells and neurons in vitro.

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

    PubMed

    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.

  1. Salidroside induces rat mesenchymal stem cells to differentiate into dopaminergic neurons.

    PubMed

    Zhao, Hong-Bin; Ma, Hui; Ha, Xiao-Qin; Zheng, Ping; Li, Xiao-Yun; Zhang, Ming; Dong, Ju-Zi; Yang, Yin-Shu

    2014-04-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterised by the loss of substantia nigra dopaminergic neurons that leads to a reduction in striatal dopamine (DA) levels. Replacing lost cells by transplanting dopaminergic neurons has potential value to repair the damaged brain. Salidroside (SD), a phenylpropanoid glycoside isolated from plant Rhodiola rosea, is neuroprotective. We examined whether salidroside can induce mesenchymal stem cells (MSCs) to differentiate into neuron-like cells, and convert MSCs into dopamine neurons that can be applied in clinical use. Salidroside induced rMSCs to adopt a neuronal morphology, upregulated the expression of neuronal marker molecules, such as gamma neuronal enolase 2 (Eno2/NSE), microtubule-associated protein 2 (Map2), and beta 3 class III tubulin (Tubb3/β-tubulin III). It also increased expression of brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and nerve growth factor (NGF) mRNAs, and promoted the secretion of these growth factors. The expression of dopamine neurons markers, such as dopamine-beta-hydroxy (DBH), dopa decarboxylase (DDC) and tyrosine hydroxylase (TH), was significantly upregulated after treatment with salidroside for 1-12 days. DA steadily increased after treatment with salidroside for 1-6 days. Thus salidroside can induce rMSCs to differentiate into dopaminergic neurons. © 2014 The Authors Cell Biology International Published by John Wiley & Sons Ltd on behalf of International Federation of Cell Biology.

  2. Novel stem/progenitor cells with neuronal differentiation potential reside in the leptomeningeal niche

    PubMed Central

    Bifari, Francesco; Decimo, Ilaria; Chiamulera, Christian; Bersan, Emanuela; Malpeli, Giorgio; Johansson, Jan; Lisi, Veronica; Bonetti, Bruno; Fumagalli, Guido; Pizzolo, Giovanni; Krampera, Mauro

    2009-01-01

    Stem cells capable of generating neural differentiated cells are recognized by the expression of nestin and reside in specific regions of the brain, namely, hippocampus, subventricular zone and olfactory bulb. For other brain structures, such as leptomeninges, which contribute to the correct cortex development and functions, there is no evidence so far that they may contain stem/precursor cells. In this work, we show for the first time that nestin-positive cells are present in rat leptomeninges during development up to adulthood. The newly identified nestin-positive cells can be extracted and expanded in vitro both as neurospheres, displaying high similarity with subventricular zone–derived neural stem cells, and as homogeneous cell population with stem cell features. In vitro expanded stem cell population can differentiate with high efficiency into excitable cells with neuronal phenotype and morphology. Once injected into the adult brain, these cells survive and differentiate into neurons, thus showing that their neuronal differentiation potential is operational also in vivo. In conclusion, our data provide evidence that a specific population of immature cells endowed of neuronal differentiation potential is resident in the leptomeninges throughout the life. As leptomeninges cover the entire central nervous system, these findings could have relevant implications for studies on cortical development and for regenerative medicine applied to neurological disorders. PMID:19228261

  3. Efficient induction of dopaminergic neuron differentiation from induced pluripotent stem cells reveals impaired mitophagy in PARK2 neurons.

    PubMed

    Suzuki, Sadafumi; Akamatsu, Wado; Kisa, Fumihiko; Sone, Takefumi; Ishikawa, Kei-Ichi; Kuzumaki, Naoko; Katayama, Hiroyuki; Miyawaki, Atsushi; Hattori, Nobutaka; Okano, Hideyuki

    2017-01-29

    Patient-specific induced pluripotent stem cells (iPSCs) show promise for use as tools for in vitro modeling of Parkinson's disease. We sought to improve the efficiency of dopaminergic (DA) neuron induction from iPSCs by the using surface markers expressed in DA progenitors to increase the significance of the phenotypic analysis. By sorting for a CD184(high)/CD44(-) fraction during neural differentiation, we obtained a population of cells that were enriched in DA neuron precursor cells and achieved higher differentiation efficiencies than those obtained through the same protocol without sorting. This high efficiency method of DA neuronal induction enabled reliable detection of reactive oxygen species (ROS) accumulation and vulnerable phenotypes in PARK2 iPSCs-derived DA neurons. We additionally established a quantitative system using the mt-mKeima reporter system to monitor mitophagy in which mitochondria fuse with lysosomes and, by combining this system with the method of DA neuronal induction described above, determined that mitophagy is impaired in PARK2 neurons. These findings suggest that the efficiency of DA neuron induction is important for the precise detection of cellular phenotypes in modeling Parkinson's disease.

  4. A two-step strategy for neuronal differentiation in vitro of human dental follicle cells.

    PubMed

    Völlner, Florian; Ernst, Wolfgang; Driemel, Oliver; Morsczeck, Christian

    2009-06-01

    Human dental follicle cells (DFCs) derived from wisdom teeth are precursor cells for cementoblasts. In this study, we recognized that naïve DFCs express constitutively the early neural cell marker beta-III-tubulin. Interestingly, DFCs formed beta-III-tubulin-positive neurosphere-like cell clusters (NLCCs) on low-attachment cell culture dishes in serum-replacement medium (SRM). For a detailed examination of the neural differentiation potential, DFCs were cultivated in different compositions of SRM containing supplements such as N2, B27, G5 and the neural stem cell supplement. Moreover, these cell culture media were combined with different cell culture substrates such as gelatin, laminin, poly-L-ornithine or poly-L-lysine. After cultivation in SRM, DFCs differentiated into cells with small cell bodies and long cellular extrusions. The expression of nestin, beta-III-tubulin, neuron-specific enolase (NSE) and neurofilament was up-regulated in SRM supplemented with G5, a cell culture supplement for glial cells, and the neural stem cell supplement. DFCs formed NLCCs and demonstrated an increased gene expression of neural cell markers beta-III-tubulin, NSE, nestin and for small neuron markers such as neuropeptides galanin (GAL) and tachykinin (TAC1) after cultivation on poly-L-lysine. For a further neural differentiation NLCC-derived cells were sub-cultivated on laminin and poly-L-ornithine cell culture substrate. After 2 weeks of differentiation, DFCs exposed neural-like cell morphology with small neurite-like cell extrusions. These cells differentially express neurofilament and NSE, but only low levels of beta-III-tubulin and nestin. In conclusion, we demonstrated the differentiation of human DFCs into neuron-like cells after a two-step strategy for neuronal differentiation.

  5. Differentiation of Human Neural Stem Cells into Motor Neurons Stimulates Mitochondrial Biogenesis and Decreases Glycolytic Flux

    PubMed Central

    Keeney, Paula M.

    2015-01-01

    Differentiation of human pluripotent stem cells (hPSCs) in vitro offers a way to study cell types that are not accessible in living patients. Previous research suggests that hPSCs generate ATP through anaerobic glycolysis, in contrast to mitochondrial oxidative phosphorylation (OXPHOS) in somatic cells; however, specialized cell types have not been assessed. To test if mitobiogenesis is increased during motor neuron differentiation, we differentiated human embryonic stem cell (hESC)- and induced pluripotent stem cell-derived human neural stem cells (hNSCs) into motor neurons. After 21 days of motor neuron differentiation, cells increased mRNA and protein levels of genes expressed by postmitotic spinal motor neurons. Electrophysiological analysis revealed voltage-gated currents characteristic of excitable cells and action potential formation. Quantitative PCR revealed an increase in peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α), an upstream regulator of transcription factors involved in mitobiogenesis, and several of its downstream targets in hESC-derived cultures. This correlated with an increase in protein expression of respiratory subunits, but no increase in protein reflecting mitochondrial mass in either cell type. Respiration analysis revealed a decrease in glycolytic flux in both cell types on day 21 (D21), suggesting a switch from glycolysis to OXPHOS. Collectively, our findings suggest that mitochondrial biogenesis, but not mitochondrial mass, is increased during differentiation of hNSCs into motor neurons. These findings help us to understand human motor neuron mitobiogenesis, a process impaired in amyotrophic lateral sclerosis, a neurodegenerative disease characterized by death of motor neurons in the brain and spinal cord. PMID:25892363

  6. Arctigenin protects against neuronal hearing loss by promoting neural stem cell survival and differentiation.

    PubMed

    Huang, Xinghua; Chen, Mo; Ding, Yan; Wang, Qin

    2017-03-01

    Neuronal hearing loss has become a prevalent health problem. This study focused on the function of arctigenin (ARC) in promoting survival and neuronal differentiation of mouse cochlear neural stem cells (NSCs), and its protection against gentamicin (GMC) induced neuronal hearing loss. Mouse cochlea was used to isolate NSCs, which were subsequently cultured in vitro. The effects of ARC on NSC survival, neurosphere formation, differentiation of NSCs, neurite outgrowth, and neural excitability in neuronal network in vitro were examined. Mechanotransduction ability demonstrated by intact cochlea, auditory brainstem response (ABR), and distortion product optoacoustic emissions (DPOAE) amplitude in mice were measured to evaluate effects of ARC on GMC-induced neuronal hearing loss. ARC increased survival, neurosphere formation, neuron differentiation of NSCs in mouse cochlear in vitro. ARC also promoted the outgrowth of neurites, as well as neural excitability of the NSC-differentiated neuron culture. Additionally, ARC rescued mechanotransduction capacity, restored the threshold shifts of ABR and DPOAE in our GMC ototoxicity murine model. This study supports the potential therapeutic role of ARC in promoting both NSCs proliferation and differentiation in vitro to functional neurons, thus supporting its protective function in the therapeutic treatment of neuropathic hearing loss in vivo.

  7. Neuronal Differentiation of Embryonic Stem Cell Derived Neuronal Progenitors Can Be Regulated by Stretchable Conducting Polymers

    PubMed Central

    Srivastava, Nishit; Venugopalan, Vijay; Divya, M.S.; Rasheed, V.A.

    2013-01-01

    Electrically conducting polymers are prospective candidates as active substrates for the development of neuroprosthetic devices. The utility of these substrates for promoting differentiation of embryonic stem cells paves viable routes for regenerative medicine. Here, we have tuned the electrical and mechanical cues provided to the embryonic stem cells during differentiation by precisely straining the conducting polymer (CP) coated, elastomeric-substrate. Upon straining the substrates, the neural differentiation pattern occurs in form of aggregates, accompanied by a gradient where substrate interface reveals a higher degree of differentiation. The CP domains align under linear stress along with the formation of local defect patterns leading to disruption of actin cytoskeleton of cells, and can provide a mechano-transductive basis for the observed changes in the differentiation. Our results demonstrate that along with biochemical and mechanical cues, conductivity of the polymer plays a major role in cellular differentiation thereby providing another control feature to modulate the differentiation and proliferation of stem cells. PMID:23544950

  8. Salidroside induces rat mesenchymal stem cells to differentiate into dopaminergic neurons

    PubMed Central

    Zhao, Hong-Bin; Ma, Hui; Ha, Xiao-Qin; Zheng, Ping; Li, Xiao-Yun; Zhang, Ming; Dong, Ju-Zi; Yang, Yin-Shu

    2014-01-01

    Parkinson’s disease (PD) is a neurodegenerative disorder characterised by the loss of substantia nigra dopaminergic neurons that leads to a reduction in striatal dopamine (DA) levels. Replacing lost cells by transplanting dopaminergic neurons has potential value to repair the damaged brain. Salidroside (SD), a phenylpropanoid glycoside isolated from plant Rhodiola rosea, is neuroprotective. We examined whether salidroside can induce mesenchymal stem cells (MSCs) to differentiate into neuron-like cells, and convert MSCs into dopamine neurons that can be applied in clinical use. Salidroside induced rMSCs to adopt a neuronal morphology, upregulated the expression of neuronal marker molecules, such as gamma neuronal enolase 2 (Eno2/NSE), microtubule-associated protein 2 (Map2), and beta 3 class III tubulin (Tubb3/β-tubulin III). It also increased expression of brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and nerve growth factor (NGF) mRNAs, and promoted the secretion of these growth factors. The expression of dopamine neurons markers, such as dopamine-beta-hydroxy (DBH), dopa decarboxylase (DDC) and tyrosine hydroxylase (TH), was significantly upregulated after treatment with salidroside for 1–12 days. DA steadily increased after treatment with salidroside for 1–6 days. Thus salidroside can induce rMSCs to differentiate into dopaminergic neurons. PMID:24323403

  9. Folate antagonist, methotrexate induces neuronal differentiation of human embryonic stem cells transplanted into nude mouse retina.

    PubMed

    Hara, Akira; Taguchi, Ayako; Aoki, Hitomi; Hatano, Yuichiro; Niwa, Masayuki; Yamada, Yasuhiro; Kunisada, Takahiro

    2010-06-25

    Transplanted embryonic stem (ES) cells can be integrated into the retinas of adult mice as well-differentiated neuroretinal cells. However, the transplanted ES cells also have a tumorigenic activity as they have the ability for multipotent differentiation to various types of tissues. In the present study, human ES (hES) cells were transplanted into adult nude mouse retinas by intravitreal injections 20 h after intravitreal N-methyl-D-aspartate (NMDA) administration. After the transplantation of hES cells, the folate antagonist, methotrexate (MTX) was administrated in order to control the differentiation of the transplanted hES cells. Neuronal differentiation and teratogenic potential of hES cells were examined immunohistochemically 5 weeks after transplantation. The proliferative activity of transplanted cells was determined by both the mitotic index and the Ki-67 proliferative index. Disappearance of Oct-4-positive hES cells showing undifferentiated morphology was observed after intraperitoneal MTX treatment daily, for 15 days. Decreased mitotic and Ki-67 proliferative indices, and increased neuronal differentiation were detected in the surviving hES cells after the MTX treatment. These results suggest two important effects of intraperitoneal MTX treatment for hES cells transplanted into nude mouse retina: (1) MTX treatment following transplantation induces neuronal differentiation, and (2) MTX decreases proliferative activity and tumorigenic potential.

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

    PubMed Central

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

    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 following induction with neural differentiation medium. We performed long-term, continuous observation of cell morphology, growth, differentiation, and neuronal development using several microscopy techniques in conjunction with immunohistochemistry. We examined specific neuronal proteins and Nissl bodies involved in the differentiation process in order to determine the neuronal differentiation of bone marrow mesenchymal stem cells. The results show that bone marrow mesenchymal stem cells that differentiate on fetal bovine acellular dermal matrix display neuronal morphology with unipolar and bi/multipolar neurite elongations that express neuronal-specific proteins, including βIII tubulin. The bone marrow mesenchymal stem cells grown on fetal bovine acellular dermal matrix and induced for long periods of time with neural differentiation medium differentiated into a multilayered neural network-like structure with long nerve fibers that was composed of several parallel microfibers and neuronal cells, forming a complete neural circuit with dendrite-dendrite to axon-dendrite to dendrite-axon synapses. In addition, growth cones with filopodia were observed using scanning electron microscopy. Paraffin sectioning showed differentiated bone marrow mesenchymal stem cells with the typical features of neuronal phenotype, such as a large, round nucleus and a cytoplasm full of Nissl bodies. The data suggest that the biological scaffold fetal bovine acellular dermal matrix is capable of supporting human bone marrow mesenchymal stem cell differentiation into functional neurons and the subsequent formation of tissue engineered nerve. PMID:25598779

  11. Neuronal cell differentiation of mesenchymal stem cells originating from canine amniotic fluid.

    PubMed

    Kim, Eun Young; Lee, Kyung-Bon; Yu, Jung; Lee, Ji Hye; Kim, Keun Jung; Han, Kil-Woo; Park, Kang-Sun; Lee, Dong-Soo; Kim, Min Kyu

    2014-04-01

    The amniotic fluid contains mesenchymal stem cells (MSCs) and can be readily available for tissue engineering. Regenerative treatments such as tissue engineering, cell therapy, and transplantation show potential in clinical trials of degenerative diseases. Disease presentation and clinical responses in the Canis familiaris not only are physiologically similar to human compared with other traditional mammalian models but is also a suitable model for human diseases. The aim of this study was to investigate whether canine amniotic-fluid-derived mesenchymal stem cells (cAF-MSCs) can differentiate into neural precursor cells in vitro when exposed to neural induction reagent. During neural differentiation, cAF-MSCs progressively acquire neuron-like morphology. Messenger RNA (mRNA) expression levels of neural-specific genes, such as NEFL, NSE, and TUBB3 (βIII-tubulin) dramatically increased in the differentiated cAF-MSCs after induction. In addition, protein expression levels of nestin, βIII-tubulin, and tyrosine hydroxylase remarkably increased in differentiated cAF-MSCs. This study demonstrates that cAF-MSCs have great potential for neural precursor differentiation in vitro. Therefore, amniotic fluid may be a suitable alternative source of stem cells and can be applied to cell therapy in neurodegenerative diseases.

  12. Molecular hierarchy in neurons differentiated from mouse ES cells containing a single human chromosome 21.

    PubMed

    Wang, Chi Chiu; Kadota, Mitsutaka; Nishigaki, Ryuichi; Kazuki, Yasuhiro; Shirayoshi, Yasuaki; Rogers, Michael Scott; Gojobori, Takashi; Ikeo, Kazuho; Oshimura, Mitsuo

    2004-02-06

    Defects in neurogenesis and neuronal differentiation in the fetal brain of Down syndrome (DS) patients lead to the apparent neuropathological abnormalities and contribute to the phenotypic characters of mental retardation, and premature development of Alzheimer's disease, those being the most common phenotype in DS. In order to understand the molecular mechanism underlying the cause of phenotypic abnormalities in the DS brain, we have utilized an in vitro model of TT2F mouse embryonic stem cells containing a single human chromosome 21 (hChr21) to study neuron development and neuronal differentiation by microarray containing 15K developmentally expressed cDNAs. Defective neuronal differentiation in the presence of extra hChr21 manifested primarily the post-transcriptional and translational modification, such as Mrpl10, SNAPC3, Srprb, SF3a60 in the early neuronal stem cell stage, and Mrps18a, Eef1g, and Ubce8 in the late differentiated stage. Hierarchical clustering patterned specific expression of hChr21 gene dosage effects on neuron outgrowth, migration, and differentiation, such as Syngr2, Dncic2, Eif3sf, and Peg3.

  13. Negative regulation of neuronal cell differentiation by INHAT subunit SET/TAF-Iβ.

    PubMed

    Kim, Dong-Wook; Kim, Kee-Beom; Kim, Ji-Young; Lee, Kyu-Sun; Seo, Sang-Beom

    2010-09-24

    Epigenetic modification plays an important role in transcriptional regulation. As a subunit of the INHAT (inhibitor of histone acetyltransferases) complex, SET/TAF-Iβ evidences transcriptional repression activity. In this study, we demonstrate that SET/TAF-Iβ is abundantly expressed in neuronal tissues of Drosophila embryos. It is expressed at high levels prior to and in early stages of neuronal development, and gradually reduced as differentiation proceeds. SET/TAF-Iβ binds to the promoters of a subset of neuronal development markers and negatively regulates the transcription of these genes. The results of this study show that the knockdown of SET/TAF-Iβ by si-RNA induces neuronal cell differentiation, thus implicating SET/TAF-Iβ as a negative regulator of neuronal development.

  14. Diverse neurotoxicants target the differentiation of embryonic neural stem cells into neuronal and glial phenotypes.

    PubMed

    Slotkin, Theodore A; Skavicus, Samantha; Card, Jennifer; Levin, Edward D; Seidler, Frederic J

    2016-11-30

    The large number of compounds that needs to be tested for developmental neurotoxicity drives the need to establish in vitro models to evaluate specific neurotoxic endpoints. We used neural stem cells derived from rat neuroepithelium on embryonic day 14 to evaluate the impact of diverse toxicants on their ability to differentiate into glia and neurons: a glucocorticoid (dexamethasone), organophosphate insecticides (chlorpyrifos, diazinon, parathion), insecticides targeting the GABAA receptor (dieldrin, fipronil), heavy metals (Ni(2+), Ag(+)), nicotine and tobacco smoke extract. We found three broad groupings of effects. One diverse set of compounds, dexamethasone, the organophosphate pesticides, Ni(2+) and nicotine, suppressed expression of the glial phenotype while having little or no effect on the neuronal phenotype. The second pattern was restricted to the pesticides acting on GABAA receptors. These compounds promoted the glial phenotype and suppressed the neuronal phenotype. Notably, the actions of compounds eliciting either of these differentiation patterns were clearly unrelated to deficits in cell numbers: dexamethasone, dieldrin and fipronil all reduced cell numbers, whereas organophosphates and Ni(2+) had no effect. The third pattern, shared by Ag(+) and tobacco smoke extract, clearly delineated cytotoxicity, characterized by major cell loss with suppression of differentiation into both glial and neuronal phenotypes; but here again, there was some selectivity in that glia were suppressed more than neurons. Our results, from this survey with diverse compounds, point to convergence of neurotoxicant effects on a specific "decision node" that controls the emergence of neurons and glia from neural stem cells.

  15. Suppression of Sin3A activity promotes differentiation of pluripotent cells into functional neurons

    PubMed Central

    Halder, Debasish; Lee, Chang-Hee; Hyun, Ji Young; Chang, Gyeong-Eon; Cheong, Eunji; Shin, Injae

    2017-01-01

    Sin3 is a transcriptional corepressor for REST silencing machinery that represses multiple neuronal genes in non-neuronal cells. However, functions of Sin3 (Sin3A and Sin3B) in suppression of neuronal phenotypes are not well characterized. Herein we show that Sin3A knockdown impedes the repressive activity of REST and enhances differentiation of pluripotent P19 cells into electrophysiologically active neurons without inducing astrogenesis. It is also found that silencing Sin3B induces neurogenesis of P19 cells with a lower efficiency than Sin3A knockdown. The results suggest that Sin3A has a more profound effect on REST repressive machinery for silencing neuronal genes in P19 cells than Sin3B. Furthermore, we show that a peptide inhibitor of Sin3A-REST interactions promotes differentiation of P19 cells into functional neurons. Observations made in studies using genetic deletion and a synthetic inhibitor suggests that Sin3A plays an important role in the repression of neuronal genes by the REST regulatory mechanism. PMID:28303954

  16. In vitro differentiation of bone marrow stromal cells into neurons and glial cells and differential protein expression in a two-compartment bone marrow stromal cell/neuron co-culture system.

    PubMed

    Qi, Xu; Shao, Ming; Peng, Haisheng; Bi, Zhenggang; Su, Zhiqiang; Li, Hulun

    2010-07-01

    This study was performed to establish a bone marrow stromal cell (BMSC)/neuron two-compartment co-culture model in which differentiation of BMSCs into neurons could occur without direct contact between the two cell types, and to investigate protein expression changes during differentiation of this entirely BMSC-derived population. Cultured BMSCs isolated from Wistar rats were divided into three groups: BMSC culture, BMSC/neuron co-culture and BMSC/neuron two-compartment co-culture. Cells were examined for neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) expression. The electrophysiological behavior of the BMSCs was examined using patch clamping. Proteins that had significantly different expression levels in BMSCs cultured alone and co-cultured with neurons were studied using a protein chip-mass spectroscopy technique. Expression of NSE and GFAP were significantly higher in co-culture cells than in two-compartment co-culture cells, and significantly higher in both co-culture groups than in BMSCs cultured alone. Five proteins showed significant changes in expression during differentiation: TIP39_RAT and CALC_RAT underwent increases, and INSL6_RAT, PNOC_RAT and PCSK1_RAT underwent decreases in expression. We conclude that BMSCs can differentiate into neurons during both contact co-culture with neurons and two-compartment co-culture with neurons. The rate at which BMSCs differentiated into neurons was higher in contact co-culture than in non-contact co-culture.

  17. Human Dental Pulp Cells Differentiate toward Neuronal Cells and Promote Neuroregeneration in Adult Organotypic Hippocampal Slices In Vitro.

    PubMed

    Xiao, Li; Ide, Ryoji; Saiki, Chikako; Kumazawa, Yasuo; Okamura, Hisashi

    2017-08-11

    The adult mammalian central nerve system has fundamental difficulties regarding effective neuroregeneration. The aim of this study is to investigate whether human dental pulp cells (DPCs) can promote neuroregeneration by (i) being differentiated toward neuronal cells and/or (ii) stimulating local neurogenesis in the adult hippocampus. Using immunostaining, we demonstrated that adult human dental pulp contains multipotent DPCs, including STRO-1, CD146 and P75-positive stem cells. DPC-formed spheroids were able to differentiate into neuronal, vascular, osteogenic and cartilaginous lineages under osteogenic induction. However, under neuronal inductive conditions, cells in the DPC-formed spheroids differentiated toward neuronal rather than other lineages. Electrophysiological study showed that these cells consistently exhibit the capacity to produce action potentials, suggesting that they have a functional feature in neuronal cells. We further co-cultivated DPCs with adult mouse hippocampal slices on matrigel in vitro. Immunostaining and presto blue assay showed that DPCs were able to stimulate the growth of neuronal cells (especially neurons) in both the CA1 zone and the edges of the hippocampal slices. Brain-derived neurotrophic factor (BDNF), was expressed in co-cultivated DPCs. In conclusion, our data demonstrated that DPCs are well-suited to differentiate into the neuronal lineage. They are able to stimulate neurogenesis in the adult mouse hippocampus through neurotrophic support in vitro.

  18. Directed differentiation of neural-stem cells and subtype-specific neurons from hESCs

    PubMed Central

    Hu, Bao-Yang; Zhang, Su-Chun

    2010-01-01

    We describe a chemically defined protocol for efficient differentiation of human embryonic stem cells (hESCs) to neural epithelial cells and then to functional spinal motor neurons. This protocol comprises four major steps. Human ESCs are differentiated without morphogens into neuroepithelial cells that form neural tube-like rosettes in the first two weeks. The neuroepithelial cells are then specified to Olig2-expressing motoneuorn progenitors in the presence of retinoic acid (RA) and sonic hedgehog (SHH) in the following 2 weeks. These OLIG2 progenitors generate post-mitotic, HB9 expressing motoneurons at the 5th week and mature to functional motor neurons thereafter. The protein factor SHH can be replaced by a small molecule purmorphamine in the entire process, which may facilitate potential clinical applications. This protocol has been shown equally effective in generating motor neurons from human induced pluropotent stem (iPS) cells. PMID:20336520

  19. Neuroprotection and neuronal differentiation studies using substantia nigra dopaminergic cells derived from transgenic mouse embryos.

    PubMed

    Son, J H; Chun, H S; Joh, T H; Cho, S; Conti, B; Lee, J W

    1999-01-01

    The major pathological lesion of Parkinson's disease (PD) is the selective cell death of dopaminergic (DA) neurons in substantia nigra (SN). Although the initial cause and subsequent molecular signaling mechanisms leading to DA cell death underlying the PD process remain elusive, brain-derived neurotrophic factor (BDNF) is thought to exert neuroprotective as well as neurotrophic roles for the survival and differentiation of DA neurons in SN. Addressing molecular mechanisms of BDNF action in both primary embryonic mesencephalic cultures and in vivo animal models has been technically difficult because DA neurons in SN are relatively rare and present with many heterogeneous cell populations in midbrain. We have developed and characterized a DA neuronal cell line of embryonic SN origin that is more accessible to molecular analysis and can be used as an in vitro model system for studying SN DA neurons. A clonal SN DA neuronal progenitor cell line SN4741, arrested at an early DA developmental stage, was established from transgenic mouse embryos containing the targeted expression of the thermolabile SV40Tag in SN DA neurons. The phenotypic and morphological differentiation of the SN4741 cells could be manipulated by environmental cues in vitro. Exogenous BDNF treatment produced significant neuroprotection against 1-methyl-4-phenylpyridinium, glutamate, and nitric oxide-induced neurotoxicity in the SN4741 cells. Simultaneous phosphorylation of receptor tyrosine kinase B accompanied the neuroprotection. This SN DA neuronal cell line provides a unique model system to circumvent the limitations associated with primary mesencephalic cultures for the elucidation of molecular mechanisms of BDNF action on DA neurons of the SN.

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

    PubMed

    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.

  1. Decreased Neuronal Differentiation of Newly Generated Cells Underlies Reduced Hippocampal Neurogenesis in Chronic Temporal Lobe Epilepsy

    PubMed Central

    Hattiangady, Bharathi; Shetty, Ashok K.

    2009-01-01

    Hippocampal neurogenesis declines substantially in chronic temporal lobe epilepsy (TLE). However, it is unclear whether this decline is linked to altered production of new cells and/or diminished survival and neuronal fate-choice decision of newly born cells. We quantified different components of hippocampal neurogenesis in rats exhibiting chronic TLE. Through intraperitoneal administration of 5'-bromodeoxyuridine (BrdU) for 12 days, we measured numbers of newly born cells in the subgranular zone-granule cell layer (SGZ-GCL) at 24 hours and 2.5 months post-BrdU administration. Furthermore, the differentiation of newly added cells into neurons and glia was quantified via dual immunofluorescence for BrdU and various markers of neurons and glia. Addition of new cells to the SGZ-GCL over 12 days was comparable between the chronically epileptic hippocampus and the age-matched intact hippocampus. Furthermore, comparison of BrdU+ cells measured at 24 hours and 2.5 months post-BrdU administration revealed similar survival of newly born cells between the two groups. However, only 4-5% of newly born cells (i.e. BrdU+ cells) differentiated into neurons in the chronically epileptic hippocampus, in comparison to 73-80% of such cells exhibiting neuronal differentiation in the intact hippocampus. Moreover, differentiation of newly born cells into S-100β+ astrocytes or NG2+ oligodendrocyte progenitors increased to ~79% in the chronically epileptic hippocampus from ~25% observed in the intact hippocampus. Interestingly, the extent of proliferation of astrocytes and microglia (identified through Ki-67 & S-100β and Ki-67 & OX-42 dual immunofluorescence) in the SGZ-GCL was similar between the chronically epileptic hippocampus and the age-matched intact hippocampus, implying that the proliferation of neural stem/progenitor cells in the SGZ-GCL of the chronically epileptic hippocampus was not obscured by an increased division of glia. Thus, severely diminished DG neurogenesis in

  2. Retinoic-acid-mediated HRas stabilization induces neuronal differentiation of neural stem cells during brain development.

    PubMed

    Park, Jong-Chan; Jeong, Woo-Jeong; Kim, Mi-Yeon; Min, DoSik; Choi, Kang-Yell

    2016-08-01

    Ras signaling is tightly regulated during neural stem cell (NSC) differentiation, and defects in this pathway result in aberrant brain development. However, the mechanism regulating Ras signaling during NSC differentiation was unknown. Here, we show that stabilized HRas specifically induces neuronal differentiation of NSCs. Lentivirus-mediated HRas overexpression and knockdown resulted in stimulation and inhibition, respectively, of NSC differentiation into neuron in the ex vivo embryo. Retinoic acid, an active metabolite of vitamin A, promoted neuronal differentiation of NSCs by stabilizing HRas, and HRas knockdown blocked the retinoic acid effect. Vitamin-A-deficient mice displayed abnormal brain development with reduced HRas levels and a reduced thickness of the postmitotic region containing differentiated neurons. All of these abnormal phenotypes were rescued with the restoration of HRas protein levels achieved upon feeding with a retinoic-acid-supplemented diet. In summary, this study shows that retinoic acid stabilizes HRas protein during neurogenesis, and that this is required for NSC differentiation into neurons and murine brain development. © 2016. Published by The Company of Biologists Ltd.

  3. Rapid, efficient, and simple motor neuron differentiation from human pluripotent stem cells.

    PubMed

    Shimojo, Daisuke; Onodera, Kazunari; Doi-Torii, Yukiko; Ishihara, Yasuharu; Hattori, Chinatsu; Miwa, Yukino; Tanaka, Satoshi; Okada, Rina; Ohyama, Manabu; Shoji, Masanobu; Nakanishi, Atsushi; Doyu, Manabu; Okano, Hideyuki; Okada, Yohei

    2015-12-01

    Human pluripotent stem cells (hPSCs) are being applied in regenerative medicine and for the in vitro modeling of human intractable disorders. In particular, neural cells derived from disease-specific human induced pluripotent stem cells (hiPSCs) established from patients with neurological disorders have been used as in vitro disease models to recapitulate in vivo pathogenesis because neural cells cannot be usually obtained from patients themselves. In this study, we established a rapid, efficient, and simple method for efficiently deriving motor neurons from hPSCs that is useful for pathophysiological analysis and the development of drugs to treat motor neuron diseases. Treatment with GSK3β inhibitors during the initial phase of differentiation in combination with dual SMAD inhibition was sufficient to induce PAX6 (+) and SOX1 (+) neural progenitors within 1 week, and subsequent treatment with retinoic acid (RA) and purmorphamine, which activates sonic hedgehog (SHH) signaling, resulted in the highly efficient induction of HB9(+) and ISL-1(+) motor neurons within 2 weeks. After 4 weeks of monolayer differentiation in motor neuron maturation medium, hPSC-derived motor neurons were shown to mature, displaying larger somas and clearer staining for the mature motor neuron marker choline acetyltransferase (ChAT). Moreover, hPSC-derived motor neurons were able to form neuromuscular junctions with human myotubes in vitro and induced acetylcholine receptor (AChR) clustering, as detected by Alexa 555-conjugated α-Bungarotoxin (α-BTX), suggesting that these hPSC-derived motor neurons formed functional contacts with skeletal muscles. This differentiation system is simple and is reproducible in several hiPSC clones, thereby minimizing clonal variation among hPSC clones. We also established a system for visualizing motor neurons with a lentiviral reporter for HB9 (HB9 (e438) ::Venus). The specificity of this reporter was confirmed through immunocytochemistry and

  4. RARβ regulates neuronal cell death and differentiation in the avian ciliary ganglion

    PubMed Central

    Boerries, Melanie; Busch, Hauke

    2015-01-01

    ABSTRACT Programmed cell death during chicken ciliary ganglion (CG) development is mostly discussed as an extrinsically regulated process, guided either by the establishment of a functional balance between preganglionic and postganglionic activity or the availability of target‐derived neurotrophic factors. We found that the expression of the gene coding for the nuclear retinoic acid receptor β (RARB) is transiently upregulated prior to and during the execution phase of cell death in the CG. Using retroviral vectors, the expression of RARB was knocked down during embryonic development in ovo. The knockdown led to a significant increase in CG neuron number after the cell death phase. BrdU injections and active caspase‐3 staining revealed that this increase in neuron number was due to an inhibition of apoptosis during the normal cell death phase. Furthermore, apoptotic neuron numbers were significantly increased at a stage when cell death is normally completed. While the cholinergic phenotype of the neurons remained unchanged after RARB knockdown, the expression of the proneural gene Cash1 was increased, but somatostatin‐like immunoreactivity, a hallmark of the mature choroid neuron population, was decreased. Taken together, these results point toward a delay in neuronal differentiation as well as cell death. The availability of nuclear retinoic acid receptor β (RARβ) and RARβ‐induced transcription of genes could therefore be a new intrinsic cue for the maturation of CG neurons and their predisposition to undergo cell death. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1204–1218, 2015 PMID:25663354

  5. Mouse embryonic stem cell-derived cells reveal niches that support neuronal differentiation in the adult rat brain.

    PubMed

    Maya-Espinosa, Guadalupe; Collazo-Navarrete, Omar; Millán-Aldaco, Diana; Palomero-Rivero, Marcela; Guerrero-Flores, Gilda; Drucker-Colín, René; Covarrubias, Luis; Guerra-Crespo, Magdalena

    2015-02-01

    A neurogenic niche can be identified by the proliferation and differentiation of its naturally residing neural stem cells. However, it remains unclear whether "silent" neurogenic niches or regions suitable for neural differentiation, other than the areas of active neurogenesis, exist in the adult brain. Embryoid body (EB) cells derived from embryonic stem cells (ESCs) are endowed with a high potential to respond to specification and neuralization signals of the embryo. Hence, to identify microenvironments in the postnatal and adult rat brain with the capacity to support neuronal differentiation, we transplanted dissociated EB cells to conventional neurogenic and non-neurogenic regions. Our results show a neuronal differentiation pattern of EB cells that was dependent on the host region. Efficient neuronal differentiation of EB cells occurred within an adjacent region to the rostral migratory stream. EB cell differentiation was initially patchy and progressed toward an even distribution along the graft by 15-21 days post-transplantation, giving rise mostly to GABAergic neurons. EB cells in the striatum displayed a lower level of neuronal differentiation and derived into a significant number of astrocytes. Remarkably, when EB cells were transplanted to the striatum of adult rats after a local ischemic stroke, increased number of neuroblasts and neurons were observed. Unexpectedly, we determined that the adult substantia nigra pars compacta, considered a non-neurogenic area, harbors a robust neurogenic environment. Therefore, neurally uncommitted cells derived from ESCs can detect regions that support neuronal differentiation within the adult brain, a fundamental step for the development of stem cell-based replacement therapies. © 2014 AlphaMed Press.

  6. Progesterone increases dopamine neuron number in differentiating mouse embryonic stem cells

    PubMed Central

    Díaz, Néstor F.; Díaz-Martínez, Néstor E.; Velasco, Iván; Camacho-Arroyo, Ignacio

    2009-01-01

    Progesterone participates in the regulation of several functions in mammals including brain differentiation and dopaminergic transmission but the role of progesterone in dopaminergic cell differentiation is unknown. We investigated the effects of progesterone on dopaminergic differentiation of embryonic stem cells using a 5-stage protocol. Cells were incubated with different progesterone concentrations during the proliferation (stage 4) or differentiation (stage 5) phases. Progesterone added at 1, 10 and 100 nM during stage 4 increased 72, 80 and 62% respectively the number of dopamine neurons at stage 5 as compared to control group. The administration of progesterone at stage 5 did not induce significant changes in the number of dopamine neurons. These actions were not mediated by the activation of intracellular progesterone receptors, because RU 486 did not block the positive effects of progesterone on differentiation to dopaminergic neurons. Our results suggest that progesterone should be useful to produce higher proportions of dopamine neurons from embryonic stem cells aimed for treating Parkinson's disease. PMID:19500215

  7. Progesterone increases dopamine neurone number in differentiating mouse embryonic stem cells.

    PubMed

    Díaz, N F; Díaz-Martínez, N E; Velasco, I; Camacho-Arroyo, I

    2009-08-01

    Progesterone participates in the regulation of several functions in mammals, including brain differentiation and dopaminergic transmission, but the role of progesterone in dopaminergic cell differentiation is unknown. We investigated the effects of progesterone on dopaminergic differentiation of embryonic stem cells using a five-stage protocol. Cells were incubated with different progesterone concentrations during the proliferation (stage 4) or differentiation (stage 5) phases. Progesterone added at 1, 10 and 100 nm during stage 4 increased the number of dopamine neurones at stage 5 by 72%, 80% and 62%, respectively, compared to the control group. The administration of progesterone at stage 5 did not induce significant changes in the number of dopamine neurones. These actions were not mediated by the activation of intracellular progesterone receptors because RU 486 did not block the positive effects of progesterone on differentiation to dopaminergic neurones. The results obtained suggest that progesterone should prove useful with respect to producing higher proportions of dopamine neurones from embryonic stem cells in the treatment of Parkinson's disease.

  8. Effects of extremely low frequency magnetic fields on NGF induced neuronal differentiation of PC12 cells.

    PubMed

    Jung, In-Soo; Kim, Hyun-Jung; Noh, Ran; Kim, Soo-Chan; Kim, Chan-Wha

    2014-10-01

    Extremely low-frequency magnetic fields (ELF-MFs) affect various cellular processes and systems, such as cell proliferation, differentiation and metabolic pathways. The present study investigated ELF-MFs effect on nerve growth factor (NGF) induced neuronal differentiation of PC12 cells using proteomic applications to understand its role in the enhancement of neuronal differentiation. After 50 Hz, 1 mT ELF-MFs 5-day exposure on NGF induced PC12 cells, it was observed to increase neurite length as well as an increase in the number of neurite bearing cells. It was also discovered that there was a decrease in proliferation activity, which is associated with an increase in differentiated cells. Neuronal differentiation related mRNA levels and protein levels were increased in NGF induced PC12 cells. Compared with NGF induced group, ELF-MFs stimulated PC12 cells had different protein expression as measured with two-dimensional electrophoresis (2-DE) gels. Consequently six differentially expressed spots were detected between the 2-DE maps, which were identified by electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-Q-TOF LC/MS/MS) as: peripherin, neurosecretory protein nerve growth factor inducible (VGF8a) precursor, dnaK-type molecular chaperone sp72-ps1 (HSP72-psI), low molecular weight (Mr) phosphotyrosine protein phosphatase isoenzyme AcP1 (LMW-PTP/ACP1), Tubulin alpha-1A (TUBA1A) chain, outcome predictor in acute leukemia 1 homolog (OPA1L). The identification of these proteins provides clues to the mechanism of ELF-MFs stimulation on NGF induced PC12 cells that occur during neuronal differentiation and may contribute to the development novel treatments for neurodegenerative diseases. © 2014 The Authors. Bioelectromagnetics Published by Wiley Periodicals, Inc.

  9. Encapsulated neural stem cell neuronal differentiation in fluorinated methacrylamide chitosan hydrogels.

    PubMed

    Li, Hang; Wijekoon, Asanka; Leipzig, Nic D

    2014-07-01

    Neural stem/progenitor cells (NSPCs) are able to differentiate into the primary cell types (neurons, oligodendrocytes and astrocytes) of the adult nervous system. This attractive property of NSPCs offers a potential solution for neural regeneration. 3D implantable scaffolds should mimic the microstructure and dynamic properties found in vivo, enabling the natural exchange of oxygen, nutrients, and growth factors for cell survival and differentiation. We have previously reported a new class of materials consisting of perfluorocarbons (PFCs) conjugated to methacrylamide chitosan (MAC), which possess the ability to repeatedly take-up and release oxygen at beneficial levels for favorable cell metabolism and proliferation. In this study, the neuronal differentiation responses of NSPCs to fluorinated methacrylamide chitosan (MACF) hydrogels were studied for 8 days. Two treatments, with oxygen reloading or without oxygen reloading, were performed during culture. Oxygen concentration distributions within cell-seeded MACF hydrogels were found to have higher concentrations of oxygen at the edge of the hydrogels and less severe drops in O2 gradient as compared with MAC hydrogel controls. Total cell number was enhanced in MACF hydrogels as the number of conjugated fluorines via PFC substitution increased. Additionally, all MACF hydrogels supported significantly more cells than MAC controls (p < 0.001). At day 8, MACF hydrogels displayed significantly greater neuronal differentiation than MAC controls (p = 0.001), and among MACF groups methacrylamide chitosan with 15 fluorines per addition (MAC(Ali15)F) demonstrated the best ability to promote NSPC differentiation.

  10. Neurogenic Radial Glia-like Cells in Meninges Migrate and Differentiate into Functionally Integrated Neurons in the Neonatal Cortex.

    PubMed

    Bifari, Francesco; Decimo, Ilaria; Pino, Annachiara; Llorens-Bobadilla, Enric; Zhao, Sheng; Lange, Christian; Panuccio, Gabriella; Boeckx, Bram; Thienpont, Bernard; Vinckier, Stefan; Wyns, Sabine; Bouché, Ann; Lambrechts, Diether; Giugliano, Michele; Dewerchin, Mieke; Martin-Villalba, Ana; Carmeliet, Peter

    2016-11-17

    Whether new neurons are added in the postnatal cerebral cortex is still debated. Here, we report that the meninges of perinatal mice contain a population of neurogenic progenitors formed during embryonic development that migrate to the caudal cortex and differentiate into Satb2(+) neurons in cortical layers II-IV. The resulting neurons are electrically functional and integrated into local microcircuits. Single-cell RNA sequencing identified meningeal cells with distinct transcriptome signatures characteristic of (1) neurogenic radial glia-like cells (resembling neural stem cells in the SVZ), (2) neuronal cells, and (3) a cell type with an intermediate phenotype, possibly representing radial glia-like meningeal cells differentiating to neuronal cells. Thus, we have identified a pool of embryonically derived radial glia-like cells present in the meninges that migrate and differentiate into functional neurons in the neonatal cerebral cortex.

  11. Plasticity of marrow mesenchymal stem cells from human first-trimester fetus: from single-cell clone to neuronal differentiation.

    PubMed

    Zhang, Yihua; Shen, Wenzheng; Sun, Bingjie; Lv, Changrong; Dou, Zhongying

    2011-02-01

    Recent results have shown that bone marrow mesenchymal stem cells (BMSCs) from human first-trimester abortus (hfBMSCs) are closer to embryonic stem cells and perform greater telomerase activity and faster propagation than mid- and late-prophase fetal and adult BMSCs. However, no research has been done on the plasticity of hfBMSCs into neuronal cells using single-cell cloned strains without cell contamination. In this study, we isolated five single cells from hfBMSCs and obtained five single-cell cloned strains, and investigated their biological property and neuronal differentiation potential. We found that four of the five strains showed similar expression profile of surface antigen markers to hfBMSCs, and most of them differentiated into neuron-like cells expressing Nestin, Pax6, Sox1, β-III Tubulin, NF-L, and NSE under induction. One strain showed different expression profile of surface antigen markers from the four strains and hfBMSCs, and did not differentiate toward neuronal cells. We demonstrated for the first time that some of single-cell cloned strains from hfBMSCs can differentiate into nerve tissue-like cell clusters under induction in vitro, and that the plasticity of each single-cell cloned strain into neuronal cells is different.

  12. Potential role of culture mediums for successful isolation and neuronal differentiation of amniotic fluid stem cells.

    PubMed

    Orciani, M; Emanuelli, M; Martino, C; Pugnaloni, A; Tranquilli, A L; Di Primio, R

    2008-01-01

    In recent years, the use of stem cells has generated increasing interest in regenerative medicine and cancer therapies. The most potent stem cells derive from the inner cell mass during embryonic development and their use yields serious ethical and methodological problems. Recently, a number of reports suggests that another suitable source of multipotent stem cells may be the amniotic fluid. Amniotic fluid mesenchymal stem cells (AFMSCs) are capable of extensive self-renewal, able to differentiate in specialized cells representative of all three germ layers, do not show ethical restriction, and display minimal risks of teratomas and a very low immunogenity. For all these reasons, amniotic fluid appears as a promising alternative source for stem cell therapy. Their recent discovery implies a lack of knowledge of their specific features as well as the existence of a protocol universally recognized as the most suitable for their isolation, growth and long-term conservation. In this study, we isolated stem cells from six amniotic fluids; these cells were cultured with three different culture mediums (Mesenchymal Stem Cell Medium (MSCGM), PC-1 and RPMI-1640), characterized by cytofluorimetric analysis, and then either frozen or induced to neuronal differentiation. Even if the immunophenotype seemed not to be influenced by culture medium (all six samples cultured in the above-mentioned mediums expressed surface antigens commonly found on stem cells), cells showed different abilities to differentiate into neuron-like cells and to re-start the culture after short/long-term storage. Cells isolated and cultured in MSCGM showed the highest proliferation rate, and formed neuron-like cells when sub-plated with neuronal differentiation medium. Cells from PC-1, on the contrary, displayed an increased ability to re-start culture after short/long term storage. Finally, cells from RPMI-1640, even if expressing stem cells markers, were not able to differentiate in neuron-like cells

  13. Activin A directs striatal projection neuron differentiation of human pluripotent stem cells

    PubMed Central

    Arber, Charles; Precious, Sophie V.; Cambray, Serafí; Risner-Janiczek, Jessica R.; Kelly, Claire; Noakes, Zoe; Fjodorova, Marija; Heuer, Andreas; Ungless, Mark A.; Rodríguez, Tristan A.; Rosser, Anne E.; Dunnett, Stephen B.; Li, Meng

    2015-01-01

    The efficient generation of striatal neurons from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) is fundamental for realising their promise in disease modelling, pharmaceutical drug screening and cell therapy for Huntington's disease. GABAergic medium-sized spiny neurons (MSNs) are the principal projection neurons of the striatum and specifically degenerate in the early phase of Huntington's disease. Here we report that activin A induces lateral ganglionic eminence (LGE) characteristics in nascent neural progenitors derived from hESCs and hiPSCs in a sonic hedgehog-independent manner. Correct specification of striatal phenotype was further demonstrated by the induction of the striatal transcription factors CTIP2, GSX2 and FOXP2. Crucially, these human LGE progenitors readily differentiate into postmitotic neurons expressing the striatal projection neuron signature marker DARPP32, both in culture and following transplantation in the adult striatum in a rat model of Huntington's disease. Activin-induced neurons also exhibit appropriate striatal-like electrophysiology in vitro. Together, our findings demonstrate a novel route for efficient differentiation of GABAergic striatal MSNs from human pluripotent stem cells. PMID:25804741

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

    PubMed

    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

  15. Ketamine-Induced Toxicity in Neurons Differentiated from Neural Stem Cells.

    PubMed

    Slikker, William; Liu, Fang; Rainosek, Shuo W; Patterson, Tucker A; Sadovova, Natalya; Hanig, Joseph P; Paule, Merle G; Wang, Cheng

    2015-10-01

    Ketamine is used as a general anesthetic, and recent data suggest that anesthetics can cause neuronal damage when exposure occurs during development. The precise mechanisms are not completely understood. To evaluate the degree of ketamine-induced neuronal toxicity, neural stem cells were isolated from gestational day 16 rat fetuses. On the eighth day in culture, proliferating neural stem cells were exposed for 24 h to ketamine at 1, 10, 100, and 500 μM. To determine the effect of ketamine on differentiated stem cells, separate cultures of neural stem cells were maintained in transition medium (DIV 6) for 1 day and kept in differentiation medium for another 3 days. Differentiated neural cells were exposed for 24 h to 10 μM ketamine. Markers of cellular proliferation and differentiation, mitochondrial health, cell death/damage, and oxidative damage were monitored to determine: (1) the effects of ketamine on neural stem cell proliferation and neural stem cell differentiation; (2) the nature and degree of ketamine-induced toxicity in proliferating neural stem cells and differentiated neural cells; and (3) to provide information regarding receptor expression and possible mechanisms underlying ketamine toxicity. After ketamine exposure at a clinically relevant concentration (10 μM), neural stem cell proliferation was not significantly affected and oxidative DNA damage was not induced. No significant effect on mitochondrial viability (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay) in neural stem cell cultures (growth medium) was observed at ketamine concentrations up to 500 μM. However, quantitative analysis shows that the number of differentiated neurons was substantially reduced in 10 μM ketamine-exposed cultures in differentiation medium, compared with the controls. No significant changes in the number of GFAP-positive astrocytes and O4-positive oligodendrocytes (in differentiation medium) were detected from ketamine-exposed cultures

  16. TDP-43 causes differential pathology in neuronal versus glial cells in the mouse brain

    PubMed Central

    Yan, Sen; Wang, Chuan-En; Wei, Wenjie; Gaertig, Marta A.; Lai, Liangxue; Li, Shihua; Li, Xiao-Jiang

    2014-01-01

    Mutations in TAR DNA-binding protein 43 (TDP-43) are associated with familial forms of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Although recent studies have revealed that mutant TDP-43 in neuronal and glial cells is toxic, how mutant TDP-43 causes primarily neuronal degeneration in an age-dependent manner remains unclear. Using adeno-associated virus (AAV) that expresses mutant TDP-43 (M337V) ubiquitously, we found that mutant TDP-43 accumulates preferentially in neuronal cells in the postnatal mouse brain. We then ubiquitously or selectively expressed mutant TDP-43 in neuronal and glial cells in the striatum of adult mouse brains via stereotaxic injection of AAV vectors and found that it also preferentially accumulates in neuronal cells. Expression of mutant TDP-43 in neurons in the striatum causes more severe degeneration, earlier death and more robust symptoms in mice than expression of mutant TDP-43 in glial cells; however, aging increases the expression of mutant TDP-43 in glial cells, and expression of mutant TDP-43 in older mice caused earlier onset of phenotypes and more severe neuropathology than that in younger mice. Although expression of mutant TDP-43 in glial cells via stereotaxic injection does not lead to robust neurological phenotypes, systemic inhibition of the proteasome activity via MG132 in postnatal mice could exacerbate glial TDP-43-mediated toxicity and cause mice to die earlier. Consistently, this inhibition increases the expression of mutant TDP-43 in glial cells in mouse brains. Thus, the differential accumulation of mutant TDP-43 in neuronal versus glial cells contributes to the preferential toxicity of mutant TDP-43 in neuronal cells and age-dependent pathology. PMID:24381309

  17. TDP-43 causes differential pathology in neuronal versus glial cells in the mouse brain.

    PubMed

    Yan, Sen; Wang, Chuan-En; Wei, Wenjie; Gaertig, Marta A; Lai, Liangxue; Li, Shihua; Li, Xiao-Jiang

    2014-05-15

    Mutations in TAR DNA-binding protein 43 (TDP-43) are associated with familial forms of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Although recent studies have revealed that mutant TDP-43 in neuronal and glial cells is toxic, how mutant TDP-43 causes primarily neuronal degeneration in an age-dependent manner remains unclear. Using adeno-associated virus (AAV) that expresses mutant TDP-43 (M337V) ubiquitously, we found that mutant TDP-43 accumulates preferentially in neuronal cells in the postnatal mouse brain. We then ubiquitously or selectively expressed mutant TDP-43 in neuronal and glial cells in the striatum of adult mouse brains via stereotaxic injection of AAV vectors and found that it also preferentially accumulates in neuronal cells. Expression of mutant TDP-43 in neurons in the striatum causes more severe degeneration, earlier death and more robust symptoms in mice than expression of mutant TDP-43 in glial cells; however, aging increases the expression of mutant TDP-43 in glial cells, and expression of mutant TDP-43 in older mice caused earlier onset of phenotypes and more severe neuropathology than that in younger mice. Although expression of mutant TDP-43 in glial cells via stereotaxic injection does not lead to robust neurological phenotypes, systemic inhibition of the proteasome activity via MG132 in postnatal mice could exacerbate glial TDP-43-mediated toxicity and cause mice to die earlier. Consistently, this inhibition increases the expression of mutant TDP-43 in glial cells in mouse brains. Thus, the differential accumulation of mutant TDP-43 in neuronal versus glial cells contributes to the preferential toxicity of mutant TDP-43 in neuronal cells and age-dependent pathology.

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

    PubMed Central

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

    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 marker β-tubulin III, were dramatically increased at 7 days in the co-culture condition. Blocking the effects of brain-derived neurotrophic factor (BDNF) with an anti-BDNF antibody reduced the number of neurons differentiated from NSCs when co-cultured with protoplasmic astrocytes. In fact, the content of BDNF in the supernatant obtained from protoplasmic astrocytes and NSCs co-culture media was significantly greater than that from control media conditions. These results indicate that protoplasmic astrocytes promote neuronal differentiation of NSCs, which is driven, at least in part, by BDNF. PMID:22693605

  19. Functional immobilization of interferon-gamma induces neuronal differentiation of neural stem cells.

    PubMed

    Leipzig, Nic D; Xu, Changchang; Zahir, Tasneem; Shoichet, Molly S

    2010-05-01

    Stem cell transplantation provides significant promise to regenerative strategies after injury in the central nervous system. Neural stem/progenitor cells (NSPCs) have been studied in terms of their regenerative capacity and their ability to differentiate into neurons when exposed to various soluble factors. In this study, interferon-gamma (IFN-gamma) was compared with brain-derived neurotrophic factor (BDNF) and erythropoietin and was shown to be the best single growth factor for inducing neuronal differentiation from adult rat brain-derived NSPCs. Next, IFN-gamma was surface immobilized to a methacrylamide chitosan (MAC) scaffold that was specifically designed to match the modulus of brain tissue and neuronal differentiation of NSPCs was examined in vitro by immunohistochemistry. Bioactive IFN-gamma was successfully immobilized and quantified by ELISA. Both soluble and immobilized IFN-gamma on MAC surfaces showed dose dependent neuronal differentiation with soluble saturation occurring at 100 ng/mL and the most effective immobilized IFN-gamma dose at 37.5 ng/cm(2), where significantly more neurons resulted compared with controls including soluble IFN-gamma.

  20. Wnt1 from cochlear schwann cells enhances neuronal differentiation of transplanted neural stem cells in a rat spiral ganglion neuron degeneration model.

    PubMed

    He, Ya; Zhang, Peng-Zhi; Sun, Dong; Mi, Wen-Juan; Zhang, Xin-Yi; Cui, Yong; Jiang, Xing-Wang; Mao, Xiao-Bo; Qiu, Jian-Hua

    2014-04-01

    Although neural stem cell (NSC) transplantation is widely expected to become a therapy for nervous system degenerative diseases and injuries, the low neuronal differentiation rate of NSCs transplanted into the inner ear is a major obstacle for the successful treatment of spiral ganglion neuron (SGN) degeneration. In this study, we validated whether the local microenvironment influences the neuronal differentiation of transplanted NSCs in the inner ear. Using a rat SGN degeneration model, we demonstrated that transplanted NSCs were more likely to differentiate into microtubule-associated protein 2 (MAP2)-positive neurons in SGN-degenerated cochleae than in control cochleae. Using real-time quantitative PCR and an immunofluorescence assay, we also proved that the expression of Wnt1 (a ligand of Wnt signaling) increases significantly in Schwann cells in the SGN-degenerated cochlea. We further verified that NSC cultures express receptors and signaling components for Wnts. Based on these expression patterns, we hypothesized that Schwann cell-derived Wnt1 and Wnt signaling might be involved in the regulation of the neuronal differentiation of transplanted NSCs. We verified our hypothesis in vitro using a coculture system. We transduced a lentiviral vector expressing Wnt1 into cochlear Schwann cell cultures and cocultured them with NSC cultures. The coculture with Wnt1-expressing Schwann cells resulted in a significant increase in the percentage of NSCs that differentiated into MAP2-positive neurons, whereas this differentiation-enhancing effect was prevented by Dkk1 (an inhibitor of the Wnt signaling pathway). These results suggested that Wnt1 derived from cochlear Schwann cells enhanced the neuronal differentiation of transplanted NSCs through Wnt signaling pathway activation. Alterations of the microenvironment deserve detailed investigation because they may help us to conceive effective strategies to overcome the barrier of the low differentiation rate of transplanted

  1. Strategies to promote differentiation of newborn neurons into mature functional cells in Alzheimer brain.

    PubMed

    Schaeffer, Evelin L; Novaes, Barbara A; da Silva, Emanuelle R; Skaf, Heni D; Mendes-Neto, Alvaro G

    2009-10-01

    Adult neurogenesis occurs in the subgranular zone (SGZ) and subventricular zone (SVZ). New SGZ neurons migrate into the granule cell layer of the dentate gyrus (DG). New SVZ neurons seem to enter the association neocortex and entorhinal cortex besides the olfactory bulb. Alzheimer disease (AD) is characterized by neuron loss in the hippocampus (DG and CA1 field), entorhinal cortex, and association neocortex, which underlies the learning and memory deficits. We hypothesized that, if the AD brain can support neurogenesis, strategies to stimulate the neurogenesis process could have therapeutic value in AD. We reviewed the literature on: (a) the functional significance of adult-born neurons; (b) the occurrence of endogenous neurogenesis in AD; and (c) strategies to stimulate the adult neurogenesis process. We found that: (a) new neurons in the adult DG contribute to memory function; (b) new neurons are generated in the SGZ and SVZ of AD brains, but they fail to differentiate into mature neurons in the target regions; and (c) numerous strategies (Lithium, Glatiramer Acetate, nerve growth factor, environmental enrichment) can enhance adult neurogenesis and promote maturation of newly generated neurons. Such strategies might help to compensate for the loss of neurons and improve the memory function in AD.

  2. Axonal alignment and enhanced neuronal differentiation of neural stem cells on graphene-nanoparticle hybrid structures.

    PubMed

    Solanki, Aniruddh; Chueng, Sy-Tsong Dean; Yin, Perry T; Kappera, Rajesh; Chhowalla, Manish; Lee, Ki-Bum

    2013-10-11

    Human neural stem cells (hNSCs) cultured on graphene-nanoparticle hybrid structures show a unique behavior wherein the axons from the differentiating hNSCs show enhanced growth and alignment. We show that the axonal alignment is primarily due to the presence of graphene and the underlying nanoparticle monolayer causes enhanced neuronal differentiation of the hNSCs, thus having great implications of these hybrid-nanostructures for neuro-regenerative medicine.

  3. Transplantation of Unique Subpopulation of Fibroblasts, Muse Cells, Ameliorates Experimental Stroke Possibly via Robust Neuronal Differentiation.

    PubMed

    Uchida, Hiroki; Morita, Takahiro; Niizuma, Kuniyasu; Kushida, Yoshihiro; Kuroda, Yasumasa; Wakao, Shohei; Sakata, Hiroyuki; Matsuzaka, Yoshiya; Mushiake, Hajime; Tominaga, Teiji; Borlongan, Cesario V; Dezawa, Mari

    2016-01-01

    Muse cells reside as pre-existing pluripotent-like stem cells within the fibroblasts, are nontumorigenic, exhibit differentiation capacity into triploblastic-lineage cells, and replenish lost cells when transplanted in injury models. Cell fate and function of human skin fibroblast-derived Muse cells were evaluated in a rat stroke model. Muse cells (30,000), collected by pluripotent surface marker stage-specific embryonic antigen-3, were injected stereotaxically into three deposits within the rat ischemic cortex at 2 days after transient middle cerebral artery occlusion, and the cells' biological effects were examined for more than 84 days. Muse cells spontaneously and promptly committed to neural/neuronal-lineage cells when cocultured with stroke brain slices. Muse-transplanted stroke rats exhibited significant improvements in neurological and motor functions compared to control groups at chronic days 70 and 84, without a reduction in the infarct size. Muse cells survived in the host brain for up to 84 days and differentiated into NeuN (∼ 65%), MAP-2 (∼ 32%), calbindin (∼ 28%), and GST-π (∼ 25%)-positive cells in the cortex, but glial fibrillary acidic protein-positive cells were rare. Tumor formation was not observed. Muse cells integrated into the sensory-motor cortex, extended their neurites into cervical spinal cord, and displayed normalized hind limb somatosensory evoked potentials. Muse cells are unique from other stem cells in that they differentiate with high ratio into neuronal cells after integration with host brain microenvironment, possibly reconstructing the neuronal circuit to mitigate stroke symptoms. Human fibroblast-derived Muse cells pose as a novel source of transplantable stem cells, circumventing the need for gene manipulations, especially when contemplating autologous cell therapy for stroke. © 2015 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

  4. Proneural Transcription Factor Atoh1 Drives Highly Efficient Differentiation of Human Pluripotent Stem Cells Into Dopaminergic Neurons

    PubMed Central

    Sagal, Jonathan; Zhan, Xiping; Xu, Jinchong; Tilghman, Jessica; Karuppagounder, Senthilkumar S.; Chen, Li; Dawson, Valina L.; Dawson, Ted M.; Laterra, John

    2014-01-01

    Human pluripotent stem cells (PSCs) are a promising cell resource for various applications in regenerative medicine. Highly efficient approaches that differentiate human PSCs into functional lineage-specific neurons are critical for modeling neurological disorders and testing potential therapies. Proneural transcription factors are crucial drivers of neuron development and hold promise for driving highly efficient neuronal conversion in PSCs. Here, we study the functions of proneural transcription factor Atoh1 in the neuronal differentiation of PSCs. We show that Atoh1 is induced during the neuronal conversion of PSCs and that ectopic Atoh1 expression is sufficient to drive PSCs into neurons with high efficiency. Atoh1 induction, in combination with cell extrinsic factors, differentiates PSCs into functional dopaminergic (DA) neurons with >80% purity. Atoh1-induced DA neurons recapitulate key biochemical and electrophysiological features of midbrain DA neurons, the degeneration of which is responsible for clinical symptoms in Parkinson’s disease (PD). Atoh1-induced DA neurons provide a reliable disease model for studying PD pathogenesis, such as neurotoxin-induced neurodegeneration in PD. Overall, our results determine the role of Atoh1 in regulating neuronal differentiation and neuron subtype specification of human PSCs. Our Atoh1-mediated differentiation approach will enable large-scale applications of PD patient-derived midbrain DA neurons in mechanistic studies and drug screening for both familial and sporadic PD. PMID:24904172

  5. Neuronal precursors within the adult rat subventricular zone differentiate into dopaminergic neurons after substantia nigra lesion and chromaffin cell transplant.

    PubMed

    Arias-Carrión, Oscar; Hernández-López, Salvador; Ibañez-Sandoval, Osvaldo; Bargas, José; Hernández-Cruz, Arturo; Drucker-Colín, René

    2006-11-15

    Neurogenesis in the adult mammalian brain continues in the subventricular zone (SVZ). Neuronal precursors from the SVZ migrate along the rostral migratory stream to replace olfactory bulb interneurons. After the destruction of the nigro-striatal pathway (SN-lesion), some SVZ precursors begin to express tyrosine hydroxylase (TH) and neuronal markers (NeuN). Grafting of chromaffin cells (CCs) into the denervated striatum increases the number of TH+ cells (SVZ TH+ cells; Arias-Carrión et al., 2004). This study examines the functional properties of these newly differentiating TH+ cells. Under whole-cell patch-clamp, most SVZ cells recorded from lesioned and grafted animals (either TH+ or TH-) were non-excitable. Nevertheless, a small percentage of SVZ TH+ cells had the electrophysiologic phenotype of mature dopaminergic neurons and showed spontaneous postsynaptic potentials. Dopamine (DA) release was measured in SVZ and striatum from both control and SN-lesioned rats. As expected, 12 weeks after SN lesion, DA release decreased drastically. Nevertheless, 8 weeks after CCs graft, release from the SVZ of SN-lesioned rats recovered, and even surpassed that from control SVZ, suggesting that newly formed SVZ TH+ cells release DA. This study shows for the first time that in response to SN-lesions and CC grafts neural precursors within the SVZ change their developmental program, by not only expressing TH, but more importantly by acquiring excitable properties of mature dopaminergic neurons. Additionally, the release of DA in a Ca(2+)-dependent manner and the attraction of synaptic afferents from neighboring neuronal networks gives further significance to the overall findings, whose potential importance is discussed.

  6. Protein hairy enhancer of split-1 expression during differentiation of muscle-derived stem cells into neuron-like cells.

    PubMed

    Huang, Mina; Guo, Zhanpeng; Liu, Kun; Mei, Xifan; Fang, Shiqiang; Zeng, Jinhao; Wang, Yansong; Yuan, Yajiang

    2012-10-05

    Muscle-derived stem cells were isolated from the skeletal muscle of Sprague-Dawley neonatal rats aged 3 days old. Cells at passage 5 were incubated in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum, 20 μg/L nerve growth factor, 20 μg/L basic fibroblast growth factor and 1% (v/v) penicillin for 6 days. Cells presented with long processes, similar to nerve cells. Connections were formed between cell processes. Immunocytochemical staining with neuron specific enolase verified that cells differentiated into neuron-like cells. Immunofluorescence cytochemistry and western blot results revealed that the expression of protein hairy enhancer of split-1 was significantly reduced. These results indicate that low expression of protein hairy enhancer of split-1 participates in the differentiation of muscle-derived stem cells into neuron-like cells.

  7. Comparative study of efficacy of dopaminergic neuron differentiation between embryonic stem cell and protein-based induced pluripotent stem cell.

    PubMed

    Kwon, Yoo-Wook; Chung, Yeon-Ju; Kim, Joonoh; Lee, Ho-Jae; Park, Jihwan; Roh, Tae-Young; Cho, Hyun-Jai; Yoon, Chang-Hwan; Koo, Bon-Kwon; Kim, Hyo-Soo

    2014-01-01

    In patients with Parkinson's disease (PD), stem cells can serve as therapeutic agents to restore or regenerate injured nervous system. Here, we differentiated two types of stem cells; mouse embryonic stem cells (mESCs) and protein-based iPS cells (P-iPSCs) generated by non-viral methods, into midbrain dopaminergic (mDA) neurons, and then compared the efficiency of DA neuron differentiation from these two cell types. In the undifferentiated stage, P-iPSCs expressed pluripotency markers as ES cells did, indicating that protein-based reprogramming was stable and authentic. While both stem cell types were differentiated to the terminally-matured mDA neurons, P-iPSCs showed higher DA neuron-specific markers' expression than ES cells. To investigate the mechanism of the superior induction capacity of DA neurons observed in P-iPSCs compared to ES cells, we analyzed histone modifications by genome-wide ChIP sequencing analysis and their corresponding microarray results between two cell types. We found that Wnt signaling was up-regulated, while SFRP1, a counter-acting molecule of Wnt, was more suppressed in P-iPSCs than in mESCs. In PD rat model, transplantation of neural precursor cells derived from both cell types showed improved function. The present study demonstrates that P-iPSCs could be a suitable cell source to provide patient-specific therapy for PD without ethical problems or rejection issues.

  8. Thermo-responsive polymeric nanoparticles for enhancing neuronal differentiation of human induced pluripotent stem cells.

    PubMed

    Seo, Hye In; Cho, Ann-Na; Jang, Jiho; Kim, Dong-Wook; Cho, Seung-Woo; Chung, Bong Geun

    2015-10-01

    We report thermo-responsive retinoic acid (RA)-loaded poly(N-isopropylacrylamide)-co-acrylamide (PNIPAM-co-Am) nanoparticles for directing human induced pluripotent stem cell (hiPSC) fate. Fourier transform infrared spectroscopy and (1)H nuclear magnetic resonance analysis confirmed that RA was efficiently incorporated into PNIAPM-co-Am nanoparticles (PCANs). The size of PCANs dropped with increasing temperatures (300-400 nm at room temperature, 80-90 nm at 37°C) due to its phase transition from hydrophilic to hydrophobic. Due to particle shrinkage caused by this thermo-responsive property of PCANs, RA could be released from nanoparticles in the cells upon cellular uptake. Immunocytochemistry and quantitative real-time polymerase chain reaction analysis demonstrated that neuronal differentiation of hiPSC-derived neuronal precursors was enhanced after treatment with 1-2 μg/ml RA-loaded PCANs. Therefore, we propose that this PCAN could be a potentially powerful carrier for effective RA delivery to direct hiPSC fate to neuronal lineage. The use of induced pluripotent stem cells (iPSCs) has been at the forefront of research in the field of regenerative medicine, as these cells have the potential to differentiate into various terminal cell types. In this article, the authors utilized a thermo-responsive polymer, Poly(N-isopropylacrylamide) (PNIPAM), as a delivery platform for retinoic acid. It was shown that neuronal differentiation could be enhanced in hiPSC-derived neuronal precursor cells. This method may pave a way for future treatment of neuronal diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

    PubMed Central

    Munro, Kathryn M.; Kennedy, Matthew J.; Gunnersen, Jenny M.

    2014-01-01

    In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (“antibody feeding”) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available. PMID:24561550

  10. Thyroid Hormone-Otx2 Signaling Is Required for Embryonic Ventral Midbrain Neural Stem Cells Differentiated into Dopamine Neurons

    PubMed Central

    Chen, Chunhai; Ma, Qinglong; Chen, Xiaowei; Zhong, Min; Deng, Ping; Zhu, Gang; Zhang, Yanwen; Zhang, Lei; Yang, Zhiqi; Zhang, Kuan; Guo, Lu; Wang, Liting; Yu, Zhengping

    2015-01-01

    Midbrain dopamine (DA) neurons are essential for maintaining multiple brain functions. These neurons have also been implicated in relation with diverse neurological disorders. However, how these neurons are developed from neuronal stem cells (NSCs) remains largely unknown. In this study, we provide both in vivo and in vitro evidence that the thyroid hormone, an important physiological factor for brain development, promotes DA neuron differentiation from embryonic ventral midbrain (VM) NSCs. We find that thyroid hormone deficiency during development reduces the midbrain DA neuron number, downregulates the expression of tyrosine hydroxylase (TH) and the dopamine transporter (DAT), and impairs the DA neuron-dependent motor behavior. In addition, thyroid hormone treatment during VM NSC differentiation in vitro increases the production of DA neurons and upregulates the expression of TH and DAT. We also found that the thyroid hormone enhances the expression of Otx2, an important determinant of DA neurogenesis, during DA neuron differentiation. Our in vitro gene silencing experiments indicate that Otx2 is required for thyroid hormone-dependent DA neuron differentiation from embryonic VM NSCs. Finally, we revealed both in vivo and in vitro that the thyroid hormone receptor alpha 1 is expressed in embryonic VM NSCs. Furthermore, it participates in the effects of thyroid hormone-induced Otx2 upregulation and DA neuron differentiation. These data demonstrate the role and molecular mechanisms of how the thyroid hormone regulates DA neuron differentiation from embryonic VM NSCs, particularly providing new mechanisms and a potential strategy for generating dopamine neurons from NSCs. PMID:25867707

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

    PubMed Central

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

    2013-01-01

    Introduction Microfluidics systems usually consist of materials like PMMA - poly(methyl methacrylate) and PDMS - poly(dimethylsiloxane) and not polystyrene (PS), which is usually used for cell culture. Cellular and molecular responses in cells grown on PS are well characterized due to decades of 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 neuronal-like cells was investigated using cell viability, cell cycle distribution, morphology, and gene expression analysis. Results/Conclusions After differentiation, the morphology, viability and cell cycle distribution of PC12 cells grown on PS, PMMA with and without PDMS underneath was the same. By 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 function. However, there were also many markers for neuronal cell development and functions that were expressed similarly in cells differentiating on PS, PMMA and PMMA with PDMS underneath. In conclusion, it was shown that PMMA has a minor impact and PDMS a major impact on gene expression in PC12 cells. PMID:23301028

  12. Isolation of neural precursor cells from skeletal muscle tissues and their differentiation into neuron-like cells.

    PubMed

    Park, Jung Sik; Kim, Soyeon; Han, Dong Keun; Lee, Ji Youl; Ghil, Sung Ho

    2007-08-31

    Skeletal muscle contains several precursor cells that generate muscle, bone, cartilage and blood cells. Although there are reports that skeletal muscle-derived cells can trans-differentiate into neural-lineage cells, methods for isolating precursor cells, and procedures for successful neural induction have not been fully established. Here, we show that the preplate cell isolation method, which separates cells based on their adhesion characteristics, permits separation of cells possessing neural precursor characteristics from other cells of skeletal muscle tissues. We term these isolated cells skeletal muscle-derived neural precursor cells (SMNPs). The isolated SMNPs constitutively expressed neural stem cell markers. In addition, we describe effective neural induction materials permitting the neuron-like cell differentiation of SMNPs. Treatment with retinoic acid or forskolin facilitated morphological changes in SMNPs; they differentiated into neuron-like cells that possessed specific neuronal markers. These results suggest that the preplate isolation method, and treatment with retinoic acid or forskolin, may provide vital assistance in the use of SMNPs in cell-based therapy of neuronal disease.

  13. Ketamine induces toxicity in human neurons differentiated from embryonic stem cells via mitochondrial apoptosis pathway

    PubMed Central

    Bosnjak, Zeljko J.; Yan, Yasheng; Canfield, Scott; Muravyeva, Maria Y.; Kikuchi, Chika; Wells, Clive; Corbett, John; Bai, Xiaowen

    2013-01-01

    Ketamine is widely used for anesthesia in pediatric patients. Growing evidence indicates that ketamine causes neurotoxicity in a variety of developing animal models. Our understanding of anesthesia neurotoxicity in humans is currently limited by difficulties in obtaining neurons and performing developmental toxicity studies in fetal and pediatric populations. It may be possible to overcome these challenges by obtaining neurons from human embryonic stem cells (hESCs) in vitro. hESCs are able to replicate indefinitely and differentiate into every cell type. In this study, we investigated the toxic effect of ketamine on neurons differentiated from hESCs. Two-week-old neurons were treated with different doses and durations of ketamine with or without the reactive oxygen species (ROS) scavenger, Trolox. Cell viability, ultrastructure, mitochondrial membrane potential (ΔΨm), cytochrome c distribution within cells, apoptosis, and ROS production were evaluated. Here we show that ketamine induced ultrastructural abnormalities and dose- and time-dependently caused cell death. In addition, ketamine decreased ΔΨm and increased cytochrome c release from mitochondria. Ketamine also increased ROS production and induced differential expression of oxidative stress-related genes. Specifically, abnormal ultrastructural and ΔΨm changes occurred earlier than cell death in the ketamine-induced toxicity process. Furthermore, Trolox significantly decreased ROS generation and attenuated cell death caused by ketamine in a dose-dependent manner. In conclusion, this study illustrates that ketamine time- and dose-dependently induces human neurotoxicity via ROS-mediated mitochondrial apoptosis pathway and that these side effects can be prevented by the antioxidant agent Trolox. Thus, hESC-derived neurons might provide a promising tool for studying anesthetic-induced developmental neurotoxicity and prevention strategies. PMID:22873495

  14. Real-Time Discrimination between Proliferation and Neuronal and Astroglial Differentiation of Human Neural Stem Cells

    PubMed Central

    Lee, Rimi; Kim, Il-Sun; Han, Nalae; Yun, Seokhwan; Park, Kook In; Yoo, Kyung-Hwa

    2014-01-01

    Neural stem cells (NSCs) are characterized by a capacity for self-renewal, differentiation into multiple neural lineages, all of which are considered to be promising components for neural regeneration. However, for cell-replacement therapies, it is essential to monitor the process of in vitro NSC differentiation and identify differentiated cell phenotypes. We report a real-time and label-free method that uses a capacitance sensor array to monitor the differentiation of human fetal brain-derived NSCs (hNSCs) and to identify the fates of differentiated cells. When hNSCs were placed under proliferation or differentiation conditions in five media, proliferating and differentiating hNSCs exhibited different frequency and time dependences of capacitance, indicating that the proliferation and differentiation status of hNSCs may be discriminated in real-time using our capacitance sensor. In addition, comparison between real-time capacitance and time-lapse optical images revealed that neuronal and astroglial differentiation of hNSCs may be identified in real-time without cell labeling. PMID:25204726

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

    SciTech Connect

    Han, Jeong-Ho; Yu, Tae-Hoon; Ryu, Hyun-Hee; Jun, Mi-Hee; Ban, Byung-Kwan; Jang, Deok-Jin; Lee, Jin-A

    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.

  16. Differentiation and Characterization of Dopaminergic Neurons From Baboon Induced Pluripotent Stem Cells

    PubMed Central

    Grow, Douglas A.; Simmons, DeNard V.; Gomez, Jorge A.; Wanat, Matthew J.; McCarrey, John R.; Paladini, Carlos A.

    2016-01-01

    The progressive death of dopamine producing neurons in the substantia nigra pars compacta is the principal cause of symptoms of Parkinson’s disease (PD). Stem cells have potential therapeutic use in replacing these cells and restoring function. To facilitate development of this approach, we sought to establish a preclinical model based on a large nonhuman primate for testing the efficacy and safety of stem cell-based transplantation. To this end, we differentiated baboon fibroblast-derived induced pluripotent stem cells (biPSCs) into dopaminergic neurons with the application of specific morphogens and growth factors. We confirmed that biPSC-derived dopaminergic neurons resemble those found in the human midbrain based on cell type-specific expression of dopamine markers TH and GIRK2. Using the reverse transcriptase quantitative polymerase chain reaction, we also showed that biPSC-derived dopaminergic neurons express PAX6, FOXA2, LMX1A, NURR1, and TH genes characteristic of this cell type in vivo. We used perforated patch-clamp electrophysiology to demonstrate that biPSC-derived dopaminergic neurons fired spontaneous rhythmic action potentials and high-frequency action potentials with spike frequency adaption upon injection of depolarizing current. Finally, we showed that biPSC-derived neurons released catecholamines in response to electrical stimulation. These results demonstrate the utility of the baboon model for testing and optimizing the efficacy and safety of stem cell-based therapeutic approaches for the treatment of PD. Significance Functional dopamine neurons were produced from baboon induced pluripotent stem cells, and their properties were compared to baboon midbrain cells in vivo. The baboon has advantages as a clinically relevant model in which to optimize the efficacy and safety of stem cell-based therapies for neurodegenerative diseases, such as Parkinson's disease. Baboons possess crucial neuroanatomical and immunological similarities to humans, and

  17. REST Regulates Non-Cell-Autonomous Neuronal Differentiation and Maturation of Neural Progenitor Cells via Secretogranin II.

    PubMed

    Kim, Hyung Joon; Denli, Ahmet M; Wright, Rebecca; Baul, Tithi D; Clemenson, Gregory D; Morcos, Ari S; Zhao, Chunmei; Schafer, Simon T; Gage, Fred H; Kagalwala, Mohamedi N

    2015-11-04

    RE-1 silencing transcription factor (REST), a master negative regulator of neuronal differentiation, controls neurogenesis by preventing the differentiation of neural stem cells. Here we focused on the role of REST in the early steps of differentiation and maturation of adult hippocampal progenitors (AHPs). REST knockdown promoted differentiation and affected the maturation of rat AHPs. Surprisingly, REST knockdown cells enhanced the differentiation of neighboring wild-type AHPs, suggesting that REST may play a non-cell-autonomous role. Gene expression analysis identified Secretogranin II (Scg2) as the major secreted REST target responsible for the non-cell-autonomous phenotype. Loss-of-function of Scg2 inhibited differentiation in vitro, and exogenous SCG2 partially rescued this phenotype. Knockdown of REST in neural progenitors in mice led to precocious maturation into neurons at the expense of mushroom spines in vivo. In summary, we found that, in addition to its cell-autonomous function, REST regulates differentiation and maturation of AHPs non-cell-autonomously via SCG2. Our results reveal that REST regulates differentiation and maturation of neural progenitor cells in vitro by orchestrating both cell-intrinsic and non-cell-autonomous factors and that Scg2 is a major secretory target of REST with a differentiation-enhancing activity in a paracrine manner. In vivo, REST depletion causes accelerated differentiation of newborn neurons at the expense of spine defects, suggesting a potential role for REST in the timing of the maturation of granule neurons. Copyright © 2015 the authors 0270-6474/15/3514872-13$15.00/0.

  18. Denervated hippocampus provides a favorable microenvironment for neuronal differentiation of endogenous neural stem cells

    PubMed Central

    Zhang, Lei; Han, Xiao; Cheng, Xiang; Tan, Xue-feng; Zhao, He-yan; Zhang, Xin-hua

    2016-01-01

    Fimbria-fornix transection induces both exogenous and endogenous neural stem cells to differentiate into neurons in the hippocampus. This indicates that the denervated hippocampus provides an environment for neuronal differentiation of neural stem cells. However, the pathways and mechanisms in this process are still unclear. Seven days after fimbria fornix transection, our reverse transcription polymerase chain reaction, western blot assay, and enzyme linked immunosorbent assay results show a significant increase in ciliary neurotrophic factor mRNA and protein expression in the denervated hippocampus. Moreover, neural stem cells derived from hippocampi of fetal (embryonic day 17) Sprague-Dawley rats were treated with ciliary neurotrophic factor for 7 days, with an increased number of microtubule associated protein-2-positive cells and decreased number of glial fibrillary acidic protein-positive cells detected. Our results show that ciliary neurotrophic factor expression is up-regulated in the denervated hippocampus, which may promote neuronal differentiation of neural stem cells in the denervated hippocampus. PMID:27212920

  19. Ampakine CX546 increases proliferation and neuronal differentiation in subventricular zone stem/progenitor cell cultures.

    PubMed

    Schitine, Clarissa; Xapelli, Sara; Agasse, Fabienne; Sardà-Arroyo, Laura; Silva, Ana P; De Melo Reis, Ricardo A; de Mello, Fernando G; Malva, João O

    2012-06-01

    Ampakines are chemical compounds known to modulate the properties of ionotropic α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA)-subtype glutamate receptors. The functional effects attributed to ampakines involve plasticity and the increase in synaptic efficiency of neuronal circuits, a process that may be intimately associated with differentiation of newborn neurons. The subventricular zone (SVZ) is the main neurogenic niche of the brain, containing neural stem cells with brain repair potential. Accordingly, the identification of new pharmaceutical compounds with neurogenesis-enhancing properties is important as a tool to promote neuronal replacement based on the use of SVZ cells. The purpose of the present paper is to examine the possible proneurogenic effects of ampakine CX546 in cell cultures derived from the SVZ of early postnatal mice. We observed that CX546 (50 μm) treatment triggered an increase in proliferation, evaluated by BrdU incorporation assay, in the neuroblast lineage. Moreover, by using a cell viability assay (TUNEL) we found that, in contrast to AMPA, CX546 did not cause cell death. Also, both AMPA and CX546 stimulated neuronal differentiation as evaluated morphologically through neuronal nuclear protein (NeuN) immunocytochemistry and functionally by single-cell calcium imaging. Accordingly, short exposure to CX546 increased axonogenesis, as determined by the number and length of tau-positive axons co-labelled for the phosphorylated form of SAPK/JNK (P-JNK), and dendritogenesis (MAP2-positive neurites). Altogether, this study shows that ampakine CX546 promotes neurogenesis in SVZ cell cultures and thereby may have potential for future stem cell-based therapies.

  20. Silicon nanowires enhanced proliferation and neuronal differentiation of neural stem cell with vertically surface microenvironment.

    PubMed

    Yan, Qiuting; Fang, Lipao; Wei, Jiyu; Xiao, Guipeng; Lv, Meihong; Ma, Quanhong; Liu, Chunfeng; Wang, Wang

    2017-09-01

    Owing to its biocompatibility, noncytotoxicity, biodegradability and three-dimensional structure, vertically silicon nanowires (SiNWs) arrays are a promising scaffold material for tissue engineering, regenerative medicine and relevant medical applications. Recently, its osteogenic differentiation effects, reorganization of cytoskeleton and regulation of the fate on stem cells have been demonstrated. However, it still remains unknown whether SiNWs arrays could affect the proliferation and neuronal differentiation of neural stem cells (NSCs) or not. In the present study, we have employed vertically aligned SiNWs arrays as culture systems for NSCs and proved that the scaffold material could promote the proliferation and neuronal differentiation of NSCs while maintaining excellent cell viability and stemness. Immunofluorescence imaging analysis, Western blot and RT-PCR results reveal that NSCs proliferation and neuronal differentiation efficiency on SiNWs arrays are significant greater than that on silicon wafers. These results implicate SiNWs arrays could offer a powerful platform for NSCs research and NSCs-based therapy in the field of neural tissue engineering.

  1. Affect of antidepressants on the in vitro differentiation of rat bone marrow mesenchymal stem cells into neuronal cells.

    PubMed

    Borkowska, Paulina; Kowalska, Joanna; Fila-Danilow, Anna; Bielecka, Anna Maria; Paul-Samojedny, Monika; Kowalczyk, Malgorzata; Kowalski, Jan

    2015-06-20

    Bone marrow is a valuable source of mesenchymal stem cells (MSCs) that can be used in regenerative medicine. MSCs are able to differentiate into cells from all three germ layers under specific conditions. The aim of the current work was to study the differentiation of rat MSCs (rMSCs) into neuron-like cells. We investigated how the antidepressants imipramine, desipramine, fluoxetine and tianeptine affect the differentiation of rMSCs. Furthermore, we present differentiation cocktails using a cortex astrocyte-conditioned medium (CACM) separately or in conjunction with each of the antidepressants and investigated their additive effect on the efficiency of differentiation. We also observed how various differentiation conditions affect the number of primary dendrites and branching dendrites per cell. Gene expression for an early neuronal marker (β-III-tubulin) and markers that are typical for adult neurons such as Th, Htr2A and Slc6a4 were observed. The Tubb3 and Htr2A gene expression were up-regulated, Th decreased slightly and Slc6a4 was down-regulated after differentiation We observed a two-fold higher percentage of β-III-tubulin positive cells after treatment with antidepressants and two-fold increase of neuron-like cells after using CACM with imipramine or fluoxetine simultaneously. Differentiation using imipramine or in conjunction with CACM and desipramine or fluoxetine simultaneously increased the number of cell dendrites. The results that were obtained are completely new and need further investigations in the nearest future. These results suggest that antidepressants improve differentiation efficiency of rMSCs and may be useful in the preparation of rMSCs for transplantation. Differentiation efficiency is higher after long-term exposure to antidepressants, than after a 24-h exposure. Nearly additive effect of CACM and imipramine or fluoxetine suggests a beneficial role of antidepressants after transplantation. Copyright © 2015 Elsevier B.V. All rights

  2. The adult CNS retains the potential to direct region-specific differentiation of a transplanted neuronal precursor cell line.

    PubMed

    Shihabuddin, L S; Hertz, J A; Holets, V R; Whittemore, S R

    1995-10-01

    The chronic survival and differentiation of the conditionally immortalized neuronal cell line, RN33B, was examined following transplantation into the adult and neonatal rat hippocampus and cerebral cortex. In clonal culture, differentiated RN33B cells express p75NTR and trkB mRNA and protein, and respond to brain-derived neurotrophic factor treatment by inducing c-fos mRNA. Transplanted cells, identified using immunohistochemistry to detect beta-galactosidase expression, were seen in most animals up to 24 weeks posttransplantation (the latest time point examined). Stably integrated cells with various morphologies consistent with their transplantation site were observed. In the cerebral cortex, many RN33B cells differentiated with morphologies similar to pyramidal neurons and stellate cells. In the hippocampal formation, many RN33B cells assumed morphologies similar to pyramidal neurons characteristic of CA1 and CA3 regions, granular cell layer neurons of the dentate gyrus, and polymorphic neurons of the hilar region. Identical morphologies were observed in both adult and neonatal hosts, although a greater percentage of beta-galactosidase immunoreactive cells had differentiated in the neonatal brains. These results suggest that RN33B cells have the developmental plasticity to respond to local microenvironmental signals and that the adult brain retains the capacity to direct the differentiation of neuronal precursor cells in a direction that is consistent with that of endogenous neurons.

  3. Neurotrophic factor mediated neuronal differentiation of human cord blood mesenchymal stem cells and their applicability to assess the developmental neurotoxicity.

    PubMed

    Jahan, Sadaf; Kumar, Dipak; Kumar, Ashvini; Rajpurohit, Chetan Singh; Singh, Shripriya; Srivastava, Akriti; Pandey, Ankita; Pant, A B

    2017-01-22

    Plasticity and developmental capacity of stem cells have now been established as a promising tool to restore the degenerative disorders. The linearity differentiation of human mesenchymal stem cells (hMSCs) into adipogenic, chondrogenic, osteogenic and even in neuronal subtypes has been demonstrated. The number of xenobiotics such as dexamethasone, insulin, isobutyl 1-methyle xanthine and retinoic acid has been reported for the potential to differentiate hMSCs into neuronal subtypes. But, the applicability of indigenous neurotrophic factor-nerve growth factor (NGF) has not been explored for the purpose. Thus, the present investigations were carried out to study the NGF induced neuronal differentiation of hMSCs. Following the isolation, purification and characterization of hMSCs were allowed to differentiate into neuronal subtypes under the influence of NGF (50 ng/mL). At various concentrations of NGF, the neuronal makers were analysed at both mRNA and protein levels. Cells, exposed with NGF were showing the significant and gradual increase in the neuronal markers in differentiating cells. The magnitude of expression of markers was maximum at day 4 of differentiation. NGF at 50 ng/mL concentration was found to induce neuronal differentiation of hMSCs into neuronal subtypes. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    PubMed

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

    2015-11-04

    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.

  5. RBM4 Regulates Neuronal Differentiation of Mesenchymal Stem Cells by Modulating Alternative Splicing of Pyruvate Kinase M.

    PubMed

    Su, Chun-Hao; Hung, Kuan-Yang; Hung, Shih-Chieh; Tarn, Woan-Yuh

    2017-02-01

    RBM4 promotes differentiation of neuronal progenitor cells and neurite outgrowth of cultured neurons via its role in splicing regulation. In this study, we further explored the role of RBM4 in neuronal differentiation. During neuronal differentiation, energy production shifts from glycolysis to oxidative phosphorylation. We found that the splice isoform change of the metabolic enzyme pyruvate kinase M (PKM) from PKM2 to PKM1 occurs during brain development and is impaired in RBM4-deficient brains. The PKM isoform change could be recapitulated in human mesenchymal stem cells (MSCs) during neuronal induction. Using a PKM minigene, we demonstrated that RBM4 plays a direct role in regulating alternative splicing of PKM. Moreover, RBM4 antagonized the function of the splicing factor PTB and induced the expression of a PTB isoform with attenuated splicing activity in MSCs. Overexpression of RBM4 or PKM1 induced the expression of neuronal genes, increased the mitochondrial respiration capacity in MSCs, and, accordingly, promoted neuronal differentiation. Finally, we demonstrated that RBM4 is induced and is involved in the PKM splicing switch and neuronal gene expression during hypoxia-induced neuronal differentiation. Hence, RBM4 plays an important role in the PKM isoform switch and the change in mitochondrial energy production during neuronal differentiation. Copyright © 2017 American Society for Microbiology.

  6. Early phosphorylation of MARCKS at Ser25 in migrating precursor cells and differentiating peripheral neurons.

    PubMed

    Ruiz-Perera, Lucía M; Arruti, Cristina; Zolessi, Flavio R

    2013-06-07

    MARCKS is a ubiquitous actin-binding protein, with special functions in the development of the central nervous system. We have previously described a neuronal-specific isoform, phosphorylated at serine 25 (S25p-MARCKS), which is present very early during neuronal differentiation in the chick retina. However, very little is known about MARCKS expression or functions in the peripheral nervous system (PNS). In the present work, we analyzed migrating PNS precursor cells in the chick embryo, particularly those originating from the neural crest, and found that they all express a high amount of MARCKS and that a subpopulation of them also contained S25p-MARCKS from early developmental stages. MARCKS protein was also found in dorsal root and trigeminal ganglia during embryo development. Not only is the protein present in these structures but it is also phosphorylated in differentiating neurons with a maximal signal on the ganglion periphery, where neurogenesis is occurring. In conclusion, MARCKS is present and phosphorylated at early stages during the differentiation of PNS cells and precursors, indicating that it might also be important for the differentiation of these tissues. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

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

    PubMed

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

    2014-09-01

    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 (10nM) decreased DG stem cell self-renewal, while 1nM 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 10nM) did not induce cell death. In accordance with the impairment of self-renewal capacity, METH (10nM) 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 (10μM). Moreover, METH (10nM) 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.

  8. Electrospun biomaterial scaffolds with varied topographies for neuronal differentiation of human-induced pluripotent stem cells.

    PubMed

    Mohtaram, Nima Khadem; Ko, Junghyuk; King, Craig; Sun, Lin; Muller, Nathan; Jun, Martin Byung-Guk; Willerth, Stephanie M

    2015-08-01

    In this study, we investigated the effect of micro and nanoscale scaffold topography on promoting neuronal differentiation of human induced pluripotent stem cells (iPSCs) and directing the resulting neuronal outgrowth in an organized manner. We used melt electrospinning to fabricate poly (ε-caprolactone) (PCL) scaffolds with loop mesh and biaxial aligned microscale topographies. Biaxial aligned microscale scaffolds were further functionalized with retinoic acid releasing PCL nanofibers using solution electrospinning. These scaffolds were then seeded with neural progenitors derived from human iPSCs. We found that smaller diameter loop mesh scaffolds (43.7 ± 3.9 µm) induced higher expression of the neural markers Nestin and Pax6 compared to thicker diameter loop mesh scaffolds (85 ± 4 µm). The loop mesh and biaxial aligned scaffolds guided the neurite outgrowth of human iPSCs along the topographical features with the maximum neurite length of these cells being longer on the biaxial aligned scaffolds. Finally, our novel bimodal scaffolds also supported the neuronal differentiation of human iPSCs as they presented both physical and chemical cues to these cells, encouraging their differentiation. These results give insight into how physical and chemical cues can be used to engineer neural tissue.

  9. MicroRNA-125b Promotes Neuronal Differentiation in Human Cells by Repressing Multiple Targets▿ †

    PubMed Central

    Le, Minh T. N.; Xie, Huangming; Zhou, Beiyan; Chia, Poh Hui; Rizk, Pamela; Um, Moonkyoung; Udolph, Gerald; Yang, Henry; Lim, Bing; Lodish, Harvey F.

    2009-01-01

    MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression at the posttranscriptional level. Research on miRNAs has highlighted their importance in neural development, but the specific functions of neurally enriched miRNAs remain poorly understood. We report here the expression profile of miRNAs during neuronal differentiation in the human neuroblastoma cell line SH-SY5Y. Six miRNAs were significantly upregulated during differentiation induced by all-trans-retinoic acid and brain-derived neurotrophic factor. We demonstrated that the ectopic expression of either miR-124a or miR-125b increases the percentage of differentiated SH-SY5Y cells with neurite outgrowth. Subsequently, we focused our functional analysis on miR-125b and demonstrated the important role of this miRNA in both the spontaneous and induced differentiations of SH-SH5Y cells. miR-125b is also upregulated during the differentiation of human neural progenitor ReNcell VM cells, and miR-125b ectopic expression significantly promotes the neurite outgrowth of these cells. To identify the targets of miR-125b regulation, we profiled the global changes in gene expression following miR-125b ectopic expression in SH-SY5Y cells. miR-125b represses 164 genes that contain the seed match sequence of the miRNA and/or that are predicted to be direct targets of miR-125b by conventional methods. Pathway analysis suggests that a subset of miR-125b-repressed targets antagonizes neuronal genes in several neurogenic pathways, thereby mediating the positive effect of miR-125b on neuronal differentiation. We have further validated the binding of miR-125b to the miRNA response elements of 10 selected mRNA targets. Together, we report here for the first time the important role of miR-125b in human neuronal differentiation. PMID:19635812

  10. MicroRNA-125b promotes neuronal differentiation in human cells by repressing multiple targets.

    PubMed

    Le, Minh T N; Xie, Huangming; Zhou, Beiyan; Chia, Poh Hui; Rizk, Pamela; Um, Moonkyoung; Udolph, Gerald; Yang, Henry; Lim, Bing; Lodish, Harvey F

    2009-10-01

    MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression at the posttranscriptional level. Research on miRNAs has highlighted their importance in neural development, but the specific functions of neurally enriched miRNAs remain poorly understood. We report here the expression profile of miRNAs during neuronal differentiation in the human neuroblastoma cell line SH-SY5Y. Six miRNAs were significantly upregulated during differentiation induced by all-trans-retinoic acid and brain-derived neurotrophic factor. We demonstrated that the ectopic expression of either miR-124a or miR-125b increases the percentage of differentiated SH-SY5Y cells with neurite outgrowth. Subsequently, we focused our functional analysis on miR-125b and demonstrated the important role of this miRNA in both the spontaneous and induced differentiations of SH-SH5Y cells. miR-125b is also upregulated during the differentiation of human neural progenitor ReNcell VM cells, and miR-125b ectopic expression significantly promotes the neurite outgrowth of these cells. To identify the targets of miR-125b regulation, we profiled the global changes in gene expression following miR-125b ectopic expression in SH-SY5Y cells. miR-125b represses 164 genes that contain the seed match sequence of the miRNA and/or that are predicted to be direct targets of miR-125b by conventional methods. Pathway analysis suggests that a subset of miR-125b-repressed targets antagonizes neuronal genes in several neurogenic pathways, thereby mediating the positive effect of miR-125b on neuronal differentiation. We have further validated the binding of miR-125b to the miRNA response elements of 10 selected mRNA targets. Together, we report here for the first time the important role of miR-125b in human neuronal differentiation.

  11. Substantial Differentiation of Human Neural Stem Cells Into Motor Neurons on a Biomimetic Polyurea.

    PubMed

    Yun, Donghwa; Lee, Young M; Laughter, Melissa R; Freed, Curt R; Park, Daewon

    2015-09-01

    To find the first restorative treatment for spinal cord injury (SCI), researchers have focused on stem cell therapies. However, one obstacle is the lack of an implantable cell scaffold that can support efficient motor neuron (MN) differentiation and proliferation. We aimed to overcome this through the use of an RGD functionalized novel biomimetic polyurea, optimized to encourage efficient differentiation of MNs. Images taken after 14-days showed increased differentiation (∼40%) of hNSCs into MNs as well as increased cell count on the biomimetic polymer compared to PDL-Laminin coating, indicating that the RGD-polyurea provides a favorable microenvironment for hNSC survival, having promising implications for future SCI therapies.

  12. Substantial Differentiation of Human Neural Stem Cells Into Motor Neurons on a Biomimetic Polyureaa

    PubMed Central

    Yun, Donghwa; Lee, Young M.; Laughter, Melissa R.; Freed, Curt R.

    2015-01-01

    To find the first restorative treatment for spinal cord injury (SCI), researchers have focused on stem cell therapies. However, one obstacle is the lack of an implantable cell scaffold that can support efficient motor neuron (MN) differentiation and proliferation. We aimed to overcome this through the use of an RGD functionalized novel biomimetic polyurea, optimized to encourage efficient differentiation of MNs. Images taken after 14-days showed increased differentiation (~40%) of hNSCs into MNs as well as increased cell count on the biomimetic polymer compared to PDL-Laminin coating, indicating that the RGD-polyurea provides a favorable microenvironment for hNSC survival, having promising implications for future SCI therapies. PMID:26033933

  13. Effect of a dominant inhibitory Ha-ras mutation on neuronal differentiation of PC12 cells.

    PubMed Central

    Szeberényi, J; Cai, H; Cooper, G M

    1990-01-01

    A dominant inhibitory mutation of Ha-ras which changes Ser-17 to Asn-17 in the gene product p21 [p21 (Asn-17)Ha-ras] has been used to investigate the role of ras in neuronal differentiation of PC12 cells. The growth of PC12 cells, in contrast to NIH 3T3 cells, was not inhibited by p21(Asn-17)Ha-ras expression. However, PC12 cells expressing the mutant Ha-ras protein showed a marked inhibition of morphological differentiation induced by nerve growth factor (NGF) or fibroblast growth factor (FGF). These cells, however, were still able to respond with neurite outgrowth to dibutyryl cyclic AMP and 12-O-tetradecanoylphorbol-13-acetate (TPA). Induction of early-response genes (fos, jun, and zif268) by NGF and FGF but not by TPA was also inhibited by high levels of p21(Asn-17)Ha-ras. However, lower levels of p21(Asn-17) expression were sufficient to block neuronal differentiation without inhibiting induction of these early-response genes. Induction of the secondary-response genes SCG10 and transin by NGF, like morphological differentiation, was inhibited by low levels of p21(Asn-17) whether or not induction of early-response genes was blocked. Therefore, although inhibition of ras function can inhibit early-response gene induction, this is not required to block morphological differentiation or secondary-response gene expression. These results suggest that ras proteins are involved in at least two different pathways of signal transduction from the NGF receptor, which can be distinguished by differential sensitivity to p21(Asn-17)Ha-ras. In addition, ras and protein kinase C can apparently induce early-response gene expression by independent pathways in PC12 cells. Images PMID:2118994

  14. Evaluation of Motor Neuron-Like Cell Differentiation of hEnSCs on Biodegradable PLGA Nanofiber Scaffolds.

    PubMed

    Ebrahimi-Barough, Somayeh; Norouzi Javidan, Abbas; Saberi, Hoshangh; Joghataei, Mohammad Tghi; Rahbarghazi, Reza; Mirzaei, Esmaeil; Faghihi, Faezeh; Shirian, Sadegh; Ai, Armin; Ai, Jafar

    2015-12-01

    Human endometrium is a high-dynamic tissue that contains human endometrial stem cells (hEnSCs) which can be differentiated into a number of cell lineages. The differentiation of hEnSCs into many cell lineages such as osteoblast, adipocyte, and neural cells has been investigated previously. However, the differentiation of these stem cells into motor neuron-like cells has not been investigated yet. Different biochemical and topographical cues can affect the differentiation of stem cells into a specific cell. The aim of this study was to investigate the capability of hEnSCs to be differentiated into motor neuron-like cells under biochemical and topographical cues. The biocompatible and biodegradable poly(lactic-co-glycolic acid) (PLGA) electrospun nanofibrous scaffold was used as a topographical cue. Human EnSCs were cultured on the PLGA scaffold and tissue culture polystyrene (TCP), then differentiation of hEnSCs into motor neuron-like cells under induction media including retinoic acid (RA) and sonic hedgehog (Shh) were evaluated for 15 days. The proliferation rate of cells was assayed by using MTT assay. The morphology of cells was studied by scanning electron microscopy imaging, and the expression of motor neuron-specific markers by real-time PCR and immunocytochemistry. Results showed that survival and differentiation of hEnSCs into motor neuron-like cells on the PLGA scaffold were better than those on the TCP group. Taken together, the results suggest that differentiated hEnSCs on PLGA can provide a suitable, three-dimensional situation for neuronal survival and outgrowth for regeneration of the central nervous system, and these cells may be a potential candidate in cellular therapy for motor neuron diseases.

  15. The redox function of APE1 is involved in the differentiation process of stem cells toward a neuronal cell fate.

    PubMed

    Domenis, Rossana; Bergamin, Natascha; Gianfranceschi, Giuseppe; Vascotto, Carlo; Romanello, Milena; Rigo, Silvia; Vagnarelli, Giovanna; Faggiani, Massimo; Parodi, Piercamillo; Kelley, Mark R; Beltrami, Carlo Alberto; Cesselli, Daniela; Tell, Gianluca; Beltrami, Antonio Paolo

    2014-01-01

    Low-to-moderate levels of reactive oxygen species (ROS) govern different steps of neurogenesis via molecular pathways that have been decrypted only partially. Although it has been postulated that redox-sensitive molecules are involved in neuronal differentiation, the molecular bases for this process have not been elucidated yet. The aim of this work was therefore to study the role played by the redox-sensitive, multifunctional protein APE1/Ref-1 (APE1) in the differentiation process of human adipose tissue-derived multipotent adult stem cells (hAT-MASC) and embryonic carcinoma stem cells (EC) towards a neuronal phenotype. Applying a definite protocol, hAT-MASC can adopt a neural fate. During this maturation process, differentiating cells significantly increase their intracellular Reactive Oxygen Species (ROS) levels and increase the APE1 nuclear fraction bound to chromatin. This latter event is paralleled by the increase of nuclear NF-κB, a transcription factor regulated by APE1 in a redox-dependent fashion. Importantly, the addition of the antioxidant N-acetyl cysteine (NAC) to the differentiation medium partially prevents the nuclear accumulation of APE1, increasing the neuronal differentiation of hAT-MASC. To investigate the involvement of APE1 in the differentiation process, we employed E3330, a specific inhibitor of the APE1 redox function. The addition of E3330, either to the neurogenic embryonic carcinoma cell line NT2-D1or to hAT-MASC, increases the differentiation of stem cells towards a neural phenotype, biasing the differentiation towards specific subtypes, such as dopaminergic cells. In conclusion, during the differentiation process of stem cells towards a neuroectodermic phenotype, APE1 is recruited, in a ROS-dependent manner, to the chromatin. This event is associated with an inhibitory effect of APE1 on neurogenesis that may be reversed by E3330. Therefore, E3330 may be employed both to boost neural differentiation and to bias the differentiation

  16. Cell reprogramming and neuronal differentiation applied to neurodegenerative diseases: Focus on Parkinson's disease.

    PubMed

    Wenker, Shirley D; Casalía, Mariana; Candedo, Verónica Cavaliere; Casabona, Juan Cruz; Pitossi, Fernando J

    2015-11-14

    Adult cells from patients can be reprogrammed to induced pluripotent stem cells (iPSCs) which successively can be used to obtain specific cells such as neurons. This remarkable breakthrough represents a new way of studying diseases and brought new therapeutic perspectives in the field of regenerative medicine. This is particular true in the neurology field, where few techniques are amenable to study the affected tissue of the patient during illness progression, in addition to the lack of neuroprotective therapies for many diseases. In this review we discuss the advantages and unresolved issues of cell reprogramming and neuronal differentiation. We reviewed evidence using iPSCs-derived neurons from neurological patients. Focusing on data obtained from Parkinson's disease (PD) patients, we show that iPSC-derived neurons possess morphological and functional characteristics of this disease and build a case for the use of this technology to study PD and other neuropathologies while disease is in progress. These data show the enormous impact that this new technology starts to have on different purposes such as the study and design of future therapies of neurological disease, especially PD.

  17. Neuronal-like cell differentiation of non-adherent bone marrow cell-derived mesenchymal stem cells.

    PubMed

    Wu, Yuxin; Zhang, Jinghan; Ben, Xiaoming

    2013-08-05

    Non-adherent bone marrow cell-derived mesenchymal stem cells from C57BL/6J mice were separated and cultured using the "pour-off" method. Non-adherent bone marrow cell-derived mesenchymal stem cells developed colony-forming unit-fibroblasts, and could be expanded by supplementation with epidermal growth factor. Immunocytochemistry showed that the non-adherent bone marrow cell-derived mesenchymal stem cells exposed to basic fibroblast growth factor/epidermal growth factor/nerve growth factor expressed the neuron specific markers, neurofilament-200 and NeuN, in vitro. Non-adherent bone marrow cell-derived mesenchymal stem cells from β-galactosidase transgenic mice were also transplanted into focal ischemic brain (right corpus striatum) of C57BL/6J mice. At 8 weeks, cells positive for LacZ and β-galactosidase staining were observed in the ischemic tissues, and cells co-labeled with both β-galactosidase and NeuN were seen by double immunohistochemical staining. These findings suggest that the non-adherent bone marrow cell-derived mesenchymal stem cells could differentiate into neuronal-like cells in vitro and in vivo.

  18. Differentiation of human induced pluripotent stem cells to mature functional Purkinje neurons

    PubMed Central

    Wang, Shuyan; Wang, Bin; Pan, Na; Fu, Linlin; Wang, Chaodong; Song, Gongru; An, Jing; Liu, Zhongfeng; Zhu, Wanwan; Guan, Yunqian; Xu, Zhi-Qing David; Chan, Piu; Chen, Zhiguo; Zhang, Y. Alex

    2015-01-01

    It remains a challenge to differentiate human induced pluripotent stem cells (iPSCs) or embryonic stem (ES) cells to Purkinje cells. In this study, we derived iPSCs from human fibroblasts and directed the specification of iPSCs first to Purkinje progenitors, by adding Fgf2 and insulin to the embryoid bodies (EBs) in a time-sensitive manner, which activates the endogenous production of Wnt1 and Fgf8 from EBs that further patterned the cells towards a midbrain-hindbrain-boundary tissue identity. Neph3-positive human Purkinje progenitors were sorted out by using flow cytometry and cultured either alone or with granule cell precursors, in a 2-dimensional or 3-dimensional environment. However, Purkinje progenitors failed to mature further under above conditions. By co-culturing human Purkinje progenitors with rat cerebellar slices, we observed mature Purkinje-like cells with right morphology and marker expression patterns, which yet showed no appropriate membrane properties. Co-culture with human fetal cerebellar slices drove the progenitors to not only morphologically correct but also electrophysiologically functional Purkinje neurons. Neph3-posotive human cells could also survive transplantation into the cerebellum of newborn immunodeficient mice and differentiate to L7- and Calbindin-positive neurons. Obtaining mature human Purkinje cells in vitro has significant implications in studying the mechanisms of spinocerebellar ataxias and other cerebellar diseases. PMID:25782665

  19. Nanofibrous scaffold-mediated REST knockdown to enhance neuronal differentiation of stem cells.

    PubMed

    Low, Wei Ching; Rujitanaroj, Pim-On; Lee, Dong-Keun; Messersmith, Phillip B; Stanton, Lawrence W; Goh, Eyleen; Chew, Sing Yian

    2013-05-01

    At present, the recovery prospect for patients with chronic neurodegenerative diseases or acute trauma in the central nervous system is sub-optimal. The controlled differentiation of neural stem/progenitor cells (NPCs) to functional neurons is a possible treatment strategy. In contrast to the classical approach of biochemicals supplementation for guided stem cell commitment, this study explores the feasibility of directing neuronal differentiation through synergistic integration of three-dimensional nanofibrous topographical cues and scaffold-mediated knockdown of RE-1 silencing transcription factor (REST) in mouse NPCs. Taking advantage of the strong adhesive property and latent reactivity of mussel-inspired polydopamine (PD) coating, electrospun polycaprolactone (PCL) nanofibers were successfully functionalized with REST siRNAs (denoted as siREST PD-fiber). Sustained REST knockdown in NPCs was achieved for up to five days in vitro and the silencing efficiency was significantly higher than that mediated through siRNA adsorption onto non-PD coated sample controls. The silencing of REST, together with nanofiber topographical effect, significantly enhanced NPC neuronal commitment (57.5% Map2(+) cells in siREST PD-fiber vs. 43.5% in siREST PD-film vs. 50% in PD-fiber controls, p < 0.05) while reducing astrocytic and oligodendrocytic differentiation (10.7% O4(+) cells vs. ∼30% in siREST PD-film, p < 0.01). Taken together, the synergistic effects of scaffold-mediated REST knockdown and topographical cues from PD-modified nanofibers may be a useful strategy for generating functional neurons for therapeutic purposes. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. Safrole oxide induced neuronal differentiation of rat bone-marrow mesenchymal stem cells by elevating Hsp70.

    PubMed

    Zhao, YanChun; Xin, Jie; Sun, ChunHui; Zhao, BaoXiang; Zhao, Jing; Su, Le

    2012-11-01

    In a previous study, we found that at low concentrations, safrole oxide (SFO) could induce vascular endothelial cell (VEC) transdifferentiation into neuron-like cells; however, whether SFO could induce bone-marrow mesenchymal stem cell (BMSC) neural differentiation was unknown. Here, we found that SFO could effectively induce BMSC neural differentiation in the presence of serum and fibroblast growth factor 2 and did not affect cell viability at low concentrations. The levels of neuron-specific enolase and neurofilament-L were increased greatly, but that of glial fibrillary acidic protein was absent with SFO treatment for 48h. Furthermore, SFO could increase the level of heat shock protein 70 (Hsp70), an important factor in neuronal differentiation. Knockdown of Hsp70 by its small interfering RNA blocked SFO-induced BMSC differentiation. Thus, SFO is a novel inducer of BMSC differentiation to neuron-like cells and Hsp70 is implicated in the differentiation process. We provide a new tool for obtaining neuron-like cells from BMSCs and for further investigating the new effect of Hsp70 on BMSC neuronal differentiation. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. Muscle and neuronal differentiation in primary cell culture of larval Mytilus trossulus (Mollusca: Bivalvia).

    PubMed

    Odintsova, Nelly A; Dyachuk, Vyacheslav A; Nezlin, Leonid P

    2010-03-01

    Molluscan in vitro technology allows the study of the differentiation of isolated cells undergoing experimental manipulations. We have used the immunofluorescence technique and laser scanning microscopy to investigate the organization of muscle proteins (actin, myosin, paramyosin, and twitchin) and the localization of neurotransmitters (serotonin and FMRFamide) in cultured mussel larval cells. Differentiation into muscle and neuron-like cells occurs during the cultivation of mussel cells from premyogenic and prenervous larval stages. Muscle proteins are colocalized in contractile cells through all stages of cultivation. The cultivation of mussel cells on various substrates and the application of integrin receptor blockers suggest that an integrin-dependent mechanism is involved in cell adhesion and differentiation. Dissociated mussel cells aggregate and become self-organized in culture. After 20 days of cultivation, they form colonies in which serotonin- and FMRFamide-immunoreactive cells are located centrally, whereas muscle cells form a contractile network at the periphery. The pattern of thick and thin filaments in cultivated mussel cells changes according to the scenario of muscle arrangement in vivo: initially, a striated pattern of muscle filaments forms but is then replaced by a smooth muscle pattern with a diffuse distribution of muscle proteins, typical of muscles of adult molluscs. Myogenesis in molluscs thus seems to be a highly dynamic and potentially variable process. Such a "flexible" developmental program can be regarded as a prerequisite for the evolution of the wide variety of striated and smooth muscles in larval and adult molluscs.

  2. Synergistic effects of CoCl(2) and ROCK inhibition on mesenchymal stem cell differentiation into neuron-like cells.

    PubMed

    Pacary, Emilie; Legros, Hélène; Valable, Samuel; Duchatelle, Pascal; Lecocq, Myriam; Petit, Edwige; Nicole, Olivier; Bernaudin, Myriam

    2006-07-01

    Bone-marrow-derived mesenchymal stem cells (MSCs) constitute an interesting cellular source to promote brain regeneration after neurodegenerative diseases. Recently, several studies suggested that oxygen-dependent gene expression is of crucial importance in governing the essential steps of neurogenesis such as cell proliferation, survival and differentiation. In this context, we analysed the effect of the HIF-1 (hypoxia inducible factor-1) activation-mimicking agent CoCl(2) on MSCs. CoCl(2) treatment increased the expression of the anti-proliferative gene BTG2/PC3 and decreased cyclin D1 expression. Expression of HIF-1alpha and its target genes EPO, VEGF and p21 was also upregulated. These changes were followed by inhibition of cell proliferation and morphological changes resulting in neuron-like cells, which had increased neuronal marker expression and responded to neurotransmitters. Echinomycin, a molecule inhibiting HIF-1 DNA-binding activity, blocked the CoCl(2) effect on MSCs. Additionally, by using Y-27632, we demonstrated that Rho kinase (ROCK) inhibition potentiated CoCl(2)-induced MSC differentiation in particular into dopaminergic neuron-like cells as attested by its effect on tyrosine hydroxylase expression. Altogether, these results support the ability of MSCs to differentiate into neuron-like cells in response to CoCl(2), an effect that might act, in part, through HIF-1 activation and cell-cycle arrest, and which is potentiated by inhibition of ROCK.

  3. Synthesis of Novel Synthetic Vitamin K Analogues Prepared by Introduction of a Heteroatom and a Phenyl Group That Induce Highly Selective Neuronal Differentiation of Neuronal Progenitor Cells.

    PubMed

    Kimura, Kimito; Hirota, Yoshihisa; Kuwahara, Shigefumi; Takeuchi, Atsuko; Tode, Chisato; Wada, Akimori; Osakabe, Naomi; Suhara, Yoshitomo

    2017-03-03

    We synthesized novel vitamin K2 analogues that incorporated a heteroatom and an aromatic ring in the side chain and evaluated their effect on the selective differentiation of neuronal progenitor cells into neurons in vitro. The results showed that a menaquinone-2 analogue bearing a p-fluoroaniline had the most potent activity, which was more than twice as great as the control. In addition, the neuronal selectivity was more than 3 times greater than the control.

  4. Neutralization of LINGO-1 during in vitro differentiation of neural stem cells results in proliferation of immature neurons.

    PubMed

    Lööv, Camilla; Fernqvist, Maria; Walmsley, Adrian; Marklund, Niklas; Erlandsson, Anna

    2012-01-01

    Identifying external factors that can be used to control neural stem cells division and their differentiation to neurons, astrocytes and oligodendrocytes is of high scientific and clinical interest. Here we show that the Nogo-66 receptor interacting protein LINGO-1 is a potent regulator of neural stem cell maturation to neurons. LINGO-1 is expressed by cortical neural stem cells from E14 mouse embryos and inhibition of LINGO-1 during the first days of neural stem cell differentiation results in decreased neuronal maturation. Compared to neurons in control cultures, which after 6 days of differentiation have long extending neurites, neurons in cultures treated with anti-LINGO-1 antibodies retain an immature, round phenotype with only very short processes. Furthermore, neutralization of LINGO-1 results in a threefold increase in βIII tubulin-positive cells compared to untreated control cultures. By using BrdU incorporation assays we show that the immature neurons in LINGO-1 neutralized cultures are dividing neuroblasts. In contrast to control cultures, in which no cells were double positive for βIII tubulin and BrdU, 36% of the neurons in cultures treated with anti-LINGO-1 antibodies were proliferating after three days of differentiation. TUNEL assays revealed that the amount of cells going through apoptosis during the early phase of differentiation was significantly decreased in cultures treated with anti-LINGO-1 antibodies compared to untreated control cultures. Taken together, our results demonstrate a novel role for LINGO-1 in neural stem cell differentiation to neurons and suggest a possibility to use LINGO-1 inhibitors to compensate for neuronal cell loss in the injured brain.

  5. Uni-directional differentiation of mouse embryonic stem cells into neurons by the neural stem sphere method.

    PubMed

    Otsu, Masahiro; Sai, Tomoaki; Nakayama, Takashi; Murakami, Koji; Inoue, Nobuo

    2011-04-01

    We previously showed that our neural stem sphere (NSS) method promotes the neuronal differentiation of mouse, monkey and human embryonic stem (ES) cells. Here we analyzed changes in expression of marker genes and proteins during neuronal differentiation. When cultured in astrocyte-conditioned medium (ACM) under free-floating conditions, colonies of ES cells formed floating cell spheres, which, within 4 days, gave rise to NSSs. In the spheres, the expression of ES cell marker genes was consistently down-regulated, while expression of an epiblast marker was transiently up-regulated, beginning on day 2, and the expression of neuroectoderm, neural stem cell and neuron markers was up-regulated, beginning on days 3, 4 and 6, respectively. The expression of the marker genes was consistent with that of marker proteins. The time course of expression of these markers in the spheres resembled that of neuronal differentiation from the inner cell mass (ICM) cells of blastula. In contrast, the expression of endoderm, mesoderm, epidermis, astrocyte and oligodendrocyte markers was low and not up-regulated during differentiation. Only a small number of apoptotic cells were present in the spheres. These results suggest that mouse ES cells uni-directionally differentiate into neurons via epiblast cells, neuroectodermal cells and neural stem cells.

  6. Progressive and spatially differentiated stability of microtubules in developing neuronal cells

    PubMed Central

    1989-01-01

    The establishment of neural circuits requires both stable and plastic properties in the neuronal cytoskeleton. In this study we show that properties of stability and lability reside in microtubules and these are governed by cellular differentiation and intracellular location. After culture for 3, 7, and 14 d in nerve growth factor-containing medium, PC-12 cells were microinjected with X-rhodamine-labeled tubulin. 8-24 h later, cells were photobleached with a laser microbeam at the cell body, neurite shaft, and growth cone. Replacement of fluorescence in bleached zones was monitored by digital video microscopy. In 3-d cultures, fluorescence recovery in all regions occurred by 26 +/- 17 min. Similarly, in older cultures, complete fluorescence recovery at the cell body and growth cone occurred by 10- 30 min. However, in neurite shafts, fluorescence recovery was markedly slower (71 +/- 48 min for 7-d and 201 +/- 94 min for 14-d cultures). This progressive increase in the stability of microtubules in the neurite shafts correlated with an increase of acetylated microtubules. Acetylated microtubules were present specifically in the neurite shaft and not in the regions of fast microtubule turnover, the cell body and growth cone. During the recovery of fluorescence, bleached zones did not move with respect to the cell body. We conclude that the microtubule component of the neuronal cytoskeleton is differentially dynamic but stationary. PMID:2745551

  7. Spiral Ganglion Stem Cells Can Be Propagated and Differentiated Into Neurons and Glia

    PubMed Central

    Zecha, Veronika; Wagenblast, Jens; Arnhold, Stefan; Edge, Albert S. B.; Stöver, Timo

    2014-01-01

    Abstract The spiral ganglion is an essential functional component of the peripheral auditory system. Most types of hearing loss are associated with spiral ganglion cell degeneration which is irreversible due to the inner ear's lack of regenerative capacity. Recent studies revealed the existence of stem cells in the postnatal spiral ganglion, which gives rise to the hope that these cells might be useful for regenerative inner ear therapies. Here, we provide an in-depth analysis of sphere-forming stem cells isolated from the spiral ganglion of postnatal mice. We show that spiral ganglion spheres have characteristics similar to neurospheres isolated from the brain. Importantly, spiral ganglion sphere cells maintain their major stem cell characteristics after repeated propagation, which enables the culture of spheres for an extended period of time. In this work, we also demonstrate that differentiated sphere-derived cell populations not only adopt the immunophenotype of mature spiral ganglion cells but also develop distinct ultrastructural features of neurons and glial cells. Thus, our work provides further evidence that self-renewing spiral ganglion stem cells might serve as a promising source for the regeneration of lost auditory neurons. PMID:24940560

  8. A neural extracellular matrix-based method for in vitro hippocampal neuron culture and dopaminergic differentiation of neural stem cells

    PubMed Central

    2013-01-01

    Background The ability to recreate an optimal cellular microenvironment is critical to understand neuronal behavior and functionality in vitro. An organized neural extracellular matrix (nECM) promotes neural cell adhesion, proliferation and differentiation. Here, we expanded previous observations on the ability of nECM to support in vitro neuronal differentiation, with the following goals: (i) to recreate complex neuronal networks of embryonic rat hippocampal cells, and (ii) to achieve improved levels of dopaminergic differentiation of subventricular zone (SVZ) neural progenitor cells. Methods Hippocampal cells from E18 rat embryos were seeded on PLL- and nECM-coated substrates. Neurosphere cultures were prepared from the SVZ of P4-P7 rat pups, and differentiation of neurospheres assayed on PLL- and nECM-coated substrates. Results When seeded on nECM-coated substrates, both hippocampal cells and SVZ progenitor cells showed neural expression patterns that were similar to their poly-L-lysine-seeded counterparts. However, nECM-based cultures of both hippocampal neurons and SVZ progenitor cells could be maintained for longer times as compared to poly-L-lysine-based cultures. As a result, nECM-based cultures gave rise to a more branched neurite arborization of hippocampal neurons. Interestingly, the prolonged differentiation time of SVZ progenitor cells in nECM allowed us to obtain a purer population of dopaminergic neurons. Conclusions We conclude that nECM-based coating is an efficient substrate to culture neural cells at different stages of differentiation. In addition, neural ECM-coated substrates increased neuronal survival and neuronal differentiation efficiency as compared to cationic polymers such as poly-L-lysine. PMID:23594371

  9. Chemogenomic analysis of neuronal differentiation with pathway changes in PC12 cells.

    PubMed

    Lin, Jack Yu-Shih; Wu, Chien Liang; Liao, Chia Nan; Higuchi, Akon; Ling, Qing-Dong

    2016-01-01

    The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database creates networks from interrelations between molecular biology and underlying chemical elements. This allows for analysis of biologic networks, genomic information, and higher-order functional information at a system level. Through high throughput experiments and system biology analysis, we investigated the genes and pathways associated with NGF induced neuronal differentiation. We performed microarray experiments and used the KEGG database, system biology analysis, and annotation of pathway functions to study NGF-induced differentiation in PC12 cells. We identified 2020 NGF-induced genes with altered expressions over time. Cross-matching with the KEGG database revealed 830 genes; among which, 395 altered genes were found to have a 2-fold increase in gene expression over a two-hour period. We then identified 191 associated biologic pathways in the KEGG database; the top 15 pathways showed correlation with neural differentiation. These included the neurotrophin pathways, mitogen-activated protein kinase (MAPK) pathways, genes associated with axonal guidance and the Wnt pathways. The activation of these pathways synchronized with nerve growth factor (NGF)-induced differentiation in PC12 cells. In summary, we have established a model system that allows one to systematically characterize the functional pathway changes in a group of neuronal population after an external stimulus.

  10. In vitro differentiation of neural stem cells derived from human olfactory bulb into dopaminergic-like neurons.

    PubMed

    Alizadeh, Rafieh; Hassanzadeh, Gholamreza; Joghataei, Mohammad Taghi; Soleimani, Mansoureh; Moradi, Fatemeh; Mohammadpour, Shahram; Ghorbani, Jahangir; Safavi, Ali; Sarbishegi, Maryam; Pirhajati Mahabadi, Vahid; Alizadeh, Leila; Hadjighassem, Mahmoudreza

    2017-03-01

    This study describes a new accessible source of neuronal stem cells that can be used in Parkinson's disease cell transplant. The human olfactory bulb contains neural stem cells (NSCs) that are responsible for neurogenesis in the brain and the replacement of damaged cellular components throughout life. NSCs are capable of differentiating into neuronal and glial cells. We isolated NSCs from the olfactory bulb of brain-death donors and differentiated them into dopaminergic neurons. The olfactory bulb tissues obtained were cultured in Dulbecco's modified Eagle's medium/nutrient mixture F12, B27 supplemented with basic fibroblast growth factor, epidermal growth factor and leukemia inhibitory factor. The NSCs and proliferation markers were assessed. The multipotentiality of olfactory bulb NSCs was demonstrated by their capacity to differentiate into neurons, oligodendrocytes and astrocytes. To generate dopaminergic neurons, olfactory bulb NSCs were differentiated in neurobasal medium, supplemented with B27, and treated with sonic hedgehog, fibroblast growth factor 8 and glial cell-derived neurotrophic factor from the 7th to the 21st day, followed by detection of dopaminergic neuronal markers including tyrosine hydroxylase and aromatic l-amino acid decarboxylase. The cells were expanded, established in continuous cell lines and differentiated into the two classical neuronal phenotypes. The percentage of co-positive cells (microtubule-associated protein 2 and tyrosine hydroxylase; aromatic l-amino acid decarboxylase and tyrosine hydroxylase) in the treated cells was significantly higher than in the untreated cells. These results illustrate the existence of multipotent NSCs in the adult human olfactory bulb that are capable of differentiating toward putative dopaminergic neurons in the presence of trophic factors. Taken together, our data encourage further investigations of the possible use of olfactory bulb NSCs as a promising cell-based therapeutic strategy for Parkinson

  11. Synthetic Glycopolymers for Highly Efficient Differentiation of Embryonic Stem Cells into Neurons: Lipo- or Not?

    PubMed

    Liu, Qi; Lyu, Zhonglin; Yu, You; Zhao, Zhen-Ao; Hu, Shijun; Yuan, Lin; Chen, Gaojian; Chen, Hong

    2017-04-05

    To realize the potential application of embryonic stem cells (ESCs) for the treatment of neurodegenerative diseases, it is a prerequisite to develop an effective strategy for the neural differentiation of ESCs so as to obtain adequate amount of neurons. Considering the efficacy of glycosaminoglycans (GAG) and their disadvantages (e.g., structure heterogeneity and impurity), GAG-mimicking glycopolymers (designed polymers containing functional units similar to natural GAG) with or without phospholipid groups were synthesized in the present work and their ability to promote neural differentiation of mouse ESCs (mESCs) was investigated. It was found that the lipid-anchored GAG-mimicking glycopolymers (lipo-pSGF) retained on the membrane of mESCs rather than being internalized by cells after 1 h of incubation. Besides, lipo-pSGF showed better activity in promoting neural differentiation. The expression of the neural-specific maker β3-tubulin in lipo-pSGF-treated cells was ∼3.8- and ∼1.9-fold higher compared to natural heparin- and pSGF-treated cells at day 14. The likely mechanism involved in lipo-pSGF-mediated neural differentiation was further investigated by analyzing its effect on fibroblast growth factor 2 (FGF2)-mediated extracellular signal-regulated kinases 1 and 2 (ERK1/2) signaling pathway which is important for neural differentiation of ESCs. Lipo-pSGF was found to efficiently bind FGF2 and enhance the phosphorylation of ERK1/2, thus promoting neural differentiation. These findings demonstrated that engineering of cell surface glycan using our synthetic lipo-glycopolymer is a highly efficient approach for neural differentiation of ESCs and this strategy can be applied for the regulation of other cellular activities mediated by cell membrane receptors.

  12. Cell-Specific Fine-Tuning of Neuronal Excitability by Differential Expression of Modulator Protein Isoforms

    PubMed Central

    Jepson, James; Sheldon, Amanda; Shahidullah, Mohammad; Fei, Hong; Koh, Kyunghee

    2013-01-01

    SLOB (SLOWPOKE-binding protein) modulates the Drosophila SLOWPOKE calcium-activated potassium channel. We have shown previously that SLOB deletion or RNAi knockdown decreases excitability of neurosecretory pars intercerebralis (PI) neurons in the adult Drosophila brain. In contrast, we found that SLOB deletion/knockdown enhances neurotransmitter release from motor neurons at the fly larval neuromuscular junction, suggesting an increase in excitability. Because two prominent SLOB isoforms, SLOB57 and SLOB71, modulate SLOWPOKE channels in opposite directions in vitro, we investigated whether divergent expression patterns of these two isoforms might underlie the differential modulation of excitability in PI and motor neurons. By performing detailed in vitro and in vivo analysis, we found strikingly different modes of regulatory control by the slob57 and slob71 promoters. The slob71, but not slob57, promoter contains binding sites for the Hunchback and Mirror transcriptional repressors. Furthermore, several core promoter elements that are absent in the slob57 promoter coordinately drive robust expression of a luciferase vector by the slob71 promoter in vitro. In addition, we visualized the expression patterns of the slob57 and slob71 promoters in vivo and found clear spatiotemporal differences in promoter activity. SLOB57 is expressed prominently in adult PI neurons, whereas larval motor neurons exclusively express SLOB71. In contrast, at the larval neuromuscular junction, SLOB57 expression appears to be restricted mainly to a subset of glial cells. Our results illustrate how the use of alternative transcriptional start sites within an ion channel modulator locus coupled with functionally relevant alternative splicing can be used to fine-tune neuronal excitability in a cell-specific manner. PMID:24133277

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

    PubMed Central

    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

  14. Kv3.1 channels stimulate adult neural precursor cell proliferation and neuronal differentiation.

    PubMed

    Yasuda, Takahiro; Cuny, Hartmut; Adams, David J

    2013-05-15

    Adult neural stem/precursor cells (NPCs) play a pivotal role in neuronal plasticity throughout life. Among ion channels identified in adult NPCs, voltage-gated delayed rectifier K(+) (KDR) channels are dominantly expressed. However, the KDR channel subtype and its physiological role are still undefined. We used real-time quantitative RT-PCR and gene knockdown techniques to identify a major functional KDR channel subtype in adult NPCs. Dominant mRNA expression of Kv3.1, a high voltage-gated KDR channel, was quantitatively confirmed. Kv3.1 gene knockdown with specific small interfering RNAs (siRNA) for Kv3.1 significantly inhibited Kv3.1 mRNA expression by 63.9% (P < 0.001) and KDR channel currents by 52.2% (P < 0.001). This indicates that Kv3.1 is the subtype responsible for producing KDR channel outward currents. Resting membrane properties, such as resting membrane potential, of NPCs were not affected by Kv3.1 expression. Kv3.1 knockdown with 300 nm siRNA inhibited NPC growth (increase in cell numbers) by 52.9% (P < 0.01). This inhibition was attributed to decreased cell proliferation, not increased cell apoptosis. We also established a convenient in vitro imaging assay system to evaluate NPC differentiation using NPCs from doublecortin-green fluorescent protein transgenic mice. Kv3.1 knockdown also significantly reduced neuronal differentiation by 31.4% (P < 0.01). We have demonstrated that Kv3.1 is a dominant functional KDR channel subtype expressed in adult NPCs and plays key roles in NPC proliferation and neuronal lineage commitment during differentiation.

  15. Establishment of cholinergic neuron-like cell lines with differential vulnerability to nitrosative stress.

    PubMed

    Personett, David A; Williams, Katrina; Baskerville, Karen A; McKinney, Michael

    2007-05-01

    Cholinergic cell lines were established by fusion of embryonic day 17 wild-type neurons from rat basal forebrain (BF) and upper brainstem (BS) with N18tg neuroblastoma cells. Isolated clones expressed choline acetyltransferase (ChAT) and neuronal nitric oxide synthase (nNOS) activities that were increased upon differentiation with retinoic acid. Clones from the BF expressed high levels of the tyrosine kinase type A (TrkA) receptor expression and activation of the mitogen-activated kinase ERK2 upon treatment with nerve growth factor. Like wild-type cholinergic populations, the six clones studied were variably resistant to nitric oxide (NO) excess from addition of S-nitroso-N-acetyl-D, L-penicillamine (SNAP). Of these, the BS2 clone exhibited resistance like in vivo BS cholinergic neurons, while the MS10 clone mimicked in vivo BF vulnerability. Apoptosis in response to NO excess was preceded by increases in mitochondrial responses bax/bcl-2 ratios, but cytochrome C was not released. Mitochondrial levels of apoptosis initiating factor (AIF) were either unchanged or increased, and only in MS clones was endonuclease G (EndoG) released. Microarray data indicated the existence of endoplasmic reticular (ER) stress and caspase-4 and caspase-12 were involved in the pathway to DNA fragmentation. The array data also indicated a survival role for mdm2, and its blockade rendered vulnerable the brainstem survivor clone BS2. Akt and ERK1/2 pathways were activated in response to NO and their blockade increased DNA fragmentation. Blockade of GSK-3 alpha/beta, a downstream target of Akt, reduced SNAP toxicity and this was more prominent in basal forebrain clones. We have identified two cholinergic cell lines useful for molecular studies of cholinergic vulnerability. We hypothesize that, in cholinergic neurons, control of ER stress signaling may be a major factor in differential vulnerability.

  16. EVALUATION OF PROTEIN MARKERS FOR NEURONAL DIFFERENTIATION IN PC12 CELLS.

    EPA Science Inventory

    Chemical-induced injury of the developing nervous system can be manifested as a change in the differentiation or growth of neurons. The present study evaluated the use of proteins associated with axonal growth and synaptogenesis as markers for neuronal differentiation in vitro. ...

  17. EVALUATION OF PROTEIN MARKERS FOR NEURONAL DIFFERENTIATION IN PC12 CELLS.

    EPA Science Inventory

    Chemical-induced injury of the developing nervous system can be manifested as a change in the differentiation or growth of neurons. The present study evaluated the use of proteins associated with axonal growth and synaptogenesis as markers for neuronal differentiation in vitro. ...

  18. Zhichan decoction induces differentiation of dopaminergic neurons in Parkinson's disease rats after neural stem cell transplantation

    PubMed Central

    Shi, Huifen; Song, Jie; Yang, Xuming

    2014-01-01

    The goal of this study was to increase the dopamine content and reduce dopaminergic metabolites in the brain of Parkinson's disease rats. Using high-performance liquid chromatography, we found that dopamine and dopaminergic metabolite (dihydroxyphenylacetic acid and homovanillic acid) content in the midbrain of Parkinson's disease rats was increased after neural stem cell transplantation + Zhichan decoction, compared with neural stem cell transplantation alone. Our genetic algorithm results show that dihydroxyphenylacetic acid and homovanillic acid levels achieve global optimization. Neural stem cell transplantation + Zhichan decoction increased dihydroxyphenylacetic acid levels up to 10-fold, while transplantation alone resulted in a 3-fold increment. Homovanillic acid levels showed no apparent change. Our experimental findings show that after neural stem cell transplantation in Parkinson's disease rats, Zhichan decoction can promote differentiation of neural stem cells into dopaminergic neurons. PMID:25206914

  19. Linking asymmetric cell division to the terminal differentiation program of postmitotic neurons in C. elegans

    PubMed Central

    Bertrand, Vincent; Hobert, Oliver

    2009-01-01

    SUMMARY How asymmetric divisions are connected to the terminal differentiation program of neuronal subtypes is poorly understood. In C. elegans, two homeodomain transcription factors, TTX-3 (a LHX2/9 orthologue) and CEH-10 (a CHX10 orthologue), directly activate a large battery of terminal differentiation genes in the cholinergic interneuron AIY. We establish here a transcriptional cascade linking asymmetric division to this differentiation program. A transient lineage-specific input formed by the Zic factor REF-2 and the bHLH factor HLH-2 directly activates ttx-3 expression in the AIY mother. During the terminal division of the AIY mother, an asymmetric Wnt/β-catenin pathway cooperates with TTX-3 to directly restrict ceh-10 expression to only one of the two daughter cells. TTX-3 and CEH-10 automaintain their expression, thereby locking in the differentiation state. Our study establishes how transient lineage and asymmetric division inputs are integrated and suggests that the Wnt/β-catenin pathway is widely used to control the identity of neuronal lineages. PMID:19386265

  20. Conditional induction of Math1 specifies embryonic stem cells to cerebellar granule neuron lineage and promotes differentiation into mature granule neurons.

    PubMed

    Srivastava, Rupali; Kumar, Manoj; Peineau, Stéphane; Csaba, Zsolt; Mani, Shyamala; Gressens, Pierre; El Ghouzzi, Vincent

    2013-04-01

    Directing differentiation of embryonic stem cells (ESCs) to specific neuronal subtype is critical for modeling disease pathology in vitro. An attractive means of action would be to combine regulatory differentiation factors and extrinsic inductive signals added to the culture medium. In this study, we have generated mature cerebellar granule neurons by combining a temporally controlled transient expression of Math1, a master gene in granule neuron differentiation, with inductive extrinsic factors involved in cerebellar development. Using a Tetracyclin-On transactivation system, we overexpressed Math1 at various stages of ESCs differentiation and found that the yield of progenitors was considerably increased when Math1 was induced during embryonic body stage. Math1 triggered expression of Mbh1 and Mbh2, two target genes directly involved in granule neuron precursor formation and strong expression of early cerebellar territory markers En1 and NeuroD1. Three weeks after induction, we observed a decrease in the number of glial cells and an increase in that of neurons albeit still immature. Combining Math1 induction with extrinsic factors specifically increased the number of neurons that expressed Pde1c, Zic1, and GABAα6R characteristic of mature granule neurons, formed "T-shaped" axons typical of granule neurons, and generated synaptic contacts and action potentials in vitro. Finally, in vivo implantation of Math1-induced progenitors into young adult mice resulted in cell migration and settling of newly generated neurons in the cerebellum. These results show that conditional induction of Math1 drives ESCs toward the cerebellar fate and indicate that acting on both intrinsic and extrinsic factors is a powerful means to modulate ESCs differentiation and maturation into a specific neuronal lineage.

  1. BM88 is an early marker of proliferating precursor cells that will differentiate into the neuronal lineage.

    PubMed

    Koutmani, Yassemi; Hurel, Catherine; Patsavoudi, Evangelia; Hack, Michael; Gotz, Magdalena; Thomaidou, Dimitra; Matsas, Rebecca

    2004-11-01

    Progression of progenitor cells towards neuronal differentiation is tightly linked with cell cycle control and the switch from proliferative to neuron-generating divisions. We have previously shown that the neuronal protein BM88 drives neuroblastoma cells towards exit from the cell cycle and differentiation into a neuronal phenotype in vitro. Here, we explored the role of BM88 during neuronal birth, cell cycle exit and the initiation of differentiation in vivo. By double- and triple-labelling with the S-phase marker BrdU or the late G2 and M-phase marker cyclin B1, antibodies to BM88 and markers of the neuronal or glial cell lineages, we demonstrate that in the rodent forebrain, BM88 is expressed in multipotential progenitor cells before terminal mitosis and in their neuronal progeny during the neurogenic interval, as well as in the adult. Further, we defined at E16 a cohort of proliferative progenitors that exit S phase in synchrony, and by following their fate for 24 h we show that BM88 is associated with the dynamics of neuron-generating divisions. Expression of BM88 was also evident in cycling cortical radial glial cells, which constitute the main neurogenic population in the cerebral cortex. In agreement, BM88 expression was markedly reduced and restricted to a smaller percentage of cells in the cerebral cortex of the Small eye mutant mice, which lack functional Pax6 and exhibit severe neurogenesis defects. Our data show an interesting correlation between BM88 expression and the progression of progenitor cells towards neuronal differentiation during the neurogenic interval.

  2. Bällchen participates in proliferation control and prevents the differentiation of Drosophila melanogaster neuronal stem cells

    PubMed Central

    Yakulov, Toma; Günesdogan, Ufuk; Jäckle, Herbert; Herzig, Alf

    2014-01-01

    ABSTRACT Stem cells continuously generate differentiating daughter cells and are essential for tissue homeostasis and development. Their capacity to self-renew as undifferentiated and actively dividing cells is controlled by either external signals from a cellular environment, the stem cell niche, or asymmetric distribution of cell fate determinants during cell division. Here we report that the protein kinase Bällchen (BALL) is required to prevent differentiation as well as to maintain normal proliferation of neuronal stem cells of Drosophila melanogaster, called neuroblasts. Our results show that the brains of ball mutant larvae are severely reduced in size, which is caused by a reduced proliferation rate of the neuroblasts. Moreover, ball mutant neuroblasts gradually lose the expression of the neuroblast determinants Miranda and aPKC, suggesting their premature differentiation. Our results indicate that BALL represents a novel cell intrinsic factor with a dual function regulating the proliferative capacity and the differentiation status of neuronal stem cells during development. PMID:25190057

  3. Retrovirus delivered neurotrophin-3 promotes survival, proliferation and neuronal differentiation of human fetal neural stem cells in vitro.

    PubMed

    Lu, Haixia; Li, Minjie; Song, Tusheng; Qian, Yihua; Xiao, Xinli; Chen, Xinlin; Zhang, Pengbo; Feng, Xinshun; Parker, Terence; Liu, Yong

    2008-10-22

    Poor survival and insufficient neuronal differentiation are the main obstacles to neural stem cell (NSC) transplantation therapy. Genetic modification of NSCs with neurotrophins is considered a promising approach to overcome these difficulties. In this study, the effects on survival, proliferation and neuronal differentiation of human fetal NSCs (hfNSCs) were observed after infection by a neurotrophin-3 (NT-3) recombinant retrovirus. The hfNSCs, from 12-week human fetal brains formed neurospheres, expressed the stem cell marker nestin and differentiated into the three main cell types of the nervous system. NT-3 recombinant retrovirus (Retro-NT-3) infected hfNSCs efficiently expressed NT-3 gene for at least 8 weeks, presented an accelerated proliferation, and therefore produced an increased number of neurospheres and after differentiation in vitro, contained a higher percentage of neuronal cells. Eight weeks after infection, 37.9+/-4.2% of hfNSCs in the Retro-NT-3 infection group expressed the neuronal marker, this was significantly higher than the control and mock infection groups. NT-3 transduced hfNSCs also displayed longer protruding neurites compared with other groups. Combined these results demonstrate that NT-3 modification promote the survival/proliferation, neuronal differentiation and growth of neurites of hfNSCs in vitro. This study proposes recombinant retrovirus mediated NT-3 modification may provide a promising means to resolve the poor survival and insufficient neuronal differentiation of NSCs.

  4. CHANGES IN THE CONTENT OF ESTROGEN α AND PROGESTERONE RECEPTORS DURING DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS TO DOPAMINE NEURONS

    PubMed Central

    Díaz, Néstor F.; Guerra-Arraiza, Christian; Díaz-Martínez, Néstor E.; Salazar, Patricia; Molina-Hernández, Anayansi; Camacho-Arroyo, Ignacio; Velasco, Ivan

    2007-01-01

    Embryonic stem cells (ESC) can differentiate to derivatives of the three embryonic germ layers. Dopamine neurons have been produced from mouse and human ESC. This in vitro induction mimics the developmental program followed by dopaminergic cells in vivo. Production of dopamine neurons might have clinical applications for Parkinson’s disease, which has a higher incidence in men than in women, suggesting a protective role for sex hormones, particularly progesterone and estradiol. These hormones exert many of their effects through the interaction with their nuclear receptors. In this study, we used a described 5-stage protocol for dopamine neuron differentiation of ESC, allowing neuronal commitment as evidenced by specific markers and by behavioural recovery of hemiparkinsonian rats after grafting. We studied the expression of steroid hormone receptors by immunoblot during this procedure and found an increase in the content of both A and B isoforms of progesterone receptor (PR) and a decrease in estrogen receptor α (ER-α) when cells were at the neural/neuronal stages, when compared with the amount found in initial pluripotent conditions. We also found the same pattern of PR and ER-α expression by immunocytochemistry. Ninety-two percent of dopamine neurons expressed progesterone receptors and only 19% of these neurons co-expressed tyrosine hydroxylase and ER-α. These results show a differential expression pattern of ER-α and PR isoforms during neuronal differentiation of ESC. PMID:17499639

  5. Human fetal striatum-derived neural stem (NS) cells differentiate to mature neurons in vitro and in vivo.

    PubMed

    Monni, Emanuela; Cusulin, Carlo; Cavallaro, Maurizio; Lindvall, Olle; Kokaia, Zaal

    2014-01-01

    Clonogenic neural stem (NS) cell lines grown in adherent cultures have previously been established from embryonic stem cells and fetal and adult CNS in rodents and from human fetal brain and spinal cord. Here we describe the isolation of a new cell line from human fetal striatum (hNS cells). These cells showed properties of NS cells in vitro such as monolayer growth, high proliferation rate and expression of radial glia markers. The hNS cells expressed an early neuronal marker while being in the proliferative state. Under appropriate conditions, the hNS cells were efficiently differentiated to neurons, and after 4 weeks about 50% of the cells were βIII tubulin positive. They also expressed the mature neuronal marker NeuN and markers of neuronal subtypes, GABA, calbindin, and DARPP32. After intrastriatal implantation into newborn rats, the hNS cells survived and many of them migrated outside the transplant core into the surrounding tissue. A high percentage of cells in the grafts expressed the neuroblast marker DCX, indicating their neurogenic potential, and some of the cells differentiated to NeuN+ mature neurons. The human fetal striatum-derived NS cell line described here should be a useful tool for studies on cell replacement strategies in models of the striatal neuronal loss occurring in Huntington's disease and stroke.

  6. Expression of early developmental markers predicts the efficiency of embryonic stem cell differentiation into midbrain dopaminergic neurons.

    PubMed

    Salti, Ahmad; Nat, Roxana; Neto, Sonya; Puschban, Zoe; Wenning, Gregor; Dechant, Georg

    2013-02-01

    Dopaminergic neurons derived from pluripotent stem cells are among the best investigated products of in vitro stem cell differentiation owing to their potential use for neurorestorative therapy of Parkinson's disease. However, the classical differentiation protocols for both mouse and human pluripotent stem cells generate a limited percentage of dopaminergic neurons and yield a considerable cellular heterogeneity comprising numerous scarcely characterized cell populations. To improve pluripotent stem cell differentiation protocols for midbrain dopaminergic neurons, we established extensive and strictly quantitative gene expression profiles, including markers for pluripotent cells, neural progenitors, non-neural cells, pan-neuronal and glial cells, neurotransmitter phenotypes, midbrain and nonmidbrain populations, floor plate and basal plate populations, as well as for Hedgehog, Fgf, and Wnt signaling pathways. The profiles were applied to discrete stages of in vitro differentiation of mouse embryonic stem cells toward the dopaminergic lineage and after transplantation into the striatum of 6-hydroxy-dopamine-lesioned rats. The comparison of gene expression in vitro with stages in the developing ventral midbrain between embryonic day 11.5 and 13.5 ex vivo revealed dynamic changes in the expression of transcription factors and signaling molecules. Based on these profiles, we propose quantitative gene expression milestones that predict the efficiency of dopaminergic differentiation achieved at the end point of the protocol, already at earlier stages of differentiation.

  7. Atoh1 inhibits neuronal differentiation and collaborates with Gli1 to generate medulloblastoma-initiating cells

    PubMed Central

    Ayrault, Olivier; Zhao, Haotian; Zindy, Frederique; Qu, Chunxu; Sherr, Charles J.; Roussel, Martine F.

    2010-01-01

    The morphogen and mitogen Sonic Hedgehog activates a Gli1-dependent transcription program that drives proliferation of granule neuron progenitors (GNPs) within the external germinal layer of the postnatally developing cerebellum. Medulloblastomas with mutations activating the Sonic Hedgehog signaling pathway preferentially arise within the external germinal layer, and the tumor cells closely resemble GNPs. Atoh1/Math1, a basic helix-loop-helix transcription factor essential for GNP histogenesis, does not induce medulloblastomas when expressed in primary mouse GNPs that are explanted from the early postnatal cerebellum and transplanted back into the brains of naïve mice. However, enforced expression of Atoh1 in primary GNPs enhances the oncogenicity of cells overexpressing Gli1 by almost three orders of magnitude. Unlike Gli1, Atoh1 cannot support GNP proliferation in the absence of Sonic Hedgehog signaling and does not govern expression of canonical cell cycle genes. Instead, Atoh1 maintains GNPs in a Sonic Hedgehog-responsive state by regulating genes that trigger neuronal differentiation, including many expressed in response to bone morphogenic protein-4. Therefore, by targeting multiple genes regulating the differentiation state of GNPs, Atoh1 collaborats with the pro-proliferative Gli1-dependent transcriptional program to influence medulloblastoma development. PMID:20516124

  8. Cobalt chloride induces neuronal differentiation of human mesenchymal stem cells through upregulation of microRNA-124a.

    PubMed

    Jeon, Eun Su; Shin, Jin Hee; Hwang, Su Jin; Moon, Gyeong Joon; Bang, Oh Young; Kim, Hyeon Ho

    2014-02-21

    Human mesenchymal stem cells (hMSCs) are known to have the capacity to differentiate into various cell types, including neurons. To examine our hypothesis that miRNA was involved in neuronal differentiation of hMSCs, CoCl2, a hypoxia-mimicking agent was used to induce neuronal differentiation, which was assessed by determining the expression of neuronal markers such as nestin and Tuj1. Treatment of hMSCs with CoCl2 led to increased expression of miR-124a, a neuron-specific miRNA. HIF-1α silencing and JNK inhibition abolished CoCl2-induced miR-124a expression, suggesting that JNK and HIF-1α signals were required for the miR-124a expression induced by CoCl2 in hMSCs. Overexpression of miR-124a or CoCl2 treatment suppressed the expression of anti-neural proteins such as SCP1 and SOX9. Silencing of both SCP1 and SOX9 induced neuronal differentiation of hMSCs, indicating that suppression of miR-124a targets is important for CoCl2-induced neuronal differentiation of hMSCs. Knockdown of HIF-1α or inhibition of JNK restored the expression of SCP1 and SOX9 in CoCl2-treated cells. Inhibition of miR-124a blocked CoCl2-induced suppression of SCP1 and SOX9 and abolished CoCl2-induced neuronal differentiation of hMSCs. Taken together, we demonstrate that miR-124a is critically regulates CoCl2-induced neuronal differentiation of hMSCs by suppressing the expression of SCP1 and SOX9.

  9. Taurine Protected Against the Impairments of Neural Stem Cell Differentiated Neurons Induced by Oxygen-Glucose Deprivation.

    PubMed

    Xiao, Bo; Liu, Huazhen; Gu, Zeyun; Liu, Sining; Ji, Cheng

    2015-11-01

    Cell transplantation of neural stem cells (NSCs) is a promising approach for neurological recovery both structurally and functionally. However, one big obstacle is to promote differentiation of NSCs into neurons and the followed maturation. In the present study, we aimed to investigate the protective effect of taurine on the differentiation of NSCs and subsequent maturation of their neuronal lineage, when exposed to oxygen-glucose deprivation (OGD). The results suggested that taurine (5-20 mM) promoted the viability and proliferation of NSCs, and it protected against 8 h of OGD induced impairments. Furthermore, 20 mM taurine promoted NSCs to differentiate into neurons after 7 days of culture, and it also protected against the suppressive impairments of 8 h of OGD. Consistently, taurine (20 mM) promoted the neurite sprouting and outgrowth of the NSC differentiated neurons after 14 days of differentiation, which were significantly inhibited by OGD (8 h). At D21, the mushroom spines and spine density were promoted or restored by 20 mM taurine. Taken together, the enhanced viability and proliferation of NSCs, more differentiated neurons and the promoted maturation of neurons by 20 mM taurine support its therapeutic application during stem cell therapy to enhance neurological recovery. Moreover, it protected against the impairments induced by OGD, which may highlight its role for a more direct therapeutic application especially in an ischemic stroke environment.

  10. Morin hydrate promotes inner ear neural stem cell survival and differentiation and protects cochlea against neuronal hearing loss.

    PubMed

    He, Qiang; Jia, Zhanwei; Zhang, Ying; Ren, Xiumin

    2017-03-01

    We aimed to investigate the effect of morin hydrate on neural stem cells (NSCs) isolated from mouse inner ear and its potential in protecting neuronal hearing loss. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and bromodeoxyuridine incorporation assays were employed to assess the effect of morin hydrate on the viability and proliferation of in vitro NSC culture. The NSCs were then differentiated into neurons, in which neurosphere formation and differentiation were evaluated, followed by neurite outgrowth and neural excitability measurements in the subsequent in vitro neuronal network. Mechanotransduction of cochlea ex vivo culture and auditory brainstem responses threshold and distortion product optoacoustic emissions amplitude in mouse ototoxicity model were also measured following gentamicin treatment to investigate the protective role of morin hydrate against neuronal hearing loss. Morin hydrate improved viability and proliferation, neurosphere formation and neuronal differentiation of inner ear NSCs, and promoted in vitro neuronal network functions. In both ex vivo and in vivo ototoxicity models, morin hydrate prevented gentamicin-induced neuronal hearing loss. Morin hydrate exhibited potent properties in promoting growth and differentiation of inner ear NSCs into functional neurons and protecting from gentamicin ototoxicity. Our study supports its clinical potential in treating neuronal hearing loss.

  11. Human neuroblastoma cell lines as models for the in vitro study of neoplastic and neuronal cell differentiation.

    PubMed Central

    Abemayor, E; Sidell, N

    1989-01-01

    Neuroblastoma is a childhood solid tumor composed of primitive cells derived from precursors of the autonomic nervous system. This neoplasm has the highest rate of spontaneous regression of all cancer types and has been noted to undergo spontaneous and chemically induced differentiation into elements resembling mature nervous tissue. As such, neuroblastoma has been a prime model system for the study of neuronal differentiation and the process of cancer cell maturation. In this paper we review those agents that have been described to induce the differentiation of neuroblastoma, with an emphasis on the effects and possible mechanisms of action of a group of related compounds, the retinoids. With this model system and the availability of subclones that are both responsive and resistant to chemically induced differentiation, fundamental questions regarding the mechanisms and processes underlying cell maturation have become more amenable to in vitro study. Images FIGURE 1. A FIGURE 1. B FIGURE 1. C FIGURE 2. A FIGURE 2. B PMID:2538324

  12. The long noncoding RNA Pnky regulates neuronal differentiation of embryonic and postnatal neural stem cells

    PubMed Central

    Liu, Siyuan John; Nowakowski, Tomasz Jan; Hong, Sung Jun; Gertz, Caitlyn; Salinas, Ryan D.; Zarabi, Hosniya; Kriegstein, Arnold R.; Lim, Daniel A.

    2015-01-01

    Summary While thousands of long noncoding RNAs (lncRNAs) have been identified, few lncRNAs that control neural stem cell (NSC) behavior are known. Here, we identify Pinky (Pnky) as a neural-specific lncRNA that regulates neurogenesis from NSCs in the embryonic and postnatal brain. In postnatal NSCs, Pnky knockdown potentiates neuronal lineage commitment and expands the transit-amplifying cell population, increasing neuron production several-fold. Pnky is evolutionarily conserved and expressed in NSCs of the developing human brain. In the embryonic mouse cortex, Pnky knockdown increases neuronal differentiation and depletes the NSC population. Pnky interacts with the splicing regulator PTBP1, and PTBP1 knockdown also enhances neurogenesis. In NSCs, Pnky and PTBP1 regulate the expression and alternative splicing of a core set of transcripts that relates to the cellular phenotype. These data thus unveil Pnky as a conserved lncRNA that interacts with a key RNA processing factor and regulates neurogenesis from embryonic and postnatal NSC populations. PMID:25800779

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

    PubMed Central

    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

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

    PubMed

    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.

  15. Differentiation potential of human CD133 positive hematopoietic stem cells into motor neuron- like cells, in vitro.

    PubMed

    Moghaddam, Sepideh Alavi; Yousefi, Behnam; Sanooghi, Davood; Faghihi, Faezeh; Hayati Roodbari, Nasim; Bana, Nikoo; Joghataei, Mohammad Taghi; Pooyan, Paria; Arjmand, Babak

    2017-07-25

    Spinal cord injuries and motor neuron-related disorders impact on life of many patients around the world. Since pharmacotherapy and surgical approaches were not efficient to regenerate these types of defects; stem cell therapy as a good strategy to restore the lost cells has become the focus of interest among the scientists. Umbilical cord blood CD133(+) hematopoietic stem cells (UCB- CD133(+) HSCs) with self- renewal property and neural lineage differentiation capacity are ethically approved cell candidate for use in regenerative medicine. In this regard the aim of this study was to quantitatively evaluate the capability of these cells to differentiate into motor neuron-like cells (MNL), in vitro. CD133(+) HSCs were isolated from human UCB using MACS system. After cell characterization using flow cytometry, the cells were treated with a combination of Retinoic acid, Sonic hedgehog, Brain derived neurotrophic factor, and B27 through a 2- step procedure for two weeks. The expression of MN-specific markers was examined using qRT- PCR, flow cytometry and immunocytochemistry. By the end of the two-week differentiation protocol, CD133(+) cells acquired unipolar MNL morphology with thin and long neurites. The expression of Isl-1(62.15%), AChE (41.83%), SMI-32 (21.55%) and Nestin (17.46%) was detected using flow cytometry and immunocytochemistry. The analysis of the expression of PAX6, ISL-1, ACHE, CHAT and SMI-32 revealed that MNLs present these neural markers at levels comparable with undifferentiated cells. In Conclusion Human UCB- CD133(+) HSCs are remarkably potent cell candidates to transdifferentiate into motor neuron-like cells, in vitro. Copyright © 2017. Published by Elsevier B.V.

  16. A potent and selective small molecule inhibitor of sirtuin 1 promotes differentiation of pluripotent P19 cells into functional neurons

    PubMed Central

    Kim, Beom Seok; Lee, Chang-Hee; Chang, Gyeong-Eon; Cheong, Eunji; Shin, Injae

    2016-01-01

    Sirtuin 1 (SIRT1) is known to suppress differentiation of pluripotent/multipotent cells and neural progenitor cells into neurons by blocking activation of transcription factors critical for neurogenesis. EX-527 is a highly selective and potent inhibitor against SIRT1 and has been used as a chemical probe that modulates SIRT1-associated biological processes. However, the effect of EX-527 on neuronal differentiation in pluripotent cells has not been well elucidated. Here, we report an examination of EX-527 effects on neurogenesis of pluripotent P19 cells. The results showed that EX-527 greatly accelerated differentiation of P19 cells into neurons without generation of cardiac cells and astrocytes. Importantly, neurons derived from P19 cells treated with EX-527 generated voltage-dependent sodium currents and depolarization-induced action potentials. The findings indicate that the differentiated cells have electrophysiological properties. The present study suggests that the selective SIRT1 inhibitor could have the potential of being employed as a chemical inducer to generate functionally active neurons. PMID:27680533

  17. Neuronal differentiation of PC12 and embryonic stem cells in two- and three-dimensional in vitro culture.

    PubMed

    Sadri, Soheil; Khazaei, Mozafar; Ghanbari, Ali; Khazaei, Mohammad Rasool; Shah, Palak

    2014-04-01

    The quality of neuronal differentiation and reduction in apoptosis that occurred in two-dimensional (2D) and three-dimensional (3D) culture conditions is compared. PC12 and embryonic stem cells are two commonly utilized cell lines for the study of neuronal regeneration. These cells were induced to neuronally differentiate by adding NGF and retinoic acid respectively. Total neurite length and expression of neuronal markers (MAP-2 and beta-tubulin) was assessed by morphometry and immunocytochemistry. Also, TUNEL assay was used to detect apoptosis. Upon exposure to a differentiation media in the 3D fibrin gel, PC12 and embryonic stem cells stopped dividing, had increased adhesion to the substratum, extended neurite processes and expressed neuronal markers. The same results, however, were not observedwith the 2D culture. Also, the apoptosis index performed by TUNEL a ss ay demonstrated a reduction in th e degree of apoptosis in the 3D culture compared to 2D culture. Fibrin matrix supports growth and n euronal differentiation of PC12 andembryonic stem cells. In addition, the 3D culture enhanced cellular resistance to apoptosis when compared to the 2D culture. It appears as if a 3D culture system may offer a better technique for future neuronal tissue engineering investigations.

  18. cAMP initiates early phase neuron-like morphology changes and late phase neural differentiation in mesenchymal stem cells

    PubMed Central

    Zhang, Linxia; Seitz, Linsey C.; Abramczyk, Amy M.; Liu, Li

    2010-01-01

    The intracellular second messenger cAMP is frequently used in induction media to induce mesenchymal stem cells (MSCs) into neural lineage cells. To date, an understanding of the role cAMP exerts on MSCs and whether cAMP can induce MSCs into functional neurons is still lacking. We found cAMP initiated neuron-like morphology changes early and neural differentiation much later. The early phase changes in morphology were due to cell shrinkage, which subsequently rendered some cells apoptotic. While the morphology changes occurred prior to the expression of neural markers, it is not required for neural marker expression and the two processes are differentially regulated downstream of cAMP-activated protein kinase A. cAMP enabled MSCs to gain neural marker expressions with neuronal function, such as, calcium rise in response to neuronal activators, dopamine, glutamate, and potassium chloride. However, only some of the cells induced by cAMP responded to the three neuronal activators and further lack the neuronal morphology, suggesting that although cAMP is able to direct MSCs towards neural differentiation, they do not achieve terminal differentiation. PMID:20725762

  19. The Ubiquitin Ligase Praja1 Reduces NRAGE Expression and Inhibits Neuronal Differentiation of PC12 Cells

    PubMed Central

    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

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

    PubMed

    Parthsarathy, Vadivel; Hölscher, Christian

    2013-01-01

    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.

  1. Rapid differentiation of human pluripotent stem cells into functional neurons by mRNAs encoding transcription factors

    PubMed Central

    Goparaju, Sravan Kumar; Kohda, Kazuhisa; Ibata, Keiji; Soma, Atsumi; Nakatake, Yukhi; Akiyama, Tomohiko; Wakabayashi, Shunichi; Matsushita, Misako; Sakota, Miki; Kimura, Hiromi; Yuzaki, Michisuke; Ko, Shigeru B. H.; Ko, Minoru S. H.

    2017-01-01

    Efficient differentiation of human pluripotent stem cells (hPSCs) into neurons is paramount for disease modeling, drug screening, and cell transplantation therapy in regenerative medicine. In this manuscript, we report the capability of five transcription factors (TFs) toward this aim: NEUROG1, NEUROG2, NEUROG3, NEUROD1, and NEUROD2. In contrast to previous methods that have shortcomings in their speed and efficiency, a cocktail of these TFs as synthetic mRNAs can differentiate hPSCs into neurons in 7 days, judged by calcium imaging and electrophysiology. They exhibit motor neuron phenotypes based on immunostaining. These results indicate the establishment of a novel method for rapid, efficient, and footprint-free differentiation of functional neurons from hPSCs. PMID:28205555

  2. Gallium nitride induces neuronal differentiation markers in neural stem/precursor cells derived from rat cerebral cortex.

    PubMed

    Chen, Chi-Ruei; Li, Yi-Chen; Young, Tai-Horng

    2009-09-01

    In the present study, gallium nitride (GaN) was used as a substrate to culture neural stem/precursor cells (NSPCs), isolated from embryonic rat cerebral cortex, to examine the effect of GaN on the behavior of NSPCs in the presence of basic fibroblast growth factor (bFGF) in serum-free medium. Morphological studies showed that neurospheres maintained their initial shape and formed many long and thick processes with the fasciculate feature on GaN. Immunocytochemical characterization showed that GaN could induce the differentiation of NSPCs into neurons and astrocytes. Compared to poly-d-lysine (PDL), the most common substrate used for culturing neurons, there was considerable expression of synapsin I for differentiated neurons on GaN, suggesting GaN could induce the differentiation of NSPCs towards the mature differentiated neurons. Western blot analysis showed that the suppression of glycogen synthase kinase-3beta (GSK-3beta) activity was one of the effects of GaN-promoted NSPC differentiation into neurons. Finally, compared to PDL, GaN could significantly improve cell survival to reduce cell death after long-term culture. These results suggest that GaN potentially has a combination of electric characteristics suitable for developing neuron and/or NSPC chip systems.

  3. A High-content screen identifies compounds promoting the neuronal differentiation and the midbrain dopamine neuron specification of human neural progenitor cells

    PubMed Central

    Rhim, Ji heon; Luo, Xiangjian; Xu, Xiaoyun; Gao, Dongbing; Zhou, Tieling; Li, Fuhai; Qin, Lidong; Wang, Ping; Xia, Xiaofeng; Wong, Stephen T. C.

    2015-01-01

    Small molecule compounds promoting the neuronal differentiation of stem/progenitor cells are of pivotal importance to regenerative medicine. We carried out a high-content screen to systematically characterize known bioactive compounds, on their effects on the neuronal differentiation and the midbrain dopamine (mDA) neuron specification of neural progenitor cells (NPCs) derived from the ventral mesencephalon of human fetal brain. Among the promoting compounds three major pharmacological classes were identified including the statins, TGF-βRI inhibitors, and GSK-3 inhibitors. The function of each class was also shown to be distinct, either to promote both the neuronal differentiation and mDA neuron specification, or selectively the latter, or promote the former but suppress the latter. We then carried out initial investigation on the possible mechanisms underlying, and demonstrated their applications on NPCs derived from human pluripotent stem cells (PSCs). Our study revealed the potential of several small molecule compounds for use in the directed differentiation of human NPCs. The screening result also provided insight into the signaling network regulating the differentiation of human NPCs. PMID:26542303

  4. Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays.

    PubMed

    Frega, Monica; van Gestel, Sebastianus H C; Linda, Katrin; van der Raadt, Jori; Keller, Jason; Van Rhijn, Jon-Ruben; Schubert, Dirk; Albers, Cornelis A; Nadif Kasri, Nael

    2017-01-08

    Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-2(12). This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (>95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

  5. Differentiation-Dependent Energy Production and Metabolite Utilization: A Comparative Study on Neural Stem Cells, Neurons, and Astrocytes

    PubMed Central

    Jády, Attila Gy.; Nagy, Ádám M.; Kőhidi, Tímea; Ferenczi, Szilamér; Tretter, László

    2016-01-01

    While it is evident that the metabolic machinery of stem cells should be fairly different from that of differentiated neurons, the basic energy production pathways in neural stem cells (NSCs) or in neurons are far from clear. Using the model of in vitro neuron production by NE-4C NSCs, this study focused on the metabolic changes taking place during the in vitro neuronal differentiation. O2 consumption, H+ production, and metabolic responses to single metabolites were measured in cultures of NSCs and in their neuronal derivatives, as well as in primary neuronal and astroglial cultures. In metabolite-free solutions, NSCs consumed little O2 and displayed a higher level of mitochondrial proton leak than neurons. In stem cells, glycolysis was the main source of energy for the survival of a 2.5-h period of metabolite deprivation. In contrast, stem cell-derived or primary neurons sustained a high-level oxidative phosphorylation during metabolite deprivation, indicating the consumption of own cellular material for energy production. The stem cells increased O2 consumption and mitochondrial ATP production in response to single metabolites (with the exception of glucose), showing rapid adaptation of the metabolic machinery to the available resources. In contrast, single metabolites did not increase the O2 consumption of neurons or astrocytes. In “starving” neurons, neither lactate nor pyruvate was utilized for mitochondrial ATP production. Gene expression studies also suggested that aerobic glycolysis and rapid metabolic adaptation characterize the NE-4C NSCs, while autophagy and alternative glucose utilization play important roles in the metabolism of stem cell-derived neurons. PMID:27116891

  6. Differentiation-Dependent Energy Production and Metabolite Utilization: A Comparative Study on Neural Stem Cells, Neurons, and Astrocytes.

    PubMed

    Jády, Attila Gy; Nagy, Ádám M; Kőhidi, Tímea; Ferenczi, Szilamér; Tretter, László; Madarász, Emília

    2016-07-01

    While it is evident that the metabolic machinery of stem cells should be fairly different from that of differentiated neurons, the basic energy production pathways in neural stem cells (NSCs) or in neurons are far from clear. Using the model of in vitro neuron production by NE-4C NSCs, this study focused on the metabolic changes taking place during the in vitro neuronal differentiation. O2 consumption, H(+) production, and metabolic responses to single metabolites were measured in cultures of NSCs and in their neuronal derivatives, as well as in primary neuronal and astroglial cultures. In metabolite-free solutions, NSCs consumed little O2 and displayed a higher level of mitochondrial proton leak than neurons. In stem cells, glycolysis was the main source of energy for the survival of a 2.5-h period of metabolite deprivation. In contrast, stem cell-derived or primary neurons sustained a high-level oxidative phosphorylation during metabolite deprivation, indicating the consumption of own cellular material for energy production. The stem cells increased O2 consumption and mitochondrial ATP production in response to single metabolites (with the exception of glucose), showing rapid adaptation of the metabolic machinery to the available resources. In contrast, single metabolites did not increase the O2 consumption of neurons or astrocytes. In "starving" neurons, neither lactate nor pyruvate was utilized for mitochondrial ATP production. Gene expression studies also suggested that aerobic glycolysis and rapid metabolic adaptation characterize the NE-4C NSCs, while autophagy and alternative glucose utilization play important roles in the metabolism of stem cell-derived neurons.

  7. Concomitant inhibition of prolyl hydroxylases and ROCK initiates differentiation of mesenchymal stem cells and PC12 towards the neuronal lineage.

    PubMed

    Pacary, Emilie; Petit, Edwige; Bernaudin, Myriam

    2008-12-12

    This study demonstrates that a prolyl hydroxylase inhibitor, FG-0041, is able, in combination with the ROCK inhibitor, Y-27632, to initiate differentiation of mesenchymal stem cells (MSCs) into neuron-like cells. FG-0041/Y-27632 co-treatment provokes morphological changes into neuron-like cells, increases neuronal marker expression and provokes modifications of cell cycle-related gene expression consistent with a cell cycle arrest of MSC, three events showing the engagement of MSC towards the neuronal lineage. Moreover, as we observed in our previous studies with cobalt chloride and desferroxamine, the activation of HIF-1 by this prolyl hydroxylase inhibitor is potentiated by Y-27632 which could explain at least in part the effect of this co-treatment on MSC neuronal differentiation. In addition, we show that this co-treatment enhances neurite outgrowth and tyrosine hydroxylase expression in PC12 cells. Altogether, these results evidence that concomitant inhibition of prolyl hydroxylases and ROCK represents a relevant protocol to initiate neuronal differentiation.

  8. Ferulic acid promotes survival and differentiation of neural stem cells to prevent gentamicin-induced neuronal hearing loss.

    PubMed

    Gu, Lintao; Cui, Xinhua; Wei, Wei; Yang, Jia; Li, Xuezhong

    2017-09-12

    Neural stem cells (NSCs) have exhibited promising potential in therapies against neuronal hearing loss. Ferulic acid (FA) has been widely reported to enhance neurogenic differentiation of different stem cells. We investigated the role of FA in promoting NSC transplant therapy to prevent gentamicin-induced neuronal hearing loss. NSCs were isolated from mouse cochlear tissues to establish in vitro culture, which were then treated with FA. The survival and differentiation of NSCs were evaluated. Subsequently, neurite outgrowth and excitability of the in vitro neuronal network were assessed. Gentamicin was used to induce neuronal hearing loss in mice, in the presence and absence of FA, followed by assessments of auditory brainstem response (ABR) and distortion product optoacoustic emissions (DPOAE) amplitude. FA promoted survival, neurosphere formation and differentiation of NSCs, as well as neurite outgrowth and excitability of in vitro neuronal network. Furthermore, FA restored ABR threshold shifts and DPOAE in gentamicin-induced neuronal hearing loss mouse model in vivo. Our data, for the first time, support potential therapeutic efficacy of FA in promoting survival and differentiation of NSCs to prevent gentamicin-induced neuronal hearing loss. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Induced pluripotent stem cells from familial Alzheimer's disease patients differentiate into mature neurons with amyloidogenic properties.

    PubMed

    Mahairaki, Vasiliki; Ryu, Jiwon; Peters, Ann; Chang, Qing; Li, Tong; Park, Tea Soon; Burridge, Paul W; Talbot, Conover C; Asnaghi, Laura; Martin, Lee J; Zambidis, Elias T; Koliatsos, Vassilis E

    2014-12-15

    Although the majority of Alzheimer's disease (AD) cases are sporadic, about 5% of cases are inherited in an autosomal dominant pattern as familial AD (FAD) and manifest at an early age. Mutations in the presenilin 1 (PSEN1) gene account for the majority of early-onset FAD. Here, we describe the generation of virus-free human induced pluripotent stem cells (hiPSCs) derived from fibroblasts of patients harboring the FAD PSEN1 mutation A246E and fibroblasts from healthy age-matched controls using nonintegrating episomal vectors. We have differentiated these hiPSC lines to the neuronal lineage and demonstrated that hiPSC-derived neurons have mature phenotypic and physiological properties. Neurons from mutant hiPSC lines express PSEN1-A246E mutations themselves and show AD-like biochemical features, that is, amyloidogenic processing of amyloid precursor protein (APP) indicated by an increase in β-amyloid (Aβ)42/Aβ40 ratio. FAD hiPSCs harboring disease properties can be used as humanized models to test novel diagnostic methods and therapies and explore novel hypotheses for AD pathogenesis.

  10. Multiple Sclerosis Patient-Specific Primary Neurons Differentiated from Urinary Renal Epithelial Cells via Induced Pluripotent Stem Cells.

    PubMed

    Massa, Megan G; Gisevius, Barbara; Hirschberg, Sarah; Hinz, Lisa; Schmidt, Matthias; Gold, Ralf; Prochnow, Nora; Haghikia, Aiden

    2016-01-01

    As multiple sclerosis research progresses, it is pertinent to continue to develop suitable paradigms to allow for ever more sophisticated investigations. Animal models of multiple sclerosis, despite their continuing contributions to the field, may not be the most prudent for every experiment. Indeed, such may be either insufficient to reflect the functional impact of human genetic variations or unsuitable for drug screenings. Thus, we have established a cell- and patient-specific paradigm to provide an in vitro model within which to perform future genetic investigations. Renal proximal tubule epithelial cells were isolated from multiple sclerosis patients' urine and transfected with pluripotency-inducing episomal factors. Subsequent induced pluripotent stem cells were formed into embryoid bodies selective for ectodermal lineage, resulting in neural tube-like rosettes and eventually neural progenitor cells. Differentiation of these precursors into primary neurons was achieved through a regimen of neurotrophic and other factors. These patient-specific primary neurons displayed typical morphology and functionality, also staining positive for mature neuronal markers. The development of such a non-invasive procedure devoid of permanent genetic manipulation during the course of differentiation, in the context of multiple sclerosis, provides an avenue for studies with a greater cell- and human-specific focus, specifically in the context of genetic contributions to neurodegeneration and drug discovery.

  11. Multiple Sclerosis Patient-Specific Primary Neurons Differentiated from Urinary Renal Epithelial Cells via Induced Pluripotent Stem Cells

    PubMed Central

    Massa, Megan G.; Gisevius, Barbara; Hirschberg, Sarah; Hinz, Lisa; Schmidt, Matthias; Gold, Ralf; Prochnow, Nora; Haghikia, Aiden

    2016-01-01

    As multiple sclerosis research progresses, it is pertinent to continue to develop suitable paradigms to allow for ever more sophisticated investigations. Animal models of multiple sclerosis, despite their continuing contributions to the field, may not be the most prudent for every experiment. Indeed, such may be either insufficient to reflect the functional impact of human genetic variations or unsuitable for drug screenings. Thus, we have established a cell- and patient-specific paradigm to provide an in vitro model within which to perform future genetic investigations. Renal proximal tubule epithelial cells were isolated from multiple sclerosis patients’ urine and transfected with pluripotency-inducing episomal factors. Subsequent induced pluripotent stem cells were formed into embryoid bodies selective for ectodermal lineage, resulting in neural tube-like rosettes and eventually neural progenitor cells. Differentiation of these precursors into primary neurons was achieved through a regimen of neurotrophic and other factors. These patient-specific primary neurons displayed typical morphology and functionality, also staining positive for mature neuronal markers. The development of such a non-invasive procedure devoid of permanent genetic manipulation during the course of differentiation, in the context of multiple sclerosis, provides an avenue for studies with a greater cell- and human-specific focus, specifically in the context of genetic contributions to neurodegeneration and drug discovery. PMID:27158987

  12. [NEURONAL DIFFERENTIATION OF PC12 CELL LINE AND MURINE NEURAL STEM CELLS ON THE CARBON NANOTUBES FILMS].

    PubMed

    Posypanova, G A; Gaiduchenko, A I; Moskaleva, E Yu; Fedorov, G E

    2016-01-01

    The study of the interaction of nerve cells with specially designed substrates (scaffolds) with different surface characteristics at the nanoscale is a necessary step in the development of methods of stimulation of regeneration of nervous tissues, as well as to create next generation of bioelectronic devices. A promising material for such scaffolds may be carbon nanotubes (CNT) that are flexible films of graphene rolled into nano-sized cylindrical tubes. CNT were produced by chemical deposition from the gas phase. The analysis of the PC12 cells cultivated on quartz glass coated by carbon nanotubes films using electron and light microscopy has shown that CNT stimulate the proliferation and do not inhibit neuronal differentiation of PC12 cells. We have found that it is possible to obtain differentiated neurons from murine neural stem cells on the quartz glasses covered with CNT films. The data obtained indicate that the CNT films produced by chemical deposition from the gas phase onto quartz glass may be used as the electro conductive scaffold to obtain and study the functions of neural cells and possibly of mature neurons.

  13. Comparison of Capability of Human Bone Marrow Mesenchymal Stem Cells and Endometrial Stem Cells to Differentiate into Motor Neurons on Electrospun Poly(ε-caprolactone) Scaffold.

    PubMed

    Shirian, Sadegh; Ebrahimi-Barough, Somayeh; Saberi, Hooshang; Norouzi-Javidan, Abbas; Mousavi, Sayed Mostafa Modarres; Derakhshan, Mohammad Ali; Arjmand, Babak; Ai, Jafar

    2016-10-01

    Human endometrial and bone marrow-derived mesenchymal stem cells can be differentiated into a number of cell lineages. Mesenchymal stem cells (MSCs) are potential candidates for cellular therapy. The differentiation of human bone marrow MSCs (hBM-MSCs) and endometrial stem cells (hEnSCs) into motor neuron-like cells has been rarely investigated previously; however, the comparison between these stem cells when they are differentiated into motor neuron-like cell is yet to be studied. The aim of this study was therefore to investigate and compare the capability of hBM-MSCs and hEnSCs cultured on tissue culture polystyrene (TCP) and poly ε-caprolactone (PCL) nanofibrous scaffold to differentiate into motor neuron-like cells in the presence of neural inductive molecules. Engineered hBM-MSCs and hEnSCs seeded on PCL nanofibrous scaffold were differentiated into beta-tubulin III, islet-1, Neurofilament-H (NF-H), HB9, Pax6, and choactase-positive motor neurons by immunostaining and real-time PCR, in response to the signaling molecules. The data obtained from PCR and immunostaining showed that the expression of motor neuron markers of both hBM-MSCs and hEnSCs differentiated cells on PCL scaffold are significantly higher than that of the control group. The expression of these markers in hEnSCs differentiated cells was higher than that in hBM-MSCs. However, this difference was not statistically significant. In conclusion, differentiated hBM-MSCs and hEnSCs on PCL can provide a suitable three-dimensional situation for neuronal survival and outgrowth for regeneration of the central nervous system. Both cells may be potential candidates for cellular therapy in motor neuron disorders. However, differentiation of hEnSCs into motor neuron-like cells was better than hBM-MSCs.

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

    PubMed

    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.

  15. Differential effects of glucocorticoids and gonadal steroids on glutathione levels in neuronal and glial cell systems.

    PubMed

    Schmidt, A J; Krieg, J -C; Vedder, H

    2002-02-15

    The aim of the present study was to investigate the short- and long-term effects of glucocorticoids [corticosterone (CORT), dexamethasone (DEX), 6-methylprednisolone (6-MP)] and gonadal steroids [17beta-estradiol (E(2)), progesterone (PROG), testosterone (TEST)] on the levels of the antioxidant glutathione (GSH) in different cell systems of the CNS (neuronal hippocampal HT22 cells, primary hippocampal and neocortical brain cells, and C(6) glioma cells). In HT22 cells, steroids exerted mainly long-term effects. Significant increases of GSH levels were detectable after a 24 hr treatment with 10(-7) M of DEX (122% +/- 5%), 6-MP (208% +/- 32%), E(2) (134% +/- 10%), and TEST (155% +/- 17%). A significant decrease occurred after incubation with PROG for 24 hr (79% +/- 9%). In primary hippocampal cultures, a 24 hr treatment with DEX (140% +/- 8%), E(2) (123% +/- 6%), and PROG (118% +/- 5%) led to significant increases of the GSH levels, whereas, in neocortical primary cultures, only an incubation with E(2) increased GSH (149% +/- 8%). In C(6) cells, hormone treatment led to both significant short-term (1 hr: CORT 114% +/- 5%, DEX 90% +/- 3%, E(2) 88% +/- 3%; 3 hr: DEX 115% +/- 5%, E(2) 122% +/- 6%, TEST 78% +/- 4%) and significant long-term (24 hr: CORT 74% +/- 4%, 6-MP 84% +/- 5%, E(2) 115% +/- 6%, PROG 91% +/- 4%, TEST 116% +/- 5%) effects. In summary, we were able to demonstrate differential effects of steroids on GSH levels in different cellular CNS models, showing an important influence of steroids and especially E(2) on antioxidative cellular functions in neuronal and glial cells.

  16. Nerve growth factor (NGF)-conjugated electrospun nanostructures with topographical cues for neuronal differentiation of mesenchymal stem cells.

    PubMed

    Cho, Young Il; Choi, Ji Suk; Jeong, Seo Young; Yoo, Hyuk Sang

    2010-12-01

    Mesenchymal stem cells (MSCs) were cultivated on the surface of nerve growth factor (NGF)-conjugated aligned nanofibrous meshes for neuronal differentiation. Amine-terminated poly(ethylene glycol) was conjugated to poly(ε-caprolactone) to prepare amine-functionalized block copolymers. The synthesized polymer was electrospun in a rotating drum to prepare aligned nanofibrous meshes. A nerve growth factor was chemically immobilized on the surface-exposed amine groups of the electrospun nanofibrous meshes in the aqueous phase. In vitro release profiles of the nerve growth factor were investigated for NGF-immobilized nanofibrous meshes. The conjugated nerve growth factor was not released for 7 days, while the growth factor physically adsorbed on the nanofibrous meshes showed an initial burst release. MSCs were cultivated on the NGF-conjugated nanofibrous meshes for 5 days, and total RNA was extracted from the cultivated cells. mRNA was extracted from cells for measuring expression levels of neuronal differentiation markers, including nestin, tubulin βIII and map2, in the cultivated stem cells. The conjugation of NGF significantly increased the expression levels of the marker proteins for neuron cells while physically adsorbed NGFs on nanofibrous meshes showed low expression of these marker genes. Furthermore, alignments of nanofibrous meshes clearly increased the expression levels of neuronal makers while the nanofibrous mesh without the topographical cue did not affect neuronal differentiation of the cultivated stem cells. Confocal microscopy revealed that the stem cells on the NGF-conjugated aligned nanofibrous meshes showed intense staining with antibodies against neuronal makers as well as elongated morphology compared to other groups. Thus, the NGF-conjugated nanofibrous meshes with topographical cues significantly increased the neuronal differentiation of mesenchymal stem cells in comparison to NGF-adsorbed nanofibrous meshes.

  17. Tailless-like (TLX) protein promotes neuronal differentiation of dermal multipotent stem cells and benefits spinal cord injury in rats.

    PubMed

    Wang, Tao; Ren, Xiaobao; Xiong, Jianqiong; Zhang, Lei; Qu, Jifu; Xu, Wenyue

    2011-04-01

    Spinal cord injury (SCI) remains a formidable challenge in the clinic. In the current study, we examined the effects of the TLX gene on the proliferation and neuronal differentiation of dermal multipotent stem cells (DMSCs) in vitro and the potential of these cells to improve SCI in rats in vivo. DMSCs were stably transfected with TLX-expressing plasmid (TLX/DMSCs). Cell proliferation was examined using the MTT assay, and neuronal differentiation was characterized by morphological observation combined with immunocytochemical/immunofluorescent staining. The in vivo functions of these cells were evaluated by transplantation into rats with SCI, followed by analysis of hindlimb locomotion and post-mortem histology. Compared to parental DMSCs, TLX/DMSCs showed enhanced proliferation and preferential differentiation into NF200-positive neurons in contrast to GFAP-positive astrocytes. When the undifferentiated cells were transplanted into rats with SCI injury, TLX/DMSCs led to significant improvement in locomotor recovery and healing of SCI, as evidenced by reduction in scar tissues and cavities, increase in continuous nerve fibers/axons and enrichment of NF200-positive neurons on the histological level. In conclusion, TLX promotes the proliferation and neuronal differentiation of DMSCs and thus, may serve as a promising therapy for SCI in the clinic.

  18. Balancing cell numbers during organogenesis: Six1a differentially affects neurons and sensory hair cells in the inner ear

    PubMed Central

    Bricaud, Olivier; Collazo, Andres

    2011-01-01

    While genes involved in the differentiation of the mechanosensory hair cells and the neurons innervating them have been identified, genes involved in balancing their relative numbers remain unknown. Six1a plays a dual role by promoting hair cell fate while inhibiting neuronal fate in these two lineages. Genes homologous to six1a act as either transcriptional activators or repressors, depending on the partners with which they interact. By assaying the in vivo and in vitro effects of mutations in presumptive protein-protein interacting and DNA-binding domains of Six1a, we show that, in the developing zebrafish inner ear, Six1a promotes hair cell fate by acting as a transcriptional activator and inhibits neuronal fate by acting as a transcriptional repressor. We also identify several potential partners for Six1a that differ between these two lineages. The dual role of Six1a in the developing otocyst provides a mechanism for balancing the relative number of hair cells and neurons during organogenesis of the inner ear. PMID:21745464

  19. Some Rat Sensory Neurons in Culture Express Characteristics of Differentiated Pain Sensory Cells

    NASA Astrophysics Data System (ADS)

    Baccaglini, Paola I.; Hogan, Patrick G.

    1983-01-01

    Sensory neurons were dissociated from trigeminal ganglia or from dorsal root ganglia of rats, grown in culture, and examined for expression of properties of pain sensory cells. Many sensory neurons in culture are excited by low concentrations of capsaicin, reportedly a selective stimulus for pain sensory neurons. Many are excited by bradykinin, sensitized by prostaglandin E2, or specifically stained by an antiserum against substance P. These experiments provide a basis for the study of pain mechanisms in cell culture.

  20. Maintenance and neuronal cell differentiation of neural stem cells C17.2 correlated to medium availability sets design criteria in microfluidic systems.

    PubMed

    Wang, Bu; Jedlicka, Sabrina; Cheng, Xuanhong

    2014-01-01

    Neural stem cells (NSCs) play an important role in developing potential cell-based therapeutics for neurodegenerative disease. Microfluidics has proven a powerful tool in mechanistic studies of NSC differentiation. However, NSCs are prone to differentiate when the nutrients are limited, which occurs unfavorable by fast medium consumption in miniaturized culture environment. For mechanistic studies of NSCs in microfluidics, it is vital that neuronal cell differentiation is triggered by controlled factors only. Thus, we studied the correlation between available cell medium and spontaneous neuronal cell differentiation of C17.2 NSCs in standard culture medium, and proposed the necessary microfluidic design criteria to prevent undesirable cell phenotype changes. A series of microchannels with specific geometric parameters were designed to provide different amount of medium to the cells over time. A medium factor (MF, defined as the volume of stem cell culture medium divided by total number of cells at seeding and number of hours between medium replacement) successfully correlated the amount of medium available to each cell averaged over time to neuronal cell differentiation. MF smaller than 8.3×10(4) µm3/cell⋅hour produced significant neuronal cell differentiation marked by cell morphological change and significantly more cells with positive β-tubulin-III and MAP2 staining than the control. When MF was equal or greater than 8.3×10(4) µm3/cell⋅hour, minimal spontaneous neuronal cell differentiation happened relative to the control. MF had minimal relation with the average neurite length. MFs can be controlled easily to maintain the stem cell status of C17.2 NSCs or to induce spontaneous neuronal cell differentiation in standard stem cell culture medium. This finding is useful in designing microfluidic culture platforms for controllable NSC maintenance and differentiation. This study also offers insight about consumption rate of serum molecules involved in

  1. Maintenance and Neuronal Cell Differentiation of Neural Stem Cells C17.2 Correlated to Medium Availability Sets Design Criteria in Microfluidic Systems

    PubMed Central

    Wang, Bu; Jedlicka, Sabrina; Cheng, Xuanhong

    2014-01-01

    Background Neural stem cells (NSCs) play an important role in developing potential cell-based therapeutics for neurodegenerative disease. Microfluidics has proven a powerful tool in mechanistic studies of NSC differentiation. However, NSCs are prone to differentiate when the nutrients are limited, which occurs unfavorable by fast medium consumption in miniaturized culture environment. For mechanistic studies of NSCs in microfluidics, it is vital that neuronal cell differentiation is triggered by controlled factors only. Thus, we studied the correlation between available cell medium and spontaneous neuronal cell differentiation of C17.2 NSCs in standard culture medium, and proposed the necessary microfluidic design criteria to prevent undesirable cell phenotype changes. Methodology/Principal Findings A series of microchannels with specific geometric parameters were designed to provide different amount of medium to the cells over time. A medium factor (MF, defined as the volume of stem cell culture medium divided by total number of cells at seeding and number of hours between medium replacement) successfully correlated the amount of medium available to each cell averaged over time to neuronal cell differentiation. MF smaller than 8.3×104 µm3/cell⋅hour produced significant neuronal cell differentiation marked by cell morphological change and significantly more cells with positive β-tubulin-III and MAP2 staining than the control. When MF was equal or greater than 8.3×104 µm3/cell⋅hour, minimal spontaneous neuronal cell differentiation happened relative to the control. MF had minimal relation with the average neurite length. Significance MFs can be controlled easily to maintain the stem cell status of C17.2 NSCs or to induce spontaneous neuronal cell differentiation in standard stem cell culture medium. This finding is useful in designing microfluidic culture platforms for controllable NSC maintenance and differentiation. This study also offers insight about

  2. Pleiotrophin antagonizes Brd2 during neuronal differentiation

    PubMed Central

    Garcia-Gutierrez, Pablo; Juarez-Vicente, Francisco; Wolgemuth, Debra J.; Garcia-Dominguez, Mario

    2014-01-01

    ABSTRACT Bromodomain-containing protein 2 (Brd2) is a BET family chromatin adaptor required for expression of cell-cycle-associated genes and therefore involved in cell cycle progression. Brd2 is expressed in proliferating neuronal progenitors, displays cell-cycle-stimulating activity and, when overexpressed, impairs neuronal differentiation. Paradoxically, Brd2 is also detected in differentiating neurons. To shed light on the role of Brd2 in the transition from cell proliferation to differentiation, we had previously looked for proteins that interacted with Brd2 upon induction of neuronal differentiation. Surprisingly, we identified the growth factor pleiotrophin (Ptn). Here, we show that Ptn antagonized the cell-cycle-stimulating activity associated with Brd2, thus enhancing induced neuronal differentiation. Moreover, Ptn knockdown reduced neuronal differentiation. We analyzed Ptn-mediated antagonism of Brd2 in a cell differentiation model and in two embryonic processes associated with the neural tube: spinal cord neurogenesis and neural crest migration. Finally, we investigated the mechanisms of Ptn-mediated antagonism and determined that Ptn destabilizes the association of Brd2 with chromatin. Thus, Ptn-mediated Brd2 antagonism emerges as a modulation system accounting for the balance between cell proliferation and differentiation in the vertebrate nervous system. PMID:24695857

  3. Pleiotrophin antagonizes Brd2 during neuronal differentiation.

    PubMed

    Garcia-Gutierrez, Pablo; Juarez-Vicente, Francisco; Wolgemuth, Debra J; Garcia-Dominguez, Mario

    2014-06-01

    Bromodomain-containing protein 2 (Brd2) is a BET family chromatin adaptor required for expression of cell-cycle-associated genes and therefore involved in cell cycle progression. Brd2 is expressed in proliferating neuronal progenitors, displays cell-cycle-stimulating activity and, when overexpressed, impairs neuronal differentiation. Paradoxically, Brd2 is also detected in differentiating neurons. To shed light on the role of Brd2 in the transition from cell proliferation to differentiation, we had previously looked for proteins that interacted with Brd2 upon induction of neuronal differentiation. Surprisingly, we identified the growth factor pleiotrophin (Ptn). Here, we show that Ptn antagonized the cell-cycle-stimulating activity associated with Brd2, thus enhancing induced neuronal differentiation. Moreover, Ptn knockdown reduced neuronal differentiation. We analyzed Ptn-mediated antagonism of Brd2 in a cell differentiation model and in two embryonic processes associated with the neural tube: spinal cord neurogenesis and neural crest migration. Finally, we investigated the mechanisms of Ptn-mediated antagonism and determined that Ptn destabilizes the association of Brd2 with chromatin. Thus, Ptn-mediated Brd2 antagonism emerges as a modulation system accounting for the balance between cell proliferation and differentiation in the vertebrate nervous system. © 2014. Published by The Company of Biologists Ltd.

  4. Conditioned medium from human amniotic epithelial cells may induce the differentiation of human umbilical cord blood mesenchymal stem cells into dopaminergic neuron-like cells.

    PubMed

    Yang, Shu; Sun, Hai-Mei; Yan, Ji-Hong; Xue, Hong; Wu, Bo; Dong, Fang; Li, Wen-Shuai; Ji, Feng-Qing; Zhou, De-Shan

    2013-07-01

    Dopaminergic (DA) neuron therapy has been established as a new clinical tool for treating Parkinson's disease (PD). Prior to cell transplantation, there are two primary issues that must be resolved: one is the appropriate seed cell origin, and the other is the efficient inducing technique. In the present study, human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) were used as the available seed cells, and conditioned medium from human amniotic epithelial cells (ACM) was used as the inducing reagent. Results showed that the proportion of DA neuron-like cells from hUCB-MSCs was significantly increased after cultured in ACM, suggested by the upregulation of DAT, TH, Nurr1, and Pitx3. To identify the process by which ACM induces DA neuron differentiation, we pretreated hUCB-MSCs with k252a, the Trk receptor inhibitor of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), and found that the proportion of DA neuron-like cells was significantly decreased compared with ACM-treated hUCB-MSCs, suggesting that NGF and BDNF in ACM were involved in the differentiation process. However, we could not rule out the involvement of other unidentified factors in the ACM, because ACM + k252a treatment does not fully block DA neuron-like cell differentiation compared with control. The transplantation of ACM-induced hUCB-MSCs could ameliorate behavioral deficits in PD rats, which may be associated with the survival of engrafted DA neuron-like cells. In conclusion, we propose that hUCB-MSCs are a good source of DA neuron-like cells and that ACM is a potential inducer to obtain DA neuron-like cells from hUCB-MSCs in vitro for an ethical and legal cell therapy for PD.

  5. The effects of diazinon and cypermethrin on the differentiation of neuronal and glial cell lines

    SciTech Connect

    Flaskos, J.; Harris, W.; Sachana, M.; Munoz, D.; Tack, J.; Hargreaves, A.J. . E-mail: alan.hargreaves@ntu.ac.uk

    2007-03-15

    Diazinon and cypermethrin are pesticides extensively used in sheep dipping. Diazinon is a known anti-cholinesterase, but there is limited information regarding its molecular mechanism of action. This paper describes the effects of diazinon and cypermethrin at a morphological and molecular level on differentiating mouse N2a neuroblastoma and rat C6 glioma cell lines. Concentrations up to 10 {mu}M of both compounds and their mixture had no effect on the viability of either cell line, as determined by methyl blue tetrazolium reduction and total protein assays. Microscopic analysis revealed that 1 {mu}M and 10 {mu}M diazinon but not cypermethrin inhibited the outgrowth of axon-like processes in N2a cells after a 24-h exposure but neither compound affected process outgrowth by differentiating C6 cells at these concentrations. Under these conditions, 10 {mu}M diazinon inhibited AChE slightly compared to the control after a 4-h exposure but not after 24 h. Western blotting analysis showed that morphological changes were associated with reduced cross-reactivity with antibodies that recognize the neurofilament heavy chain (NFH), microtubule associated protein MAP 1B and HSP-70 compared to control cell extracts, whereas reactivity with anti-{alpha}-tubulin antibodies was unchanged. Aggregation of NFH was observed in cell bodies of diazinon-treated N2a cells, as determined by indirect immunofluorescence staining. These data demonstrate that diazinon specifically targets neurite outgrowth in neuronal cells and that this effect is associated with disruption of axonal cytoskeleton proteins, whereas cypermethrin has no effect on the same parameters.

  6. Genome-wide characterisation of Foxa1 binding sites reveals several mechanisms for regulating neuronal differentiation in midbrain dopamine cells.

    PubMed

    Metzakopian, Emmanouil; Bouhali, Kamal; Alvarez-Saavedra, Matías; Whitsett, Jeffrey A; Picketts, David J; Ang, Siew-Lan

    2015-04-01

    Midbrain dopamine neuronal progenitors develop into heterogeneous subgroups of neurons, such as substantia nigra pars compacta, ventral tegmental area and retrorubal field, that regulate motor control, motivated and addictive behaviours. The development of midbrain dopamine neurons has been extensively studied, and these studies indicate that complex cross-regulatory interactions between extrinsic and intrinsic molecules regulate a precise temporal and spatial programme of neurogenesis in midbrain dopamine progenitors. To elucidate direct molecular interactions between multiple regulatory factors during neuronal differentiation in mice, we characterised genome-wide binding sites of the forkhead/winged helix transcription factor Foxa1, which functions redundantly with Foxa2 to regulate the differentiation of mDA neurons. Interestingly, our studies identified a rostral brain floor plate Neurog2 enhancer that requires direct input from Otx2, Foxa1, Foxa2 and an E-box transcription factor for its transcriptional activity. Furthermore, the chromatin remodelling factor Smarca1 was shown to function downstream of Foxa1 and Foxa2 to regulate differentiation from immature to mature midbrain dopaminergic neurons. Our genome-wide Foxa1-bound cis-regulatory sequences from ChIP-Seq and Foxa1/2 candidate target genes from RNA-Seq analyses of embryonic midbrain dopamine cells also provide an excellent resource for probing mechanistic insights into gene regulatory networks involved in the differentiation of midbrain dopamine neurons. © 2015. Published by The Company of Biologists Ltd.

  7. Sexually Dimorphic Differentiation of a C. elegans Hub Neuron Is Cell Autonomously Controlled by a Conserved Transcription Factor.

    PubMed

    Serrano-Saiz, Esther; Oren-Suissa, Meital; Bayer, Emily A; Hobert, Oliver

    2017-01-23

    Functional and anatomical sexual dimorphisms in the brain are either the result of cells that are generated only in one sex or a manifestation of sex-specific differentiation of neurons present in both sexes. The PHC neuron pair of the nematode C. elegans differentiates in a strikingly sex-specific manner. In hermaphrodites the PHC neurons display a canonical pattern of synaptic connectivity similar to that of other sensory neurons, but in males PHC differentiates into a densely connected hub sensory neuron/interneuron, integrating a large number of male-specific synaptic inputs and conveying them to both male-specific and sex-shared circuitry. We show that the differentiation into such a hub neuron involves the sex-specific scaling of several components of the synaptic vesicle machinery, including the vesicular glutamate transporter eat-4/VGLUT, induction of neuropeptide expression, changes in axonal projection morphology, and a switch in neuronal function. We demonstrate that these molecular and anatomical remodeling events are controlled cell autonomously by the phylogenetically conserved Doublesex homolog dmd-3, which is both required and sufficient for sex-specific PHC differentiation. Cellular specificity of dmd-3 action is ensured by its collaboration with non-sex-specific terminal selector-type transcription factors, whereas the sex specificity of dmd-3 action is ensured by the hermaphrodite-specific transcriptional master regulator of hermaphroditic cell identity tra-1, which represses the transcription of dmd-3 in hermaphrodite PHC. Taken together, our studies provide mechanistic insights into how neurons are specified in a sexually dimorphic manner.

  8. HMGB4 is expressed by neuronal cells and affects the expression of genes involved in neural differentiation

    PubMed Central

    Rouhiainen, Ari; Zhao, Xiang; Vanttola, Päivi; Qian, Kui; Kulesskiy, Evgeny; Kuja-Panula, Juha; Gransalke, Kathleen; Grönholm, Mikaela; Unni, Emmanual; Meistrich, Marvin; Tian, Li; Auvinen, Petri; Rauvala, Heikki

    2016-01-01

    HMGB4 is a new member in the family of HMGB proteins that has been characterized in sperm cells, but little is known about its functions in somatic cells. Here we show that HMGB4 and the highly similar rat Transition Protein 4 (HMGB4L1) are expressed in neuronal cells. Both proteins had slow mobility in nucleus of living NIH-3T3 cells. They interacted with histones and their differential expression in transformed cells of the nervous system altered the post-translational modification statuses of histones in vitro. Overexpression of HMGB4 in HEK 293T cells made cells more susceptible to cell death induced by topoisomerase inhibitors in an oncology drug screening array and altered variant composition of histone H3. HMGB4 regulated over 800 genes in HEK 293T cells with a p-value ≤0.013 (n = 3) in a microarray analysis and displayed strongest association with adhesion and histone H2A –processes. In neuronal and transformed cells HMGB4 regulated the expression of an oligodendrocyte marker gene PPP1R14a and other neuronal differentiation marker genes. In conclusion, our data suggests that HMGB4 is a factor that regulates chromatin and expression of neuronal differentiation markers. PMID:27608812

  9. Light-controlled astrocytes promote human mesenchymal stem cells toward neuronal differentiation and improve the neurological deficit in stroke rats.

    PubMed

    Tu, Jie; Yang, Fan; Wan, Jun; Liu, Yunhui; Zhang, Jie; Wu, Bifeng; Liu, Yafeng; Zeng, Shaoqun; Wang, Liping

    2014-01-01

    Astrocytes are key components of the central nervous system (CNS) and release factors to support neural stem cell proliferation, differentiation, and migration. Adenosine 5'-triphosphate (ATP) is one of the key factors released upon activation of astrocytes that regulates the neural stem cell's function. However, it is not clear whether ATP derived from the depolarized astrocytes plays a vital role in promoting the neuronal differentiation of mesenchymal stem cells (MSCs) in vitro and in vivo. Herein, for the first time, we co-cultured MSCs with light-stimulated-channelrhodopsin-2 (ChR2)-astrocytes, and observed that the neuronal differentiation of MSCs was enhanced by expressing more neuronal markers, Tuj1 and NeuN. The ChR2-astrocyte-conditioned medium also stimulated MSCs differentiating into neuronal lineage cells by expressing more Tuj1 and Pax6, which was blocked by the P2X receptor antagonist, TNP-ATP. Then we found that light-depolarization of astrocytes significantly increased ATP accumulation in their bathing medium without impairing the cell membrane. We further found that ATP up-regulated the Tuj1, Pax6, FZD8 and β-catenin mRNA levels of MSCs, which could be reversed by application of TNP-ATP. Together these in vitro data provided convergent evidence that ATP from light-depolarized-astrocytes activated the wnt/β-catenin signaling of MSCs through binding to the P2X receptors, and promoted the neuronal differentiation of MSCs. Finally but importantly, our study also demonstrated in stroke rats that light-controlled astrocytes stimulated endogenous ATP release into the ischemic area to influence the transplanted MSCs, resulting in promoting the MSCs towards neuronal differentiation and improvements of neurological deficit. Copyright © 2013 Wiley Periodicals, Inc.

  10. Schwann cells promote the capability of neural stem cells to differentiate into neurons and secret neurotrophic factors.

    PubMed

    Yu, Ziwei; Men, Yongzhi; Dong, Pin

    2017-05-01

    The present study investigated whether co-culturing Schwann cells (SCs) with neural stem cells (NSCs) improves viability, direction of differentiation and secretion of brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) in NSCs. The three groups assessed were as follows: SCs, NSCs, and a co-culture of SCs and NSCs. Cellular morphological changes were observed under an inverted phase contrast microscope and quantified. Cells were identified by immunofluorescence staining: S100 for SCs, Nestin for NSCs, microtubule associated protein (Map) 2 and NeuN for neurons and glial fibrillary acidic protein for astrocytes. Cell viability was evaluated by MTT assay. Secretion of BDNF and GDNF was quantified; mRNA expression was quantified by reverse transcription-quantitative polymerase chain reaction. The majority of NSCs in the co-cultured group differentiated into neurons. The cell survival rate of the co-culture group was significantly higher than the other groups on days 3, 5 and 10 (P<0.01). The secretion of BDNF in the co-culture group was significantly higher than NSCs on days 3, 5 and 7 (P<0.05), while the amount of GDNF in co-culture was significantly higher than both NSCs and SCs on day 1 (P<0.05). BDNF and GDNF gene expression in the co-culture group was significantly higher than SCs (P<0.01). Gene expression of Map2 in co-culture group was also significantly higher than both NSC and SC groups (P<0.01). Therefore, co-cultured SCs and NSCs promote differentiation of NSCs into neurons and secrete higher levels of neurotropic factors including BDNF and GDNF.

  11. All-Trans-Retinoid Acid Induces the Differentiation of Encapsulated Mouse Embryonic Stem Cells into GABAergic Neurons

    PubMed Central

    Addae, Cynthia; Yi, Xiaoping; Gernapudi, Ramkishore; Cheng, Henrique; Musto, Alberto; Martinez-Ceballos, Eduardo

    2012-01-01

    Embryonic stem (ES) cells are pluripotent cells that can differentiate into all three main germ layers: endoderm, mesoderm, and ectoderm. Although a number of methods have been developed to differentiate ES cells into neuronal phenotypes such as sensory and motor neurons, the efficient generation of GABAergic interneurons from ES cells still presents an ongoing challenge. Because the main output of inhibitory GABAergic interneurons is the gamma-aminobutyric-acid (GABA), a neurotransmitter whose controlled homeostasis is required for normal brain function, the efficient generation in culture of functional interneurons may have future implications on the treatment of neurological disorders such as epilepsy, autism, and schizophrenia. The goal of this work was to examine the generation of GABAergic neurons from mouse ES cells by comparing an embryoid body-based methodology versus a hydrogel-based encapsulation protocol that involves the use of all-trans-retinoid acid (RA). We observed that 1) there was a 2-fold increase in neuronal differentiation in encapsulated versus non-encapsulated cells and 2) there was an increase in the specificity for interneuronal differentiation in encapsulated cells, as assessed by mRNA expression and electrophysiology approaches. Furthermore, our results indicate that most of the neurons obtained from encapsulated mouse ES cells are GABA-positive (~87%). Thus, these results suggest that combining encapsulation of ES cells and RA treatment provide a more efficient and scalable differentiation strategy for the generation in culture of functional GABAergic interneurons. This technology may have implications for future cell replacement therapies and the treatment of CNS disorders. PMID:22466603

  12. Neuronal differentiation of human mesenchymal stem cells: changes in the expression of the Alzheimer's disease-related gene seladin-1.

    PubMed

    Benvenuti, Susanna; Saccardi, Riccardo; Luciani, Paola; Urbani, Serena; Deledda, Cristiana; Cellai, Ilaria; Francini, Fabio; Squecco, Roberta; Rosati, Fabiana; Danza, Giovanna; Gelmini, Stefania; Greeve, Isabell; Rossi, Matteo; Maggi, Roberto; Serio, Mario; Peri, Alessandro

    2006-08-01

    Seladin-1 (SELective Alzheimer's Disease INdicator-1) is an anti-apoptotic gene, which is down-regulated in brain regions affected by Alzheimer's disease (AD). In addition, seladin-1 catalyzes the conversion of desmosterol into cholesterol. Disruption of cholesterol homeostasis in neurons may increase cell susceptibility to toxic agents. Because the hippocampus and the subventricular zone, which are affected in AD, are the unique regions containing stem cells with neurogenic potential in the adult brain, it might be hypothesized that this multipotent cell compartment is the predominant source of seladin-1 in normal brain. In the present study, we isolated and characterized human mesenchymal stem cells (hMSC) as a model of cells with the ability to differentiate into neurons. hMSC were then differentiated toward a neuronal phenotype (hMSC-n). These cells were thoroughly characterized and proved to be neurons, as assessed by molecular and electrophysiological evaluation. Seladin-1 expression was determined and found to be significantly reduced in hMSC-n compared to undifferentiated cells. Accordingly, the total content of cholesterol was decreased after differentiation. These original results demonstrate for the first time that seladin-1 is abundantly expressed by stem cells and appear to suggest that reduced expression in AD might be due to an altered pool of multipotent cells.

  13. Differentiation of rat adipose tissue-derived stem cells into neuron-like cells by valproic acid, a histone deacetylase inhibitor.

    PubMed

    Okubo, Takumi; Hayashi, Daiki; Yaguchi, Takayuki; Fujita, Yudai; Sakaue, Motoharu; Suzuki, Takehito; Tsukamoto, Atsushi; Murayama, Ohoshi; Lynch, Jonathan; Miyazaki, Yoko; Tanaka, Kazuaki; Takizawa, Tatsuya

    2016-01-01

    Valproic acid (VPA) is a widely used antiepileptic drug, which has recently been reported to modulate the neuronal differentiation of adipose tissue-derived stem cells (ASCs) in humans and dogs. However, controversy exists as to whether VPA really acts as an inducer of neuronal differentiation of ASCs. The present study aimed to elucidate the effect of VPA in neuronal differentiation of rat ASCs. One or three days of pretreatment with VPA (2 mM) followed by neuronal induction enhanced the ratio of immature neuron marker βIII-tubulin-positive cells in a time-dependent manner, where the majority of cells also had a positive signal for neurofilament medium polypeptide (NEFM), a mature neuron marker. RT-PCR analysis revealed increases in the mRNA expression of microtubule-associated protein 2 (MAP2) and NEFM mature neuron markers, even without neuronal induction. Three-days pretreatment of VPA increased acetylation of histone H3 of ASCs as revealed by immunofluorescence staining. Chromatin immunoprecipitation assay also showed that the status of histone acetylation at H3K9 correlated with the gene expression of TUBB3 in ASCs by VPA. These results indicate that VPA significantly promotes the differentiation of rat ASCs into neuron-like cells through acetylation of histone H3, which suggests that VPA may serve as a useful tool for producing transplantable cells for future applications in clinical treatments.

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

    NASA Astrophysics Data System (ADS)

    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.

  15. Transgenic GDNF Positively Influences Proliferation, Differentiation, Maturation and Survival of Motor Neurons Produced from Mouse Embryonic Stem Cells.

    PubMed

    Cortés, Daniel; Robledo-Arratia, Yolanda; Hernández-Martínez, Ricardo; Escobedo-Ávila, Itzel; Bargas, José; Velasco, Iván

    2016-01-01

    Embryonic stem cells (ESC) are pluripotent and thus can differentiate into every cell type present in the body. Directed differentiation into motor neurons (MNs) has been described for pluripotent cells. Although neurotrophic factors promote neuronal survival, their role in neuronal commitment is elusive. Here, we developed double-transgenic lines of mouse ESC (mESC) that constitutively produce glial cell line-derived neurotrophic factor (GDNF) and also contain a GFP reporter, driven by HB9, which is expressed only by postmitotic MNs. After lentiviral transduction, ESC lines integrated and expressed the human GDNF (hGDNF) gene without altering pluripotency markers before differentiation. Further, GDNF-ESC showed significantly higher spontaneous release of this neurotrophin to the medium, when compared to controls. To study MN induction, control and GDNF cell lines were grown as embryoid bodies and stimulated with retinoic acid and Sonic Hedgehog. In GDNF-overexpressing cells, a significant increase of proliferative Olig2+ precursors, which are specified as spinal MNs, was found. Accordingly, GDNF increases the yield of cells with the pan motor neuronal markers HB9, monitored by GFP expression, and Isl1. At terminal differentiation, almost all differentiated neurons express phenotypic markers of MNs in GDNF cultures, with lower proportions in control cells. To test if the effects of GDNF were present at early differentiation stages, exogenous recombinant hGDNF was added to control ESC, also resulting in enhanced MN differentiation. This effect was abolished by the co-addition of neutralizing anti-GDNF antibodies, strongly suggesting that differentiating ESC are responsive to GDNF. Using the HB9::GFP reporter, MNs were selected for electrophysiological recordings. MNs differentiated from GDNF-ESC, compared to control MNs, showed greater electrophysiological maturation, characterized by increased numbers of evoked action potentials (APs), as well as by the appearance

  16. Transgenic GDNF Positively Influences Proliferation, Differentiation, Maturation and Survival of Motor Neurons Produced from Mouse Embryonic Stem Cells

    PubMed Central

    Cortés, Daniel; Robledo-Arratia, Yolanda; Hernández-Martínez, Ricardo; Escobedo-Ávila, Itzel; Bargas, José; Velasco, Iván

    2016-01-01

    Embryonic stem cells (ESC) are pluripotent and thus can differentiate into every cell type present in the body. Directed differentiation into motor neurons (MNs) has been described for pluripotent cells. Although neurotrophic factors promote neuronal survival, their role in neuronal commitment is elusive. Here, we developed double-transgenic lines of mouse ESC (mESC) that constitutively produce glial cell line-derived neurotrophic factor (GDNF) and also contain a GFP reporter, driven by HB9, which is expressed only by postmitotic MNs. After lentiviral transduction, ESC lines integrated and expressed the human GDNF (hGDNF) gene without altering pluripotency markers before differentiation. Further, GDNF-ESC showed significantly higher spontaneous release of this neurotrophin to the medium, when compared to controls. To study MN induction, control and GDNF cell lines were grown as embryoid bodies and stimulated with retinoic acid and Sonic Hedgehog. In GDNF-overexpressing cells, a significant increase of proliferative Olig2+ precursors, which are specified as spinal MNs, was found. Accordingly, GDNF increases the yield of cells with the pan motor neuronal markers HB9, monitored by GFP expression, and Isl1. At terminal differentiation, almost all differentiated neurons express phenotypic markers of MNs in GDNF cultures, with lower proportions in control cells. To test if the effects of GDNF were present at early differentiation stages, exogenous recombinant hGDNF was added to control ESC, also resulting in enhanced MN differentiation. This effect was abolished by the co-addition of neutralizing anti-GDNF antibodies, strongly suggesting that differentiating ESC are responsive to GDNF. Using the HB9::GFP reporter, MNs were selected for electrophysiological recordings. MNs differentiated from GDNF-ESC, compared to control MNs, showed greater electrophysiological maturation, characterized by increased numbers of evoked action potentials (APs), as well as by the appearance

  17. Unrestricted somatic stem cells from human umbilical cord blood can be differentiated into neurons with a dopaminergic phenotype.

    PubMed

    Greschat, Susanne; Schira, Jessica; Küry, Patrick; Rosenbaum, Claudia; de Souza Silva, Maria Angelica; Kögler, Gesine; Wernet, Peter; Müller, Hans Werner

    2008-04-01

    Recently, it has been shown that human unrestricted somatic stem cells (USSCs) from umbilical cord blood represent pluripotent, neonatal, nonhematopoietic stem cells with the potential to differentiate into the neural lineage. However, molecular and functional characterization of the neural phenotype and evaluation of the degree of maturity of the resulting cells are still lacking. In this study, we addressed the question of neuronal differentiation and maturation induced by a defined composition of growth and differentiation factors (XXL medium). We demonstrated the expression of different neuronal markers and their enrichment in USSC cultures during XXL medium incubation. Furthermore, we showed enrichment of USSCs expressing tyrosine hydroxylase (TH), an enzyme specific for dopaminergic neurons and other catecholamine-producing neurons, accompanied by induction of Nurr1, a factor regulating dopaminergic neurogenesis. The functionality of USSCs has been analyzed by patch-clamp recordings and high-performance liquid chromatography (HPLC). Voltage-gated sodium-channels could be identified in laminin-predifferentiated USSCs. In addition, HPLC analysis revealed synthesis and release of the neurotransmitter dopamine by USSC-derived cells, thus correlating well with the detection of TH transcripts and protein. This study provides novel insight into the potential of unrestricted somatic stem cells from human umbilical cord blood to acquire a neuronal phenotype and function.

  18. Motor Neuron Transdifferentiation of Neural Stem Cell from Adipose-Derived Stem Cell Characterized by Differential Gene Expression.

    PubMed

    Darvishi, Marzieh; Tiraihi, Taki; Mesbah-Namin, Seyed A; Delshad, AliReza; Taheri, Taher

    2017-03-01

    Adipose-derived stem cells (ADSC) are adult stem cells which can be induced into motor neuron-like cells (MNLC) with a preinduction-induction protocol. The purpose of this study is to generate MNLC from neural stem cells (NSC) derived from ADSC. The latter were isolated from the perinephric regions of Sprague-Dawley rats, transdifferentiated into neurospheres (NS) using B27, EGF, and bFGF. After generating NSC from the NS, they induced into MNLC by treating them with Shh and RA, then with GDNF, CNTF, BDNF, and NT-3. The ADSC lineage was evaluated by its mesodermal differentiation and was characterized by immunostaining with CD90, CD105, CD49d, CD106, CD31, CD45, and stemness genes (Oct4, Nanog, and Sox2). The NS and the NSC were evaluated by immunostaining with nestin, NF68, and Neurod1, while the MNLC were evaluated by ISLET1, Olig2, and HB9 genes. The efficiency of MNLC generation was more than 95 ± 1.4 % (mean ± SEM). The in vitro generated myotubes were innervated by the MNLC. The induced ADSC adopted multipolar motor neuron morphology, and they expressed ISLET1, Olig2, and HB9. We conclude that ADSC can be induced into motor neuron phenotype with high efficiency, associated with differential expression of the motor neuron gene. The release of MNLC synaptic vesicles was demonstrated by FM1-43, and they were immunostained with synaptophysin. This activity was correlated with the intracellular calcium ion shift and membrane depolarization upon stimulation as was demonstrated by the calcium indicator and the voltage-sensitive dye, respectively.

  19. Geldanamycin-inspired compounds induce direct trans-differentiation of human mesenchymal stem cells to neurons.

    PubMed

    Jogula, Srinivas; Soorneedi, Anand Ram; Gaddam, Jagan; Chamakuri, Srinivas; Deora, Girdhar Singh; Indarapu, Ranjith Kumar; Ramgopal, Murali Krishna; Dravida, Subhadra; Arya, Prabhat

    2017-07-28

    Inspired from geldanamycin, the synthesis of a new series of 20-membered macrocyclic compounds is developed. The key features in our design are (i) retention of the fragment having the precise chiral functional groups of geldanamycin at C10, C11, C12 and C14, and (ii) replacement of an olefin moiety with the ester group, and the quinoid sub-structure with the triazole ring. The southern fragment needed for the macrocyclic ring formation was obtained from Evans' syn aldol as the key reaction and with the use of D-mannitol as the cheap source of a chiral starting material. For the synthesis of the northern fragment, we utilized l-ascorbic acid, which provided the desired chiral functional groups at C6 and C7. Further, the chain extension completed the synthesis of the northern fragment. In our approach, the crucial 20 membered macrocyclic ring was formed employing the click chemistry. When tested for their ability to directly trans-differentiate human mesenchymal stem cells to neurons, two novel compounds (20a and 7) from this series were identified and this was further validated by the presence of specific neuronal biomarkers (i.e. nestin, agrin and RTN4). Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  20. Why are enteric ganglia so small? Role of differential adhesion of enteric neurons and enteric neural crest cells.

    PubMed Central

    Rollo, Benjamin N.; Zhang, Dongcheng; Simkin, Johanna E.; Menheniott, Trevelyan R.; Newgreen, Donald F.

    2015-01-01

    The avian enteric nervous system (ENS) consists of a vast number of unusually small ganglia compared to other peripheral ganglia. Each ENS ganglion at mid-gestation has a core of neurons and a shell of mesenchymal precursor/glia-like enteric neural crest (ENC) cells. To study ENS cell ganglionation we isolated midgut ENS cells by HNK-1 fluorescence-activated cell sorting (FACS) from E5 and E8 quail embryos, and from E9 chick embryos. We performed cell-cell aggregation assays which revealed a developmentally regulated functional increase in ENS cell adhesive function, requiring both Ca 2+ -dependent and independent adhesion. This was consistent with N-cadherin and NCAM labelling. Neurons sorted to the core of aggregates, surrounded by outer ENC cells, showing that neurons had higher adhesion than ENC cells. The outer surface of aggregates became relatively non-adhesive, correlating with low levels of NCAM and N-cadherin on this surface of the outer non-neuronal ENC cells. Aggregation assays showed that ENS cells FACS selected for NCAM-high and enriched for enteric neurons formed larger and more coherent aggregates than unsorted ENS cells. In contrast, ENS cells of the NCAM-low FACS fraction formed small, disorganised aggregates.  This suggests a novel mechanism for control of ENS ganglion morphogenesis where i) differential adhesion of ENS neurons and ENC cells controls the core/shell ganglionic structure and ii) the ratio of neurons to ENC cells dictates the equilibrium ganglion size by generation of an outer non-adhesive surface. PMID:26064478

  1. Amniotic fluid derived stem cells give rise to neuron-like cells without a further differentiation potential into retina-like cells.

    PubMed

    Hartmann, K; Raabe, O; Wenisch, S; Arnhold, S

    2013-01-01

    Amniotic fluid contains heterogeneous cell types and has become an interesting source for obtaining fetal stem cells. These stem cells have a high proliferative capacity and a good differentiation potential and may thus be suitable for regenerative medicine. As there is increasing evidence, that these stem cells are also able to be directed into the neural lineage, in our study we investigated the neuronal and glial differentiation potential of these cells, so that they may also be applied to cure degenerative diseases of the retina. Mesenchymal stem cells were isolated from routine prenatal amniocentesis at 15 to 18 weeks of pregnancy of human amniotic fluid and expanded in the cell culture. Cells were cultivated according to standard procedures for mesenchymal stem cells and were differentiated along the neural lineage using various protocols. Furthermore, it was also tried to direct them into cell types of the retina as well as into endothelial cells. Cells of more than 72 amniotic fluid samples were collected and characterized. While after induction neural-like phenotypes could actually be detected, which was confirmed using neural marker proteins such as GFAP and ßIII tubulina further differentiation into retinal like cells could not reliably be shown. These data suggest that amniotic fluid derived cells are an interesting cell source, which may also give rise to neural-like cells. However, a more specific differentiation into neuronal and glial cells could not unequivocally be shown, so that further investigations have to becarried out.

  2. Reverse engineering a mouse embryonic stem cell-specific transcriptional network reveals a new modulator of neuronal differentiation

    PubMed Central

    De Cegli, Rossella; Iacobacci, Simona; Flore, Gemma; Gambardella, Gennaro; Mao, Lei; Cutillo, Luisa; Lauria, Mario; Klose, Joachim; Illingworth, Elizabeth; Banfi, Sandro; di Bernardo, Diego

    2013-01-01

    Gene expression profiles can be used to infer previously unknown transcriptional regulatory interaction among thousands of genes, via systems biology ‘reverse engineering’ approaches. We ‘reverse engineered’ an embryonic stem (ES)-specific transcriptional network from 171 gene expression profiles, measured in ES cells, to identify master regulators of gene expression (‘hubs’). We discovered that E130012A19Rik (E13), highly expressed in mouse ES cells as compared with differentiated cells, was a central ‘hub’ of the network. We demonstrated that E13 is a protein-coding gene implicated in regulating the commitment towards the different neuronal subtypes and glia cells. The overexpression and knock-down of E13 in ES cell lines, undergoing differentiation into neurons and glia cells, caused a strong up-regulation of the glutamatergic neurons marker Vglut2 and a strong down-regulation of the GABAergic neurons marker GAD65 and of the radial glia marker Blbp. We confirmed E13 expression in the cerebral cortex of adult mice and during development. By immuno-based affinity purification, we characterized protein partners of E13, involved in the Polycomb complex. Our results suggest a role of E13 in regulating the division between glutamatergic projection neurons and GABAergic interneurons and glia cells possibly by epigenetic-mediated transcriptional regulation. PMID:23180766

  3. Reverse engineering a mouse embryonic stem cell-specific transcriptional network reveals a new modulator of neuronal differentiation.

    PubMed

    De Cegli, Rossella; Iacobacci, Simona; Flore, Gemma; Gambardella, Gennaro; Mao, Lei; Cutillo, Luisa; Lauria, Mario; Klose, Joachim; Illingworth, Elizabeth; Banfi, Sandro; di Bernardo, Diego

    2013-01-01

    Gene expression profiles can be used to infer previously unknown transcriptional regulatory interaction among thousands of genes, via systems biology 'reverse engineering' approaches. We 'reverse engineered' an embryonic stem (ES)-specific transcriptional network from 171 gene expression profiles, measured in ES cells, to identify master regulators of gene expression ('hubs'). We discovered that E130012A19Rik (E13), highly expressed in mouse ES cells as compared with differentiated cells, was a central 'hub' of the network. We demonstrated that E13 is a protein-coding gene implicated in regulating the commitment towards the different neuronal subtypes and glia cells. The overexpression and knock-down of E13 in ES cell lines, undergoing differentiation into neurons and glia cells, caused a strong up-regulation of the glutamatergic neurons marker Vglut2 and a strong down-regulation of the GABAergic neurons marker GAD65 and of the radial glia marker Blbp. We confirmed E13 expression in the cerebral cortex of adult mice and during development. By immuno-based affinity purification, we characterized protein partners of E13, involved in the Polycomb complex. Our results suggest a role of E13 in regulating the division between glutamatergic projection neurons and GABAergic interneurons and glia cells possibly by epigenetic-mediated transcriptional regulation.

  4. Markers of Pluripotency in Human Amniotic Epithelial Cells and Their Differentiation to Progenitor of Cortical Neurons

    PubMed Central

    García-Castro, Irma Lydia; García-López, Guadalupe; Ávila-González, Daniela; Flores-Herrera, Héctor; Molina-Hernández, Anayansi; Portillo, Wendy; Ramón-Gallegos, Eva; Díaz, Néstor Fabián

    2015-01-01

    Human pluripotent stem cells (hPSC) have promise for regenerative medicine due to their auto-renovation and differentiation capacities. Nevertheless, there are several ethical and methodological issues about these cells that have not been resolved. Human amniotic epithelial cells (hAEC) have been proposed as source of pluripotent stem cells. Several groups have studied hAEC but have reported inconsistencies about their pluripotency properties. The aim of the present study was the in vitro characterization of hAEC collected from a Mexican population in order to identify transcription factors involved in the pluripotency circuitry and to determine their epigenetic state. Finally, we evaluated if these cells differentiate to cortical progenitors. We analyzed qualitatively and quantitatively the expression of the transcription factors of pluripotency (OCT4, SOX2, NANOG, KLF4 and REX1) by RT-PCR and RT-qPCR in hAEC. Also, we determined the presence of OCT4, SOX2, NANOG, SSEA3, SSEA4, TRA-1-60, E-cadherin, KLF4, TFE3 as well as the proliferation and epigenetic state by immunocytochemistry of the cells. Finally, hAEC were differentiated towards cortical progenitors using a protocol of two stages. Here we show that hAEC, obtained from a Mexican population and cultured in vitro (P0-P3), maintained the expression of several markers strongly involved in pluripotency maintenance (OCT4, SOX2, NANOG, TFE3, KLF4, SSEA3, SSEA4, TRA-1-60 and E-cadherin). Finally, when hAEC were treated with growth factors and small molecules, they expressed markers characteristic of cortical progenitors (TBR2, OTX2, NeuN and β-III-tubulin). Our results demonstrated that hAEC express naïve pluripotent markers (KLF4, REX1 and TFE3) as well as the cortical neuron phenotype after differentiation. This highlights the need for further investigation of hAEC as a possible source of hPSC. PMID:26720151

  5. Effect of the Environmental Pollutant Hexachlorobenzene (HCB) on the Neuronal Differentiation of Mouse Embryonic Stem Cells

    PubMed Central

    Addae, Cynthia; Cheng, Henrique; Martinez-Ceballos, Eduardo

    2013-01-01

    Exposure to persistent environmental pollutants may constitute an important factor on the onset of a number of neurological disorders such as autism, Parkinson’s disease, and Attention Deficit Disorder (ADD), which have also been linked to reduced GABAergic neuronal function. GABAergic neurons produce γ-aminobutyric acid (GABA), which is the main inhibitory neurotransmitter in the brain. However, the lack of appropriate models has hindered the study of suspected environmental pollutants on GABAergic function. In this work, we have examined the effect of hexachlorobenzene (HCB), a persistent and bioaccumulative environmental pollutant, on the function and morphology of GABAergic neurons generated in vitro from mouse embryonic stem (ES) cells. We observed that: (1) treatment with 0.5 nM HCB did not affect cell viability, but affected the neuronal differentiation of ES cells; (2) HCB induced the production of reactive oxygen species (ROS); and (3) HCB repressed neurite outgrowth in GABAergic neurons, but this effect was reversed by the ROS scavenger N-acetylcysteine (NAC). Our study also revealed that HCB did not significantly interfere with the function of K+ ion channels in the neuronal soma, which indicates that this pollutant does not affect the maturation of the GABAergic neuronal soma. Our results suggest a mechanism by which environmental pollutants interfere with normal GABAergic neuronal function and may promote the onset of a number of neurological disorders such as autism and ADD. PMID:24157519

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

    PubMed

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

    2014-06-15

    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 stem cells and neurons. miR-124 expression was substantially reduced in bone marrow-derived mesenchymal stem cells compared with the other cell types. We constructed a lentiviral vector overexpressing miR-124 and transfected it into bone marrow-derived mesenchymal stem cells. Intracellular expression levels of the neuronal early markers β-III tubulin and microtubule-associated protein-2 were significantly 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 results suggest that miR-124 plays an important role in the differentiation of bone marrow-derived mesenchymal stem cells into neurons. Our findings should facilitate the development of novel strategies for enhancing the therapeutic efficacy of bone marrow-derived mesenchymal stem cell transplantation for spinal cord injury.

  7. Large-scale generation of human iPSC-derived neural stem cells/early neural progenitor cells and their neuronal differentiation

    PubMed Central

    D’Aiuto, Leonardo; Zhi, Yun; Kumar Das, Dhanjit; Wilcox, Madeleine R; Johnson, Jon W; McClain, Lora; MacDonald, Matthew L; Di Maio, Roberto; Schurdak, Mark E; Piazza, Paolo; Viggiano, Luigi; Sweet, Robert; Kinchington, Paul R; Bhattacharjee, Ayantika G; Yolken, Robert; Nimgaonka, Vishwajit L

    2014-01-01

    Induced pluripotent stem cell (iPSC)-based technologies offer an unprecedented opportunity to perform high-throughput screening of novel drugs for neurological and neurodegenerative diseases. Such screenings require a robust and scalable method for generating large numbers of mature, differentiated neuronal cells. Currently available methods based on differentiation of embryoid bodies (EBs) or directed differentiation of adherent culture systems are either expensive or are not scalable. We developed a protocol for large-scale generation of neuronal stem cells (NSCs)/early neural progenitor cells (eNPCs) and their differentiation into neurons. Our scalable protocol allows robust and cost-effective generation of NSCs/eNPCs from iPSCs. Following culture in neurobasal medium supplemented with B27 and BDNF, NSCs/eNPCs differentiate predominantly into vesicular glutamate transporter 1 (VGLUT1) positive neurons. Targeted mass spectrometry analysis demonstrates that iPSC-derived neurons express ligand-gated channels and other synaptic proteins and whole-cell patch-clamp experiments indicate that these channels are functional. The robust and cost-effective differentiation protocol described here for large-scale generation of NSCs/eNPCs and their differentiation into neurons paves the way for automated high-throughput screening of drugs for neurological and neurodegenerative diseases. PMID:25629202

  8. Differentiation of Wharton's Jelly-Derived Mesenchymal Stem Cells into Motor Neuron-Like Cells on Three-Dimensional Collagen-Grafted Nanofibers.

    PubMed

    Bagher, Zohreh; Azami, Mahmoud; Ebrahimi-Barough, Somayeh; Mirzadeh, Hamid; Solouk, Atefeh; Soleimani, Mansooreh; Ai, Jafar; Nourani, Mohammad Reza; Joghataei, Mohammad Taghi

    2016-05-01

    Cell transplantation strategies have provided potential therapeutic approaches for treatment of neurodegenerative diseases. Mesenchymal stem cells from Wharton's jelly (WJMSCs) are abundant and available adult stem cells with low immunological incompatibility, which could be considered for cell replacement therapy in the future. However, MSC transplantation without any induction or support material causes poor control of cell viability and differentiation. In this study, we investigated the effect of the nanoscaffolds on WJMSCs differentiation into motor neuronal lineages in the presence of retinoic acid (RA) and sonic hedgehog (Shh). Surface properties of scaffolds have been shown to significantly influence cell behaviors such as adhesion, proliferation, and differentiation. Therefore, polycaprolactone (PCL) nanofibers were constructed via electrospinning, surface modified by plasma treatment, and grafted by collagen. Characterization of the scaffolds by means of ATR-FTIR, contact angel, and Bradford proved grafting of the collagen on the surface of the scaffolds. WJMSCs were seeded on nanofibrous and tissue culture plate (TCP) and viability of WJMSCs were measured by MTT assay and then induced to differentiate into motor neuron-like cells for 15 days. Differentiated cells were evaluated morphologically, and real-time PCR and immunocytochemistry methods were done to evaluate expression of motor neuron-like cell markers in mRNA and protein levels. Our results showed that obtained cells could express motor neuron biomarkers at both RNA and protein levels, but the survival and differentiation of WJMSCs into motor neuron-like cells on the PCL/collagen scaffold were higher than cultured cells in the TCP and PCL groups. Taken together, WJMSCs are an attractive stem cell source for inducing into motor neurons in vitro especially when grown on nanostructural scaffolds and PCL/collagen scaffolds can provide a suitable, three-dimensional situation for neuronal survival and

  9. Lingo-1 shRNA and Notch signaling inhibitor DAPT promote differentiation of neural stem/progenitor cells into neurons.

    PubMed

    Wang, Jue; Ye, Zhizhong; Zheng, Shuhui; Chen, Luming; Wan, Yong; Deng, Yubin; Yang, Ruirui

    2016-03-01

    Determination of the exogenous factors that regulate differentiation of neural stem/progenitor cells into neurons, oligodendrocytes and astrocytes is an important step in the clinical therapy of spinal cord injury (SCI). The Notch pathway inhibits the differentiation of neural stem/progenitor cells and Lingo-1 is a strong negative regulator for myelination and axon growth. While Lingo-1 shRNA and N-[N-(3, 5-difluorophenacetyl)-1-alanyl]-S-Phenylglycinet-butylester (DAPT), a Notch pathway inhibitor, have been used separately to help repair SCI, the results have been unsatisfactory. Here we investigated and elucidated the preliminary mechanism for the effect of Lingo-1 shRNA and DAPT on neural stem/progenitor cells differentiation. We found that neural stem/progenitor cells from E14 rat embryos expressed Nestin, Sox-2 and Lingo-1, and we optimized the transduction of neural stem/progenitor cells using lentiviral vectors encoding Lingo-1 shRNA. The addition of DAPT decreased the expression of Notch intracellular domain (NICD) as well as the downstream genes Hes1 and Hes5. Expression of NeuN, CNPase and GFAP in DAPT treated cells and expression of NeuN in Lingo-1 shRNA treated cells confirmed differentiation of neural stem/progenitor cells into neurons, oligodendrocytes and astrocytes. These results revealed that while Lingo-1 shRNA and Notch signaling inhibitor DAPT both promoted differentiation of neural stem cells into neurons, only DAPT was capable of driving neural stem/progenitor cells differentiation into oligodendrocytes and astrocytes. Since we were able to show that both Lingo-1 shRNA and DAPT could drive neural stem/progenitor cells differentiation, our data might aid the development of more effective SCI therapies using Lingo-1 shRNA and DAPT.

  10. TRF2 dysfunction elicits DNA damage responses associated with senescence in proliferating neural cells and differentiation of neurons.

    PubMed

    Zhang, Peisu; Furukawa, Katsutoshi; Opresko, Patricia L; Xu, Xiangru; Bohr, Vilhelm A; Mattson, Mark P

    2006-04-01

    Telomeres are specialized structures at the ends of chromosomes that consist of tandem repeats of the DNA sequence TTAGGG and several proteins that protect the DNA and regulate the plasticity of the telomeres. The telomere-associated protein TRF2 (telomeric repeat binding factor 2) is critical for the control of telomere structure and function; TRF2 dysfunction results in the exposure of the telomere ends and activation of ATM (ataxia telangiectasin mutated)-mediated DNA damage response. Recent findings suggest that telomere attrition can cause senescence or apoptosis of mitotic cells, but the function of telomeres in differentiated neurons is unknown. Here, we examined the impact of telomere dysfunction via TRF2 inhibition in neurons (primary embryonic hippocampal neurons) and mitotic neural cells (astrocytes and neuroblastoma cells). We demonstrate that telomere dysfunction induced by adenovirus-mediated expression of dominant-negative TRF2 (DN-TRF2) triggers a DNA damage response involving the formation of nuclear foci containing phosphorylated histone H2AX and activated ATM in each cell type. In mitotic neural cells DN-TRF2 induced activation of both p53 and p21 and senescence (as indicated by an up-regulation of beta-galactosidase). In contrast, in neurons DN-TRF2 increased p21, but neither p53 nor beta-galactosidase was induced. In addition, TRF2 inhibition enhanced the morphological, molecular and biophysical differentiation of hippocampal neurons. These findings demonstrate divergent molecular and physiological responses to telomere dysfunction in mitotic neural cells and neurons, indicate a role for TRF2 in regulating neuronal differentiation, and suggest a potential therapeutic application of inhibition of TRF2 function in the treatment of neural tumors.

  11. Possible promotion of neuronal differentiation in fetal rat brain neural progenitor cells after sustained exposure to static magnetism.

    PubMed

    Nakamichi, Noritaka; Ishioka, Yukichi; Hirai, Takao; Ozawa, Shusuke; Tachibana, Masaki; Nakamura, Nobuhiro; Takarada, Takeshi; Yoneda, Yukio

    2009-08-15

    We have previously shown significant potentiation of Ca(2+) influx mediated by N-methyl-D-aspartate receptors, along with decreased microtubules-associated protein-2 (MAP2) expression, in hippocampal neurons cultured under static magnetism without cell death. In this study, we investigated the effects of static magnetism on the functionality of neural progenitor cells endowed to proliferate for self-replication and differentiate into neuronal, astroglial, and oligodendroglial lineages. Neural progenitor cells were isolated from embryonic rat neocortex and hippocampus, followed by culture under static magnetism at 100 mT and subsequent determination of the number of cells immunoreactive for a marker protein of particular progeny lineages. Static magnetism not only significantly decreased proliferation of neural progenitor cells without affecting cell viability, but also promoted differentiation into cells immunoreactive for MAP2 with a concomitant decrease in that for an astroglial marker, irrespective of the presence of differentiation inducers. In neural progenitors cultured under static magnetism, a significant increase was seen in mRNA expression of several activator-type proneural genes, such as Mash1, Math1, and Math3, together with decreased mRNA expression of the repressor type Hes5. These results suggest that sustained static magnetism could suppress proliferation for self-renewal and facilitate differentiation into neurons through promoted expression of activator-type proneural genes by progenitor cells in fetal rat brain.

  12. Evaluation of motor neuron differentiation potential of human umbilical cord blood- derived mesenchymal stem cells, in vitro.

    PubMed

    Yousefi, Behnam; Sanooghi, Davood; Faghihi, Faezeh; Joghataei, Mohammad Taghi; Latifi, Nourahmad

    2017-04-01

    Many people suffer from spinal cord injuries annually. These deficits usually threaten the quality of life of patients. As a postpartum medically waste product, human Umbilical Cord Blood (UCB) is a rich source of stem cells with self- renewal properties and neural differentiation capacity which made it useful in regenerative medicine. Since there is no report on potential of human umbilical cord blood-derived mesenchymal stem cells into motor neurons, we set out to evaluate the differentiation properties of these cells into motor neuron-like cells through administration of Retinoic Acid(RA), Sonic Hedgehog(Shh) and BDNF using a three- step in vitro procedure. The results were evaluated using Real-time PCR, Flowcytometry and Immunocytochemistry for two weeks. Our data showed that the cells changed into bipolar morphology and could express markers related to motor neuron; including Hb-9, Pax-6, Islet-1, NF-H, ChAT at the level of mRNA and protein. We could also quantitatively evaluate the expression of Islet-1, ChAT and NF-H at 7 and 14days post- induction using flowcytometry. It is concluded that human UCB-MSCs is potent to express motor neuron- related markers in the presence of RA, Shh and BDNF through a three- step protocol; thus it could be a suitable cell candidate for regeneration of motor neurons in spinal cord injuries. Copyright © 2017. Published by Elsevier B.V.

  13. Modulation of the Mesenchymal Stem Cell Secretome Using Computer-Controlled Bioreactors: Impact on Neuronal Cell Proliferation, Survival and Differentiation.

    PubMed

    Teixeira, Fábio G; Panchalingam, Krishna M; Assunção-Silva, Rita; Serra, Sofia C; Mendes-Pinheiro, Bárbara; Patrício, Patrícia; Jung, Sunghoon; Anjo, Sandra I; Manadas, Bruno; Pinto, Luísa; Sousa, Nuno; Behie, Leo A; Salgado, António J

    2016-06-15

    In recent years it has been shown that the therapeutic benefits of human mesenchymal stem/stromal cells (hMSCs) in the Central Nervous System (CNS) are mainly attributed to their secretome. The implementation of computer-controlled suspension bioreactors has shown to be a viable route for the expansion of these cells to large numbers. As hMSCs actively respond to their culture environment, there is the hypothesis that one can modulate its secretome through their use. Herein, we present data indicating that the use of computer-controlled suspension bioreactors enhanced the neuroregulatory profile of hMSCs secretome. Indeed, higher levels of in vitro neuronal differentiation and NOTCH1 expression in human neural progenitor cells (hNPCs) were observed when these cells were incubated with the secretome of dynamically cultured hMSCs. A similar trend was also observed in the hippocampal dentate gyrus (DG) of rat brains where, upon injection, an enhanced neuronal and astrocytic survival and differentiation, was observed. Proteomic analysis also revealed that the dynamic culturing of hMSCs increased the secretion of several neuroregulatory molecules and miRNAs present in hMSCs secretome. In summary, the appropriate use of dynamic culture conditions can represent an important asset for the development of future neuro-regenerative strategies involving the use of hMSCs secretome.

  14. Modulation of the Mesenchymal Stem Cell Secretome Using Computer-Controlled Bioreactors: Impact on Neuronal Cell Proliferation, Survival and Differentiation

    PubMed Central

    Teixeira, Fábio G.; Panchalingam, Krishna M.; Assunção-Silva, Rita; Serra, Sofia C.; Mendes-Pinheiro, Bárbara; Patrício, Patrícia; Jung, Sunghoon; Anjo, Sandra I.; Manadas, Bruno; Pinto, Luísa; Sousa, Nuno; Behie, Leo A.; Salgado, António J.

    2016-01-01

    In recent years it has been shown that the therapeutic benefits of human mesenchymal stem/stromal cells (hMSCs) in the Central Nervous System (CNS) are mainly attributed to their secretome. The implementation of computer-controlled suspension bioreactors has shown to be a viable route for the expansion of these cells to large numbers. As hMSCs actively respond to their culture environment, there is the hypothesis that one can modulate its secretome through their use. Herein, we present data indicating that the use of computer-controlled suspension bioreactors enhanced the neuroregulatory profile of hMSCs secretome. Indeed, higher levels of in vitro neuronal differentiation and NOTCH1 expression in human neural progenitor cells (hNPCs) were observed when these cells were incubated with the secretome of dynamically cultured hMSCs. A similar trend was also observed in the hippocampal dentate gyrus (DG) of rat brains where, upon injection, an enhanced neuronal and astrocytic survival and differentiation, was observed. Proteomic analysis also revealed that the dynamic culturing of hMSCs increased the secretion of several neuroregulatory molecules and miRNAs present in hMSCs secretome. In summary, the appropriate use of dynamic culture conditions can represent an important asset for the development of future neuro-regenerative strategies involving the use of hMSCs secretome. PMID:27301770

  15. SVCT2 Is Expressed by Cerebellar Precursor Cells, Which Differentiate into Neurons in Response to Ascorbic Acid.

    PubMed

    Oyarce, Karina; Silva-Alvarez, Carmen; Ferrada, Luciano; Martínez, Fernando; Salazar, Katterine; Nualart, Francisco

    2017-01-17

    Ascorbic acid (AA) is a known antioxidant that participates in a wide range of processes, including stem cell differentiation. It enters the cell through the sodium-ascorbate co-transporter SVCT2, which is mainly expressed by neurons in the adult brain. Here, we have characterized SVCT2 expression in the postnatal cerebellum in situ, a model used for studying neurogenesis, and have identified its expression in granular precursor cells and mature neurons. We have also detected SVCT2 expression in the cerebellar cell line C17.2 and in postnatal cerebellum-derived neurospheres in vitro and have identified a tight relationship between SVCT2 expression and that of the stem cell-like marker nestin. AA supplementation potentiates the neuronal phenotype in cerebellar neural stem cells by increasing the expression of the neuronal marker β III tubulin. Stable over-expression of SVCT2 in C17.2 cells enhances β III tubulin expression, but it also increases cell death, suggesting that AA transporter levels must be finely tuned during neural stem cell differentiation.

  16. Microcarrier-Expanded Neural Progenitor Cells Can Survive, Differentiate, and Innervate Host Neurons Better When Transplanted as Aggregates.

    PubMed

    Qiu, Lifeng; Lim, Yu Ming; Chen, Allen K; Reuveny, Shaul; Oh, Steve K W; Tan, Eng King; Zeng, Li

    2016-01-01

    Neuronal progenitor cells (NPCs) derived from human embryonic stem cells (hESCs) are an excellent cell source for transplantation therapy due to their availability and ethical acceptability. However, the traditional method of expansion and differentiation of hESCs into NPCs in monolayer cultures requires a long time, and the cell yield is low. A microcarrier (MC) platform can improve the expansion of hESCs and increase the yield of NPCs. In this study, for the first time, we transplanted microcarrier-expanded hESC-derived NPCs into the striatum of adult NOD-SCID IL2Rgc null mice, either as single cells or as cell aggregates. The recipient mice were perfused, and the in vivo survival, differentiation, and targeted innervation of the transplanted cells were assessed by immunostaining. We found that both the transplanted single NPCs and aggregate NPCs were able to survive 1 month posttransplantation, as revealed by human-specific neural cell adhesion molecule (NCAM) and human nuclear antigen staining. Compared to the single cells, the transplanted cell aggregates showed better survival over a 3-month period. In addition, both the transplanted single NPCs and the aggregate NPCs were able to differentiate into DCX-positive immature neurons and Tuj1-positive neurons in vivo by 1 month posttransplantation. However, only the transplantation of aggregate NPCs was shown to result in mature neurons at 3 months posttransplantation. Furthermore, we found that the cell aggregates were able to send long axons to innervate their targets. Our study provides preclinical evidence that the use of MCs to expand and differentiate hESC-derived NPCs and transplantation of these cells as aggregates produce longer survival in vivo.

  17. Fifty-Hertz electromagnetic fields facilitate the induction of rat bone mesenchymal stromal cells to differentiate into functional neurons.

    PubMed

    Bai, Wen-Fang; Xu, Wei-Cheng; Feng, Yu; Huang, Hong; Li, Xin-Ping; Deng, Chun-Yu; Zhang, Ming-Sheng

    2013-08-01

    Research results have shown that bone mesenchymal stromal cells (BMSC) can different into neural cells. Electromagnetic fields (EMF) play a role in regulating cell proliferation and differentiation, but the mechanisms behind this are unknown. In the present study, we explored the efficacy of EMF on the induction of rat BMSC differentiation into neurons in vitro. First, rat BMSC were induced in a nerve cell culture environment and divided into three groups: an EMF induction treatment group (frequency of 50 Hz, magnetic induction of 5 mT, 60 min per day for 12 days), an induction-only group and a control group. Second, we observed cell phenotypes in a confocal microscope, tested gene expression through the use of reverse transcriptase-polymerase chain reaction, and detected postsynaptic currents by means of a cell patch-clamp. We analyzed the cell cycles and the portion of cells expressing neural cell markers with the use of flow cytometry. The results indicated that EMF can facilitate BMSC differentiation into neural cells, which expressed neuronal-specific markers and genes; they formed synaptic junctions and pulsed excitatory postsynaptic currents. At the same time, the G0-G1 phase ratio recorded by means of flow cytometry gradually decreased under the EMF treatment, whereas there was an increase of S-phase ratio, and the portion of cells expressing neuronal-specific markers increased. Given that a noninvasive treatment of 50-Hz EMF could significantly facilitate BMSC to differentiate into functional neurons, EMF appears to be a promising clinical option for stem cell transplantation therapies to combat central nervous system diseases. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  18. Limited Ca2+ and PKA-pathway dependent neurogenic differentiation of human adult mesenchymal stem cells as compared to fetal neuronal stem cells.

    PubMed

    Lepski, Guilherme; Jannes, Cinthia Elim; Maciaczyk, Jaroslaw; Papazoglou, Anna; Mehlhorn, Alexander T; Kaiser, Stefan; Teixeira, Manoel Jacobsen; Marie, Suely K N; Bischofberger, Josef; Nikkhah, Guido

    2010-01-15

    The ability of mesenchymal stem cells to generate functional neurons in culture is still a matter of controversy. In order to assess this issue, we performed a functional comparison between neuronal differentiation of human MSCs and fetal-derived neural stem cells (NSCs) based on morphological, immunocytochemical, and electrophysiological criteria. Furthermore, possible biochemical mechanisms involved in this process were presented. NF200 immunostaining was used to quantify the yield of differentiated cells after exposure to cAMP. The addition of a PKA inhibitor and Ca(2+) blockers to the differentiation medium significantly reduced the yield of differentiated cells. Activation of CREB was also observed on MSCs during maturation. Na(+)-, K(+)-, and Ca(2+)-voltage-dependent currents were recorded from MSCs-derived cells. In contrast, significantly larger Na(+) currents, firing activity, and spontaneous synaptic currents were recorded from NSCs. Our results indicate that the initial neuronal differentiation of MSCs is induced by cAMP and seems to be dependent upon Ca(2+) and the PKA pathway. However, compared to fetal neural stem cells, adult mesenchymal counterparts are limited in their neurogenic potential. Despite the similar yield of neuronal cells, NSCs achieved a more mature functional state. Description of the underlying mechanisms that govern MSCs' differentiation toward a stable neuronal phenotype and their limitations provides a unique opportunity to enhance our understanding of stem cell plasticity.

  19. Tumour necrosis factor-alpha impairs neuronal differentiation but not proliferation of hippocampal neural precursor cells: Role of Hes1.

    PubMed

    Keohane, Aoife; Ryan, Sinead; Maloney, Eimer; Sullivan, Aideen M; Nolan, Yvonne M

    2010-01-01

    Tumour necrosis factor-alpha (TNFalpha) is a pro-inflammatory cytokine, which influences neuronal survival and function yet there is limited information available on its effects on hippocampal neural precursor cells (NPCs). We show that TNFalpha treatment during proliferation had no effect on the percentage of proliferating cells prepared from embryonic rat hippocampal neurosphere cultures, nor did it affect cell fate towards either an astrocytic or neuronal lineage when cells were then allowed to differentiate. However, when cells were differentiated in the presence of TNFalpha, significantly reduced percentages of newly born and post-mitotic neurons, significantly increased percentages of astrocytes and increased expression of TNFalpha receptors, TNF-R1 and TNF-R2, as well as expression of the anti-neurogenic Hes1 gene, were observed. These data indicate that exposure of hippocampal NPCs to TNFalpha when they are undergoing differentiation but not proliferation has a detrimental effect on their neuronal lineage fate, which may be mediated through increased expression of Hes1.

  20. Coculture of dorsal root ganglion neurons and differentiated human corneal stromal stem cells on silk-based scaffolds.

    PubMed

    Wang, Siran; Ghezzi, Chiara E; White, James D; Kaplan, David L

    2015-10-01

    Corneal tissue displays the highest peripheral nerve density in the human body. Engineering of biomaterials to promote interactions between neurons and corneal tissue could provide tissue models for nerve/cornea development, platforms for drug screening, as well as innovative opportunities to regenerate cornea tissue. The focus of this study was to develop a coculture system for differentiated human corneal stromal stem cells (dhCSSCs) and dorsal root ganglion neurons (DRG) to mimic the human cornea tissue interactions. Axon extension, connectivity, and neuron cell viability were studied. DRG neurons developed longer axons when cocultured with dhCSSCs in comparison to neuron cultures alone. To assess the mechanism involved in the coculture response, nerve growth factors (NGF) secreted by dhCSSCs including NGF, brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), and neurotrophin-3 were characterized with greater focus on BDNF secretion. DhCSSCs also secreted collagen type I, an extracellular matrix molecule favorable for neuronal outgrowth. This coculture system provides a slowly degrading silk matrix to study neuronal responses in concert with hCSSCs related to innervation of corneal tissue with utility toward human corneal nerve regeneration and associated diseases. © 2015 Wiley Periodicals, Inc.

  1. Surface-Functionalized Silk Fibroin Films as a Platform To Guide Neuron-like Differentiation of Human Mesenchymal Stem Cells.

    PubMed

    Manchineella, Shivaprasad; Thrivikraman, Greeshma; Basu, Bikramjit; Govindaraju, T

    2016-09-07

    Surface interactions at the biomaterial-cellular interface determine the proliferation and differentiation of stem cells. Manipulating such interactions through the surface chemistry of scaffolds renders control over directed stem cell differentiation into the cell lineage of interest. This approach is of central importance for stem cell-based tissue engineering and regenerative therapy applications. In the present study, silk fibroin films (SFFs) decorated with integrin-binding laminin peptide motifs (YIGSR and GYIGSR) were prepared and employed for in vitro adult stem cell-based neural tissue engineering applications. Functionalization of SFFs with short peptides showcased the peptide sequence and nature of functionalization-dependent differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs). Intriguingly, covalently functionalized SFFs with GYIGSR hexapeptide (CL2-SFF) supported hMSC proliferation and maintenance in an undifferentiated pluripotent state and directed the differentiation of hMSCs into neuron-like cells in the presence of a biochemical cue, on-demand. The observed morphological changes were further corroborated by the up-regulation of neuronal-specific marker gene expression (MAP2, TUBB3, NEFL), confirmed through semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis. The enhanced proliferation and on-demand directed differentiation of adult stem cells (hMSCs) by the use of an economically viable short recognition peptide (GYIGSR), as opposed to the integrin recognition protein laminin, establishes the potential of SFFs for neural tissue engineering and regenerative therapy applications.

  2. Integrated transcriptome analysis of human iPS cells derived from a fragile X syndrome patient during neuronal differentiation.

    PubMed

    Lu, Ping; Chen, Xiaolong; Feng, Yun; Zeng, Qiao; Jiang, Cizhong; Zhu, Xianmin; Fan, Guoping; Xue, Zhigang

    2016-11-01

    Fragile X syndrome (FXS) patients carry the expansion of over 200 CGG repeats at the promoter of fragile X mental retardation 1 (FMR1), leading to decreased or absent expression of its encoded fragile X mental retardation protein (FMRP). However, the global transcriptional alteration by FMRP deficiency has not been well characterized at single nucleotide resolution, i.e., RNA-seq. Here, we performed in-vitro neuronal differentiation of human induced pluripotent stem (iPS) cells that were derived from fibroblasts of a FXS patient (FXS-iPSC). We then performed RNA-seq and examined the transcriptional misregulation at each intermediate stage during in-vitro differentiation of FXS-iPSC into neurons. After thoroughly analyzing the transcriptomic data and integrating them with those from other platforms, we found up-regulation of many genes encoding TFs for neuronal differentiation (WNT1, BMP4, POU3F4, TFAP2C, and PAX3), down-regulation of potassium channels (KCNA1, KCNC3, KCNG2, KCNIP4, KCNJ3, KCNK9, and KCNT1) and altered temporal regulation of SHANK1 and NNAT in FXS-iPSC derived neurons, indicating impaired neuronal differentiation and function in FXS patients. In conclusion, we demonstrated that the FMRP deficiency in FXS patients has significant impact on the gene expression patterns during development, which will help to discover potential targeting candidates for the cure of FXS symptoms.

  3. The effect of magnetic nanoparticles on neuronal differentiation of induced pluripotent stem cell-derived neural precursors

    PubMed Central

    Jiráková, Klára; Šeneklová, Monika; Jirák, Daniel; Turnovcová, Karolína; Vosmanská, Magda; Babič, Michal; Horák, Daniel; Veverka, Pavel; Jendelová, Pavla

    2016-01-01

    Introduction Magnetic resonance (MR) imaging is suitable for noninvasive long-term tracking. We labeled human induced pluripotent stem cell-derived neural precursors (iPSC-NPs) with two types of iron-based nanoparticles, silica-coated cobalt zinc ferrite nanoparticles (CZF) and poly-l-lysine-coated iron oxide superparamagnetic nanoparticles (PLL-coated γ-Fe2O3) and studied their effect on proliferation and neuronal differentiation. Materials and methods We investigated the effect of these two contrast agents on neural precursor cell proliferation and differentiation capability. We further defined the intracellular localization and labeling efficiency and analyzed labeled cells by MR. Results Cell proliferation was not affected by PLL-coated γ-Fe2O3 but was slowed down in cells labeled with CZF. Labeling efficiency, iron content and relaxation rates measured by MR were lower in cells labeled with CZF when compared to PLL-coated γ-Fe2O3. Cytoplasmic localization of both types of nanoparticles was confirmed by transmission electron microscopy. Flow cytometry and immunocytochemical analysis of specific markers expressed during neuronal differentiation did not show any significant differences between unlabeled cells or cells labeled with both magnetic nanoparticles. Conclusion Our results show that cells labeled with PLL-coated γ-Fe2O3 are suitable for MR detection, did not affect the differentiation potential of iPSC-NPs and are suitable for in vivo cell therapies in experimental models of central nervous system disorders. PMID:27920532

  4. The effect of magnetic nanoparticles on neuronal differentiation of induced pluripotent stem cell-derived neural precursors.

    PubMed

    Jiráková, Klára; Šeneklová, Monika; Jirák, Daniel; Turnovcová, Karolína; Vosmanská, Magda; Babič, Michal; Horák, Daniel; Veverka, Pavel; Jendelová, Pavla

    Magnetic resonance (MR) imaging is suitable for noninvasive long-term tracking. We labeled human induced pluripotent stem cell-derived neural precursors (iPSC-NPs) with two types of iron-based nanoparticles, silica-coated cobalt zinc ferrite nanoparticles (CZF) and poly-l-lysine-coated iron oxide superparamagnetic nanoparticles (PLL-coated γ-Fe2O3) and studied their effect on proliferation and neuronal differentiation. We investigated the effect of these two contrast agents on neural precursor cell proliferation and differentiation capability. We further defined the intracellular localization and labeling efficiency and analyzed labeled cells by MR. Cell proliferation was not affected by PLL-coated γ-Fe2O3 but was slowed down in cells labeled with CZF. Labeling efficiency, iron content and relaxation rates measured by MR were lower in cells labeled with CZF when compared to PLL-coated γ-Fe2O3. Cytoplasmic localization of both types of nanoparticles was confirmed by transmission electron microscopy. Flow cytometry and immunocytochemical analysis of specific markers expressed during neuronal differentiation did not show any significant differences between unlabeled cells or cells labeled with both magnetic nanoparticles. Our results show that cells labeled with PLL-coated γ-Fe2O3 are suitable for MR detection, did not affect the differentiation potential of iPSC-NPs and are suitable for in vivo cell therapies in experimental models of central nervous system disorders.

  5. Developmentally coordinated extrinsic signals drive human pluripotent stem cell differentiation toward authentic DARPP-32+ medium-sized spiny neurons.

    PubMed

    Delli Carri, Alessia; Onorati, Marco; Lelos, Mariah J; Castiglioni, Valentina; Faedo, Andrea; Menon, Ramesh; Camnasio, Stefano; Vuono, Romina; Spaiardi, Paolo; Talpo, Francesca; Toselli, Mauro; Martino, Gianvito; Barker, Roger A; Dunnett, Stephen B; Biella, Gerardo; Cattaneo, Elena

    2013-01-15

    Medium-sized spiny neurons (MSNs) are the only neostriatum projection neurons, and their degeneration underlies some of the clinical features of Huntington's disease. Using knowledge of human developmental biology and exposure to key neurodevelopmental molecules, human pluripotent stem (hPS) cells were induced to differentiate into MSNs. In a feeder-free adherent culture, ventral telencephalic specification is induced by BMP/TGFβ inhibition and subsequent SHH/DKK1 treatment. The emerging FOXG1(+)/GSX2(+) telencephalic progenitors are then terminally differentiated, resulting in the systematic line-independent generation of FOXP1(+)/FOXP2(+)/CTIP2(+)/calbindin(+)/DARPP-32(+) MSNs. Similar to mature MSNs, these neurons carry dopamine and A2a receptors, elicit a typical firing pattern and show inhibitory postsynaptic currents, as well as dopamine neuromodulation and synaptic integration ability in vivo. When transplanted into the striatum of quinolinic acid-lesioned rats, hPS-derived neurons survive and differentiate into DARPP-32(+) neurons, leading to a restoration of apomorphine-induced rotation behavior. In summary, hPS cells can be efficiently driven to acquire a functional striatal fate using an ontogeny-recapitulating stepwise method that represents a platform for in vitro human developmental neurobiology studies and drug screening approaches.

  6. Nanofiber Matrices Promote the Neuronal Differentiation of Human Embryonic Stem Cell-Derived Neural Precursors In Vitro

    PubMed Central

    Lim, Shawn H.; Christopherson, Gregory T.; Xu, Leyan; Nasonkin, Igor; Yu, Christopher; Mao, Hai-Quan; Koliatsos, Vassilis E.

    2011-01-01

    The potential of human embryonic stem (ES) cells as experimental therapies for neuronal replacement has recently received considerable attention. In view of the organization of the mature nervous system into distinct neural circuits, key challenges of such therapies are the directed differentiation of human ES cell-derived neural precursors (NPs) into specific neuronal types and the directional growth of axons along specified trajectories. In the present study, we cultured human NPs derived from the NIH-approved ES line BGO1 on polycaprolactone fiber matrices of different diameter (i.e., nanofibers and microfibers) and orientation (i.e., aligned and random); fibers were coated with poly-L-ornithine/laminin to mimic the extracellular matrix and support the adhesion, viability, and differentiation of NPs. On aligned fibrous meshes, human NPs adopt polarized cell morphology with processes extending along the axis of the fiber, whereas NPs on plain tissue culture surfaces or random fiber substrates form nonpolarized neurite networks. Under differentiation conditions, human NPs cultured on aligned fibrous substrates show a higher rate of neuronal differentiation than other matrices; 62% and 86% of NPs become TUJ1 (+) early neurons on aligned micro- and nanofibers, respectively, whereas only 32% and 27% of NPs acquire the same fate on random micro- and nanofibers. Metabolic cell activity/viability studies reveal that fiber alignment and diameter also have an effect on NP viability, but only in the presence of mitogens. Our findings demonstrate that fibrous substrates serve as an artificial extracellular matrix and provide a microenviroment that influences key aspects of the neuronal differentiation of ES-derived NPs. PMID:20973749

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

    SciTech Connect

    Ivanov, Vladimir N.; Hei, Tom K.

    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.

  8. Nanomechanics controls neuronal precursors adhesion and differentiation.

    PubMed

    Migliorini, Elisa; Ban, Jelena; Grenci, Gianluca; Andolfi, Laura; Pozzato, Alessandro; Tormen, Massimo; Torre, Vincent; Lazzarino, Marco

    2013-08-01

    The ability to control the differentiation of stem cells into specific neuronal types has a tremendous potential for the treatment of neurodegenerative diseases. In vitro neuronal differentiation can be guided by the interplay of biochemical and biophysical cues. Different strategies to increase the differentiation yield have been proposed, focusing everything on substrate topography, or, alternatively on substrate stiffness. Both strategies demonstrated an improvement of the cellular response. However it was often impossible to separate the topographical and the mechanical contributions. Here we investigate the role of the mechanical properties of nanostructured substrates, aiming at understanding the ultimate parameters which govern the stem cell differentiation. To this purpose a set of different substrates with controlled stiffness and with or without nanopatterning are used for stem cell differentiation. Our results show that the neuronal differentiation yield depends mainly on the substrate mechanical properties while the geometry plays a minor role. In particular nanostructured and flat polydimethylsiloxane (PDMS) substrates with comparable stiffness show the same neuronal yield. The improvement in the differentiation yield obtained through surface nanopatterning in the submicrometer scale could be explained as a consequence of a substrate softening effect. Finally we investigate by single cell force spectroscopy the neuronal precursor adhesion on the substrate immediately after seeding, as a possible critical step governing the neuronal differentiation efficiency. We observed that neuronal precursor adhesion depends on substrate stiffness but not on surface structure, and in particular it is higher on softer substrates. Our results suggest that cell-substrate adhesion forces and mechanical response are the key parameters to be considered for substrate design in neuronal regenerative medicine.

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

    PubMed

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

    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

  10. Non-neuronal acetylcholine as an endogenous regulator of proliferation and differentiation of Lgr5-positive stem cells in mice.

    PubMed

    Takahashi, Toshio; Ohnishi, Hiroe; Sugiura, Yuki; Honda, Kurara; Suematsu, Makoto; Kawasaki, Takashi; Deguchi, Tomonori; Fujii, Takeshi; Orihashi, Kaoru; Hippo, Yoshitaka; Watanabe, Takehiro; Yamagaki, Tohru; Yuba, Shunsuke

    2014-10-01

    Non-neuronal acetylcholine (ACh) is predicted to function as a local cell signaling molecule. However, the physiological significance of the synthesis of non-neuronal ACh in the intestine remains unclear. Here, experiments using crypt-villus organoids that lack nerve and immune cells in culture led us to suggest that endogenous ACh is synthesized in the intestinal epithelium to evoke growth and differentiation of the organoids through activation of muscarinic ACh receptors (mAChRs). The extracts of the cultured organoids showed a noticeable capacity for ACh synthesis that was sensitive to a potent inhibitor of choline acetyltransferase. Imaging MS revealed endogenous ACh localized in the epithelial layer in mouse small intestinal epithelium in vivo, suggesting that there are non-neuronal resources of ACh. Treatment of organoids with carbachol downregulated the growth of organoids and the expression of marker genes for epithelial cells. On the other hand, antagonists for mAChRs enhanced the growth and differentiation of organoids, indicating the involvement of mAChRs in regulating the proliferation and differentiation of Lgr5-positive stem cells. Collectively, our data provide evidence that endogenous ACh released from intestinal epithelium maintains homeostasis of intestinal epithelial cell growth and differentiation via mAChRs in mice.

  11. Tumor necrosis factor-alpha modulates survival, proliferation, and neuronal differentiation in neonatal subventricular zone cell cultures.

    PubMed

    Bernardino, Liliana; Agasse, Fabienne; Silva, Bruno; Ferreira, Raquel; Grade, Sofia; Malva, João O

    2008-09-01

    Tumor necrosis factor (TNF)-alpha has been reported to modulate brain injury, but remarkably, little is known about its effects on neurogenesis. We report that TNF-alpha strongly influences survival, proliferation, and neuronal differentiation in cultured subventricular zone (SVZ) neural stem/progenitor cells derived from the neonatal P1-3 C57BL/6 mice. By using single-cell calcium imaging, we developed a method, based on cellular response to KCl and/or histamine, that allows the functional evaluation of neuronal differentiation. Exposure of SVZ cultures to 1 and 10 ng/ml mouse or 1 ng/ml human recombinant TNF-alpha resulted in increased differentiation of cells displaying a neuronal-like profile of [Ca2+](i) responses, compared with the predominant profile of immature cells observed in control, nontreated cultures. Moreover, by using neutralizing antibodies for each TNF-alpha receptor, we found that the proneurogenic effect of 1 ng/ml TNF-alpha is mediated via tumor necrosis factor receptor 1 activation. Accordingly, the percentage of neuronal nuclear protein-positive neurons was increased following exposure to mouse TNF-alpha. Interestingly, exposure of SVZ cultures to 1 ng/ml TNF-alpha induced cell proliferation, whereas 10 and 100 ng/ml TNF-alpha induced apoptotic cell death. Moreover, we found that exposure of SVZ cells to TNF-alpha for 15 minutes or 6 hours caused an increase in the phospho-stress-activated protein kinase/c-Jun N-terminal kinase immunoreactivity initially in the nucleus and then in growing axons, colocalizing with tau, consistent with axonogenesis. Taken together, these results show that TNF-alpha induces neurogenesis in neonatal SVZ cell cultures of mice. TNF-alpha, a proinflammatory cytokine and a proneurogenic factor, may play a central role in promoting neurogenesis and brain repair in response to brain injury and infection.

  12. Neuronal growth and differentiation on biodegradable membranes.

    PubMed

    Morelli, Sabrina; Piscioneri, Antonella; Messina, Antonietta; Salerno, Simona; Al-Fageeh, Mohamed B; Drioli, Enrico; De Bartolo, Loredana

    2015-02-01

    Semipermeable polymeric membranes with appropriate morphological, physicochemical and transport properties are relevant to inducing neural regeneration. We developed novel biodegradable membranes to support neuronal differentiation. In particular, we developed chitosan, polycaprolactone and polyurethane flat membranes and a biosynthetic blend between polycaprolactone and polyurethane by phase-inversion techniques. The biodegradable membranes were characterized in order to evaluate their morphological, physicochemical, mechanical and degradation properties. We investigated the efficacy of these different membranes to promote the adhesion and differentiation of neuronal cells. We employed as model cell system the human neuroblastoma cell line SHSY5Y, which is a well-established system for studying neuronal differentiation. The investigation of viability and specific neuronal marker expression allowed assessment that the correct neuronal differentiation and the formation of neuronal network had taken place in vitro in the cells seeded on different biodegradable membranes. Overall, this study provides evidence that neural cell responses depend on the nature of the biodegradable polymer used to form the membranes, as well as on the dissolution, hydrophilic and, above all, mechanical membrane properties. PCL-PU membranes exhibit mechanical properties that improve neurite outgrowth and the expression of specific neuronal markers.

  13. Leucine-Rich Repeat Kinase 2 Modulates Retinoic Acid-Induced Neuronal Differentiation of Murine Embryonic Stem Cells

    PubMed Central

    Schulz, Cathrin; Paus, Marie; Frey, Katharina; Schmid, Ramona; Kohl, Zacharias; Mennerich, Detlev; Winkler, Jürgen; Gillardon, Frank

    2011-01-01

    Background Dominant mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most prevalent cause of Parkinson's disease, however, little is known about the biological function of LRRK2 protein. LRRK2 is expressed in neural precursor cells suggesting a role in neurodevelopment. Methodology/Principal Findings In the present study, differential gene expression profiling revealed a faster silencing of pluripotency-associated genes, like Nanog, Oct4, and Lin28, during retinoic acid-induced neuronal differentiation of LRRK2-deficient mouse embryonic stem cells compared to wildtype cultures. By contrast, expression of neurotransmitter receptors and neurotransmitter release was increased in LRRK2+/− cultures indicating that LRRK2 promotes neuronal differentiation. Consistently, the number of neural progenitor cells was higher in the hippocampal dentate gyrus of adult LRRK2-deficient mice. Alterations in phosphorylation of the putative LRRK2 substrates, translation initiation factor 4E binding protein 1 and moesin, do not appear to be involved in altered differentiation, rather there is indirect evidence that a regulatory signaling network comprising retinoic acid receptors, let-7 miRNA and downstream target genes/mRNAs may be affected in LRRK2-deficient stem cells in culture. Conclusion/Significance Parkinson's disease-linked LRRK2 mutations that associated with enhanced kinase activity may affect retinoic acid receptor signaling during neurodevelopment and/or neuronal maintenance as has been shown in other mouse models of chronic neurodegenerative diseases. PMID:21695257

  14. Pharmacological inhibition of DNA methyltransferase 1 promotes neuronal differentiation from rodent and human nasal olfactory stem/progenitor cell cultures.

    PubMed

    Franco, I; Ortiz-López, L; Roque-Ramírez, B; Ramírez-Rodríguez, G B; Lamas, M

    2017-05-01

    Nasal olfactory stem and neural progenitor cells (NOS/PCs) are considered possible tools for regenerative stem cell therapies in neurodegenerative diseases. Neurogenesis is a complex process regulated by extrinsic and intrinsic signals that include DNA-methylation and other chromatin modifications that could be experimentally manipulated in order to increase neuronal differentiation. The aim of the present study was the characterization of primary cultures and consecutive passages (P2-P10) of NOS/PCs isolated from male Swiss-Webster (mNOS/PCs) or healthy humans (hNOS/PCs). We evaluated and compared cellular morphology, proliferation rates and the expression pattern of pluripotency-associated markers and DNA methylation-associated gene expression in these cultures. Neuronal differentiation was induced by exposure to all-trans retinoic acid and forskolin for 7 days and evaluated by morphological analysis and immunofluorescence against neuronal markers MAP2, NSE and MAP1B. In response to the inductive cues mNOS/PCs expressed NSE (75.67%) and MAP2 (35.34%); whereas the majority of the hNOS/PCs were immunopositive to MAP1B. Treatment with procainamide, a specific inhibitor of DNA methyltransferase 1 (DNMT1), increases in the number of forskolin'/retinoic acid-induced mature neuronal marker-expressing mNOS/PCs cells and enhances neurite development in hNOS/PCs. Our results indicate that mice and human nasal olfactory stem/progenitors cells share pluripotency-related gene expression suggesting that their application for stem cell therapy is worth pursuing and that DNA methylation inhibitors could be efficient tools to enhance neuronal differentiation from these cells.

  15. Assembly of polyelectrolyte multilayer films on supported lipid bilayers to induce neural stem/progenitor cell differentiation into functional neurons.

    PubMed

    Lee, I-Chi; Wu, Yu-Chieh

    2014-08-27

    The key factors affecting the success of neural engineering using neural stem/progenitor cells (NSPCs) are the neuron quantity, the guidance of neurite outgrowth, and the induction of neurons to form functional synapses at synaptic junctions. Herein, a biomimetic material comprising a supported lipid bilayer (SLB) with adsorbed sequential polyelectrolyte multilayer (PEM) films was fabricated to induce NSPCs to form functional neurons without the need for serum and growth factors in a short-term culture. SLBs are suitable artificial substrates for neural engineering due to their structural similarity to synaptic membranes. In addition, PEM film adsorption provides protection for the SLB as well as the ability to vary the surface properties to evaluate the effects of physical and mechanical signals on NSPC differentiation. Our results revealed that NSPCs were inducible on SLB-PEM films consisting of up to eight alternating layers. In addition, the process outgrowth length, the percentage of differentiated neurons, and the synaptic function were regulated by the number of layers and the surface charge of the outermost layer. The average process outgrowth length was greater than 500 μm on SLB-PLL/PLGA (n = 7.5) after only 3 days of culture. Moreover, the quantity and quality of the differentiated neurons were obviously enhanced on the SLB-PEM system compared with those on the PEM-only substrates. These results suggest that the PEM films can induce NSPC adhesion and differentiation and that an SLB base may enhance neuron differentiation and trigger the formation of functional synapses.

  16. Hippo/YAP-mediated rigidity-dependent motor neuron differentiation of human pluripotent stem cells

    NASA Astrophysics Data System (ADS)

    Sun, Yubing; Yong, Koh Meng Aw; Villa-Diaz, Luis G.; Zhang, Xiaoli; Chen, Weiqiang; Philson, Renee; Weng, Shinuo; Xu, Haoxing; Krebsbach, Paul H.; Fu, Jianping

    2014-06-01

    Our understanding of the intrinsic mechanosensitive properties of human pluripotent stem cells (hPSCs), in particular the effects that the physical microenvironment has on their differentiation, remains elusive. Here, we show that neural induction and caudalization of hPSCs can be accelerated by using a synthetic microengineered substrate system consisting of poly(dimethylsiloxane) micropost arrays (PMAs) with tunable mechanical rigidities. The purity and yield of functional motor neurons derived from hPSCs within 23 days of culture using soft PMAs were improved more than fourfold and tenfold, respectively, compared with coverslips or rigid PMAs. Mechanistic studies revealed a multi-targeted mechanotransductive process involving Smad phosphorylation and nucleocytoplasmic shuttling, regulated by rigidity-dependent Hippo/YAP activities and actomyosin cytoskeleton integrity and contractility. Our findings suggest that substrate rigidity is an important biophysical cue influencing neural induction and subtype specification, and that microengineered substrates can thus serve as a promising platform for large-scale culture of hPSCs.

  17. Role for Egr1 in the Transcriptional Program Associated with Neuronal Differentiation of PC12 Cells

    PubMed Central

    Kletsov, Sergey; Lamm, Ryan J.; Elman, Jessica S.; Mullenbrock, Steven; Cooper, Geoffrey M.

    2017-01-01

    PC12 cells are a well-established model to study how differences in signal transduction duration can elicit distinct cell behaviors. Epidermal growth factor (EGF) activates transient ERK signaling in PC12 cells that lasts 30–60 min, which in turn promotes proliferation; nerve growth factor (NGF) activates more sustained ERK signaling that lasts 4–6 h, which in turns induces neuronal differentiation. Data presented here extend a previous study by Mullenbrock et al. (2011) that demonstrated that sustained ERK signaling in response to NGF induces preferential expression of a 69-member gene set compared to transient ERK signaling in response to EGF and that the transcription factors AP-1 and CREB play a major role in the preferential expression of several genes within the set. Here, we examined whether the Egr family of transcription factors also contributes to the preferential expression of the gene set in response to NGF. Our data demonstrate that NGF causes transient induction of all Egr family member transcripts, but a corresponding induction of protein was detected for only Egr1 and 2. Chromatin immunoprecipitation experiments provided clearest evidence that, after induction, Egr1 binds 12 of the 69 genes that are preferentially expressed during sustained ERK signaling. In addition, Egr1 expression and binding upstream of its target genes were both sustained in response to NGF versus EGF within the same timeframe that its targets are preferentially expressed. These data thus provide evidence that Egr1 contributes to the transcriptional program activated by sustained ERK signaling in response to NGF, specifically by contributing to the preferential expression of its target genes identified here. PMID:28076410

  18. Expression pattern of neurotrophins and their receptors during neuronal differentiation of adipose-derived stem cells in simulated microgravity condition

    PubMed Central

    Zarrinpour, Vajiheh; Hajebrahimi, Zahra; Jafarinia, Mojtaba

    2017-01-01

    Objective(s): Studies have confirmed that microgravity, as a mechanical factor, influences both differentiation and function of mesenchymal stem cells. Here we investigated the effects of simulated microgravity on neural differentiation of human adipose-derived stem cells (ADSCs). Materials and Methods: We have used a fast rotating clinostat (clinorotation) to simulate microgravity condition. Real-time PCR and flow cytometry analysis were used to evaluate the regulation of neurotrophins, their receptors, and neural markers by simulated microgravity and their impact on neural differentiation of cells. Results: Our data revealed that simulation microgravity up-regulated the expression of MAP-2, BDNF, TrkB, NT-3, and TrkC both before and after neural differentiation. Also, the neural cells derived from ADSCs in microgravity condition expressed more MAP-2, GFAP, and synaptophysin protein in comparison to the 1G control. Conclusion: We showed that simulated microgravity can enhance the differentiation of mesenchymal stem cells into neurons. Our findings provide a new strategy for differentiation of ADSCs to neural-like cells and probably other cell lineages. Meanwhile, microgravity simulation had no adverse effects on the viability of the cells and could be used as a new environment to successfully manipulate cells. PMID:28293395

  19. Flash photo stimulation of human neural stem cells on graphene/TiO2 heterojunction for differentiation into neurons

    NASA Astrophysics Data System (ADS)

    Akhavan, Omid; Ghaderi, Elham

    2013-10-01

    For the application of human neural stem cells (hNSCs) in neural regeneration and brain repair, it is necessary to stimulate hNSC differentiation towards neurons rather than glia. Due to the unique properties of graphene in stem cell differentiation, here we introduce reduced graphene oxide (rGO)/TiO2 heterojunction film as a biocompatible flash photo stimulator for effective differentiation of hNSCs into neurons. Using the stimulation, the number of cell nuclei on rGO/TiO2 increased by a factor of ~1.5, while on GO/TiO2 and TiO2 it increased only ~48 and 24%, respectively. Moreover, under optimum conditions of flash photo stimulation (10 mW cm-2 flash intensity and 15.0 mM ascorbic acid in cell culture medium) not only did the number of cell nuclei and neurons differentiated on rGO/TiO2 significantly increase (by factors of ~2.5 and 3.6), but also the number of glial cells decreased (by a factor of ~0.28). This resulted in a ~23-fold increase in the neural to glial cell ratio. Such highly accelerated differentiation was assigned to electron injection from the photoexcited TiO2 into the cells on the rGO through Ti-C and Ti-O-C bonds. The role of ascorbic acid, as a scavenger of the photoexcited holes, in flash photo stimulation was studied at various concentrations and flash intensities.

  20. Elevated IKKα Accelerates the Differentiation of Human Neuronal Progenitor Cells and Induces MeCP2-Dependent BDNF Expression

    PubMed Central

    Khoshnan, Ali; Patterson, Paul H.

    2012-01-01

    The IκB kinase α (IKKα) is implicated in the differentiation of epithelial and immune cells. We examined whether IKKα also plays a role in the differentiation and maturation of embryonic human neuronal progenitor cells (NPCs). We find that expression of an extra copy of IKKα (IKKα+) blocks self-renewal and accelerates the differentiation of NPCs. This coincides with reduced expression of the Repressor Element Silencing Transcription Factor/Neuron-Restrictive Silencing Factor (REST/NRSF), which is a prominent inhibitor of neurogenesis, and subsequent induction of the pro-differentiation non-coding RNA, miR-124a. However, the effects of IKKα on REST/NRSF and miR-124a expression are likely to be indirect. IKKα+ neurons display extensive neurite outgrowth and accumulate protein markers of neuronal maturation such as SCG10/stathmin-2, postsynaptic density 95 (PSD95), syntaxin, and methyl-CpG binding protein 2 (MeCP2). Interestingly, IKKα associates with MeCP2 in the nuclei of human neurons and can phosphorylate MeCP2 in vitro. Using chromatin immunoprecipitation assays, we find that IKKα is recruited to the exon-IV brain-derived neurotrophic factor (BDNF) promoter, which is a well-characterized target of MeCP2 activity. Moreover, IKKα induces the transcription of BDNF and knockdown expression of MeCP2 interferes with this event. These studies highlight a role for IKKα in accelerating the differentiation of human NPCs and identify IKKα as a potential regulator of MeCP2 function and BDNF expression. PMID:22848609

  1. Effect of Glutamate and Riluzole on Manganese-Induced Apoptotic Cell Signaling in Neuronally Differentiated Mouse P19 Cells

    PubMed Central

    Roth, Jerome A.; Sridhar, Swetha; Singleton, Steven T.

    2012-01-01

    Excess exposure to Mn causes a neurological disorder known as manganism which is similar to dystonic movements associated with Parkinson’s disease. Manganism is largely restricted to occupations in which high atmospheric levels are prevalent which include Mn miners, welders and those employed in the ferroalloy processing or related industrial settings. T1 weighted MRI images reveal that Mn is deposited to the greatest extent in the globus pallidus, an area of the brain that is presumed to be responsible for the major CNS associated symptoms. Neurons within the globus pallidus receive glutamatergic input from the subthalamic nuclei which has been suggested to be involved in the toxic actions of Mn. The neurotoxic actions of Mn and glutamate are similar in that they both affect calcium accumulation in the mitochondria leading to apoptotic cell death. In this paper we demonstrate that the combination of Mn and glutamate potentiates toxicity of neuronally differentiated P19 cells over that observed with either agent alone. Apoptotic signals ROS, caspase 3 and JNK were increased in an additive fashion when the two neurotoxins were combined. The anti-glutamatergic drug, riluzole, was shown to attenuate these apoptotic signals and prevent P19 cell death. Results of this study confirm, for the first time, that Mn toxicity is potentiated in the presence of glutamate and that riluzole is an effective antioxidant which protects against both Mn and glutamate toxicity. PMID:22543103

  2. Differential expression of connexins in trigeminal ganglion neurons and satellite glial cells in response to chronic or acute joint inflammation.

    PubMed

    Garrett, Filip G; Durham, Paul L

    2008-11-01

    Trigeminal nerve activation in response to inflammatory stimuli has been shown to increase neuron-glia communication via gap junctions in trigeminal ganglion. The goal of this study was to identify changes in the expression of gap junction proteins, connexins (Cxs), in trigeminal ganglia in response to acute or chronic joint inflammation. Although mRNA for Cxs 26, 36, 40 and 43 was detected under basal conditions, protein expression of only Cxs 26, 36 and 40 increased following capsaicin or complete Freund's adjuvant (CFA) injection into the temporomandibular joint (TMJ). While Cx26 plaque formation between neurons and satellite glia was transiently increased following capsaicin injections, Cx26 plaque formation between neurons and satellite glia was sustained in response to CFA. Interestingly, levels of Cx36 and Cx40 were only elevated in neurons following capsaicin or CFA injections, but the temporal response was similar to that observed for Cx26. In contrast, Cx43 expression was not increased in neurons or satellite glial cells in response to CFA or capsaicin. Thus, trigeminal ganglion neurons and satellite glia can differentially regulate Cx expression in response to the type and duration of inflammatory stimuli, which likely facilitates increased neuron-glia communication during acute and chronic inflammation and pain in the TMJ.

  3. Wnt signalling in neuronal differentiation and development.

    PubMed

    Inestrosa, Nibaldo C; Varela-Nallar, Lorena

    2015-01-01

    Wnts are secreted glycoproteins that play multiple roles in early development, including the differentiation of precursor cells. During this period, gradients of Wnts and other morphogens are formed and regulate the differentiation and migration of neural progenitor cells. Afterwards, Wnt signalling cascades participate in the formation of neuronal circuits, playing roles in dendrite and axon development, dendritic spine formation and synaptogenesis. Finally, in the adult brain, Wnts control hippocampal plasticity, regulating synaptic transmission and neurogenesis. In this review, we summarize the reported roles of Wnt signalling cascades in these processes with a particular emphasis on the role of Wnts in neuronal differentiation and development.

  4. RAF Kinase Activity Regulates Neuroepithelial Cell Proliferation and Neuronal Progenitor Cell Differentiation during Early Inner Ear Development

    PubMed Central

    Magariños, Marta; Aburto, María R.; Sánchez-Calderón, Hortensia; Muñoz-Agudo, Carmen; Rapp, Ulf R.; Varela-Nieto, Isabel

    2010-01-01

    Background Early inner ear development requires the strict regulation of cell proliferation, survival, migration and differentiation, coordinated by the concerted action of extrinsic and intrinsic factors. Deregulation of these processes is associated with embryonic malformations and deafness. We have shown that insulin-like growth factor I (IGF-I) plays a key role in embryonic and postnatal otic development by triggering the activation of intracellular lipid and protein kinases. RAF kinases are serine/threonine kinases that regulate the highly conserved RAS-RAF-MEK-ERK signaling cascade involved in transducing the signals from extracellular growth factors to the nucleus. However, the regulation of RAF kinase activity by growth factors during development is complex and still not fully understood. Methodology/Principal Findings By using a combination of qRT-PCR, Western blotting, immunohistochemistry and in situ hybridization, we show that C-RAF and B-RAF are expressed during the early development of the chicken inner ear in specific spatiotemporal patterns. Moreover, later in development B-RAF expression is associated to hair cells in the sensory patches. Experiments in ex vivo cultures of otic vesicle explants demonstrate that the influence of IGF-I on proliferation but not survival depends on RAF kinase activating the MEK-ERK phosphorylation cascade. With the specific RAF inhibitor Sorafenib, we show that blocking RAF activity in organotypic cultures increases apoptosis and diminishes the rate of cell proliferation in the otic epithelia, as well as severely impairing neurogenesis of the acoustic-vestibular ganglion (AVG) and neuron maturation. Conclusions/Significance We conclude that RAF kinase activity is essential to establish the balance between cell proliferation and death in neuroepithelial otic precursors, and for otic neuron differentiation and axonal growth at the AVG. PMID:21203386

  5. The mental retardation associated protein, srGAP3 negatively regulates VPA-induced neuronal differentiation of Neuro2A cells.

    PubMed

    Chen, Keng; Mi, Ya-Jing; Ma, Yue; Fu, Hua-Lin; Jin, Wei-Lin

    2011-07-01

    The Slit-Robo GTPase-activating proteins (srGAPs) are important multifunctional adaptor proteins involved in various aspects of neuronal development, including axon guidance, neuronal migration, neurite outgrowth, dendritic morphology and synaptic plasticity. Among them, srGAP3, also named MEGAP (Mental disorder-associated GTPase-activating protein), plays a putative role in severe mental retardation. SrGAP3 expression in ventricular zones of neurogenesis indicates its involvement in early stage of neuronal development and differentiation. Here, we show that overexpression of srGAP3 inhibits VPA (valproic acid)-induced neurite initiation and neuronal differentiation in Neuro2A neuroblastoma cells, whereas knockdown of srGAP3 facilitates the neuronal differentiation in this cell line. In contrast to the wild type, overexpression of srGAP3 harboring an artificially mutation R542A within the functionally important RhoGAP domain does not exert a visible inhibitory effect on neuronal differentiation. The endogenous srGAP3 selectively binds to activated form of Rac1 in a RhoGAP pull-down assay. We also show that constitutively active (CA) Rac1 can rescue the effect of srGAP3 on attenuating neuronal differentiation. Furthermore, change in expression and localization of endogenous srGAP3 is observed in neuronal differentiated Neuro2A cells. Together, our data suggest that srGAP3 could regulate neuronal differentiation in a Rac1-dependent manner.

  6. Isolation of Multipotent Nestin-Expressing Stem Cells Derived from the Epidermis of Elderly Humans and TAT-VHL Peptide-Mediated Neuronal Differentiation of These Cells

    PubMed Central

    Kanno, Hiroshi; Kubo, Atsuhiko; Yoshizumi, Tetsuya; Mikami, Taro; Maegawa, Jiro

    2013-01-01

    A specialized population of cells residing in the hair follicle is quiescent but shows pluripotency for differentiating into epithelial-mesenchymal lineage cells. Therefore, such cells are hoped to be useful as implantable donor cells for regenerative therapy. Recently, it was reported that intracellular delivery of TAT-VHL peptide induces neuronal differentiation of skin-derived precursors. In the present study, we successfully isolated multipotent stem cells derived from the epidermis of elderly humans, characterized these cells as being capable of sphere formation and strong expression of nestin, fibronectin, and CD34 but not of keratin 15, and identified the niche of these cells as being the outer root sheath of the hair follicles. In addition, we showed that TAT-VHL peptide induced their neuronal differentiation in vitro, and confirmed by fluorescence immunohistochemistry the neuronal differentiation of such peptide-treated cells implanted into rodent brains. These multipotent nestin-expressing stem cells derived from human epidermis are easily accessible and should be useful as donor cells for neuronal regenerative cell therapy. PMID:23644888

  7. A Novel Protocol to Differentiate Induced Pluripotent Stem Cells by Neuronal microRNAs to Provide a Suitable Cellular Model.

    PubMed

    Zare, Mehrak; Soleimani, Masoud; Akbarzadeh, Abolfazl; Bakhshandeh, Behnaz; Aghaee-Bakhtiari, Seyed Hamid; Zarghami, Nosratollah

    2015-08-01

    Neurodegenerative diseases are one of the most challenging subjects in medicine. Investigation of their underlying genetic or epigenetic factors is hampered by lack of suitable models. Patient-specific induced pluripotent stem cells (iPS cells) represent a valuable approach to provide a proper model for poorly understood mechanisms of neuronal diseases and the related drug screenings. miR-124 and miR-128 are the two brain-enriched miRNAs with different time-points of expression during neuronal development. Herein, we transduced human iPS cells with miR-124 and miR-128 harboring lentiviruses sequentially. The transduced plasmids contained GFP and puromycin antibiotic-resistant genes for easier selection and identification. Morphological assessment and immunocytochemistry (overexpressions of beta-tubulin and neuron-specific enolase) confirmed that induced hiPS cells by miR-124 and miR-128 represent similar characteristics to those of mature neurons. In addition, the upregulation of neuron-specific enolase, beta-tubulin, Map2, GFAP, and BDNF was detected by quantitative real-time PCR. In conclusion, it seems that our novel protocol remarks the combinatorial effect of miR-124 and miR-128 on neural differentiation in the absence of any extrinsic factor. Moreover, such cellular models could be used in personalized drug screening and applied for more effective therapies.

  8. Propofol at Clinically Relevant Concentrations Increases Neuronal Differentiation but Is Not Toxic to Hippocampal Neural Precursor Cells In Vitro

    PubMed Central

    Sall, Jeffrey W.; Stratmann, Greg; Leong, Jason; Woodward, Elliott; Bickler, Philip E.

    2012-01-01

    Background Propofol in the early postnatal period has been shown to cause brain cell death. One proposed mechanism for cognitive dysfunction after anesthesia is alteration of neural stem cell function and neurogenesis. We examined the effect of propofol on neural precursor or stem cells (NPCs) grown in vitro. Methods Hippocampal derived NPCs from postnatal day 2 rats were exposed to propofol or to Diprivan. NPCs were then analyzed for bromodeoxyuridine incorporation to measure proliferation. Cell death was measured by lactate dehydrogenase release. Immunocytochemistry was used to evaluate the expression of neuronal and glial markers in differentiating NPCs exposed to propofol. Results Propofol dose dependently increases the release of lactate dehydrogenase from NPCs under both proliferating and differentiating conditions at supraclinical concentrations (> 7.1μM). Both Diprivan and propofol had the same effect on NPCs. Propofol mediated release of lactate dehydrogenase is not inhibited by blocking the γ-aminobutyric acid type A receptor or extracellular calcium influx and is not mediated by caspase-3/7. Direct γ-aminobutyric acid type A receptor activation did not have the same effect. In differentiating NPCs 6 h of propofol at 2.1 μM increased the number neurons but not glial cells 4 days later. Increased neuronal differentiation was not blocked by Bicuculline. Conclusions Only supraclinical concentrations of propofol or Diprivan kill NPCs in culture by a non-γ-aminobutyric acid type A, noncaspase 3 mechanism. Clinically relevant doses of propofol increase neuronal fate choice by a non-γ-aminobutyric acid type A mechanism. PMID:23001052

  9. Dopaminergic neurons differentiating from LRRK2 G2019S induced pluripotent stem cells show early neuritic branching defects.

    PubMed

    Borgs, Laurence; Peyre, Elise; Alix, Philippe; Hanon, Kevin; Grobarczyk, Benjamin; Godin, Juliette D; Purnelle, Audrey; Krusy, Nathalie; Maquet, Pierre; Lefebvre, Philippe; Seutin, Vincent; Malgrange, Brigitte; Nguyen, Laurent

    2016-09-19

    Some mutations of the LRRK2 gene underlie autosomal dominant form of Parkinson's disease (PD). The G2019S is a common mutation that accounts for about 2% of PD cases. To understand the pathophysiology of this mutation and its possible developmental implications, we developed an in vitro assay to model PD with human induced pluripotent stem cells (hiPSCs) reprogrammed from skin fibroblasts of PD patients suffering from the LRKK2 G2019S mutation. We differentiated the hiPSCs into neural stem cells (NSCs) and further into dopaminergic neurons. Here we show that NSCs bearing the mutation tend to differentiate less efficiently into dopaminergic neurons and that the latter exhibit significant branching defects as compared to their controls.

  10. Dopaminergic neurons differentiating from LRRK2 G2019S induced pluripotent stem cells show early neuritic branching defects

    PubMed Central

    Borgs, Laurence; Peyre, Elise; Alix, Philippe; Hanon, Kevin; Grobarczyk, Benjamin; Godin, Juliette D.; Purnelle, Audrey; Krusy, Nathalie; Maquet, Pierre; Lefebvre, Philippe; Seutin, Vincent; Malgrange, Brigitte; Nguyen, Laurent

    2016-01-01

    Some mutations of the LRRK2 gene underlie autosomal dominant form of Parkinson’s disease (PD). The G2019S is a common mutation that accounts for about 2% of PD cases. To understand the pathophysiology of this mutation and its possible developmental implications, we developed an in vitro assay to model PD with human induced pluripotent stem cells (hiPSCs) reprogrammed from skin fibroblasts of PD patients suffering from the LRKK2 G2019S mutation. We differentiated the hiPSCs into neural stem cells (NSCs) and further into dopaminergic neurons. Here we show that NSCs bearing the mutation tend to differentiate less efficiently into dopaminergic neurons and that the latter exhibit significant branching defects as compared to their controls. PMID:27640816

  11. Differentiating effects of the glucagon-like peptide-1 analogue exendin-4 in a human neuronal cell model.

    PubMed

    Luciani, Paola; Deledda, Cristiana; Benvenuti, Susanna; Cellai, Ilaria; Squecco, Roberta; Monici, Monica; Cialdai, Francesca; Luciani, Giorgia; Danza, Giovanna; Di Stefano, Chiara; Francini, Fabio; Peri, Alessandro

    2010-11-01

    Glucagon-like peptide-1 (GLP-1) is an insulinotropic peptide with neurotrophic properties, as assessed in animal cell models. Exendin-4, a GLP-1 analogue, has been recently approved for the treatment of type 2 diabetes mellitus. The aim of this study was to morphologically, structurally, and functionally characterize the differentiating actions of exendin-4 using a human neuronal cell model (i.e., SH-SY5Y cells). We found that exendin-4 increased the number of neurites paralleled by dramatic changes in intracellular actin and tubulin distribution. Electrophysiological analyses showed an increase in cell membrane surface and in stretch-activated-channels sensitivity, an increased conductance of Na(+) channels and amplitude of Ca(++) currents (T- and L-type), typical of a more mature neuronal phenotype. To our knowledge, this is the first demonstration that exendin-4 promotes neuronal differentiation in human cells. Noteworthy, our data support the claimed favorable role of exendin-4 against diabetic neuropathy as well as against different neurodegenerative diseases.

  12. Ectonucleotide pyrophosphatase/phosphodiesterase activity in Neuro-2a neuroblastoma cells: changes in expression associated with neuronal differentiation.

    PubMed

    Gómez-Villafuertes, Rosa; Pintor, Jesús; Miras-Portugal, María Teresa; Gualix, Javier

    2014-11-01

    Neuro-2a (N2a) neuroblastoma cells display an ectoenzymatic hydrolytic activity capable of degrading diadenosine polyphosphates. The Apn A-cleaving activity has been analysed with the use of the fluorogenic compound BODIPY FL guanosine 5'-O-(3-thiotriphosphate) thioester. Hydrolysis of this dinucleotide analogue showed a hyperbolic kinetic with a Km value of 4.9 ± 1.3 μM. Diadenosine pentaphosphate, diadenosine tetraphosphate, diadenosine triphosphate, and the nucleoside monophosphate AMP behaved as an inhibitor of BODIPY FL guanosine 5'-O-(3-thiotriphosphate) thioester extracellular degradation. Ectoenzymatic activity shared the typical characteristics of the ectonucleotide pyrophosphatase/phosphodiesterase family, as hydrolysis reached maximal activity at alkaline pH and was dependent on the presence of divalent cations, being strongly inhibited by EDTA and activated by Zn(2+) ions. Both NPP1 and NPP3 isozymes are expressed in N2a cells, their expression levels substantially changing when cells differentiate into a neuronal-like phenotype. In this sense, it is relevant to point the expression pattern of the NPP3 protein, whose levels were drastically reduced in the differentiated cells, being almost completely absent after 24 h of differentiation. Enzymatic activity assays carried out with differentiated N2a cells showed that NPP1 is the main isozyme involved in the extracellular degradation of dinucleotides in these cells, this enzyme reducing its activity and changing its subcellular location following neuronal differentiation. We described the presence of an ectoenzymatic activity able to hydrolyse diadenosine polyphosphates (ApnA) in N2a cells. This activity displays biochemical features that are typical of the ectonucleotide pyrophosphatase/phosphodiesterase (E-NPP) family members, as demonstrated by the use of the fluorogenic compound BODIPY-FL-GTPγS. Both NPP1 and NPP3 ectoenzymes are expressed in N2a cells, their levels dramatically changing when cells

  13. Corticosterone affects the differentiation of a neuronal cerebral cortex-derived cell line through modulation of the nicotinic acetylcholine receptor.

    PubMed

    Baier, C J; Franco, D L; Gallegos, C E; Mongiat, L A; Dionisio, L; Bouzat, C; Caviedes, P; Barrantes, F J

    2014-08-22

    Chronic exposure to stress hormones has an impact on brain structures relevant to cognition. Nicotinic acetylcholine receptors (AChRs) are involved in numerous cognitive processes including learning and memory formation. In order to better understand the molecular mechanisms of chronic stress-triggered mental disease, the effect of corticosterone (CORT) on the biology of AChRs was studied in the neuronal cell line CNh. We found that chronic treatment with CORT reduced the expression levels of the α7-type neuronal AChR and, to a lesser extent, of α4-AChR. CORT also delayed the acquisition of the mature cell phenotype in CNh cells. Chronic nicotine treatment affected the differentiation of CNh cells and exerted a synergistic effect with CORT, suggesting that AChR could participate in signaling pathways that control the cell cycle. Overexpression of α7-AChR-GFP abolished the CORT effects on the cell cycle and the specific α7-AChR inhibitor, methyllycaconitine, mimicked the proliferative action exerted by CORT. Whole-cell voltage-clamp recordings showed a significant decrease in nicotine-evoked currents in CORT-treated cells. Taken together, these observations indicate that AChRs, and the α7-AChR in particular, could act as modulators of the differentiation of CNh cells and that CORT could impair the acquisition of a mature phenotype by affecting the function of this AChR subtype. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

  14. Growth inhibition, morphological differentiation and stimulation of survival in neuronal cell type (Neuro-2a) treated with trophic molecules.

    PubMed

    Blanco, V; Lopez Camelo, J; Carri, N G

    2001-01-01

    Trophic molecules are key regulators of survival, growth and differentiation of neural cells. Neuronal cell type Neuro-2a is a good model to study development and molecules modulating this process, and retinoic acid (RA) and neurotrophins (NGF, BDNF, NT-3 and NT-4) have been shown to be active in this modulation. The purpose of the present study was the functional analysis of these trophic molecules in our short-term bioassay of Neuro-2a cells, an immortalised murine neuroblastoma cell line. Through cell counting, image process and arithmetic combination of digital parameters of treated and untreated cultures, we show that RA inhibits growth and induces morphological neuronal phenotype of treated cells. Through DNA labelling with BrdU we also show that NGF, BDNF, and NT-3 increase survival and proliferation of cells, grown in serum-deprived media. From these results we conclude that neurotrophins have manifest trophic effects on cells improving survival, growth and proliferation and we also confirm the growth arrest and differentiation properties of RA on Neuro-2a cells. Copyright 2001 Academic Press.

  15. Functional Comparison of Neuronal Cells Differentiated from Human Induced Pluripotent Stem Cell-Derived Neural Stem Cells under Different Oxygen and Medium Conditions.

    PubMed

    Yamazaki, Kazuto; Fukushima, Kazuyuki; Sugawara, Michiko; Tabata, Yoshikuni; Imaizumi, Yoichi; Ishihara, Yasuharu; Ito, Masashi; Tsukahara, Kappei; Kohyama, Jun; Okano, Hideyuki

    2016-12-01

    Because neurons are difficult to obtain from humans, generating functional neurons from human induced pluripotent stem cells (hiPSCs) is important for establishing physiological or disease-relevant screening systems for drug discovery. To examine the culture conditions leading to efficient differentiation of functional neural cells, we investigated the effects of oxygen stress (2% or 20% O2) and differentiation medium (DMEM/F12:Neurobasal-based [DN] or commercial [PhoenixSongs Biologicals; PS]) on the expression of genes related to neural differentiation, glutamate receptor function, and the formation of networks of neurons differentiated from hiPSCs (201B7) via long-term self-renewing neuroepithelial-like stem (lt-NES) cells. Expression of genes related to neural differentiation occurred more quickly in PS and/or 2% O2 than in DN and/or 20% O2, resulting in high responsiveness of neural cells to glutamate, N-methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and ( S)-3,5-dihydroxyphenylglycine (an agonist for mGluR1/5), as revealed by calcium imaging assays. NMDA receptors, AMPA receptors, mGluR1, and mGluR5 were functionally validated by using the specific antagonists MK-801, NBQX, JNJ16259685, and 2-methyl-6-(phenylethynyl)-pyridine, respectively. Multielectrode array analysis showed that spontaneous firing occurred earlier in cells cultured in 2% O2 than in 20% O2. Optimization of O2 tension and culture medium for neural differentiation of hiPSCs can efficiently generate physiologically relevant cells for screening systems.

  16. Differential effects of lipopolysaccharide on energy metabolism in murine microglial N9 and cholinergic SN56 neuronal cells.

    PubMed

    Klimaszewska-Łata, Joanna; Gul-Hinc, Sylwia; Bielarczyk, Hanna; Ronowska, Anna; Zyśk, Marlena; Grużewska, Katarzyna; Pawełczyk, Tadeusz; Szutowicz, Andrzej

    2015-04-01

    There are significant differences between acetyl-CoA and ATP levels, enzymes of acetyl-CoA metabolism, and toll-like receptor 4 contents in non-activated microglial N9 and non-differentiated cholinergic SN56 neuroblastoma cells. Exposition of N9 cells to lipopolysaccharide caused concentration-dependent several-fold increases of nitrogen oxide synthesis, accompanied by inhibition of pyruvate dehydrogenase complex, aconitase, and α-ketoglutarate dehydrogenase complex activities, and by nearly proportional depletion of acetyl-CoA, but by relatively smaller losses in ATP content and cell viability (about 5%). On the contrary, SN56 cells appeared to be insensitive to direct exposition to high concentration of lipopolysaccharide. However, exogenous nitric oxide resulted in marked inhibition pyruvate dehydrogenase and aconitase activities, depletion of acetyl-CoA, along with respective loss of SN56 cells viability. These data indicate that these two common neurodegenerative signals may differentially affect energy-acetyl-CoA metabolism in microglial and cholinergic neuronal cell compartments in the brain. Moreover, microglial cells appeared to be more resistant than neuronal cells to acetyl-CoA and ATP depletion evoked by these neurodegenerative conditions. Together, these data indicate that differential susceptibility of microglia and cholinergic neuronal cells to neurotoxic signals may result from differences in densities of toll-like receptors and degree of disequilibrium between acetyl-CoA provision in mitochondria and its utilization for energy production and acetylation reactions in each particular group of cells. There are significant differences between acetyl-CoA and ATP levels and enzymes of acetyl-CoA metabolism in non-activated microglial N9 and non-differentiated cholinergic SN56 neuroblastoma cells. Pathological stimulation of microglial toll-like receptors (TLRs) triggered excessive synthesis of microglia-derived nitric oxide (NO)/NOO radicals that

  17. SFRP2 enhanced the adipogenic and neuronal differentiation potentials of stem cells from apical papilla.

    PubMed

    Lin, Xiao; Dong, Rui; Diao, Shu; Yu, Guoxia; Wang, Liping; Li, Jun; Fan, Zhipeng

    2017-02-28

    Dental tissue-derived mesenchymal stem cells (MSCs) are easily obtained and considered as a favorable cell source for tissue engineering, but the regulation of direct differentiation is unknown, which restricts their application. The present study investigated the effect of SFRP2, a Wnt signaling modulator, on MSC differentiation using stem cells from apical papilla (SCAPs). The cells were cultured in specific inducing medium for adipogenic, neurogenic, or chondrogenic differentiation. Over-expression of SFRP2 via retroviral infection enhanced the adipogenic and neurogenic differentiation of SCAPs. While inhibit of Wnt pathway by IWR1-endo could enhance the neurogenic differentiation potentials of SCAPs, similar with the function of SFRP2. In addition, over-expression of SFRP2 up-regulated the expression of stemness-related genes SOX2 and OCT4. Furthermore, SOX2 and OCT4 expression was significantly inhibited after lentiviral silencing of SFRP2 in SCAPs. Therefore, our results suggest that SFRP2 enhances the adipogenic and neurogenic differentiation potentials of SCAPs by up-regulating SOX2 and OCT4. Moreover, the effect of SFRP2 in neurogenic differentiation of SCAPs maybe also associated with Wnt inhibition. Our results provided useful information about the molecular mechanism underlying directed differentiation in dental tissue-derived MSCs.

  18. Berberine induces neuronal differentiation through inhibition of cancer stemness and epithelial-mesenchymal transition in neuroblastoma cells.

    PubMed

    Naveen, C R; Gaikwad, Sagar; Agrawal-Rajput, Reena

    2016-06-15

    Berberine, a plant alkaloid, has been used since many years for treatment of gastrointestinal disorders. It also shows promising medicinal use against metabolic disorders, neurodegenerative disorders and cancer; however its efficacy in neuroblastoma (NB) is poorly explored. EMT is important in cancer stemness and metastasis resulting in failure to differentiate; thus targeting EMT and related pathways can have clinical benefits. Potential of berberine was investigated for (i) neuronal differentiation and cancer stemness inhibition, (ii) underlying molecular mechanisms regulating cancer-stemness and (iii) EMT reversal. Using neuro2a (N2a) neuroblastoma cells (NB); we investigated effect of berberine on neuronal differentiation, cancer-stemness, EMT and underlying signalling by immunofluorescence, RT-PCR, Western blot. High glucose-induced TGF-β mediated EMT model was used to test EMT reversal potential by Western blot and RT-PCR. STRING analysis was done to determine and validate functional protein-interaction networks. We demonstrate berberine induces neuronal differentiation accompanying increased neuronal differentiation markers like MAP2, β-III tubulin and NCAM; generated neurons were viable. Berberine attenuated cancer stemness markers CD133, β-catenin, n-myc, sox2, notch2 and nestin. Berberine potentiated G0/G1 cell cycle arrest by inhibiting proliferation, cyclin dependent kinases and cyclins resulting in apoptosis through increased bax/bcl-2 ratio. Restoration of tumor suppressor proteins, p27 and p53, indicate promising anti-cancer property. The induction of NCAM and reduction in its polysialylation indicates anti-migratory potential which is supported by down regulation of MMP-2/9. It increased epithelial marker laminin and smad and increased Hsp70 levels also suggest its protective role. Molecular insights revealed that berberine regulates EMT via downregulation of PI3/Akt and Ras-Raf-ERK signalling and subsequent upregulation of p38-MAPK. TGF

  19. Functional cross-talk between the cellular prion protein and the neural cell adhesion molecule is critical for neuronal differentiation of neural stem/precursor cells.

    PubMed

    Prodromidou, Kanella; Papastefanaki, Florentia; Sklaviadis, Theodoros; Matsas, Rebecca

    2014-06-01

    Cellular prion protein (PrP) is prominently expressed in brain, in differentiated neurons but also in neural stem/precursor cells (NPCs). The misfolding of PrP is a central event in prion diseases, yet the physiological function of PrP is insufficiently understood. Although PrP has been reported to associate with the neural cell adhesion molecule (NCAM), the consequences of concerted PrP-NCAM action in NPC physiology are unknown. Here, we generated NPCs from the subventricular zone (SVZ) of postnatal day 5 wild-type and PrP null (-/-) mice and observed that PrP is essential for proper NPC proliferation and neuronal differentiation. Moreover, we found that PrP is required for the NPC response to NCAM-induced neuronal differentiation. In the absence of PrP, NCAM not only fails to promote neuronal differentiation but also induces an accumulation of doublecortin-positive neuronal progenitors at the proliferation stage. In agreement, we noted an increase in cycling neuronal progenitors in the SVZ of PrP-/- mice compared with PrP+/+ mice, as evidenced by double labeling for the proliferation marker Ki67 and doublecortin as well as by 5-bromo-2'-deoxyuridine incorporation experiments. Additionally, fewer newly born neurons were detected in the rostral migratory stream of PrP-/- mice. Analysis of the migration of SVZ cells in microexplant cultures from wild-type and PrP-/- mice revealed no differences between genotypes or a role for NCAM in this process. Our data demonstrate that PrP plays a critical role in neuronal differentiation of NPCs and suggest that this function is, at least in part, NCAM-dependent.

  20. Stress Conditions Increase Vimentin Cleavage by Omi/HtrA2 Protease in Human Primary Neurons and Differentiated Neuroblastoma Cells.

    PubMed

    Lucotte, Bérangère; Tajhizi, Mehdi; Alkhatib, Dareen; Samuelsson, Eva-Britt; Wiehager, Birgitta; Schedin-Weiss, Sophia; Sundström, Erik; Winblad, Bengt; Tjernberg, Lars O; Behbahani, Homira

    2015-12-01

    Dysfunctional Omi/HtrA2, a mitochondrial serine protease, has been implicated in various neurodegenerative disorders. Despite the wealth of evidence on the roles of Omi/HtrA2 in apoptosis, little is known about its cytosolic targets, the cleavage of which could account for the observed morphological changes such as cytoskeletal reorganizations in axons. By proteomic analysis, vimentin was identified as a substrate for Omi/HtrA2 and we have reported increased Omi/HtrA2 protease activity in Alzheimer disease (AD) brain. Here, we investigated a possible link between Omi/HtrA2 and vimentin cleavage, and consequence of this cleavage on mitochondrial distribution in neurons. In vitro protease assays showed vimentin to be cleaved by Omi/HtrA2 protease, and proximity ligation assay demonstrated an increased interaction between Omi/HtrA2 and vimentin in human primary neurons upon stress stimuli. Using differentiated neuroblastoma SH-SY5Y cells, we showed that Omi/HtrA2 under several different stress conditions induces cleavage of vimentin in wild-type as well as SH-SY5Y cells transfected with amyloid precursor protein with the Alzheimer disease-associated Swedish mutation. After stress treatment, inhibition of Omi/HtrA2 protease activity by the Omi/HtrA2 specific inhibitor, Ucf-101, reduced the cleavage of vimentin in wild-type cells. Following altered vimentin filaments integrity by stress stimuli, mitochondria was redistributed in differentiated SH-SY5Y cells and human primary neurons. In summary, the findings outlined in this paper suggest a role of Omi/HtrA2 in modulation of vimentin filamentous structure in neurons. Our results provide important findings for understanding the biological role of Omi/HtrA2 activity during stress conditions, and give knowledge of interplay between Omi/HtrA2 and vimentin which might affect mitochondrial distribution in neurons.

  1. 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.

    PubMed

    Jiao, Yu; Palmgren, Björn; Novozhilova, Ekaterina; Englund Johansson, Ulrica; Spieles-Engemann, Anne L; Kale, Ajay; Stupp, Samuel I; Olivius, Petri

    2014-01-01

    To study possible nerve regeneration of a damaged auditory nerve by the use of stem cell transplantation. 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. 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. 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).

  2. The effect of multivalent Sonic hedgehog on differentiation of human embryonic stem cells into dopaminergic and GABAergic neurons.

    PubMed

    Vazin, Tandis; Ashton, Randolph S; Conway, Anthony; Rode, Nikhil A; Lee, Susan M; Bravo, Verenice; Healy, Kevin E; Kane, Ravi S; Schaffer, David V

    2014-01-01

    Stem cell differentiation is regulated by complex repertoires of signaling ligands which often use multivalent interactions, where multiple ligands tethered to one entity interact with multiple cellular receptors to yield oligomeric complexes. One such ligand is Sonic hedgehog (Shh), whose posttranslational lipid modifications and assembly into multimers enhance its biological potency, potentially through receptor clustering. Investigations of Shh typically utilize recombinant, monomeric protein, and thus the impact of multivalency on ligand potency is unexplored. Among its many activities, Shh is required for ventralization of the midbrain and forebrain and is therefore critical for the development of midbrain dopaminergic (mDA) and forebrain gamma-aminobutyric acid (GABA) inhibitory neurons. We have designed multivalent biomaterials presenting Shh in defined spatial arrangements and investigated the role of Shh valency in ventral specification of human embryonic stem cells (hESCs) into these therapeutically relevant cell types. Multivalent Shh conjugates with optimal valencies, compared to the monomeric Shh, increased the percentages of neurons belonging to mDA or forebrain GABAergic fates from 33% to 60% or 52% to 86%, respectively. Thus, multivalent Shh bioconjugates can enhance neuronal lineage commitment of pluripotent stem cells and thereby facilitate efficient derivation of neurons that could be used to treat Parkinson's and epilepsy patients.

  3. The effects of functional magnetic nanotubes with incorporated nerve growth factor in neuronal differentiation of PC12 cells

    NASA Astrophysics Data System (ADS)

    Xie, Jining; Chen, Linfeng; Varadan, Vijay K.; Yancey, Justin; Srivatsan, Malathi

    2008-03-01

    In this in vitro study the efficiency of magnetic nanotubes to bind with nerve growth factor (NGF) and the ability of NGF-incorporated magnetic nanotubes to release the bound NGF are investigated using rat pheochromocytoma cells (PC12 cells). It is found that functional magnetic nanotubes with NGF incorporation enabled the differentiation of PC12 cells into neurons exhibiting growth cones and neurite outgrowth. Microscope observations show that filopodia extending from neuron growth cones were in close proximity to the NGF-incorporated magnetic nanotubes, at times appearing to extend towards or into them. These results show that magnetic nanotubes can be used as a delivery vehicle for NGF and thus may be exploited in attempts to treat neurodegenerative disorders such as Parkinson's disease with neurotrophins. Further neurite outgrowth can be controlled by manipulating magnetic nanotubes with external magnetic fields, thus helping in directed regeneration.

  4. Factor-Reduced Human Induced Pluripotent Stem Cells Efficiently Differentiate into Neurons Independent of the Number of Reprogramming Factors

    PubMed Central

    Hermann, Andreas; Kim, Jeong Beom; Srimasorn, Sumitra; Zaehres, Holm; Reinhardt, Peter; Schöler, Hans R.; Storch, Alexander

    2016-01-01

    Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) by overexpression of the transcription factors OCT4, SOX2, KLF4, and c-Myc holds great promise for the development of personalized cell replacement therapies. In an attempt to minimize the risk of chromosomal disruption and to simplify reprogramming, several studies demonstrated that a reduced set of reprogramming factors is sufficient to generate iPSC. We recently showed that a reduction of reprogramming factors in murine cells not only reduces reprogramming efficiency but also may worsen subsequent differentiation. To prove whether this is also true for human cells, we compared the efficiency of neuronal differentiation of iPSC generated from fetal human neural stem cells with either one (OCT4; hiPSC1F-NSC) or two (OCT4, KLF4; hiPSC2F-NSC) reprogramming factors with iPSC produced from human fibroblasts using three (hiPSC3F-FIB) or four reprogramming factors (hiPSC4F-FIB). After four weeks of coculture with PA6 stromal cells, neuronal differentiation of hiPSC1F-NSC and hiPSC2F-NSC was as efficient as iPSC3F-FIB or iPSC4F-FIB. We conclude that a reduction of reprogramming factors in human cells does reduce reprogramming efficiency but does not alter subsequent differentiation into neural lineages. This is of importance for the development of future application of iPSC in cell replacement therapies. PMID:26977154

  5. YAP regulates neuronal differentiation through Sonic hedgehog signaling pathway

    SciTech Connect

    Lin, Yi-Ting; Ding, Jing-Ya; Li, Ming-Yang; Yeh, Tien-Shun; Wang, Tsu-Wei; Yu, Jenn-Yah

    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

  6. Diva/BclB regulates differentiation by inhibiting NDPKB/Nm23H2-mediated neuronal differentiation in PC-12 cells

    PubMed Central

    2012-01-01

    Background Diva (death inducer binding to vBcl-2 and Apaf-1)/BclB is a Bcl-2 family member, which is known for its function in apoptosis. Diva/BclB has been shown to interact with NDPKB/Nm23H2, which is involved in cellular differentiation. Thus far, there has been no direct evidence of Diva/BclB having a role in differentiation. In the present study, we investigated the expression of Diva/BclB and NDPKB/Nm23H2 during differentiation in PC-12 cell line. Results Our results show that after differentiation, Diva/BclB expression was decreased and reciprocally, NDPKB/Nm23H2 expression was increased and it translocated into the nucleus. Overexpression of NDPKB/Nm23H2 promoted PC-12 neuronal differentiation by increasing neurite outgrowth and arresting cell cycle progression. There was a concurrent downregulation of Diva/Boo when NDPKB/Nm23H2 was overexpressed, which mirrors the effect of NGF on PC-12 cell differentiation. Overexpression of Diva/BclB did not change the expression level of NDPKB/Nm23H2, but inhibited its nuclear localization. Cells that overexpressed Diva/BclB presented a decreased percentage of differentiated cells and average neurite length was shortened. This was due to an increase in the formation of Diva/BclB and NDPKB/Nm23H2 complexes as well as Diva/BclB and β-tubulin complexes. Concomitantly, there was a decrease in formation of NDPKB/Nm23H2 and β-tubulin complexes. Overexpression of Diva/BclB also resulted in a higher percentage of S-phase cells. Conclusion Our results showed a novel role for Diva/BclB in neuronal differentiation. Its downregulation during neuronal differentiation may be necessary to allow NDPKB/Nm23H2 and β-tubulin interaction that promotes NDPKB/Nm23H2 mediated differentiation. PMID:23057762

  7. Highly Efficient Differentiation and Enrichment of Spinal Motor Neurons Derived from Human and Monkey Embryonic Stem Cells

    PubMed Central

    Wada, Tamaki; Honda, Makoto; Minami, Itsunari; Tooi, Norie; Amagai, Yuji; Nakatsuji, Norio; Aiba, Kazuhiro

    2009-01-01

    Background There are no cures or efficacious treatments for severe motor neuron diseases. It is extremely difficult to obtain naïve spinal motor neurons (sMNs) from human tissues for research due to both technical and ethical reasons. Human embryonic stem cells (hESCs) are alternative sources. Several methods for MN differentiation have been reported. However, efficient production of naïve sMNs and culture cost were not taken into consideration in most of the methods. Methods/Principal Findings We aimed to establish protocols for efficient production and enrichment of sMNs derived from pluripotent stem cells. Nestin+ neural stem cell (NSC) clusters were induced by Noggin or a small molecule inhibitor of BMP signaling. After dissociation of NSC clusters, neurospheres were formed in a floating culture containing FGF2. The number of NSCs in neurospheres could be expanded more than 30-fold via several passages. More than 33% of HB9+ sMN progenitor cells were observed after differentiation of dissociated neurospheres by all-trans retinoic acid (ATRA) and a Shh agonist for another week on monolayer culture. HB9+ sMN progenitor cells were enriched by gradient centrifugation up to 80% purity. These HB9+ cells differentiated into electrophysiologically functional cells and formed synapses with myotubes during a few weeks after ATRA/SAG treatment. Conclusions and Significance The series of procedures we established here, namely neural induction, NSC expansion, sMN differentiation and sMN purification, can provide large quantities of naïve sMNs derived from human and monkey pluripotent stem cells. Using small molecule reagents, reduction of culture cost could be achieved. PMID:19701462

  8. ORF7 of Varicella-Zoster Virus Is Required for Viral Cytoplasmic Envelopment in Differentiated Neuronal Cells.

    PubMed

    Jiang, Hai-Fei; Wang, Wei; Jiang, Xuan; Zeng, Wen-Bo; Shen, Zhang-Zhou; Song, Yi-Ge; Yang, Hong; Liu, Xi-Juan; Dong, Xiao; Zhou, Jing; Sun, Jin-Yan; Yu, Fei-Long; Guo, Lin; Cheng, Tong; Rayner, Simon; Zhao, Fei; Zhu, Hua; Luo, Min-Hua

    2017-06-15

    Although a varicella-zoster virus (VZV) vaccine has been used for many years, the neuropathy caused by VZV infection is still a major health concern. Open reading frame 7 (ORF7) of VZV has been recognized as a neurotropic gene in vivo, but its neurovirulent role remains unclear. In the present study, we investigated the effect of ORF7 deletion on VZV replication cycle at virus entry, genome replication, gene expression, capsid assembly and cytoplasmic envelopment, and transcellular transmission in differentiated neural progenitor cells (dNPCs) and neuroblastoma SH-SY5Y (dSY5Y) cells. Our results demonstrate that the ORF7 protein is a component of the tegument layer of VZV virions. Deleting ORF7 did not affect viral entry, viral genome replication, or the expression of typical viral genes but clearly impacted cytoplasmic envelopment of VZV capsids, resulting in a dramatic increase of envelope-defective particles and a decrease in intact virions. The defect was more severe in differentiated neuronal cells of dNPCs and dSY5Y. ORF7 deletion also impaired transmission of ORF7-deficient virus among the neuronal cells. These results indicate that ORF7 is required for cytoplasmic envelopment of VZV capsids, virus transmission among neuronal cells, and probably the neuropathy induced by VZV infection.IMPORTANCE The neurological damage caused by varicella-zoster virus (VZV) reactivation is commonly manifested as clinical problems. Thus, identifying viral neurovirulent genes and characterizing their functions are important for relieving VZV related neurological complications. ORF7 has been previously identified as a potential neurotropic gene, but its involvement in VZV replication is unclear. In this study, we found that ORF7 is required for VZV cytoplasmic envelopment in differentiated neuronal cells, and the envelopment deficiency caused by ORF7 deletion results in poor dissemination of VZV among neuronal cells. These findings imply that ORF7 plays a role in neuropathy

  9. Spatiotemporal changes in Cx30 and Cx43 expression during neuronal differentiation of P19 EC and NT2/D1 cells.

    PubMed

    Wan, Carthur K; O'Carroll, Simon J; Kim, Sue-Ling; Green, Colin R; Nicholson, Louise F B

    2013-12-01

    While connexins (Cxs) are thought to be involved in differentiation, their expression and role has yet to be fully elucidated. We investigated the temporal expression of Cx30, Cx36 and Cx43 in two in vitro models of neuronal differentiation: human NT2/D1 and murine P19 cells, and the spatial localisation of Cx30 and Cx43 in these models. A temporal Cx43 downregulation was confirmed in both cell lines during RA-induced neuronal differentiation using RT-PCR (P < 0.05) preceding an increase in neuronal doublecortin protein. RT-PCR showed Cx36 was upregulated twofold in NT2/D1 cells (P < 0.05) and sixfold in P19 cells (P < 0.001) during neuronal differentiation. Cx30 exhibited a transient peak in expression midway through the timecourse of differentiation increasing threefold in NT2/D1 cells (P < 0.001) and eightfold in P19 cells (P < 0.01). Qualitative immunocytochemistry was used to examine spatiotemporal patterns of Cx protein distribution alongside neuronal differentiation markers. The temporal immunolabelling pattern was similar to that seen using RT-PCR. Cx43 was observed intracellularly and on cell surfaces, while Cx30 was seen as puncta. Spatially Cx43 was seen on doublecortin-negative cells, which may indicate Cx43 downregulation is requisite for differentiation in these models. Conversely, Cx30 puncta were observed on doublecortin-positive and -negative cells in NT2/D1 cells and examination of the Cx30 peak showed puncta also localized to nestin-positive cells, with few puncta on MAP2-positive cells. In P19 cells Cx30 was localized on clusters of cells surrounded by MAP2- and doublecortin-positive processes. The expression pattern of Cx30 indicates a role in neuronal differentiation; the nature of that role warrants future investigation.

  10. Dopaminergic Neuronal Differentiation from the Forebrain-Derived Human Neural Stem Cells Induced in Cultures by Using a Combination of BMP-7 and Pramipexole with Growth Factors

    PubMed Central

    Yang, HongNa; Wang, Jing; Wang, Feng; Liu, XiaoDun; Chen, Heng; Duan, WeiMing; Qu, TingYu

    2016-01-01

    Transplantation of dopaminergic (DA) neurons is considered to be the most promising therapeutic strategy for replacing degenerated dopamine cells in the midbrain of Parkinson's disease (PD), thereby restoring normal neural circuit function and slow clinical progression of the disease. Human neural stem cells (hNSCs) derived from fetal forebrain are thought to be the important cell sources for producing DA neurons because of their multipotency for differentiation and long-term expansion property in cultures. However, low DA differentiation of the forebrain-derived hNSCs limited their therapeutic potential in PD. In the current study, we explored a combined application of Pramipexole (PRX), bone morphogenetic proteins 7 (BMP-7), and growth factors, including acidic fibroblast factor (aFGF), forskolin, and phorbol-12-myristae-13-acetate (TPA), to induce differentiation of forebrain-derived hNSCs toward DA neurons in cultures. We found that DA neuron-associated genes, including Nurr1, Neurogenin2 (Ngn2), and tyrosine hydroxylase (TH) were significantly increased after 24 h of differentiation by RT-PCR analysis (p < 0.01). Fluorescent examination showed that about 25% of cells became TH-positive neurons at 24 h, about 5% of cells became VMAT2 (vascular monoamine transporter 2)-positive neurons, and less than 5% of cells became DAT (dopamine transporter)-positive neurons at 72 h following differentiation in cultures. Importantly, these TH-, VMAT2-, and DAT-expressing neurons were able to release dopamine into cultures under both of the basal and evoked conditions. Dopamine levels released by DA neurons produced using our protocol were significantly higher compared to the control groups (P < 0.01), as examined by ELISA. Our results demonstrated that the combination of PRX, BMP-7, and growth factors was able to greatly promote differentiation of the forebrain-derived hNSCs into DA-releasing neurons. PMID:27147976

  11. WNT/β-catenin pathway activation in Myc immortalised cerebellar progenitor cells inhibits neuronal differentiation and generates tumours resembling medulloblastoma.

    PubMed

    Rogers, H A; Sousa, S; Salto, C; Arenas, E; Coyle, B; Grundy, R G

    2012-09-25

    Medulloblastoma is the most common malignant childhood brain tumour. Aberrant activation of the WNT/β-catenin pathway occurs in approximately 25% of medulloblastomas. However, its role in medulloblastoma pathogenesis is not understood. We have developed a model of WNT/β-catenin pathway-activated medulloblastoma. Pathway activation was induced in a Myc immortalised cerebellar progenitor cell line through stable expression of Wnt1. In vitro and in vivo analysis was undertaken to understand the effect of pathway activation and identify the potential cell of origin. Tumours that histologically resembled classical medulloblastoma formed in vivo using cells overexpressing Wnt1, but not with the control cell line. Wnt1 overexpression inhibited neuronal differentiation in vitro, suggesting WNT/β-catenin pathway activation prevents cells terminally differentiating, maintaining them in a more 'stem-like' state. Analysis of cerebellar progenitor cell markers demonstrated the cell line resembled cells from the cerebellar ventricular zone. We have developed a cell line with the means of orthotopically modelling WNT/β-catenin pathway-activated medulloblastoma. We provide evidence of the role pathway activation is playing in tumour pathogenesis and suggest medulloblastomas can arise from cells other than granule cell progenitors. This cell line is a valuable resource to further understand the role of pathway activation in tumorigenesis and for investigation of targeted therapies.

  12. Differential distribution of glycine transporters in Müller cells and neurons in amphibian retinas.

    PubMed

    Jiang, Zheng; Li, Baoqin; Jursky, Frantisek; Shen, Wen

    2007-01-01

    Amphibian retinas are commonly used for electrophysiological studies on neural function and transduction because they share the same general properties as higher vertebrate retinas. Glycinergic synapses have been well described in amphibian retinas. However, the role of glycine transporters in the synapses is largely unknown. We studied the distribution and function of glycine transporters in the retinas from tiger salamanders, mudpuppies, and leopard frogs by immunofluorescence labeling and whole-cell recording methods. Our results indicated that GlyT1- and GlyT2-like transporters were present in Müller cells and neurons, respectively. GlyT1 labeling was present in Müller glial cells and co-localized with Glial fibrillary acidic protein (GFAP), a Müller cell marker, whereas the GlyT2 immunoreactivity was present in the somas of amacrine cells (ACs) and processes in the inner plexiform layer (IPL) and the outer plexiform layer (OPL). Because the axon processes of glycinergic interplexiform cells (IPCs) are the only source of glycine input in the OPL, GlyT2 staining revealed a spatial pattern of the axon processes of IPCs in the OPL. The function of GlyT2 in the IPCs was studied in tiger salamander retinal horizontal cells (HCs) by whole-cell gramicidin perforated recording. The results demonstrated that inhibition of GlyT2 by a specific inhibitor, amoxapine, increased a tonic glycine input to HCs. Thus, the GlyT2 transporter is responsible for uptake of synaptic glycine in the outer retina. We also compared the distribution of glycine transporters in other amphibian species: salamander, mudpuppy, and frog. The results are consistent with the general pattern that GlyT1-like transporters are present in Müller cells and GlyT2-like transporters in neurons in amphibian retinas.

  13. Differentiation Potential of Human Chorion-Derived Mesenchymal Stem Cells into Motor Neuron-Like Cells in Two- and Three-Dimensional Culture Systems.

    PubMed

    Faghihi, Faezeh; Mirzaei, Esmaeil; Ai, Jafar; Lotfi, Abolfazl; Sayahpour, Forough Azam; Ebrahimi-Barough, Somayeh; Barough, Somayeh Ebrahimi; Joghataei, Mohammad Taghi

    2016-04-01

    Many people worldwide suffer from motor neuron-related disorders such as amyotrophic lateral sclerosis and spinal cord injuries. Recently, several attempts have been made to recruit stem cells to modulate disease progression in ALS and also regenerate spinal cord injuries. Chorion-derived mesenchymal stem cells (C-MSCs), used to be discarded as postpartum medically waste product, currently represent a class of cells with self renewal property and immunomodulatory capacity. These cells are able to differentiate into mesodermal and nonmesodermal lineages such as neural cells. On the other hand, gelatin, as a simply denatured collagen, is a suitable substrate for cell adhesion and differentiation. It has been shown that electrospinning of scaffolds into fibrous structure better resembles the physiological microenvironment in comparison with two-dimensional (2D) culture system. Since there is no report on potential of human chorion-derived MSCs to differentiate into motor neuron cells in two- and three-dimensional (3D) culture systems, we set out to determine the effect of retinoic acid (RA) and sonic hedgehog (Shh) on differentiation of human C-MSCs into motor neuron-like cells cultured on tissue culture plates (2D) and electrospun nanofibrous gelatin scaffold (3D).

  14. Directed Differentiation of Human Embryonic Stem Cells Toward Placode-Derived Spiral Ganglion-Like Sensory Neurons.

    PubMed

    Matsuoka, Akihiro J; Morrissey, Zachery D; Zhang, Chaoying; Homma, Kazuaki; Belmadani, Abdelhak; Miller, Charles A; Chadly, Duncan M; Kobayashi, Shun; Edelbrock, Alexandra N; Tanaka-Matakatsu, Miho; Whitlon, Donna S; Lyass, Ljuba; McGuire, Tammy L; Stupp, Samuel I; Kessler, John A

    2017-03-01

    The ability to generate spiral ganglion neurons (SGNs) from stem cells is a necessary prerequisite for development of cell-replacement therapies for sensorineural hearing loss. We present a protocol that directs human embryonic stem cells (hESCs) toward a purified population of otic neuronal progenitors (ONPs) and SGN-like cells. Between 82% and 95% of these cells express SGN molecular markers, they preferentially extend neurites to the cochlear nucleus rather than nonauditory nuclei, and they generate action potentials. The protocol follows an in vitro stepwise recapitulation of developmental events inherent to normal differentiation of hESCs into SGNs, resulting in efficient sequential generation of nonneuronal ectoderm, preplacodal ectoderm, early prosensory ONPs, late ONPs, and cells with cellular and molecular characteristics of human SGNs. We thus describe the sequential signaling pathways that generate the early and later lineage species in the human SGN lineage, thereby better describing key developmental processes. The results indicate that our protocol generates cells that closely replicate the phenotypic characteristics of human SGNs, advancing the process of guiding hESCs to states serving inner-ear cell-replacement therapies and possible next-generation hybrid auditory prostheses. © Stem Cells Translational Medicine 2017;6:923-936.

  15. Tianma modulates proteins with various neuro-regenerative modalities in differentiated human neuronal SH-SY5Y cells.

    PubMed

    Ramachandran, Umamaheswari; Manavalan, Arulmani; Sundaramurthi, Husvinee; Sze, Siu Kwan; Feng, Zhi Wei; Hu, Jiang-Miao; Heese, Klaus

    2012-06-01

    Tianma (Rhizoma gastrodiae) is the dried rhizome of the plant Gastrodia elata Blume (Orchidaceae family). As a medicinal herb in traditional Chinese medicine (TCM) its functions are to control convulsions, pain, headache, dizziness, vertigo, seizure, epilepsy and others. In addition, tianma is frequently used for the treatment of neurodegenerative disorders though the mechanism of action is widely unknown. Accordingly, this study was designed to examine the effects of tianma on the proteome metabolism in differentiated human neuronal SH-SY5Y cells to explore its specific effects on neuronal signaling pathways. Using an iTRAQ (isobaric tags for relative and absolute quantitation)-based proteomics research approach, we identified 2390 modulated proteins, out of which 406 were found to be altered by tianma in differentiated human neuronal SH-SY5Y cells. Based on the observed data, we hypothesize that tianma promotes neuro-regenerative signaling cascades by controlling chaperone/proteasomal degradation pathways (e.g. CALR, FKBP3/4, HSP70/90) and mobilizing neuro-protective genes (such as AIP5) as well as modulating other proteins (RTN1/4, NCAM, PACSIN2, and PDLIM1/5) with various regenerative modalities and capacities related to neuro-synaptic plasticity.

  16. Niche astrocytes promote the survival, proliferation and neuronal differentiation of co-transplanted neural stem cells following ischemic stroke in rats

    PubMed Central

    Luo, Li; Guo, Kaihua; Fan, Wenguo; Lu, Yinghong; Chen, Lizhi; Wang, Yang; Shao, Yijia; Wu, Gongxiong; Xu, Jie; Lü, Lanhai

    2017-01-01

    Niche astrocytes have been reported to promote neuronal differentiation through juxtacrine signaling. However, the effects of astrocytes on neuronal differentiation following ischemic stroke are not fully understood. In the present study, transplanted astrocytes and neural stem cells (NSCs) were transplanted into the ischemic striatum of transient middle cerebral artery occlusion (MCAO) model rats 48 h following surgery. It was observed that the co-transplantation of astrocytes and NSCs resulted in a higher ratio of survival and proliferation of the transplanted NSCs, and neuronal differentiation, in MCAO rats compared with NSC transplantation alone. These results demonstrate that the co-administration of astrocytes promotes the survival and neuronal differentiation of NSCs in the ischemic brain. These results suggest that the co-transplantation of astrocytes and NSCs is more effective than NSCs alone in the production of neurons following ischemic stroke in rats. PMID:28352345

  17. Prenatal Hypoxia in Different Periods of Embryogenesis Differentially Affects Cell Migration, Neuronal Plasticity, and Rat Behavior in Postnatal Ontogenesis

    PubMed Central

    Vasilev, Dmitrii S.; Dubrovskaya, Nadezhda M.; Tumanova, Natalia L.; Zhuravin, Igor A.

    2016-01-01

    Long-term effects of prenatal hypoxia on embryonic days E14 or E18 on the number, type and localization of cortical neurons, density of labile synaptopodin-positive dendritic spines, and parietal cortex-dependent behavioral tasks were examined in the postnatal ontogenesis of rats. An injection of 5′ethynyl-2′deoxyuridine to pregnant rats was used to label neurons generated on E14 or E18 in the fetuses. In control rat pups a majority of cells labeled on E14 were localized in the lower cortical layers V-VI while the cells labeled on E18 were mainly found in the superficial cortical layers II-III. It was shown that hypoxia both on E14 and E18 results in disruption of neuroblast generation and migration but affects different cell populations. In rat pups subjected to hypoxia on E14, the total number of labeled cells in the parietal cortex was decreased while the number of labeled neurons scattered within the superficial cortical layers was increased. In rat pups subjected to hypoxia on E18, the total number of labeled cells in the parietal cortex was also decreased but the number of scattered labeled neurons was higher in the lower cortical layers. It can be suggested that prenatal hypoxia both on E14 and E18 causes a disruption in neuroblast migration but with a different outcome. Only in rats subjected to hypoxia on E14 did we observe a reduction in the total number of pyramidal cortical neurons and the density of labile synaptopodin-positive dendritic spines in the molecular cortical layer during the first month after birth which affected development of the cortical functions. As a result, rats subjected to hypoxia on E14, but not on E18, had impaired development of the whisker-placing reaction and reduced ability to learn reaching by a forepaw. The data obtained suggest that hypoxia on E14 in the period of generation of the cells, which later differentiate into the pyramidal cortical neurons of the V-VI layers and form cortical minicolumns, affects formation of

  18. Differentiation from human pluripotent stem cells of cortical neurons of the superficial layers amenable to psychiatric disease modeling and high-throughput drug screening

    PubMed Central

    Boissart, C; Poulet, A; Georges, P; Darville, H; Julita, E; Delorme, R; Bourgeron, T; Peschanski, M; Benchoua, A

    2013-01-01

    Cortical neurons of the superficial layers (II-IV) represent a pivotal neuronal population involved in the higher cognitive functions of the human and are particularly affected by psychiatric diseases with developmental manifestations such as schizophrenia and autism. Differentiation protocols of human pluripotent stem cells (PSC) into cortical neurons have been achieved, opening the way to in vitro modeling of neuropsychiatric diseases. However, these protocols commonly result in the asynchronous production of neurons typical for the different layers of the cortex within an extended period of culture, thus precluding the analysis of specific subtypes of neurons in a standardized manner. Addressing this issue, we have successfully captured a stable population of self-renewing late cortical progenitors (LCPs) that synchronously and massively differentiate into glutamatergic cortical neurons of the upper layers. The short time course of differentiation into neurons of these progenitors has made them amenable to high-throughput assays. This has allowed us to analyze the capability of LCPs at differentiating into post mitotic neurons as well as extending and branching neurites in response to a collection of selected bioactive molecules. LCPs and cortical neurons of the upper layers were successfully produced from patient-derived-induced PSC, indicating that this system enables functional studies of individual-specific cortical neurons ex vivo for disease modeling and therapeutic purposes. PMID:23962924

  19. Developmental changes in expression, subcellular distribution, and function of Drosophila N-cadherin, guided by a cell-intrinsic program during neuronal differentiation.

    PubMed

    Kurusu, Mitsuhiko; Katsuki, Takeo; Zinn, Kai; Suzuki, Emiko

    2012-06-15

    Cell adhesion molecules (CAMs) perform numerous functions during neural development. An individual CAM can play different roles during each stage of neuronal differentiation; however, little is known about how such functional switching is accomplished. Here we show that Drosophila N-cadherin (CadN) is required at multiple developmental stages within the same neuronal population and that its sub-cellular expression pattern changes between the different stages. During development of mushroom body neurons and motoneurons, CadN is expressed at high levels on growing axons, whereas expression becomes downregulated and restricted to synaptic sites in mature neurons. Phenotypic analysis of CadN mutants reveals that developing axons require CadN for axon guidance and fasciculation, whereas mature neurons for terminal growth and receptor clustering. Furthermore, we demonstrate that CadN downregulation can be achieved in cultured neurons without synaptic contact with other cells. Neuronal silencing experiments using Kir(2.1) indicate that neuronal excitability is also dispensable for CadN downregulation in vivo. Interestingly, downregulation of CadN can be prematurely induced by ectopic expression of a nonselective cation channel, dTRPA1, in developing neurons. Together, we suggest that switching of CadN expression during neuronal differentiation involves regulated cation influx within neurons. Copyright © 2012 Elsevier Inc. All rights reserved.

  20. Successful elimination of non-neural cells and unachievable elimination of glial cells by means of commonly used cell culture manipulations during differentiation of GFAP and SOX2 positive neural progenitors (NHA) to neuronal cells.

    PubMed

    Witusik, Monika; Piaskowski, Sylwester; Hulas-Bigoszewska, Krystyna; Zakrzewska, Magdalena; Gresner, Sylwia M; Azizi, S Ausim; Krynska, Barbara; Liberski, Pawel P; Rieske, Piotr

    2008-07-19

    Although extensive research has been performed to control differentiation of neural stem cells - still, the response of those cells to diverse cell culture conditions often appears to be random and difficult to predict. To this end, we strived to obtain stabilized protocol of NHA cells differentiation - allowing for an increase in percentage yield of neuronal cells. Uncommitted GFAP and SOX2 positive neural progenitors - so-called, Normal Human Astrocytes (NHA) were differentiated in different environmental conditions to: only neural cells consisted of neuronal [MAP2+, GFAP-] and glial [GFAP+, MAP2-] population, non-neural cells [CD44+, VIMENTIN+, FIBRONECTIN+, MAP2-, GFAP-, S100beta-, SOX2-], or mixture of neural and non-neural cells.In spite of successfully increasing the percentage yield of glial and neuronal vs. non-neural cells by means of environmental changes, we were not able to increase significantly the percentage of neuronal (GABA-ergic and catecholaminergic) over glial cells under several different cell culture testing conditions. Supplementing serum-free medium with several growth factors (SHH, bFGF, GDNF) did not radically change the ratio between neuronal and glial cells--i.e., 1,1:1 in medium without growth factors and 1,4:1 in medium with GDNF, respectively. We suggest that biotechnologists attempting to enrich in vitro neural cell cultures in one type of cells - such as that required for transplantology purposes, should consider the strong limiting influence of intrinsic factors upon extracellular factors commonly tested in cell culture conditions.

  1. Claulansine F promoted the neuronal differentiation of neural stem and progenitor cells through Akt/GSK-3β/β-catenin pathway.

    PubMed

    Huang, Ju-Yang; Ma, Yin-Zhong; Yuan, Yu-He; Zuo, Wei; Chu, Shi-Feng; Liu, Hang; Du, Guan-Hua; Zhang, Dong-Ming; Chen, Nai-Hong

    2016-09-05

    The persistence of neurogenesis raises the idea that neurons produced by the resident or transplanted neural stem cells could replace the neurons lost from brain injury or neurodegenerative disease. Therefore, compounds or methods for promoting neuronal differentiation become the focus of neurodegenerative disease therapy research. Claulansine F (Clau F), a newly discovered carbazole alkaloid, has been showed to induce neuritogenesis in PC12 cells. Herein, we studied the effect of Clau F on neuronal differentiation of neural stem/progenitor cells (NS/PCs). The current study demonstrated that Clau F initiated neuronal differentiation with a significant increase of TuJ1-positive cells and TuJ1 protein levels. We also found that Clau F promoted the maturity and sustainability of neurons by increasing MAP2-positive cells and MAP2 protein levels. At the same time, Clau F significantly inhibited the proliferation of NS/PCs. The underlying mechanism of Clau F was preliminary explored. Clau F treatment resulted in a profound increase of phosphorylation of Akt and GSK-3β, which led to GSK-3β inhibition and subsequently the nuclear accumulation of β-catenin. Further, the interaction between β-catenin and p300 in the nucleus was enhanced and the transcription of p300/β-catenin responsive genes were increased significantly (c-jun, fra-1) by Clau F. Importantly, the positive effect of Clau F on neuronal differentiation was abolished by Akti-1/2, a specific inhibitor of Akt-1/2 kinase, which indicated the involvement of Akt/GSK-3β in Clau F-mediated neuronal differentiation. In conclusion, these data suggested that Clau F promoted neuronal differentiation through Akt/GSK-3β/β-catenin signaling pathway in NS/PCs.

  2. Material-driven differentiation of induced pluripotent stem cells in neuron growth factor-grafted poly(ε-caprolactone)-poly(β-hydroxybutyrate) scaffolds.

    PubMed

    Kuo, Yung-Chih; Huang, Min-Jung

    2012-08-01

    The potential of constructs comprising induced pluripotent stem (iPS) cells and biopolymers can be high for neurological surgery practice, if the systematic activity of neuronal regeneration is clarified. This study shows a guided differentiation of iPS cells toward neurons in neuron growth factor (NGF)-grafted poly(ε-caprolactone) (PCL)-poly(β-hydroxybutyrate) (PHB) scaffolds. The porosity of PCL-PHB scaffolds enhanced with increasing the concentration of salt particles (porogen) and the weight percentage of PCL. An increase in the graft concentration of NGF elevated the atomic ratios of N/C and O/C on the surface of NGF-grafted PCL-PHB scaffolds. In addition, incorporating heparin and NGF promoted the adhesion and viability of iPS cells in constructs. When the weight percentage of PCL increased, the viability of iPS cells reduced; however, more PCL in constructs benefited the adhesion of iPS cells. Under the influence of heparin and NGF, a high weight percentage of PCL and a long inductive period improved iPS cells to differentiate into neuron-like cells carrying βIII tubulin and inhibited other differentiation(s). The material-driven differentiation in NGF-grafted PCL-PHB constructs can be promising in guiding iPS cells to produce neurons for nerve tissue engineering. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. Nanotubes impregnated human olfactory bulb neural stem cells promote neuronal differentiation in Trimethyltin-induced neurodegeneration rat model.

    PubMed

    Marei, Hany E; Elnegiry, Ahmed A; Zaghloul, Adel; Althani, Asma; Afifi, Nahla; Abd-Elmaksoud, Ahmed; Farag, Amany; Lashen, Samah; Rezk, Shymaa; Shouman, Zeinab; Cenciarelli, Carlo; Hasan, Anwarul

    2017-12-01

    Neural stem cells (NSCs) are multipotent self-renewing cells that could be used in cellular-based therapy for a wide variety of neurodegenerative diseases including Alzheimer's diseases (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). Being multipotent in nature, they are practically capable of giving rise to major cell types of the nervous tissue including neurons, astrocytes, and oligodendrocytes. This is in marked contrast to neural progenitor cells which are committed to a specific lineage fate. In previous studies, we have demonstrated the ability of NSCs isolated from human olfactory bulb (OB) to survive, proliferate, differentiate, and restore cognitive and motor deficits associated with AD, and PD rat models, respectively. The use of carbon nanotubes (CNTs) to enhance the survivability and differentiation potential of NSCs following their in vivo engraftment have been recently suggested. Here, in order to assess the ability of CNTs to enhance the therapeutic potential of human OBNSCs for restoring cognitive deficits and neurodegenerative lesions, we co-engrafted CNTs and human OBNSCs in TMT-neurodegeneration rat model. The present study revealed that engrafted human OBNSCS-CNTs restored cognitive deficits, and neurodegenerative changes associated with TMT-induced rat neurodegeneration model. Moreover, the CNTs seemed to provide a support for engrafted OBNSCs, with increasing their tendency to differentiate into neurons rather than into glia cells. The present study indicate the marked ability of CNTs to enhance the therapeutic potential of human OBNSCs which qualify this novel therapeutic paradigm as a promising candidate for cell-based therapy of different neurodegenerative diseases. © 2017 Wiley Periodicals, Inc.

  4. Differential induction of Toll-like receptors & type 1 interferons by Sabin attenuated & wild type 1 polioviruses in human neuronal cells

    PubMed Central

    Mohanty, Madhu C.; Deshpande, Jagadish M.

    2013-01-01

    Background & objectives: Polioviruses are the causative agent of paralytic poliomyelitis. Attenuated polioviruses (Sabin oral poliovirus vaccine strains) do not replicate efficiently in neurons as compared to the wild type polioviruses and therefore do not cause disease. This study was aimed to investigate the differential host immune response to wild type 1 poliovirus (wild PV) and Sabin attenuated type 1 poliovirus (Sabin PV) in cultured human neuronal cells. Methods: By using flow cytometry and real time PCR methods we examined host innate immune responses and compared the role of toll like receptors (TLRs) and cytoplasmic RNA helicases in cultured human neuronal cells (SK-N-SH) infected with Sabin PV and wild PV. Results: Human neuronal cells expressed very low levels of TLRs constitutively. Sabin PV infection induced significantly higher expression of TLR3, TLR7 and melanoma differentiation-associated protein-5 (MDA-5) m-RNA in neuronal cells at the beginning of infection (up to 4 h) as compared to wild PV. Further, Sabin PV also induced the expression of interferon α/β at early time point of infection. The induced expression of IFN α/β gene by Sabin PV in neuronal cells could be suppressed by inhibiting TLR7. Interpretation & conclusions: Neuronal cell innate immune response to Sabin and wild polioviruses differ significantly for TLR3, TLR7, MDA5 and type 1 interferons. Effects of TLR7 activation and interferon production and Sabin virus replication in neuronal cells need to be actively investigated in future studies. PMID:24056597

  5. Nerve growth factor (NGF) induces neuronal differentiation in neuroblastoma cells transfected with the NGF receptor cDNA

    SciTech Connect

    Matsushima, H.; Bogenmann, E. )

    1990-09-01

    Human nerve growth factor (NGF) receptor (NGFR) cDNA was transfected into a neuroblastoma cell line (HTLA 230) which does not express a functional NGF-NGFR signal transduction cascade. Short-term treatment of stably transfected cells (98-3) expressing membrane-bound NGF receptor molecules resulted in a cell cycle-dependent, transient expression of the c-fos gene upon treatment with NGF, suggesting the presence of functional high-affinity NGFR. Extensive outgrowth of neurites and cessation of DNA synthesis occurred in transfectants grown on an extracellular matrix after long-term treatment with NGF, suggesting terminal differentiation. Our data support the idea that introduction of a constitutively expressed NGFR cDNA into cells with neuronal background results in the assembly of a functional NGF-NGFR signal cascade in a permissive extracellular environment.

  6. NGF induction of the gene encoding the protease transin accompanies neuronal differentiation in PC12 cells.

    PubMed

    Machida, C M; Rodland, K D; Matrisian, L; Magun, B E; Ciment, G

    1989-06-01

    Various proteases have been found to be released by the growth cones of developing neurons in culture and have been hypothesized to play a role in the process of axon elongation. We report here that nerve growth factor (NGF) induced the gene encoding the metalloprotease transin in PC12 cells with a time course coincident with the initial appearance of neurites by these cells. Acidic and basic fibroblast growth factors also stimulated transin mRNA expression and neurite outgrowth, whereas various other agents had no effects on either of these phenomena. In contrast, dexamethasone was found to inhibit the induction of transin mRNA when added with, or following, NGF treatment. Finally, we show that sequences contained within 750 bp of the 5' untranscribed region of the transin gene confer responsiveness to NGF and dexamethasone.

  7. Magnesium Elevation Promotes Neuronal Differentiation While Suppressing Glial Differentiation of Primary Cultured Adult Mouse Neural Progenitor Cells through ERK/CREB Activation

    PubMed Central

    Liao, Wang; Jiang, Mujun; Li, Mei; Jin, Congli; Xiao, Songhua; Fan, Shengnuo; Fang, Wenli; Zheng, Yuqiu; Liu, Jun

    2017-01-01

    This study aimed to explore the influence of magnesium elevation on fate determination of adult neural progenitor cells (aNPCs) and the underlying mechanism in vitro. Adult neurogenesis, which is the generation of functional neurons from neural precursors, occurs throughout life in restricted anatomical regions in mammals. Magnesium is the fourth most abundant ion in mammals, and its elevation in the brain has been shown to enhance memory and synaptic plasticity in vivo. However, the effects of magnesium on fate determination of aNPCs, which are vital processes in neurogenesis, remain unknown. NPCs isolated from the dentate gyrus of adult C57/BL6 mice were induced to differentiate in a medium with varying magnesium concentrations (0.6, 0.8, and 1.0 mM) and extracellular signal-regulated kinase (ERK) inhibitor PD0325901. The proportion of cells that differentiated into neurons and glial cells was evaluated using immunofluorescence. Quantitative real-time polymerase chain reaction and Western blot methods were used to determine the expression of β-III tubulin (Tuj1) and glial fibrillary acidic protein (GFAP). The activation of ERK and cAMP response element-binding protein (CREB) was examined by Western blot to reveal the underlying mechanism. Magnesium elevation increased the proportion of Tju1-positive cells and decreased the proportion of GFAP-positive cells. Also, the expression of Tuj1 was upregulated, whereas the expression of GFAP was downregulated. Moreover, magnesium elevation enhanced the activation of both ERK and CREB. Treatment with PD0325901 reversed these effects in a dose-dependent manner. Magnesium elevation promoted neural differentiation while suppressing glial cell differentiation, possibly via ERK-induced CREB activation. PMID:28280456

  8. Phosphatidylcholine-specific phospholipase C/heat shock protein 70 (Hsp70)/transcription factor B-cell translocation gene 2 signaling in rat bone marrow stromal cell differentiation to cholinergic neuron-like cells.

    PubMed

    Shao, Jing; Sun, Chunhui; Su, Le; Zhao, Jing; Zhang, Shangli; Miao, Junying

    2012-12-01

    Although bone marrow stromal cells (BMSCs) can differentiate into neuron-like cells, the mechanisms underlying neuronal differentiation are not well understood. We recently found that inhibition of phosphatidylcholine-specific phospholipase C (PC-PLC) by its inhibitor D609 promoted BMSCs' differentiation into cholinergic neuron-like cells. Using the effective small molecule D609 and gene microarray technology, we investigated the change of gene expression profile to identify key mediators involved in the neuronal differentiation. We selected heat shock protein 70 (Hsp70) and transcription factor B-cell translocation gene 2 (Btg2) that were maximally up-regulated for further study. We found that functional suppression of Hsp70 blocked D609-induced increase of Btg2 expression and cholinergic neuronal differentiation of BMSCs. These results demonstrated that Hsp70 was the pivotal factor in PC-PLC-medicated neuronal differentiation of BMSCs, and Btg2 might be its downstream target. Our findings provide new clues for controlling BMSCs' differentiation into cholinergic neuron-like cells and provide a putative strategy for neurodegenerative diseases therapies. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

  9. Microtubule-Targeting Agents Eribulin and Paclitaxel Differentially Affect Neuronal Cell Bodies in Chemotherapy-Induced Peripheral Neuropathy.

    PubMed

    Benbow, Sarah J; Wozniak, Krystyna M; Kulesh, Bridget; Savage, April; Slusher, Barbara S; Littlefield, Bruce A; Jordan, Mary Ann; Wilson, Leslie; Feinstein, Stuart C

    2017-04-08

    Chemotherapy-induced peripheral neuropathy (CIPN) is a common side effect of anticancer treatment with microtubule-targeted agents (MTAs). The frequency of severe CIPN, which can be dose limiting and even life threatening, varies widely among different MTAs. For example, paclitaxel induces a higher frequency of severe CIPN than does eribulin. Different MTAs also possess distinct mechanisms of microtubule-targeted action. Recently, we demonstrated that paclitaxel and eribulin differentially affect sciatic nerve axons, with paclitaxel inducing more pronounced neurodegenerative effects and eribulin inducing greater microtubule stabilizing biochemical effects. Here, we complement and extend these axonal studies by assessing the effects of paclitaxel and eribulin in the cell bodies of sciatic nerve axons, housed in the dorsal root ganglia (DRG). Importantly, the microtubule network in cell bodies is known to be significantly more dynamic than in axons. Paclitaxel induced activating transcription factor 3 expression, a marker of neuronal stress/injury. Paclitaxel also increased expression levels of acetylated tubulin and end binding protein 1, markers of microtubule stability and growth, respectively. These effects are hypothesized to be detrimental to the dynamic microtubule network within the cell bodies. In contrast, eribulin had no significant effect on any of these parameters in the cell bodies. Taken together, DRG cell bodies and their axons, two distinct neuronal cell compartments, contain functionally distinct microtubule networks that exhibit unique biochemical responses to different MTA treatments. We hypothesize that these distinct mechanistic actions may underlie the variability seen in the initiation, progression, persistence, and recovery from CIPN.

  10. Translational regulation of NeuroD1 expression by FMRP: involvement in glutamatergic neuronal differentiation of cultured rat primary neural progenitor cells.

    PubMed

    Jeon, Se Jin; Kim, Ji-Woon; Kim, Ki Chan; Han, So Min; Go, Hyo Sang; Seo, Jung Eun; Choi, Chang Soon; Ryu, Jong Hoon; Shin, Chan Young; Song, Mi-Ryoung

    2014-03-01

    Fragile X mental retardation protein (FMRP) is encoded by Fmr1 gene in which mutation is known to cause fragile X syndrome characterized by mental impairment and other psychiatric symptoms similar to autism spectrum disorders. FMRP plays important roles in cellular mRNA biology such as transport, stability, and translation as an RNA-binding protein. In the present study, we identified potential role of FMRP in the neural differentiation, using cortical neural progenitor cells from Sprague-Dawley rat. We newly found NeuroD1, an essential regulator of glutamatergic neuronal differentiation, as a new mRNA target interacting with FMRP in co-immunoprecipitation experiments. We also identified FMRP as a regulator of neuronal differentiation by modulating NeuroD1 expression. Down-regulation of FMRP by siRNA also increased NeuroD1 expression along with increased pre- and post-synaptic development of glutamatergic neuron, as evidenced by Western blot and immunocytochemistry. On the contrary, cells harboring FMRP over-expression construct showed decreased NeuroD1 expression. Treatment of cultured neural precursor cells with a histone deacetylase inhibitor, valproic acid known as an inducer of hyper-glutamatergic neuronal differentiation, down-regulated the expression of FMRP, and induced NeuroD1 expression. Our study suggests that modulation of FMRP expression regulates neuronal differentiation by interaction with its binding target mRNA, and provides an example of the gene and environmental interaction regulating glutamatergic neuronal differentiation.

  11. Activated adult microglia influence retinal progenitor cell proliferation and differentiation toward recoverin-expressing neuron-like cells in a co-culture model.

    PubMed

    Xu, Yunhe; Balasubramaniam, Balini; Copland, David A; Liu, Jian; Armitage, M John; Dick, Andrew D

    2015-07-01

    Microglia contribute to immune homeostasis of the retina, and thus act as a potential regulator determining successful repair or retinal stem cell transplantation. We investigated the interaction between human microglia and retinal progenitor cells in cell co-culture to further our exploration on developing a new therapeutic strategy for retinal degeneration. Microglia and retinal progenitor cultures were developed using CD11b(+) and CD133(+), respectively, from adult donor retina. Microglia activation was developed using interferon-gamma and lipopolysaccharide. Retinal progenitor differentiation was analysed in co-culture with or without microglial activation. Retinal progenitor proliferation was analysed in presence of conditioned medium from activated microglia. Phenotype and function of adult human retinal cell cultures were examined using cell morphology, immunohistochemistry and real-time PCR. By morphology, neuron-like cells generated in co-culture expressed photoreceptor marker recoverin. Neurospheres derived from retinal progenitor cells showed reduced growth in the presence of conditioned medium from activated microglia. Delayed retinal progenitor cell migration and reduced cellular differentiation was observed in co-cultures with activated microglia. In independent experiments, activated microglia showed enhanced mRNA expression of CXCL10, IL-27, IL-6, and TNF-alpha compared to controls. Adult human retina retains retinal progenitors or potential to reprogram cells to then proliferate and differentiate into neuron-like cells in vitro. Human microglia support retinal progenitor differentiation into neuron-like cells, but such capacity is altered following microglial activation. Modulating microglia activity is a potential approach to promote retinal repair and facilitate success of stem-cell transplantation.

  12. Scutellarin may alleviate cognitive deficits in a mouse model of hypoxia by promoting proliferation and neuronal differentiation of neural stem cells

    PubMed Central

    Wang, Wei-Wei; Han, Jian-Hong; Wang, Lin; Bao, Tian-Hao

    2017-01-01

    Objective(s): Scutellarin, a flavonoid extracted from the medicinal herb Erigeron breviscapus Hand-Mazz, protects neurons from damage and inhibits glial activation. Here we examined whether scutellarin may also protect neurons from hypoxia-induced damage. Materials and Methods: Mice were exposed to hypoxia for 7 days and then administered scutellarin (50 mg/kg/d) or vehicle for 30 days Cognitive impairment in the two groups was assessed using the Morris water maze test, cell proliferation in the hippocampus was compared using 5-bromo-2-deoxyuridine (BrdU) immunohistochemistry, and hippocampal levels of nestin and neuronal class III β-tubulin (Tuj-1) were measured using Western blotting. These results were validated in vitro by treating cultured neural stem cells (NSCs) with scutellarin (30 μM). Results: Treating mice with scutellarin shortened escape times and increased the number of platform crossings, it increased the number of BrdU-positive proliferating cells in the hippocampus, and it up-regulated expression of nestin and Tuj-1. Treating NSC cultures with scutellarin increased the number of proliferating cells and the proportion of cells differentiating into neurons instead of astrocytes. The increase in NSC proliferation was associated with phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, while neuronal differentiation was associated with altered expression of differentiation-related genes. Conclusion: Scutellarin may alleviate cognitive impairment in a mouse model of hypoxia by promo-ting proliferation and neuronal differentiation of NSCs. PMID:28392899

  13. Acetylcholine-induced neuronal differentiation: muscarinic receptor activation regulates EGR-1 and REST expression in neuroblastoma cells.

    PubMed

    Salani, Monica; Anelli, Tonino; Tocco, Gabriella Augusti; Lucarini, Elena; Mozzetta, Chiara; Poiana, Giancarlo; Tata, Ada Maria; Biagioni, Stefano

    2009-02-01

    Neurotransmitters are considered part of the signaling system active in nervous system development and we have previously reported that acetylcholine (ACh) is capable of enhancing neuronal differentiation in cultures of sensory neurons and N18TG2 neuroblastoma cells. To study the mechanism of ACh action, in this study, we demonstrate the ability of choline acetyltransferase-transfected N18TG2 clones (e.g. 2/4 clone) to release ACh. Analysis of muscarinic receptors showed the presence of M1-M4 subtypes and the activation of both IP(3) and cAMP signal transduction pathways. Muscarinic receptor activation increases early growth response factor-1 (EGR-1) levels and treatments with agonists, antagonists, and signal transduction enzyme inhibitors suggest a role for M3 subtype in EGR-1 induction. The role of EGR-1 in the enhancement of differentiation was investigated transfecting in N18TG2 cells a construct for EGR-1. EGR-1 clones show increased neurite extension and a decrease in Repressor Element-1 silencing transcription factor (REST) expression: both these features have also been observed for the 2/4 clone. Transfection of this latter with EGR zinc-finger domain, a dominant negative inhibitor of EGR-1 action, increases REST expression, and decreases fiber outgrowth. The data reported suggest that progression of the clone 2/4 in the developmental program is dependent on ACh release and the ensuing activation of muscarinic receptors, which in turn modulate the level of EGR-1 and REST transcription factors.

  14. Gene regulatory logic of dopaminergic neuron differentiation

    PubMed Central

    Flames, Nuria; Hobert, Oliver

    2009-01-01

    Dopamine signaling regulates a variety of complex behaviors and defects in dopaminergic neuron function or survival result in severe human pathologies, such as Parkinson's disease 1. The common denominator of all dopaminergic neurons is the expression of dopamine pathway genes, which code for a set of phylogenetically conserved proteins involved in dopamine synthesis and transport. Gene regulatory mechanisms that result in the activation of dopamine pathway genes and thereby ultimately determine the identity of dopaminergic neurons are poorly understood in any system studied to date 2. We show here that a simple cis-regulatory element, the DA motif, controls the expression of all dopamine pathway genes in all dopaminergic cell types in C. elegans. The DA motif is activated by the ETS transcription factor, AST-1. Loss of ast-1 results in the failure of all distinct dopaminergic neuronal subtypes to terminally differentiate. Ectopic expression of ast-1 is sufficient to activate the dopamine production pathway in some cellular contexts. Vertebrate dopaminergic pathway genes also contain phylogenetically conserved DA motifs that can be activated by the mouse ETS transcription factor Etv1/ER81 and a specific class of dopaminergic neurons fails to differentiate in mice lacking Etv1/ER81. Moreover, ectopic Etv1/ER81 expression induces dopaminergic fate marker expression in neuronal primary cultures. Mouse Etv1/ER81 can also functionally substitute for ast-1 in C.elegans. Our studies reveal an astoundingly simple and apparently conserved regulatory logic of dopaminergic neuron terminal differentiation and may provide new entry points into the diagnosis or therapy of conditions in which dopamine neurons are defective. PMID:19287374

  15. The synergistic inhibitory actions of oxcarbazepine on voltage-gated sodium and potassium currents in differentiated NG108-15 neuronal cells and model neurons.

    PubMed

    Huang, Chin-Wei; Huang, Chao-Ching; Lin, Ming-Wei; Tsai, Jing-Jane; Wu, Sheng-Nan

    2008-08-01

    Oxcarbazepine (OXC), one of the newer anti-epileptic drugs, has been demonstrating its efficacy on wide-spectrum neuropsychiatric disorders. However, the ionic mechanism of OXC actions in neurons remains incompletely understood. With the aid of patch-clamp technology, we first investigated the effects of OXC on ion currents in NG108-15 neuronal cells differentiated with cyclic AMP. We found OXC (0.3-30 microm) caused a reversible reduction in the amplitude of voltage-gated Na+ current (INa). The IC50 value required for the inhibition of INa by OXC was 3.1 microm. OXC (3 microm) could shift the steady-state inactivation of INa to a more negative membrane potential by approximately -9 mV with no effect on the slope of the inactivation curve, and produce a significant prolongation in the recovery of INa inactivation. Additionally, OXC was effective in suppressing persistent INa (INa(P)) elicited by long ramp pulses. The blockade of INa by OXC does not simply reduce current magnitude, but alters current kinetics. Moreover, OXC could suppress the amplitude of delayed rectifier K+ current (IK(DR)), with no effect on M-type K+ current (IK(M)). In current-clamp configuration, OXC could reduce the amplitude of action potentials and prolong action-potential duration. Furthermore, the simulations, based on hippocampal pyramidal neurons (Pinsky-Rinzel model) and a network of the Hodgkin-Huxley model, were analysed to investigate the effect of OXC on action potentials. Taken together, our results suggest that the synergistic blocking effects on INa and IK(DR) may contribute to the underlying mechanisms through which OXC affects neuronal function in vivo.

  16. Amelioration of penetrating ballistic-like brain injury induced cognitive deficits after neuronal differentiation of transplanted human neural stem cells.

    PubMed

    Spurlock, Markus S; Ahmed, Aminul Islam; Rivera, Karla N; Yokobori, Shoji; Lee, Stephanie W; Sam, Pingdewinde N; Shear, Deborah A; Hefferan, Michael P; Hazel, Thomas G; Johe, Karl K; Gajavelli, Shyam; Tortella, Frank C; Bullock, Ross

    2017-03-01

    Penetrating traumatic brain injury (PTBI) is one of the major cause of death and disability worldwide. Previous studies in penetrating ballistic-like brain injury (PBBI), a PTBI rat model revealed widespread peri-lesional neurodegeneration, similar to that seen in humans following gunshot wound to head, which is unmitigated by any available therapies to date. Therefore, we evaluated human neural stem cell (hNSC) engraftment to putatively exploit the potential of cell therapy that has been seen in other central nervous system injury models. Towards this, green fluorescent protein (GFP) labeled hNSCs (400,000 per animal) were transplanted in immunosuppressed Sprague Dawley (SD), Fisher, and athymic (ATN) PBBI rats one week after injury. Tacrolimus (3mg/kg two days prior to transplantation, then 1mg/kg/day), Methylprednisolone (10mg/kg on day of transplant, 1mg/kg/week thereafter), and Mycophenolate mofetil (30mg/kg/day) for seven days following transplantation were used to confer immunosuppression. Engraftment in SD and ATN was comparable at 8-weeks post transplantation. Evaluation of hNSC differentiation and distribution revealed increased neuronal differentiation of transplanted cells with time. At 16-weeks post-transplantation neither cell proliferation nor glial lineage markers expression was detected. Transplanted cell morphology was similar to neighboring host neurons and there was relatively little migration of cells from the peri-transplant site. By 16 weeks, GFP positive processes extended both rostro-caudally and bilaterally into parenchyma, spreading along host white matter tracts, traversing internal capsule, extending ~13 mm caudally from transplantation site reaching into the brain stem. In a Morris water maze test at 8-weeks post-transplantation, animals with transplants had shorter latency to platform compared to vehicle treated animals. However, weak injury-induced cognitive deficits in the control group at the delayed time point confounded benefits

  17. Effects of nerve growth factor and basic fibroblast growth factor dual gene modification on rat bone marrow mesenchymal stem cell differentiation into neuron-like cells in vitro

    PubMed Central

    HU, YANG; ZHANG, YAN; TIAN, KANG; XUN, CHONG; WANG, SHOUYU; LV, DECHENG

    2016-01-01

    Recent studies regarding regenerative medicine have focused on bone marrow mesenchymal stem cells (BMSCs), which have the potential to undergo neural differentiation, and may be transfected with specific genes. BMSCs can differentiate into neuron-like cells in certain neurotropic circumstances in vitro. Basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) are often used to induce neural differentiation in BMSCs in vitro. However, previous studies regarding their combined actions are insufficient. The present study is the first, to the best of our knowledge, to thoroughly assess the enhancement of neural differentiation of BMSCs following transfection with bFGF and NGF. Sprague-Dawley (SD) rat BMSCs were separated through whole bone marrow adherence, and were then passaged to the third generation. The cells were subsequently divided into five groups: The control group, which consisted of untransfected BMSCs; the plv-blank-transfected BMSCs group; the plv-bFGF-trans-fected BMSCs group; the plv-NGF-transfected BMSCs group; and the plv-NGF-bFGF co-transfected BMSCs group. Cell neural differentiation was characterized in terms of stem cell molecular expression, and the neuronal morphology and expression of neural-like molecules was detected in each of the groups. A total of 72 h post-transfection, the expression levels of neuron-specific enolase, glial fibrillary acidic protein, and nestin protein, were higher in the co-transfected group, as compared with the other groups, the expression levels of β-tubulin III were also increased in the co-transfected cells, thus suggesting the maturation of differentiated neuron-like cells. Furthermore, higher neuronal proliferation was observed in the co-transfected group, as compared with the other groups at passages 2, 4, 6 and 8. Western blotting demonstrated that the transfected groups exhibited a simultaneous increase in phosphorylation of the AKT and extracellular signal-regulated kinases (ERK) signaling pathway

  18. Mesenchymal Stem Cells Increase Hippocampal Neurogenesis and Neuronal Differentiation by Enhancing the Wnt Signaling Pathway in an Alzheimer's Disease Model.

    PubMed

    Oh, Se Hee; Kim, Ha Na; Park, Hyun-Jung; Shin, Jin Young; Lee, Phil Hyu

    2015-01-01

    Neurogenesis in the subgranular zone of the hippocampal dentate gyrus may act as an endogenous repair mechanism in Alzheimer's disease (AD), and the Wnt signaling pathway has been suggested to closely modulate neurogenesis in amyloid-β (Aβ)-related AD models. The present study investigated whether mesenchymal stem cells (MSCs) would modulate hippocampal neurogenesis via modulation of the Wnt signaling pathway in a model of AD. In Aβ-treated neuronal progenitor cells (NPCs), the coculture with MSCs increased significantly the expression of Ki-67, GFAP, SOX2, nestin, and HuD compared to Aβ treatment alone. In addition, MSC treatment in Aβ-treated NPCs enhanced the expression of β-catenin and Ngn1 compared to Aβ treatment alone. MSC treatment in Aβ-treated animals significantly increased the number of BrdU-ir cells in the hippocampus at 2 and 4 weeks compared to Aβ treatment alone. In addition, quantitative analysis showed that the number of BrdU and HuD double-positive cells in the dentate gyrus was significantly higher in the MSC-treated group than in controls or after Aβ treatment alone. These results demonstrate that MSC administration significantly augments hippocampal neurogenesis and enhances the differentiation of NPCs into mature neurons in AD models by augmenting the Wnt signaling pathway. The use of MSCs to modulate endogenous adult neurogenesis may have a significant impact on future strategies for AD treatment.

  19. High neuronal/astroglial differentiation plasticity of adult rat hippocampal neural stem/progenitor cells in response to the effects of embryonic and adult cerebrospinal fluids

    PubMed Central

    Peirouvi, T.; Yekani, F.; Azarnia, M.; Massumi, M.

    2015-01-01

    Hippocampal neural stem/progenitor cells (hipp-NS/PCs) of the adult mammalian brain are important sources of neuronal and gial cell production. In this study, the main goal is to investigate the plasticity of these cells in neuronal/astroglial differentiations. To this end, the differentiation of the hipp-NS/PCs isolated from 3-month-old Wistar rats was investigated in response to the embryonic cerebrospinal fluid (E-CSF) including E13.5, E17-CSF and the adult cerebrospinal fluid (A-CSF), all extracted from rats. CSF samples were selected based on their effects on cell behavioral parameters. Primary cell culture was performed in the presence of either normal or high levels of KCL in a culture medium. High levels of KCL cause cell depolarization, and thus the activation of quiescent NSCs. Results from immunocytochemistry (ICC) and semi-quantitative RT-PCR (sRT-PCR) techniques showed that in E-CSF-treated groups, neuronal differentiation increased (E17>E13.5). In contrast, A-CSF decreased and increased neuronal and astroglial differentiations, respectively. Cell survivability and/or proliferation (S/P), evaluated by an MTT assay, increased by E13.5 CSF, but decreased by both E17 CSF and A-CSF. Based on the results, it is finally concluded that adult rat hippocampal proliferative cells are not restricted progenitors but rather show high plasticity in neuronal/astroglial differentiation according to the effects of CSF samples. In addition, using high concentrations of KCL in the primary cell culture led to an increase in the number of NSCs, which in turn resulted in the increase in neuronal or astroglial differentiations after CSF treatment. PMID:27175157

  20. High neuronal/astroglial differentiation plasticity of adult rat hippocampal neural stem/progenitor cells in response to the effects of embryonic and adult cerebrospinal fluids.

    PubMed

    Peirouvi, T; Yekani, F; Azarnia, M; Massumi, M

    2015-01-01

    Hippocampal neural stem/progenitor cells (hipp-NS/PCs) of the adult mammalian brain are important sources of neuronal and gial cell production. In this study, the main goal is to investigate the plasticity of these cells in neuronal/astroglial differentiations. To this end, the differentiation of the hipp-NS/PCs isolated from 3-month-old Wistar rats was investigated in response to the embryonic cerebrospinal fluid (E-CSF) including E13.5, E17-CSF and the adult cerebrospinal fluid (A-CSF), all extracted from rats. CSF samples were selected based on their effects on cell behavioral parameters. Primary cell culture was performed in the presence of either normal or high levels of KCL in a culture medium. High levels of KCL cause cell depolarization, and thus the activation of quiescent NSCs. Results from immunocytochemistry (ICC) and semi-quantitative RT-PCR (sRT-PCR) techniques showed that in E-CSF-treated groups, neuronal differentiation increased (E17>E13.5). In contrast, A-CSF decreased and increased neuronal and astroglial differentiations, respectively. Cell survivability and/or proliferation (S/P), evaluated by an MTT assay, increased by E13.5 CSF, but decreased by both E17 CSF and A-CSF. Based on the results, it is finally concluded that adult rat hippocampal proliferative cells are not restricted progenitors but rather show high plasticity in neuronal/astroglial differentiation according to the effects of CSF samples. In addition, using high concentrations of KCL in the primary cell culture led to an increase in the number of NSCs, which in turn resulted in the increase in neuronal or astroglial differentiations after CSF treatment.

  1. ZNF804A Transcriptional Networks in Differentiating Neurons Derived from Induced Pluripotent Stem Cells of Human Origin.

    PubMed

    Chen, Jian; Lin, Mingyan; Hrabovsky, Anastasia; Pedrosa, Erika; Dean, Jason; Jain, Swati; Zheng, Deyou; Lachman, Herbert M

    2015-01-01

    ZNF804A (Zinc Finger Protein 804A) has been identified as a candidate gene for schizophrenia (SZ), autism spectrum disorders (ASD), and bipolar disorder (BD) in replicated genome wide association studies (GWAS) and by copy number variation (CNV) analysis. Although its function has not been well-characterized, ZNF804A contains a C2H2-type zinc-finger domain, suggesting that it has DNA binding properties, and consequently, a role in regulating gene expression. To further explore the role of ZNF804A on gene expression and its downstream targets, we used a gene knockdown (KD) approach to reduce its expression in neural progenitor cells (NPCs) derived from induced pluripotent stem cells (iPSCs). KD was accomplished by RNA interference (RNAi) using lentiviral particles containing shRNAs that target ZNF804A mRNA. Stable transduced NPC lines were generated after puromycin selection. A control cell line expressing a random (scrambled) shRNA was also generated. Neuronal differentiation was induced, RNA was harvested after 14 days and transcriptome analysis was carried out using RNA-seq. 1815 genes were found to be differentially expressed at a nominally significant level (p<0.05); 809 decreased in expression in the KD samples, while 1106 increased. Of these, 370 achieved genome wide significance (FDR<0.05); 125 were lower in the KD samples, 245 were higher. Pathway analysis showed that genes involved in interferon-signaling were enriched among those that were down-regulated in the KD samples. Correspondingly, ZNF804A KD was found to affect interferon-alpha 2 (IFNA2)-mediated gene expression. The findings suggest that ZNF804A may affect a differentiating neuron's response to inflammatory cytokines, which is consistent with models of SZ and ASD that support a role for infectious disease, and/or autoimmunity in a subgroup of patients.

  2. Functional decreases in P2X7 receptors are associated with retinoic acid-induced neuronal differentiation of Neuro-2a neuroblastoma cells.

    PubMed

    Wu, Pei-Yu; Lin, Yu-Chia; Chang, Chia-Ling; Lu, Hsing-Tsen; Chin, Chia-Hsuan; Hsu, Tsan-Ting; Chu, Dachen; Sun, Synthia H

    2009-06-01

    Neuro-2a (N2a) cells are derived from spontaneous neuroblastoma of mouse and capable to differentiate into neuronal-like cells. Recently, P2X7 receptor has been shown to sustain growth of human neuroblastoma cells but its role during neuronal differentiation remains unexamined.We characterized the role of P2X7 receptors in the retinoic acid (RA)-differentiated N2a cells. RA induced N2a cells differentiation into neurite bearing and neuronal specific proteins, microtubule-associated protein 2 (MAP2) and neuronal specific nuclear protein (NeuN), expressing neuronal-like cells. Interestingly, the RA-induced neuronal differentiation was associated with decreases in the expression and function of P2X7 receptors. Functional inhibition of P2X7 receptors by P2X7 receptor selective antagonists, 5'-triphosphate, periodate-oxidized 2',3'-dialdehyde ATP (oATP), brilliant blue G (BBG) or A438079 induced neurite outgrowth. In addition, RA and oATP treatment stimulated the expression of neuron-specific class III beta-tubulin (TuJ1), and knockdown of P2X7 receptor expression by siRNA induced neurite outgrowth. To elucidate the possible mechanism, we found the levels of basal intracellular Ca2+ concentrations ([Ca2+]i) were decreased in either RA- or oATP-differentiated or P2X7receptor knockdown N2a cells. Simply cultured N2a cells in low Ca2+ medium induced a 2-fold increase in neurite length. Treatment of N2a cells with ATP hydrolase apyrase and the P2X7 receptors selective antagonist oATP or BBG decreased cell viability and cell number. Nevertheless, oATP but not BBG decreased cell proliferation and cell cycle progression. These results suggest for the first time that decreases in expression/function of P2X7 receptors are involved in neuronal differentiation.We provide additional evidence shown that the ATP release-activated P2X7 receptor is important in maintaining cell survival of N2a neuroblastoma cells.

  3. Organic Cation Transporter-Mediated Ergothioneine Uptake in Mouse Neural Progenitor Cells Suppresses Proliferation and Promotes Differentiation into Neurons

    PubMed Central

    Ishimoto, Takahiro; Nakamichi, Noritaka; Hosotani, Hiroshi; Masuo, Yusuke; Sugiura, Tomoko; Kato, Yukio

    2014-01-01

    The aim of the present study is to clarify the functional expression and physiological role in neural progenitor cells (NPCs) of carnitine/organic cation transporter OCTN1/SLC22A4, which accepts the naturally occurring food-derived antioxidant ergothioneine (ERGO) as a substrate in vivo. Real-time PCR analysis revealed that mRNA expression of OCTN1 was much higher than that of other organic cation transporters in mouse cultured cortical NPCs. Immunocytochemical analysis showed colocalization of OCTN1 with the NPC marker nestin in cultured NPCs and mouse embryonic carcinoma P19 cells differentiated into neural progenitor-like cells (P19-NPCs). These cells exhibited time-dependent [3H]ERGO uptake. These results demonstrate that OCTN1 is functionally expressed in murine NPCs. Cultured NPCs and P19-NPCs formed neurospheres from clusters of proliferating cells in a culture time-dependent manner. Exposure of cultured NPCs to ERGO or other antioxidants (edaravone and ascorbic acid) led to a significant decrease in the area of neurospheres with concomitant elimination of intracellular reactive oxygen species. Transfection of P19-NPCs with small interfering RNA for OCTN1 markedly promoted formation of neurospheres with a concomitant decrease of [3H]ERGO uptake. On the other hand, exposure of cultured NPCs to ERGO markedly increased the number of cells immunoreactive for the neuronal marker βIII-tubulin, but decreased the number immunoreactive for the astroglial marker glial fibrillary acidic protein (GFAP), with concomitant up-regulation of neuronal differentiation activator gene Math1. Interestingly, edaravone and ascorbic acid did not affect such differentiation of NPCs, in contrast to the case of proliferation. Knockdown of OCTN1 increased the number of cells immunoreactive for GFAP, but decreased the number immunoreactive for βIII-tubulin, with concomitant down-regulation of Math1 in P19-NPCs. Thus, OCTN1-mediated uptake of ERGO in NPCs inhibits cellular proliferation

  4. The MMP-1/PAR-1 Axis Enhances Proliferation and Neuronal Differentiation of Adult Hippocampal Neural Progenitor Cells

    PubMed Central

    Valente, Maria Maddalena; Allen, Megan; Bortolotto, Valeria; Lim, Seung T.; Conant, Katherine; Grilli, Mariagrazia

    2015-01-01

    Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that play a role in varied forms of developmental and postnatal neuroplasticity. MMP substrates include protease-activated receptor-1 (PAR-1), a G-protein coupled receptor expressed in hippocampus. We examined proliferation and differentiation of adult neural progenitor cells (aNPCs) from hippocampi of mice that overexpress the potent PAR-1 agonist MMP-1. We found that, as compared to aNPCs from littermate controls, MMP-1 tg aNPCs display enhanced proliferation. Under differentiating conditions, these cells give rise to a higher percentage of MAP-2+ neurons and a reduced number of oligodendrocyte precursors, and no change in the number of astrocytes. The fact that these results are MMP and PAR-1 dependent is supported by studies with distinct antagonists. Moreover, JSH-23, an inhibitor of NF-κB p65 nuclear translocation, counteracted both the proliferation and differentiation changes seen in MMP-1 tg-derived NPCs. In complementary studies, we found that the percentage of Sox2+ undifferentiated progenitor cells is increased in hippocampi of MMP-1 tg animals, compared to wt mice. Together, these results add to a growing body of data suggesting that MMPs are effectors of hippocampal neuroplasticity in the adult CNS and that the MMP-1/PAR-1 axis may play a role in neurogenesis following physiological and/or pathological stimuli. PMID:26783471

  5. Coordinated waves of gene expression during neuronal differentiation of embryonic stem cells as basis for novel approaches to developmental neurotoxicity testing

    PubMed Central

    Zimmer, B; Kuegler, P B; Baudis, B; Genewsky, A; Tanavde, V; Koh, W; Tan, B; Waldmann, T; Kadereit, S; Leist, M

    2011-01-01

    As neuronal differentiation of embryonic stem cells (ESCs) recapitulates embryonic neurogenesis, disturbances of this process may model developmental neurotoxicity (DNT). To identify the relevant steps of in vitro neurodevelopment, we implemented a differentiation protocol yielding neurons with desired electrophysiological properties. Results from focussed transcriptional profiling suggested that detection of non-cytotoxic developmental disturbances triggered by toxicants such as retinoic acid (RA) or cyclopamine was possible. Therefore, a broad transcriptional profile of the 20-day differentiation process was obtained. Cluster analysis of expression kinetics, and bioinformatic identification of overrepresented gene ontologies revealed waves of regulation relevant for DNT testing. We further explored the concept of superimposed waves as descriptor of ordered, but overlapping biological processes. The initial wave of transcripts indicated reorganization of chromatin and epigenetic changes. Then, a transient upregulation of genes involved in the formation and patterning of neuronal precursors followed. Simultaneously, a long wave of ongoing neuronal differentiation started. This was again superseded towards the end of the process by shorter waves of neuronal maturation that yielded information on specification, extracellular matrix formation, disease-associated genes and the generation of glia. Short exposure to lead during the final differentiation phase, disturbed neuronal maturation. Thus, the wave kinetics and the patterns of neuronal specification define the time windows and end points for examination of DNT. PMID:20865013

  6. Neuronal Differentiation and Extensive Migration of Human Neural Precursor Cells following Co-Culture with Rat Auditory Brainstem Slices

    PubMed Central

    Novozhilova, Ekaterina; Olivius, Petri; Siratirakun, Piyaporn; Lundberg, Cecilia; Englund-Johansson, Ulrica

    2013-01-01

    Congenital or acquired hearing loss is often associated with a progressive degeneration of the auditory nerve (AN) in the inner ear. The AN is composed of processes and axons of the bipolar spiral ganglion neurons (SGN), forming the connection between the hair cells in the inner ear cochlea and the cochlear nuclei (CN) in the brainstem (BS). Therefore, replacement of SGNs for restoring the AN to improve hearing function in patients who receive a cochlear implantation or have severe AN malfunctions is an attractive idea. A human neural precursor cell (HNPC) is an appropriate donor cell to investigate, as it can be isolated and expanded in vitro with maintained potential to form neurons and glia. We recently developed a post-natal rodent in vitro auditory BS slice culture model including the CN and the central part of the AN for initial studies of candidate cells. Here we characterized the survival, distribution, phenotypic differentiation, and integration capacity of HNPCs into the auditory circuitry in vitro. HNPC aggregates (spheres) were deposited adjacent to or on top of the BS slices or as a monoculture (control). The results demonstrate that co-cultured HNPCs compared to monocultures (1) survive better, (2) distribute over a larger area, (3) to a larger extent and in a shorter time-frame form mature neuronal and glial phenotypes. HNPC showed the ability to extend neurites into host tissue. Our findings suggest that the HNPC-BS slice co-culture is appropriate for further investigations on the integration capacity of HNPCs into the auditory circuitry. PMID:23505423

  7. Neuronal cell cycle: the neuron itself and its circumstances.

    PubMed

    Frade, José M; Ovejero-Benito, María C

    2015-01-01

    Neurons are usually regarded as postmitotic cells that undergo apoptosis in response to cell cycle reactivation. Nevertheless, recent evidence indicates the existence of a defined developmental program that induces DNA replication in specific populations of neurons, which remain in a tetraploid state for the rest of their adult life. Similarly, de novo neuronal tetraploidization has also been described in the adult brain as an early hallmark of neurodegeneration. The aim of this review is to integrate these recent developments in the context of cell cycle regulation and apoptotic cell death in neurons. We conclude that a variety of mechanisms exists in neuronal cells for G1/S and G2/M checkpoint regulation. These mechanisms, which are connected with the apoptotic machinery, can be modulated by environmental signals and the neuronal phenotype itself, thus resulting in a variety of outcomes ranging from cell death at the G1/S checkpoint to full proliferation of differentiated neurons.

  8. Modeling Human Neurological and Neurodegenerative Diseases: From Induced Pluripotent Stem Cells to Neuronal Differentiation and Its Applications in Neurotrauma

    PubMed Central

    Bahmad, Hisham; Hadadeh, Ola; Chamaa, Farah; Cheaito, Katia; Darwish, Batoul; Makkawi, Ahmad-Kareem; Abou-Kheir, Wassim

    2017-01-01

    With the help of several inducing factors, somatic cells can be reprogrammed to become induced pluripotent stem cell (iPSCs) lines. The success is in obtaining iPSCs almost identical to embryonic stem cells (ESCs), therefore various approaches have been tested and ultimately several ones have succeeded. The importance of these cells is in how they serve as models to unveil the molecular pathways and mechanisms underlying several human diseases, and also in its potential roles in the development of regenerative medicine. They further aid in the development of regenerative medicine, autologous cell therapy and drug or toxicity screening. Here, we provide a comprehensive overview of the recent development in the field of iPSCs research, specifically for modeling human neurological and neurodegenerative diseases, and its applications in neurotrauma. These are mainly characterized by progressive functional or structural neuronal loss rendering them extremely challenging to manage. Many of these diseases, including Parkinson's disease (PD), Huntington's disease (HD), Amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD) have been explored in vitro. The main purpose is to generate patient-specific iPS cell lines from the somatic cells that carry mutations or genetic instabilities for the aim of studying their differentiation potential and behavior. This new technology will pave the way for future development in the field of stem cell research anticipating its use in clinical settings and in regenerative medicine in order to treat various human diseases, including neurological and neurodegenerative diseases. PMID:28293168

  9. Healthy human CSF promotes glial differentiation of hESC-derived neural cells while retaining spontaneous activity in existing neuronal networks.

    PubMed

    Kiiski, Heikki; Aänismaa, Riikka; Tenhunen, Jyrki; Hagman, Sanna; Ylä-Outinen, Laura; Aho, Antti; Yli-Hankala, Arvi; Bendel, Stepani; Skottman, Heli; Narkilahti, Susanna

    2013-06-15

    The possibilities of human pluripotent stem cell-derived neural cells from the basic research tool to a treatment option in regenerative medicine have been well recognized. These cells also offer an interesting tool for in vitro models of neuronal networks to be used for drug screening and neurotoxicological studies and for patient/disease specific in vitro models. Here, as aiming to develop a reductionistic in vitro human neuronal network model, we tested whether human embryonic stem cell (hESC)-derived neural cells could be cultured in human cerebrospinal fluid (CSF) in order to better mimic the in vivo conditions. Our results showed that CSF altered the differentiation of hESC-derived neural cells towards glial cells at the expense of neuronal differentiation. The proliferation rate was reduced in CSF cultures. However, even though the use of CSF as the culture medium altered the glial vs. neuronal differentiation rate, the pre-existing spontaneous activity of the neuronal networks persisted throughout the study. These results suggest that it is possible to develop fully human cell and culture-based environments that can further be modified for various in vitro modeling purposes.

  10. Effects of brain-derived neurotrophic factor on cell survival, differentiation and patterning of neuronal connections and Müller glia cells in the developing retina.

    PubMed

    Pinzón-Duarte, Germán; Arango-González, Blanca; Guenther, Elke; Kohler, Konrad

    2004-03-01

    The aim of the present study was to determine the influence of brain-derived neurotrophic factor (BDNF) on survival, phenotype differentiation and network formation of retinal neurons and glia cells. To achieve a defined concentration and constant level of BDNF over several days, experiments were performed in an organotypic culture of the developing rat retina. After 6 days in vitro, apoptosis in the different cell layers was determined by TUNEL staining and cell-type-specific antibodies were used to identify distinct neuronal cell types and Müller cells. Cultured retinas treated with BDNF (100 ng BDNF/mL medium) were compared with untreated as well as with age-matched in vivo retinas. Quantitative morphometry was carried out using confocal microscopy. BDNF promoted the in vitro development and differentiation of the retina in general, i.e. the number of cells in the nuclear layers and the thickness of the plexiform layers were increased. For all neurons, the number of cells and the complexity of arborizations in the synaptic layers were clearly up-regulated by BDNF. In control cultures, the synaptic stratification of cone bipolar cells within the On- and Off-layer of the inner plexiform layer was disturbed and a strong reactivity of Müller cell glia was observed. These effects were not present in BDNF-treated cultures. Our data show that BDNF promotes the survival of retinal interneurons and plays an important role in establishing the phenotypes and the synaptic connections of a large number of neuronal types in the developing retina. Moreover, we show an effect of BDNF on Müller glia cells.

  11. Differential neurogenic effects of casein-derived opioid peptides on neuronal stem cells: implications for redox-based epigenetic changes.

    PubMed

    Trivedi, Malav; Zhang, Yiting; Lopez-Toledano, Miguel; Clarke, Andrew; Deth, Richard

    2016-11-01

    Food-derived peptides, such as β-casomorphin BCM7, have potential to cross the gastrointestinal tract and blood-brain barrier and are associated with neurological disorders and neurodevelopmental disorders. We previously established a novel mechanism through which BCM7 affects the antioxidant levels in neuronal cells leading to inflammatory consequences. In the current study, we elucidated the effects of casein-derived peptides on neuronal development by using the neurogenesis of neural stem cells (NSCs) as an experimental model. First, the transient changes in intracellular thiol metabolites during NSC differentiation (neurogenesis) were investigated. Next, the neurogenic effects of food-derived opioid peptides were measured, along with changes in intracellular thiol metabolites, redox status and global DNA methylation levels. We observed that the neurogenesis of NSCs was promoted by human BCM7 to a greater extent, followed by A2-derived BCM9 in contrast to bovine BCM7, which induced increased astrocyte formation. The effect was most apparent when human BCM7 was administered for 1day starting on 3days postplating, consistent with immunocytochemistry. Furthermore, neurogenic changes regulated by bovine BCM7 and morphine were associated with an increase in the glutathione/glutathione disulfide ratio and a decrease in the S-adenosylmethionine/S-adenosylhomocysteine ratio, indicative of changes in the redox and the methylation states. Finally, bovine BCM7 and morphine decreased DNA methylation in differentiating NSCs. In conclusion, these results suggest that food-derived opioid peptides and morphine regulated neurogenesis and differentiation of NSCs through changes in the redox state and epigenetic regulation.

  12. Dorsal root ganglia neurons and differentiated adipose-derived stem cells: an in vitro co-culture model to study peripheral nerve regeneration.

    PubMed

    de Luca, Alba C; Faroni, Alessandro; Reid, Adam J

    2015-02-26

    Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

  13. Biologically synthesized silver nanoparticles induce neuronal differentiation of SH-SY5Y cells via modulation of reactive oxygen species, phosphatases, and kinase signaling pathways.

    PubMed

    Dayem, Ahmed Abdal; Kim, BongWoo; Gurunathan, Sangiliyandi; Choi, Hye Yeon; Yang, Gwangmo; Saha, Subbroto Kumar; Han, Dawoon; Han, Jihae; Kim, Kyeongseok; Kim, Jin-Hoi; Cho, Ssang-Goo

    2014-07-01

    Nano-scale materials are noted for unique properties, distinct from those of their bulk material equivalents. In this study, we prepared spherical silver nanoparticles (AgNPs) with an average size of about 30 nm and tested their potency to induce neuronal differentiation of SH-SY5Y cells. Human neuroblastoma SH-SY5Y cells are considered an ideal in vitro model for studying neurogenesis, as they can be maintained in an undifferentiated state or be induced to differentiate into neuron-like phenotypes in vitro by several differentiation-inducing agents. Treatment of SH-SY5Y cells by biologically synthesized AgNPs led to cell morphological changes and significant increase in neurite length and enhanced the expression of neuronal differentiation markers such as Map-2, β-tubulin III, synaptophysin, neurogenin-1, Gap-43, and Drd-2. Furthermore, we observed an increase in generation of intracellular reactive oxygen species (ROS), activation of several kinases such as ERK and AKT, and downregulation of expression of dual-specificity phosphatases (DUSPs) in AgNPs-exposed SH-SY5Y cells. Our results suggest that AgNPs modulate the intracellular signaling pathways, leading to neuronal differentiation, and could be applied as promising nanomaterials for stem cell research and therapy.

  14. Adult human brain neural progenitor cells (NPCs) and fibroblast-like cells have similar properties in vitro but only NPCs differentiate into neurons.

    PubMed

    Park, Thomas In-Hyeup; Monzo, Hector; Mee, Edward W; Bergin, Peter S; Teoh, Hoon H; Montgomery, Johanna M; Faull, Richard L M; Curtis, Maurice A; Dragunow, Mike

    2012-01-01

    The ability to culture neural progenitor cells from the adult human brain has provided an exciting opportunity to develop and test potential therapies on adult human brain cells. To achieve a reliable and reproducible adult human neural progenitor cell (AhNPC) culture system for this purpose, this study fully characterized the cellular composition of the AhNPC cultures, as well as the possible changes to this in vitro system over prolonged culture periods. We isolated cells from the neurogenic subventricular zone/hippocampus (SVZ/HP) of the adult human brain and found a heterogeneous culture population comprised of several types of post-mitotic brain cells (neurons, astrocytes, and microglia), and more importantly, two distinct mitotic cell populations; the AhNPCs, and the fibroblast-like cells (FbCs). These two populations can easily be mistaken for a single population of AhNPCs, as they both proliferate under AhNPC culture conditions, form spheres and express neural progenitor cell and early neuronal markers, all of which are characteristics of AhNPCs in vitro. However, despite these similarities under proliferating conditions, under neuronal differentiation conditions, only the AhNPCs differentiated into functional neurons and glia. Furthermore, AhNPCs showed limited proliferative capacity that resulted in their depletion from culture by 5-6 passages, while the FbCs, which appear to be from a neurovascular origin, displayed a greater proliferative capacity and dominated the long-term cultures. This gradual change in cellular composition resulted in a progressive decline in neurogenic potential without the apparent loss of self-renewal in our cultures. These results demonstrate that while AhNPCs and FbCs behave similarly under proliferative conditions, they are two different cell populations. This information is vital for the interpretation and reproducibility of AhNPC experiments and suggests an ideal time frame for conducting AhNPC-based experiments.

  15. Nestin-expressing stem cells from the hair follicle can differentiate into motor neurons and reduce muscle atrophy after transplantation to injured nerves.

    PubMed

    Liu, Fang; Zhang, Chuansen; Hoffman, Robert M

    2014-02-01

    We have previously shown that nestin-expressing hair follicle stem cells from the mouse and human are multipotent and can differentiate into many cell types, including neurons and glial cells. The nestin-expressing hair follicle stem cells can effect nerve and spinal cord repair upon transplantation in mouse models. In the present study, nestin-expressing hair follicle stem cells expressing red fluorescent protein (RFP) were induced by retinoic acid and fetal bovine serum to differentiate and then transplanted together with Matrigel into the transected distal sciatic or tibial nerve stump of transgenic nude mice ubiquitously expressing green fluorescent protein (GFP). Control mice were transplanted with Matrigel only. The transplanted cells appeared neuron like, with large round nuclei and long extensions. Immunofluorescence staining showed that some of the transplanted cells in the distal nerve stump expressed the neuron marker Tuj1 as well as motor neuron markers Isl 1/2 and EN1. These transplanted cells contacted each other as well as host nerve fibers. Two weeks post-transplantation, nerve fibers in the distal sciatic nerve stump of the transplanted mice had greater expression of motor neuron markers and neurotrophic factor-3 than those in the Matrigel-only transplanted mice. Muscle fiber areas in the nestin-expressing stem cell plus Matrigel-transplanted animals were much bigger than that in the Matrigel-only transplanted animals after 4 weeks. The present results suggest that transplanted nestin-expressing hair follicle stem cells can differentiate into motor neurons and reduce muscle atrophy after sciatic nerve transection. This study demonstrates a new and accessible neuron source to reduce muscle atrophy after nerve injury.

  16. Inhibitor of PI3K/Akt Signaling Pathway Small Molecule Promotes Motor Neuron Differentiation of Human Endometrial Stem Cells Cultured on Electrospun Biocomposite Polycaprolactone/Collagen Scaffolds.

    PubMed

    Ebrahimi-Barough, Somayeh; Hoveizi, Elham; Yazdankhah, Meysam; Ai, Jafar; Khakbiz, Mehrdad; Faghihi, Faezeh; Tajerian, Roksana; Bayat, Neda

    2017-05-01

    Small molecules as useful chemical tools can affect cell differentiation and even change cell fate. It is demonstrated that LY294002, a small molecule inhibitor of phosphatidylinositol 3-kinase (PI3K)/Akt signal pathway, can inhibit proliferation and promote neuronal differentiation of mesenchymal stem cells (MSCs). The purpose of this study was to investigate the differentiation effect of Ly294002 small molecule on the human endometrial stem cells (hEnSCs) into motor neuron-like cells on polycaprolactone (PCL)/collagen scaffolds. hEnSCs were cultured in a neurogenic inductive medium containing 1 μM LY294002 on the surface of PCL/collagen electrospun fibrous scaffolds. Cell attachment and viability of cells on scaffolds were characterized by scanning electron microscope (SEM) and 3-(4,5-dimethylthiazoyl-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay. The expression of neuron-specific markers was assayed by real-time PCR and immunocytochemistry analysis after 15 days post induction. Results showed that attachment and differentiation of hEnSCs into motor neuron-like cells on the scaffolds with Ly294002 small molecule were higher than that of the cells on tissue culture plates as control group. In conclusion, PCL/collagen electrospun scaffolds with Ly294002 have potential for being used in neural tissue engineering because of its bioactive and three-dimensional structure which enhances viability and differentiation of hEnSCs into neurons through inhibition of the PI3K/Akt pathway. Thus, manipulation of this pathway by small molecules can enhance neural differentiation.

  17. Neurons generated from APP/APLP1/APLP2 triple knockout embryonic stem cells behave normally in vitro and in vivo: lack of evidence for a cell autonomous role of the amyloid precursor protein in neuronal differentiation.

    PubMed

    Bergmans, Bruno A; Shariati, S Ali M; Habets, Ron L P; Verstreken, Patrik; Schoonjans, Luc; Müller, Ulrike; Dotti, Carlos G; De Strooper, Bart

    2010-03-31

    Alzheimer's disease amyloid precursor protein (APP) has been implicated in many neurobiologic processes, but supporting evidence remains indirect. Studies are confounded by the existence of two partially redundant APP homologues, APLP1 and APLP2. APP/APLP1/APLP2 triple knockout (APP tKO) mice display cobblestone lissencephaly and are perinatally lethal. To circumvent this problem, we generated APP triple knockout embryonic stem (ES) cells and differentiated these to APP triple knockout neurons in vitro and in vivo. In comparison with wild-type (WT) ES cell-derived neurons, APP tKO neurons formed equally pure neuronal cultures, had unaltered in vitro migratory capacities, had a similar acquisition of polarity, and were capable of extending long neurites and forming active excitatory synapses. These data were confirmed in vivo in chimeric mice with APP tKO neurons expressing the enhanced green fluorescent protein (eGFP) present in a WT background brain. The results suggest that the loss of the APP family of proteins has no major effect on these critical neuronal processes and that the apparent multitude of functions in which APP has been implicated might be characterized by molecular redundancy. Our stem cell culture provides an excellent tool to circumvent the problem of lack of viability of APP/APLP triple knockout mice and will help to explore the function of this intriguing protein further in vitro and in vivo.

  18. Pre-B cell leukemia transcription factor (PBX) proteins are important mediators for retinoic acid-dependent endodermal and neuronal differentiation of mouse embryonal carcinoma P19 cells.

    PubMed

    Qin, Pu; Haberbusch, Juliet M; Zhang, Zhenping; Soprano, Kenneth J; Soprano, Dianne R

    2004-04-16

    Pre-B cell leukemia transcription factors (PBXs) act as cofactors in the transcriptional regulation mediated by Homeobox proteins during embryonic development and cellular differentition. PBX1 protein is expressed throughout murine embryonic development, and its deletion in mice disrupts chondrogenesis. PBX protein levels are also increased in mouse embryonal carcinoma P19 cells during retinoic acid (RA)-induced differentiation. To elucidate the role of PBX proteins in this process, we stably overexpressed PBX1b antisense mRNA in P19 cells (PBX1b-AS cells). PBX1b-AS cells did not differentiate to neuronal or endodermal cells following treatment with RA suggesting PBX proteins are required for both processes. Furthermore we demonstrated that PBX proteins regulate the RA-dependent induction in the mRNA levels of bone morphogenetic protein 4 (BMP4) and Decorin (DCN) in P19 cells using both PBX1b-AS cells and PBX1 small interfering RNA. Chromatin immunoprecipitation assays further demonstrated that PBX proteins directly bind to the promoter of Bmp4 and Dcn in vivo in a RA-dependent fashion. In addition, type I and type II BMP receptor mRNA levels were also increased in P19 cells following RA treatment; however, this was PBX-independent. Taken together these data demonstrate that PBX proteins are required for RA-induced differentiation of P19 cells and that PBX proteins regulate the expression of BMP4 and DCN during this differentiation process.

  19. Purmorphamine as a Shh Signaling Activator Small Molecule Promotes Motor Neuron Differentiation of Mesenchymal Stem Cells Cultured on Nanofibrous PCL Scaffold.

    PubMed

    Bahrami, Naghmeh; Bayat, Mohammad; Mohamadnia, Abdolreza; Khakbiz, Mehrdad; Yazdankhah, Meysam; Ai, Jafar; Ebrahimi-Barough, Somayeh

    2016-09-14

    There is variety of stem cell sources but problems in ethical issues, contamination, and normal karyotype cause many limitations in obtaining and using these cells. The cells in Wharton's jelly region of umbilical cord are abundant and available stem cells with low immunological incompatibility, which could be considered for cell replacement therapy. Small molecules have been presented as less expensive biologically active compounds that can regulate different developmental process. Purmorphamine (PMA) is a small molecule that, according to some studies, possesses certain differentiation effects. In this study, we investigated the effect of the PMA on Wharton's jelly mesenchymal stem cell (WJ-MSC) differentiation into motor neuronal lineages instead of sonic hedgehog (Shh) on PCL scaffold. After exposing to induction media for 15 days, the cells were characterized for expression of motor neuron markers including PAX6, NF-H, Islet1, HB9, and choline acetyl transferase (ChAT) by quantitative reverse transcription (PCR) and immunocytochemistry. Our results demonstrated that induced WJ-MSCs with PMA could significantly express motor neuron markers in RNA and protein levels 15 days post induction. These results suggested that WJ-MSCs can differentiate to motor neuron-like cells with PMA on PCL scaffold and might provide a potential source in cell therapy for nervous system.

  20. Wnts enhance neurotrophin-induced neuronal differentiation in adult bone-marrow-derived mesenchymal stem cells via canonical and noncanonical signaling pathways.

    PubMed

    Tsai, Hung-Li; Deng, Wing-Ping; Lai, Wen-Fu Thomas; Chiu, Wen-Ta; Yang, Charn-Bing; Tsai, Yu-Hui; Hwang, Shiaw-Min; Renshaw, Perry F

    2014-01-01

    Wnts were previously shown to regulate the neurogenesis of neural stem or progenitor cells. Here, we explored the underlying molecular mechanisms through which Wnt signaling regulates neurotrophins (NTs) in the NT-induced neuronal differentiation of human mesenchymal stem cells (hMSCs). NTs can increase the expression of Wnt1 and Wnt7a in hMSCs. However, only Wnt7a enables the expression of synapsin-1, a synaptic marker in mature neurons, to be induced and triggers the formation of cholinergic and dopaminergic neurons. Human recombinant (hr)Wnt7a and general neuron makers were positively correlated in a dose- and time-dependent manner. In addition, the expression of synaptic markers and neurites was induced by Wnt7a and lithium, a glycogen synthase kinase-3β inhibitor, in the NT-induced hMSCs via the canonical/β-catenin pathway, but was inhibited by Wnt inhibitors and frizzled-5 (Frz5) blocking antibodies. In addition, hrWnt7a triggered the formation of cholinergic and dopaminergic neurons via the non-canonical/c-jun N-terminal kinase (JNK) pathway, and the formation of these neurons was inhibited by a JNK inhibitor and Frz9 blocking antibodies. In conclusion, hrWnt7a enhances the synthesis of synapse and facilitates neuronal differentiation in hMSCS through various Frz receptors. These mechanisms may be employed widely in the transdifferentiation of other adult stem cells.

  1. Cell-type-specific and differentiation-status-dependent variations in cytotoxicity of tributyltin in cultured rat cerebral neurons and astrocytes.

    PubMed

    Oyanagi, Koshi; Tashiro, Tomoko; Negishi, Takayuki

    2015-08-01

    Tributyltin (TBT) is an organotin used as an anti-fouling agent for fishing nets and ships and it is a widespread environmental contaminant at present. There is an increasing concern about imperceptible but serious adverse effect(s) of exposure to chemicals existing in the environment on various organs and their physiological functions, e.g. brain and mental function. Here, so as to contribute to improvement of and/or advances in in vitro cell-based assay systems for evaluating brain-targeted adverse effect of chemicals, we tried to evaluate cell-type-specific and differentiation-status-dependent variations in the cytotoxicity of TBT towards neurons and astrocytes using the four culture systems differing in the relative abundance of these two types of cells; primary neuron culture (> 95% neurons), primary neuron-astrocyte (2 : 1) mix culture, primary astrocyte culture (> 95% astrocytes), and passaged astrocyte culture (100% proliferative astrocytes). Cell viability was measured at 48 hr after exposure to TBT in serum-free medium. IC50's of TBT were 198 nM in primary neuron culture, 288 nM in primary neuron-astrocyte mix culture, 2001 nM in primary astrocyte culture, and 1989 nM in passaged astrocyte culture. Furthermore, in primary neuron-astrocyte mix culture, vulnerability of neurons cultured along with astrocytes to TBT toxicity was lower than that of neurons cultured purely in primary neuron culture. On the other hand, astrocytes in primary neuron-astrocyte mix culture were considered to be more vulnerable to TBT than those in primary or passaged astrocyte culture. The present study demonstrated variable cytotoxicity of TBT in neural cells depending on the culture condition.

  2. Cochlear nucleus whole mount explants promote the differentiation of neuronal stem cells from the cochlear nucleus in co-culture experiments.

    PubMed

    Rak, Kristen; Völker, Johannes; Jürgens, Lukas; Völker, Christine; Frenz, Silke; Scherzad, Agmal; Schendzielorz, Philipp; Jablonka, Sibylle; Mlynski, Robert; Radeloff, Andreas; Hagen, Rudolf

    2015-08-07

    The cochlear nucleus is the first brainstem nucleus to receive sensory input from the cochlea. Depriving this nucleus of auditory input leads to cellular and molecular disorganization which may potentially be counteracted by the activation or application of stem cells. Neuronal stem cells (NSCs) have recently been identified in the neonatal cochlear nucleus and a persistent neurogenic niche was demonstrated in this brainstem nucleus until adulthood. The present work investigates whether the neurogenic environment of the cochlear nucleus can promote the survival of engrafted NSCs and whether cochlear nucleus-derived NSCs can differentiate into neurons and glia in brain tissue. Therefore, cochlear nucleus whole-mount explants were co-cultured with NSCs extracted from either the cochlear nucleus or the hippocampus and compared to a second environment using whole-mount explants from the hippocampus. Factors that are known to induce neuronal differentiation were also investigated in these NSC-explant experiments. NSCs derived from the cochlear nucleus engrafted in the brain tissue and differentiated into all cells of the neuronal lineage. Hippocampal NSCs also immigrated in cochlear nucleus explants and differentiated into neurons, astrocytes and oligodendrocytes. Laminin expression was up-regulated in the cochlear nucleus whole-mounts and regulated the in vitro differentiation of NSCs from the cochlear nucleus. These experiments confirm a neurogenic environment in the cochlear nucleus and the capacity of cochlear nucleus-derived NSCs to differentiate into neurons and glia. Consequently, the presented results provide a first step for the possible application of stem cells to repair the disorganization of the cochlear nucleus, which occurs after hearing loss. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Crosstalk between HIF-1 and ROCK pathways in neuronal differentiation of mesenchymal stem cells, neurospheres and in PC12 neurite outgrowth.

    PubMed

    Pacary, Emilie; Tixier, Emmanuelle; Coulet, Florence; Roussel, Simon; Petit, Edwige; Bernaudin, Myriam

    2007-07-01

    This study demonstrates that the Rho-kinase (ROCK) inhibitor, Y-27632, potentiates not only the effect of cobalt chloride (CoCl(2)) but also that of deferoxamine, another HIF-1 inducer, on mesenchymal stem cell (MSC) neuronal differentiation. HIF-1 is essential for CoCl(2)+/-Y-27632-induced MSC neuronal differentiation, since agents inhibiting HIF-1 abolish the changes of morphology and cell cycle arrest-related gene or protein expressions (p21, cyclin D1) and the increase of neuronal marker expressions (Tuj1, NSE). Y-27632 potentiates the CoCl(2)-induced decrease of cyclin D1 and nestin expressions, the increase of HIF-1 activation and EPO expression, and decreases pVHL expression. Interestingly, CoCl(2) decreases RhoA expression, an effect potentiated by Y-27632, revealing crosstalk between HIF-1 and RhoA/ROCK pathways. Moreover, we demonstrate a synergistic effect of CoCl(2) and Y-27632 on neurosphere differentiation into neurons and PC12 neurite outgrowth underlining that a co-treatment targeting both HIF-1 and ROCK pathways might be relevant to differentiate stem cells into neurons.

  4. Quinazoline-based tricyclic compounds that regulate programmed cell death, induce neuronal differentiation, and are curative in animal models for excitotoxicity and hereditary brain disease

    PubMed Central

    Vainshtein, A; Veenman, L; Shterenberg, A; Singh, S; Masarwa, A; Dutta, B; Island, B; Tsoglin, E; Levin, E; Leschiner, S; Maniv, I; Pe’er, L; Otradnov, I; Zubedat, S; Aga-Mizrachi, S; Weizman, A; Avital, A; Marek, I; Gavish, M

    2015-01-01

    Expanding on a quinazoline scaffold, we developed tricyclic compounds with biological activity. These compounds bind to the 18 kDa translocator protein (TSPO) and protect U118MG (glioblastoma cell line of glial origin) cells from glutamate-induced cell death. Fascinating, they can induce neuronal differentiation of PC12 cells (cell line of pheochromocytoma origin with neuronal characteristics) known to display neuronal characteristics, including outgrowth of neurites, tubulin expression, and NeuN (antigen known as ‘neuronal nuclei’, also known as Rbfox3) expression. As part of the neurodifferentiation process, they can amplify cell death induced by glutamate. Interestingly, the compound 2-phenylquinazolin-4-yl dimethylcarbamate (MGV-1) can induce expansive neurite sprouting on its own and also in synergy with nerve growth factor and with glutamate. Glycine is not required, indicating that N-methyl-D-aspartate receptors are not involved in this activity. These diverse effects on cells of glial origin and on cells with neuronal characteristics induced in culture by this one compound, MGV-1, as reported in this article, mimic the diverse events that take place during embryonic development of the brain (maintenance of glial integrity, differentiation of progenitor cells to mature neurons, and weeding out of non-differentiating progenitor cells). Such mechanisms are also important for protective, curative, and restorative processes that occur during and after brain injury and brain disease. Indeed, we found in a rat model of systemic kainic acid injection that MGV-1 can prevent seizures, counteract the process of ongoing brain damage, including edema, and restore behavior defects to normal patterns. Furthermore, in the R6-2 (transgenic mouse model for Huntington disease; Strain name: B6CBA-Tg(HDexon1)62Gpb/3J) transgenic mouse model for Huntington disease, derivatives of MGV-1 can increase lifespan by >20% and reduce incidence of abnormal movements. Also in vitro

  5. Corexit-EC9527A Disrupts Retinol Signaling and Neuronal Differentiation in P19 Embryonal Pluripotent Cells

    DOE PAGES

    Chen, Yanling; Reese, David H.; Kelly, Gregory M.

    2016-09-29

    Corexit-EC9500A and Corexit-EC9527A are two chemical dispersants that have been used to remediate the impact of the 2010 Deepwater Horizon oil spill. Both dispersants are composed primarily of organic solvents and surfactants and act by emulsifying the crude oil to facilitate biodegradation. The potential adverse effect of the Corexit chemicals on mammalian embryonic development remains largely unknown. Retinol (vitamin A) signaling, mediated by all-trans retinoic acid (RA), is essential for neural tube formation and the development of many organs in the embryo. The physiological levels of RA in cells and tissues are maintained by the retinol signaling pathway (RSP), whichmore » controls the biosynthesis of RA from dietary retinol and the catabolism of RA to polar metabolites for removal. RA is a potent activating ligand for the RAR/RXR nuclear receptors. Through RA and the receptors, the RSP modulates the expression of many developmental genes; interference with the RSP is potentially teratogenic. In this study the mouse P19 embryonal pluripotent cell, which contains a functional RSP, was used to evaluate the effects of the Corexit dispersants on retinol signaling and associated neuronal differentiation. The results showed that Corexit-EC9500A was more cytotoxic than Corexit-EC9527A to P19 cells. At non-cytotoxic doses, Corexit-EC9527A inhibited retinol-induced expression of the Hoxa1 gene, which encodes a transcription factor for the regulation of body patterning in the embryo. Such inhibition was seen in the retinol- and retinal- induced, but not RA-induced, Hoxa1 up-regulation, indicating that the Corexit chemicals primarily inhibit RA biosynthesis from retinal. In addition, Corexit-EC9527A suppressed retinol-induced P19 cell differentiation into neuronal cells, indicating potential neurotoxic effect of the chemicals under the tested conditions. In conclusion, the surfactant ingredient, dioctyl sodium sulfosuccinate (DOSS), may be a major contributor to the

  6. Corexit-EC9527A Disrupts Retinol Signaling and Neuronal Differentiation in P19 Embryonal Pluripotent Cells

    PubMed Central

    Chen, Yanling; Reese, David H.

    2016-01-01

    Corexit-EC9500A and Corexit-EC9527A are two chemical dispersants that have been used to remediate the impact of the 2010 Deepwater Horizon oil spill. Both dispersants are composed primarily of organic solvents and surfactants and act by emulsifying the crude oil to facilitate biodegradation. The potential adverse effect of the Corexit chemicals on mammalian embryonic development remains largely unknown. Retinol (vitamin A) signaling, mediated by all-trans retinoic acid (RA), is essential for neural tube formation and the development of many organs in the embryo. The physiological levels of RA in cells and tissues are maintained by the retinol signaling pathway (RSP), which controls the biosynthesis of RA from dietary retinol and the catabolism of RA to polar metabolites for removal. RA is a potent activating ligand for the RAR/RXR nuclear receptors. Through RA and the receptors, the RSP modulates the expression of many developmental genes; interference with the RSP is potentially teratogenic. In this study the mouse P19 embryonal pluripotent cell, which contains a functional RSP, was used to evaluate the effects of the Corexit dispersants on retinol signaling and associated neuronal differentiation. The results showed that Corexit-EC9500A was more cytotoxic than Corexit-EC9527A to P19 cells. At non-cytotoxic doses, Corexit-EC9527A inhibited retinol-induced expression of the Hoxa1 gene, which encodes a transcription factor for the regulation of body patterning in the embryo. Such inhibition was seen in the retinol- and retinal- induced, but not RA-induced, Hoxa1 up-regulation, indicating that the Corexit chemicals primarily inhibit RA biosynthesis from retinal. In addition, Corexit-EC9527A suppressed retinol-induced P19 cell differentiation into neuronal cells, indicating potential neurotoxic effect of the chemicals under the tested conditions. The surfactant ingredient, dioctyl sodium sulfosuccinate (DOSS), may be a major contributor to the observed effect of

  7. Corexit-EC9527A Disrupts Retinol Signaling and Neuronal Differentiation in P19 Embryonal Pluripotent Cells

    SciTech Connect

    Chen, Yanling; Reese, David H.; Kelly, Gregory M.

    2016-09-29

    Corexit-EC9500A and Corexit-EC9527A are two chemical dispersants that have been used to remediate the impact of the 2010 Deepwater Horizon oil spill. Both dispersants are composed primarily of organic solvents and surfactants and act by emulsifying the crude oil to facilitate biodegradation. The potential adverse effect of the Corexit chemicals on mammalian embryonic development remains largely unknown. Retinol (vitamin A) signaling, mediated by all-trans retinoic acid (RA), is essential for neural tube formation and the development of many organs in the embryo. The physiological levels of RA in cells and tissues are maintained by the retinol signaling pathway (RSP), which controls the biosynthesis of RA from dietary retinol and the catabolism of RA to polar metabolites for removal. RA is a potent activating ligand for the RAR/RXR nuclear receptors. Through RA and the receptors, the RSP modulates the expression of many developmental genes; interference with the RSP is potentially teratogenic. In this study the mouse P19 embryonal pluripotent cell, which contains a functional RSP, was used to evaluate the effects of the Corexit dispersants on retinol signaling and associated neuronal differentiation. The results showed that Corexit-EC9500A was more cytotoxic than Corexit-EC9527A to P19 cells. At non-cytotoxic doses, Corexit-EC9527A inhibited retinol-induced expression of the Hoxa1 gene, which encodes a transcription factor for the regulation of body patterning in the embryo. Such inhibition was seen in the retinol- and retinal- induced, but not RA-induced, Hoxa1 up-regulation, indicating that the Corexit chemicals primarily inhibit RA biosynthesis from retinal. In addition, Corexit-EC9527A suppressed retinol-induced P19 cell differentiation into neuronal cells, indicating potential neurotoxic effect of the chemicals under the tested conditions. In conclusion, the surfactant ingredient, dioctyl sodium sulfosuccinate (DOSS), may be a major

  8. ZNF804A Transcriptional Networks in Differentiating Neurons Derived from Induced Pluripotent Stem Cells of Human Origin

    PubMed Central

    Hrabovsky, Anastasia; Pedrosa, Erika; Dean, Jason; Jain, Swati; Zheng, Deyou; Lachman, Herbert M.

    2015-01-01

    ZNF804A (Zinc Finger Protein 804A) has been identified as a candidate gene for schizophrenia (SZ), autism spectrum disorders (ASD), and bipolar disorder (BD) in replicated genome wide association studies (GWAS) and by copy number variation (CNV) analysis. Although its function has not been well-characterized, ZNF804A contains a C2H2-type zinc-finger domain, suggesting that it has DNA binding properties, and consequently, a role in regulating gene expression. To further explore the role of ZNF804A on gene expression and its downstream targets, we used a gene knockdown (KD) approach to reduce its expression in neural progenitor cells (NPCs) derived from induced pluripotent stem cells (iPSCs). KD was accomplished by RNA interference (RNAi) using lentiviral particles containing shRNAs that target ZNF804A mRNA. Stable transduced NPC lines were generated after puromycin selection. A control cell line expressing a random (scrambled) shRNA was also generated. Neuronal differentiation was induced, RNA was harvested after 14 days and transcriptome analysis was carried out using RNA-seq. 1815 genes were found to be differentially expressed at a nominally significant level (p<0.05); 809 decreased in expression in the KD samples, while 1106 increased. Of these, 370 achieved genome wide significance (FDR<0.05); 125 were lower in the KD samples, 245 were higher. Pathway analysis showed that genes involved in interferon-signaling were enriched among those that were down-regulated in the KD samples. Correspondingly, ZNF804A KD was found to affect interferon-alpha 2 (IFNA2)-mediated gene expression. The findings suggest that ZNF804A may affect a differentiating neuron’s response to inflammatory cytokines, which is consistent with models of SZ and ASD that support a role for infectious disease, and/or autoimmunity in a subgroup of patients. PMID:25905630

  9. The hematopoietic factor GM-CSF (granulocyte-macrophage colony-stimulating factor) promotes neuronal differentiation of adult neural stem cells in vitro.

    PubMed

    Krüger, Carola; Laage, Rico; Pitzer, Claudia; Schäbitz, Wolf-Rüdiger; Schneider, Armin

    2007-10-22

    Granulocyte-macrophage colony stimulating factor (GM-CSF) is a hematopoietic growth factor involved in the generation of granulocytes, macrophages, and dendritic cells from hematopoietic progenitor cells. We have recently demonstrated that GM-CSF has anti-apoptotic functions on neurons, and is neuroprotective in animal stroke models. The GM-CSF receptor alpha is expressed on adult neural stem cells in the rodent brain, and in culture. Addition of GM-CSF to NSCs in vitro increased neuronal differentiation in a dose-dependent manner as determined by quantitative PCR, reporter gene assays, and FACS analysis. Similar to the hematopoietic factor Granulocyte-colony stimulating factor (G-CSF), GM-CSF stimulates neuronal differentiation of adult NSCs. These data highlight the astonishingly similar functions of major hematopoietic factors in the brain, and raise the clinical attractiveness of GM-CSF as a novel drug for neurological disorders.

  10. The cellular form of the prion protein guides the differentiation of human embryonic stem cells into neuron-, oligodendrocyte-, and astrocyte-committed lineages.

    PubMed

    Lee, Young Jin; Baskakov, Ilia V

    2014-01-01

    Prion protein, PrP(C), is a glycoprotein that is expressed on the cell surface beginning with the early stages of embryonic stem cell differentiation. Previously, we showed that ectopic expression of PrP(C) in human embryonic stem cells (hESCs) triggered differentiation toward endodermal, mesodermal, and ectodermal lineages, whereas silencing of PrP(C) suppressed differentiation toward ectodermal but not endodermal or mesodermal lineages. Considering that PrP(C) might be involved in controlling the balance between cells of different lineages, the current study was designed to test whether PrP(C) controls differentiation of hESCs into cells of neuron-, oligodendrocyte-, and astrocyte-committed lineages. PrP(C) was silenced in hESCs cultured under three sets of conditions that were previously shown to induce hESCs differentiation into predominantly neuron-, oligodendrocyte-, and astrocyte-committed lineages. We found that silencing of PrP(C) suppressed differentiation toward all three lineages. Similar results were observed in all three protocols, arguing that the effect of PrP(C) was independent of differentiation conditions employed. Moreover, switching PrP(C) expression during a differentiation time course revealed that silencing PrP(C) expression during the very initial stage that corresponds to embryonic bodies has a more significant impact than silencing at later stages of differentiation. The current work illustrates that PrP(C) controls differentiation of hESCs toward neuron-, oligodendrocyte-, and astrocyte-committed lineages and is likely involved at the stage of uncommitted neural progenitor cells rather than lineage-committed neural progenitors.

  11. Neural Precursors Derived from Embryonic Stem Cells, but Not Those from Fetal Ventral Mesencephalon, Maintain the Potential to Differentiate into Dopaminergic Neurons After Expansion In Vitro

    PubMed Central

    Chung, Sangmi; Shin, Byoung-Soo; Hwang, Michelle; Lardaro, Thomas; Kang, Un Jung; Isacson, Ole; Kima, Kwang-Soo

    2008-01-01

    Neural precursors (NPs) derived from ventral mesencephalon (VM) normally generate dopaminergic (DA) neurons in vivo but lose their potential to differentiate into DA neurons during mitogenic expansion in vitro, hampering their efficient use as a transplantable and experimental cell source. Because embryonic stem (ES) cell-derived NPs (ES NP) do not go through the same maturation process during in vitro expansion, we hypothesized that expanded ES NPs may maintain their potential to differentiate into DA neurons. To address this, we expanded NPs derived from mouse embryonic day-12.5 (E12.5) VM or ES cells and compared their developmental properties. Interestingly, expanded ES NPs fully sustain their ability to differentiate to the neuronal as well as to the DA fate. In sharp contrast, VM NPs almost completely lost their ability to become neurons and tyrosine hydroxylase-positive (TH+) neurons after expansion. Expanded ES NP-derived TH+ neurons coexpressed additional DA markers such as dopa decarboxylase and DAT (dopamine transporter). Furthermore, they also expressed other midbrain DA markers, including Nurr1 and Pitx3, and released significant amounts of DA. We also found that these ES NPs can be cryopreserved without losing their proliferative and developmental potential. Finally, we tested the in vivo characteristics of the expanded NPs derived from J1 ES cells with low passage number. When transplanted into the mouse striatum, the expanded NPs as well as control NPs efficiently generated DA neurons expressing mature DA markers, with approximately 10% tumor formation in both cases. We conclude that ES NPs maintain their developmental potential during in vitro expansion, whereas mouse E12.5 VM NPs do not. PMID:16543488

  12. Reevaluation of in vitro differentiation protocols for bone marrow stromal cells: disruption of actin cytoskeleton induces rapid morphological changes and mimics neuronal phenotype.

    PubMed

    Neuhuber, Birgit; Gallo, Gianluca; Howard, Linda; Kostura, Lisa; Mackay, Alastair; Fischer, Itzhak

    2004-07-15

    Bone marrow stromal cells (MSC), which represent a population of multipotential mesenchymal stem cells, have been reported to undergo rapid and robust transformation into neuron-like phenotypes in vitro following treatment with chemical induction medium including dimethyl sulfoxide (DMSO; Woodbury et al. [2002] J. Neurosci. Res. 96:908). In this study, we confirmed the ability of cultured rat MSC to undergo in vitro osteogenesis, chondrogenesis, and adipogenesis, demonstrating differentiation of these cells to three mesenchymal cell fates. We then evaluated the potential for in vitro neuronal differentiation of these MSC, finding that changes in morphology upon addition of the chemical induction medium were caused by rapid disruption of the actin cytoskeleton. Retraction of the cytoplasm left behind long processes, which, although strikingly resembling neurites, showed essentially no motility and no further elaboration during time-lapse studies. Similar neurite-like processes were induced by treating MSC with DMSO only or with actin filament-depolymerizing agents. Although process formation was accompanied by rapid expression of some neuronal and glial markers, the absence of other essential neuronal proteins pointed toward aberrantly induced gene expression rather than toward a sequence of gene expression as is required for neurogenesis. Moreover, rat dermal fibroblasts responded to neuronal induction by forming similar processes and expressing similar markers. These studies do not rule out the possibility that MSC can differentiate into neurons; however, we do want to caution that in vitro differentiation protocols may have unexpected, misleading effects. A dissection of molecular signaling and commitment events may be necessary to verify the ability of MSC transdifferentiation to neuronal lineages. Copyright 2004 Wiley-Liss, Inc.

  13. Elevated α-synuclein caused by SNCA gene triplication impairs neuronal differentiation and maturation in Parkinson's patient-derived induced pluripotent stem cells

    PubMed Central

    Oliveira, L M A; Falomir-Lockhart, L J; Botelho, M G; Lin, K-H; Wales, P; Koch, J C; Gerhardt, E; Taschenberger, H; Outeiro, T F; Lingor, P; Schüle, B; Arndt-Jovin, D J; Jovin, T M

    2015-01-01

    We have assessed the impact of α-synuclein overexpression on the differentiation potential and phenotypic signatures of two neural-committed induced pluripotent stem cell lines derived from a Parkinson's disease patient with a triplication of the human SNCA genomic locus. In parallel, comparative studies were performed on two control lines derived from healthy individuals and lines generated from the patient iPS-derived neuroprogenitor lines infected with a lentivirus incorporating a small hairpin RNA to knock down the SNCA mRNA. The SNCA triplication lines exhibited a reduced capacity to differentiate into dopaminergic or GABAergic neurons and decreased neurite outgrowth and lower neuronal activity compared with control cultures. This delayed maturation phenotype was confirmed by gene expression profiling, which revealed a significant reduction in mRNA for genes implicated in neuronal differentiation such as delta-like homolog 1 (DLK1), gamma-aminobutyric acid type B receptor subunit 2 (GABABR2), nuclear receptor related 1 protein (NURR1), G-protein-regulated inward-rectifier potassium channel 2 (GIRK-2) and tyrosine hydroxylase (TH). The differentiated patient cells also demonstrated increased autophagic flux when stressed with chloroquine. We conclude that a two-fold overexpression of α-synuclein caused by a triplication of the SNCA gene is sufficient to impair the differentiation of neuronal progenitor cells, a finding with implications for adult neurogenesis and Parkinson's disease progression, particularly in the context of bioenergetic dysfunction. PMID:26610207

  14. Elevated α-synuclein caused by SNCA gene triplication impairs neuronal differentiation and maturation in Parkinson's patient-derived induced pluripotent stem cells.

    PubMed

    Oliveira, L M A; Falomir-Lockhart, L J; Botelho, M G; Lin, K-H; Wales, P; Koch, J C; Gerhardt, E; Taschenberger, H; Outeiro, T F; Lingor, P; Schüle, B; Arndt-Jovin, D J; Jovin, T M

    2015-11-26

    We have assessed the impact of α-synuclein overexpression on the differentiation potential and phenotypic signatures of two neural-committed induced pluripotent stem cell lines derived from a Parkinson's disease patient with a triplication of the human SNCA genomic locus. In parallel, comparative studies were performed on two control lines derived from healthy individuals and lines generated from the patient iPS-derived neuroprogenitor lines infected with a lentivirus incorporating a small hairpin RNA to knock down the SNCA mRNA. The SNCA triplication lines exhibited a reduced capacity to differentiate into dopaminergic or GABAergic neurons and decreased neurite outgrowth and lower neuronal activity compared with control cultures. This delayed maturation phenotype was confirmed by gene expression profiling, which revealed a significant reduction in mRNA for genes implicated in neuronal differentiation such as delta-like homolog 1 (DLK1), gamma-aminobutyric acid type B receptor subunit 2 (GABABR2), nuclear receptor related 1 protein (NURR1), G-protein-regulated inward-rectifier potassium channel 2 (GIRK-2) and tyrosine hydroxylase (TH). The differentiated patient cells also demonstrated increased autophagic flux when stressed with chloroquine. We conclude that a two-fold overexpression of α-synuclein caused by a triplication of the SNCA gene is sufficient to impair the differentiation of neuronal progenitor cells, a finding with implications for adult neurogenesis and Parkinson's disease progression, particularly in the context of bioenergetic dysfunction.

  15. Moclobemide upregulated Bcl-2 expression and induced neural stem cell differentiation into serotoninergic neuron via extracellular-regulated kinase pathway

    PubMed Central

    Chiou, Shih-Hwa; Ku, Hung-Hai; Tsai, Tung-Hu; Lin, Heng-Liang; Chen, Li-Hsin; Chien, Chan-Shiu; Ho, Larry L -T; Lee, Chen-Hsen; Chang, Yuh-Lih

    2006-01-01

    Moclobemide (MB) is an antidepressant drug that selectively and reversibly inhibits monoamine oxidase-A. Recent studies have revealed that antidepressant drugs possess the characters of potent growth-promoting factors for the development of neurogenesis and improve the survival rate of serotonin (5-hydroxytrytamine; 5-HT) neurons. However, whether MB comprises neuroprotection effects or modulates the proliferation of neural stem cells (NSCs) needs to be elucidated. In this study, firstly, we used the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay to demonstrate that 50 μM MB can increase the cell viability of NSCs. The result of real-time reverse transcription–polymerase chain reaction (RT–PCR) showed that the induction of MB can upregulate the gene expressions of Bcl-2 and Bcl-xL. By using caspases 8 and 3, ELISA and terminal dUTP nick-end labeling (TUNEL) assay, our data further confirmed that 50 μM MB-treated NSCs can prevent FasL-induced apoptosis. The morphological findings also supported the evidence that MB can facilitate the dendritic development and increase the neurite expansion of NSCs. Moreover, we found that MB treatment increased the expression of Bcl-2 in NSCs through activating the extracellular-regulated kinase (ERK) phosphorylation. By using the triple-staining immunofluorescent study, the percentages of serotonin- and MAP-2-positive cells in the day 7 culture of MB-treated NSCs were significantly increased (P<0.01). Furthermore, our data supported that MB treatment increased functional production of serotonin in NSCs via the modulation of ERK1/2. In sum, the study results support that MB can upregulate Bcl-2 expression and induce the differentiation of NSCs into serotoninergic neuron via ERK pathway. PMID:16702990

  16. In Vitro Differentiation of Bone Marrow Mesenchymal Stem Cells into Neuron-Like Cells by Cerebrospinal Fluid Improves Motor Function of Middle Cerebral Artery Occlusion Rats

    PubMed Central

    Ye, Ying; Peng, Yi-ran; Hu, Shu-qun; Yan, Xian-liang; Chen, Juan; Xu, Tie

    2016-01-01

    Bone marrow mesenchymal stem cells (BMSCs) represent a promising tool for stem cell-based therapies. However, the majority of BMSC transplants only allow for limited recovery of the lost functions. We previously found that human cerebrospinal fluid (hCSF) is more potent than growth factors in differentiating human BMSCs into neuron-like cells in vitro. In this study, we studied the effect of transplantation of rat BMSC-derived neuron-like cells (BMSC-Ns) induced by hCSF into rat brain with middle cerebral artery occlusion (MCAO). The survival and differentiation of the transplanted cells were determined using immunofluorescence staining of bromodeoxyuridine. The recovery of neurological function were observed by the modified neurological severity score (modified NSS) at 4, 15, and 32 days after cell transplantation, HE staining for determination of the infarct volume at day 32 after cell transplantation. Transplantation of BMSC-Ns or BMSCs significantly improved indexes of neurological function and reduced infarct size in rats previously subjected to MCAO compared with those in the control group. Remarkably, 32 days after transplantation, rats treated with BMSC-Ns presented a smaller infarct size, higher number of neuron-specific, enolase-positive, and BrdU-positive cells, and improved neurological function compared with BMSC group. Our results demonstrate that transplantation of hCSF-treated BMSC-Ns significantly improves neurological function and reduces infarct size in rats subjected to MCAO. This study may pave a new avenue for the treatment of MCAO. PMID:27833584

  17. Overexpression of β-NGF promotes differentiation of bone marrow mesenchymal stem cells into neurons through regulation of AKT and MAPK pathway.

    PubMed

    Yuan, Jun; Huang, Guorong; Xiao, Zhe; Lin, Lvbiao; Han, Tianwang

    2013-11-01

    Bone marrow stromal stem cells (BMSCs) are fibroblastic in shape and capable of self-renewal and have the potential for multi-directional differentiation. Nerve growth factor (NGF), a homodimeric polypeptide, plays an important role in the nervous system by supporting the survival and growth of neural cells, regulating cell growth, promoting differentiation into neuron, and neuron migration. Adenoviral vectors are DNA viruses that contain 36 kb of double-stranded DNA allowing for transmission of the genes to the host nucleus but not inserting them into the host chromosome. The present study aimed to investigate the induction efficiency and differentiation of neural cells from BMSCs by β-NGF gene transfection with recombinant adenoviral vector (Ad-β-NGF) in vitro. The results of immunochemical assay confirmed the induced cells as neuron cells. Moreover, flow cytometric analysis, Annexin-V-FITC/PI, and BrdU assay revealed that chemical inducer β-mercaptoethanol (β-met) triggered apoptosis of BMSCs, as evidenced by inhibition of DNA fragmentation, nuclear condensation, translocation of phospholipid phosphatidylserine, and activation of caspase-3. Furthermore, the results of western blotting showed that β-met suppressed AKT signaling pathway and regulated the MAPKs during differentiation of BMSCs. In contrast, Ad-β-NGF effectively induced the differentiation of BMSCs without causing any cytopathic phenomenon and apoptotic cell death. Moreover, Ad-β-NGF recovered the expression level of phosphorylated AKT and MAPKs in cells exposed to chemical reagents. Taken together, these results suggest that β-NGF gene transfection promotes the differentiation of BMSCs into neurons through regulation of AKT and MAPKs signaling pathways.

  18. Epigenetic regulation of Dpp6 expression by Dnmt3b and its novel role in the inhibition of RA induced neuronal differentiation of P19 cells.

    PubMed

    Sheikh, Muhammad Abid; Malik, Yousra Saeed; Yu, Huali; Lai, Mingming; Wang, Xingzhi; Zhu, Xiaojuan

    2013-01-01

    DNA methylation is an important mechanism of gene silencing in mammals catalyzed by a group of DNA methyltransferases including Dnmt1, Dnmt3a, and Dnmt3b which are required for the establishment of genomic methylation patterns during development and differentiation. In this report, we studied the role of DNA methyltransferases during retinoic acid induced neuronal differentiation of P19 cells. We observed an increase in the mRNA and protein level of Dnmt3b, whereas the expression of Dnmt1 and Dnmt3a was decreased after RA treatment of P19 cells which indicated that Dnmt3b is more important during neuronal differentiation of P19 cells. Dnmt3b enriched chromatin library from RA treated P19 cells identified dipeptidyl peptidase 6 (Dpp6) gene as a novel target of Dnmt3b. Further, quantitative ChIP analysis showed that the amount of Dnmt3b recruited on Dpp6 promoter was equal in both RA treated as well as untreated p19 cells. Bisulfite genomic sequencing, COBRA, and methylation specific PCR analysis revealed that Dpp6 promoter was heavily methylated in both RA treated and untreated P19 cells. Dnmt3b was responsible for transcriptional silencing of Dpp6 gene as depletion of Dnmt3b resulted in increased mRNA and protein expression of Dpp6. Consequently, the average methylation of Dpp6 gene promoter was reduced to half in Dnmt3b knockdown cells. In the absence of Dnmt3b, Dnmt3a was associated with Dpp6 gene promoter and regulated its expression and methylation in P19 cells. RA induced neuronal differentiation was inhibited upon ectopic expression of Dpp6 in P19 cells. Taken together, the present study described epigenetic silencing of Dpp6 expression by DNA methylation and established that its ectopic expression can act as negative signal during RA induced neuronal differentiation of P19 cells.

  19. The Differentiation of Human Endometrial Stem Cells into Neuron-Like Cells on Electrospun PAN-Derived Carbon Nanofibers with Random and Aligned Topographies.

    PubMed

    Mirzaei, Esmaeil; Ai, Jafar; Ebrahimi-Barough, Somayeh; Verdi, Javad; Ghanbari, Hossein; Faridi-Majidi, Reza

    2016-09-01

    Electrospun carbon nanofibers (CNFs) have great potential for applications in neural tissue regeneration due to their electrical conductivity, biocompatibility, and morphological similarity to natural extracellular matrix. In this study, we cultured human endometrial stem cells (hEnSCs) on electrospun CNFs with random and aligned topographies and demonstrated that hEnSCs could attach, proliferate, and differentiate into neural cells on both random and aligned CNFs. However, the proliferation, differentiation, and morphology of cells were affected by CNF morphology. Under the proliferative condition, hEnSCs showed lower proliferation on aligned CNFs than on random CNFs and on tissue culture plate (TCP) control. When cultured on aligned CNFs in neural induction media, hEnSCs showed significant upregulation of neuronal markers, NF-H and Tuj-1, and downregulation of neural progenitor marker (nestin) compared to that on random CNFs and on TCP. In contrast, hEnSCs showed higher expression of nestin and slight upregulation of oligodendrocyte marker (OLIG-2) on random CNFs compared to that on aligned CNFs and on TCP. SEM imaging revealed that differentiated cells extended along the CNF main axis on aligned CNFs but stretched multidirectionally on random CNFs. These findings suggest electrospun CNFs as proper substrate for stem cell differentiation into specific neural cells.

  20. Immortalization of neuronal progenitors using SV40 large T antigen and differentiation towards dopaminergic neurons

    PubMed Central

    Alwin Prem Anand, A; Gowri Sankar, S; Kokila Vani, V

    2012-01-01

    Transplantation is common in clinical practice where there is availability of the tissue and organ. In the case of neurodegenerative disease such as Parkinson's disease (PD), transplantation is not possible as a result of the non-availability of tissue or organ and therefore, cell therapy is an innovation in clinical practice. However, the availability of neuronal cells for transplantation is very limited. Alternatively, immortalized neuronal progenitors could be used in treating PD. The neuronal progenitor cells can be differentiated into dopaminergic phenotype. Here in this article, the current understanding of the molecular mechanisms involved in the differentiation of dopaminergic phenotype from the neuronal progenitors immortalized with SV40 LT antigen is discussed. In addition, the methods of generating dopaminergic neurons from progenitor cells and the factors that govern their differentiation are elaborated. Recent advances in cell-therapy based transplantation in PD patients and future prospects are discussed. PMID:22863662

  1. HLXB9 Gene Expression, and Nuclear Location during In Vitro Neuronal Differentiation in the SK-N-BE Neuroblastoma Cell Line

    PubMed Central

    Leotta, Claudia Giovanna; Federico, Concetta; Brundo, Maria Violetta; Tosi, Sabrina; Saccone, Salvatore

    2014-01-01

    Different parts of the genome occupy specific compartments of the cell nucleus based on the gene content and the transcriptional activity. An example of this is the altered nuclear positioning of the HLXB9 gene in leukaemia cells observed in association with its over-expression. This phenomenon was attributed to the presence of a chromosomal translocation with breakpoint proximal to the HLXB9 gene. Before becoming an interesting gene in cancer biology, HLXB9 was studied as a developmental gene. This homeobox gene is also known as MNX1 (motor neuron and pancreas homeobox 1) and it is relevant for both motor neuronal and pancreatic beta cells development. A spectrum of mutations in this gene are causative of sacral agenesis and more broadly, of what is known as the Currarino Syndrome, a constitutional autosomal dominant disorder. Experimental work on animal models has shown that HLXB9 has an essential role in motor neuronal differentiation. Here we present data to show that, upon treatment with retinoic acid, the HLXB9 gene becomes over-expressed during the early stages of neuronal differentiation and that this corresponds to a reposition of the gene in the nucleus. More precisely, we used the SK-N-BE human neuroblastoma cell line as an in vitro model and we demonstrated a transient transcription of HLXB9 at the 4th and 5th days of differentiation that corresponded to the presence, predominantly in the cell nuclei, of the encoded protein HB9. The nuclear positioning of the HLXB9 gene was monitored at different stages: a peripheral location was noted in the proliferating cells whereas a more internal position was noted during differentiation, that is while HLXB9 was transcriptionally active. Our findings suggest that HLXB9 can be considered a marker of early neuronal differentiation, possibly involving chromatin remodeling pathways. PMID:25136833

  2. HLXB9 gene expression, and nuclear location during in vitro neuronal differentiation in the SK-N-BE neuroblastoma cell line.

    PubMed

    Leotta, Claudia Giovanna; Federico, Concetta; Brundo, Maria Violetta; Tosi, Sabrina; Saccone, Salvatore

    2014-01-01

    Different parts of the genome occupy specific compartments of the cell nucleus based on the gene content and the transcriptional activity. An example of this is the altered nuclear positioning of the HLXB9 gene in leukaemia cells observed in association with its over-expression. This phenomenon was attributed to the presence of a chromosomal translocation with breakpoint proximal to the HLXB9 gene. Before becoming an interesting gene in cancer biology, HLXB9 was studied as a developmental gene. This homeobox gene is also known as MNX1 (motor neuron and pancreas homeobox 1) and it is relevant for both motor neuronal and pancreatic beta cells development. A spectrum of mutations in this gene are causative of sacral agenesis and more broadly, of what is known as the Currarino Syndrome, a constitutional autosomal dominant disorder. Experimental work on animal models has shown that HLXB9 has an essential role in motor neuronal differentiation. Here we present data to show that, upon treatment with retinoic acid, the HLXB9 gene becomes over-expressed during the early stages of neuronal differentiation and that this corresponds to a reposition of the gene in the nucleus. More precisely, we used the SK-N-BE human neuroblastoma cell line as an in vitro model and we demonstrated a transient transcription of HLXB9 at the 4th and 5th days of differentiation that corresponded to the presence, predominantly in the cell nuclei, of the encoded protein HB9. The nuclear positioning of the HLXB9 gene was monitored at different stages: a peripheral location was noted in the proliferating cells whereas a more internal position was noted during differentiation, that is while HLXB9 was transcriptionally active. Our findings suggest that HLXB9 can be considered a marker of early neuronal differentiation, possibly involving chromatin remodeling pathways.

  3. Dickkopf 3 Promotes the Differentiation of a Rostrolateral Midbrain Dopaminergic Neuronal Subset In Vivo and from Pluripotent Stem Cells In Vitro in the Mouse.

    PubMed

    Fukusumi, Yoshiyasu; Meier, Florian; Götz, Sebastian; Matheus, Friederike; Irmler, Martin; Beckervordersandforth, Ruth; Faus-Kessler, Theresa; Minina, Eleonora; Rauser, Benedict; Zhang, Jingzhong; Arenas, Ernest; Andersson, Elisabet; Niehrs, Christof; Beckers, Johannes; Simeone, Antonio; Wurst, Wolfgang; Prakash, Nilima

    2015-09-30

    Wingless-related MMTV integration site 1 (WNT1)/β-catenin signaling plays a crucial role in the generation of mesodiencephalic dopaminergic (mdDA) neurons, including the substantia nigra pars compacta (SNc) subpopulation that preferentially degenerates in Parkinson's disease (PD). However, the precise functions of WNT1/β-catenin signaling in this context remain unknown. Stem cell-based regenerative (transplantation) therapies for PD have not been implemented widely in the clinical context, among other reasons because of the heterogeneity and incomplete differentiation of the transplanted cells. This might result in tumor formation and poor integration of the transplanted cells into the dopaminergic circuitry of the brain. Dickkopf 3 (DKK3) is a secreted glycoprotein implicated in the modulation of WNT/β-catenin signaling. Using mutant mice, primary ventral midbrain cells, and pluripotent stem cells, we show that DKK3 is necessary and sufficient for the correct differentiation of a rostrolateral mdDA neuron subset. Dkk3 transcription in the murine ventral midbrain coincides with the onset of mdDA neurogenesis and is required for the activation and/or maintenance of LMX1A (LIM homeobox transcription factor 1α) and PITX3 (paired-like homeodomain transcription factor 3) expression in the corresponding mdDA precursor subset, without affecting the proliferation or specification of their progenitors. Notably, the treatment of differentiating pluripotent stem cells with recombinant DKK3 and WNT1 proteins also increases the proportion of mdDA neurons with molecular SNc DA cell characteristics in these cultures. The specific effects of DKK3 on the differentiation of rostrolateral mdDA neurons in the murine ventral midbrain, together with its known prosurvival and anti-tumorigenic properties, make it a good candidate for the improvement of regenerative and neuroprotective strategies in the treatment of PD. Significance statement: We show here that Dickkopf 3 (DKK3), a

  4. Pax6 Is Essential for the Maintenance and Multi-Lineage Differentiation of Neural Stem Cells, and for Neuronal Incorporation into the Adult Olfactory Bulb

    PubMed Central

    Curto, Gloria G.; Nieto-Estévez, Vanesa; Hurtado-Chong, Anahí; Valero, Jorge; Gómez, Carmela; Alonso, José R.; Weruaga, Eduardo

    2014-01-01

    The paired type homeobox 6 (Pax6) transcription factor (TF) regulates multiple aspects of neural stem cell (NSC) and neuron development in the embryonic central nervous system. However, less is known about the role of Pax6 in the maintenance and differentiation of adult NSCs and in adult neurogenesis. Using the +/SeyDey mouse, we have analyzed how Pax6 heterozygosis influences the self-renewal and proliferation of adult olfactory bulb stem cells (aOBSCs). In addition, we assessed its influence on neural differentiation, neuronal incorporation, and cell death in the adult OB, both in vivo and in vitro. Our results indicate that the Pax6 mutation alters Nestin+-cell proliferation in vivo, as well as self-renewal, proliferation, and survival of aOBSCs in vitro although a subpopulation of +/SeyDey progenitors is able to expand partially similar to wild-type progenitors. This mutation also impairs aOBSC differentiation into neurons and oligodendrocytes, whereas it increases cell death while preserving astrocyte survival and differentiation. Furthermore, Pax6 heterozygosis causes a reduction in the variety of neurochemical interneuron subtypes generated from aOBSCs in vitro and in the incorporation of newly generated neurons into the OB in vivo. Our findings support an important role of Pax6 in the maintenance of aOBSCs by regulating cell death, self-renewal, and cell fate, as well as in neuronal incorporation into the adult OB. They also suggest that deregulation of the cell cycle machinery and TF expression in aOBSCs which are deficient in Pax6 may be at the origin of the phenotypes observed in this adult NSC population. PMID:25117830

  5. Induction and repression of mammalian achaete-scute homologue (MASH) gene expression during neuronal differentiation of P19 embryonal carcinoma cells.

    PubMed

    Johnson, J E; Zimmerman, K; Saito, T; Anderson, D J

    1992-01-01

    MASH1 and MASH2, mammalian homologues of the Drosophila neural determination genes achaete-scute, are members of the basic helix-loop-helix (bHLH) family of transcription factors. We show here that murine P19 embryonal carcinoma cells can be used as a model system to study the regulation and function of these genes. MASH1 and MASH2 display complementary patterns of expression during the retinoic-acid-induced neuronal differentiation of P19 cells. MASH1 mRNA is undetectable in undifferentiated P19 cells but is induced to high levels by retinoic acid coincident with neuronal differentiation. In contrast, MASH2 mRNA is expressed in undifferentiated P19 cells and is repressed by retinoic acid treatment. These complementary expression patterns suggest distinct functions for MASH1 and MASH2 in development, despite their sequence homology. In retinoic-acid-treated P19 cells, MASH1 protein expression precedes and then overlaps expression of neuronal markers. However, MASH1 is expressed by a smaller proportion of cells than expresses such markers. MASH1 immunoreactivity is not detected in differentiated cells displaying a neuronal morphology, suggesting that its expression is transient. These features of MASH1 expression are similar to those observed in vivo, and suggest that P19 cells represent a good model system in which to study the regulation of this gene. Forced expression of MASH1 was achieved in undifferentiated P19 cells by transfection of a cDNA expression construct. The transfected cells expressing exogenous MASH1 protein contained E-box-binding activity that could be super-shifted by an anti-MASH1 antibody, but exhibited no detectable phenotypic changes. Thus, unlike myogenic bHLH genes, such as MyoD, which are sufficient to induce muscle differentiation, expression of MASH1 appears insufficient to promote neurogenesis.

  6. Effects of nerve growth factor and basic fibroblast growth factor dual gene modification on rat bone marrow mesenchymal stem cell differentiation into neuron-like cells in vitro.

    PubMed

    Hu, Yang; Zhang, Yan; Tian, Kang; Xun, Chong; Wang, Shouyu; Lv, Decheng

    2016-01-01

    Recent studies regarding regenerative medicine have focused on bone marrow mesenchymal stem cells (BMSCs), which have the potential to undergo neural differentiation, and may be transfected with specific genes. BMSCs can differentiate into neuron‑like cells in certain neurotropic circumstances in vitro. Basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) are often used to induce neural differentiation in BMSCs in vitro. However, previous studies regarding their combined actions are insufficient. The present study is the first, to the best of our knowledge, to thoroughly assess the enhancement of neural differentiation of BMSCs following transfection with bFGF and NGF. Sprague‑Dawley (SD) rat BMSCs were separated through whole bone marrow adherence, and were then passaged to the third generation. The cells were subsequently divided into five groups: The control group, which consisted of untransfected BMSCs; the plv‑blank‑transfected BMSCs group; the plv‑bFGF‑transfected BMSCs group; the plv‑NGF‑transfected BMSCs group; and the plv‑NGF‑bFGF co‑transfected BMSCs group. Cell neural differentiation was characterized in terms of stem cell molecular expression, and the neuronal morphology and expression of neural‑like molecules was detected in each of the groups. A total of 72 h post‑transfection, the expression levels of neuron‑specific enolase, glial fibrillary acidic protein, and nestin protein, were higher in the co‑transfected group, as compared with the other groups, the expression levels of β‑tubulin III were also increased in the co‑transfected cells, thus suggesting the maturation of differentiated neuron‑like cells. Furthermore, higher neuronal proliferation was observed in the co‑transfected group, as compared with the other groups at passages 2, 4, 6 and 8. Western blotting demonstrated that the transfected groups exhibited a simultaneous increase in phosphorylation of the AKT and extracellular signal

  7. Effects of brain‑derived neurotrophic factor and neurotrophin‑3 on the neuronal differentiation of rat adipose‑derived stem cells.

    PubMed

    Ji, Wenchen; Zhang, Xiaowei; Ji, Le; Wang, Kunzheng; Qiu, Yusheng

    2015-10-01

    Tissue engineering is a promising method that may be used to treat spinal cord injury (SCI). The underlying repair mechanism of tissue engineering involves the stable secretion of neurotrophins from seed cells, which eventually differentiate into neurons; therefore, the selection of appropriate seed cells, which stably secrete neurotrophins that easily differentiate into neurons requires investigation. Adipose‑derived stem cells (ADSCs), which are adult SCs, are advantageous due to convenience sampling and easy expansion; therefore, ADSCs are currently the most popular type of seed cell. Brain‑derived neurotrophic factor (BDNF) and neurotrophin‑3 (NT‑3) possess superior properties, when compared with other neurotrophic factors, in the maintenance of neuronal survival and promotion of SC differentiation into neurons. The present study used two lentiviruses, which specifically express BDNF and NT‑3 [Lenti‑BDNF‑green fluorescent protein (GFP), Lenti‑NT‑3‑red fluorescent protein (RFP)], to transfect third‑generation ADSCs. Three types of seed cell were obtained: i) Seed cells overexpressing BDNF (ADSC/Lenti‑BDNF‑GFP); ii) seed cells overexpressing NT‑3 (ADSC/Lenti‑NT‑3‑RFP); and iii) seed cells overexpressing BDNF and NT‑3 (ADSC/Lenti‑BDNF‑GFP and NT‑3‑RFP). The transfected cells were then induced to differentiate into neurons and were divided into a further four groups: i) The BDNF and NT‑3 co‑overexpression group; ii) the BDNF overexpression group; iii) the NT‑3 overexpression group; and iv) the control group, which consisted of untransfected ADSCs. The results of the present study demonstrate that BDNF and NT‑3 expression was higher 10 days after induction, as detected by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blotting. Neuron‑specific enolase is a neuronal marker, the expression of which was highest in the BDNF and NT‑3 co‑overexpression group, followed by the

  8. Differentiation of human adult skin-derived neuronal precursors into mature neurons.

    PubMed

    Gingras, Marie; Champigny, Marie-France; Berthod, François

    2007-02-01

    The isolation of autologous neuronal precursors from skin-derived precursor cells extracted from adult human skin would be a very efficient source of neurons for the treatment of various neurodegenerative diseases. The purpose of this study was to demonstrate that these neuronal precursors were able to differentiate into mature neurons. We isolated neuronal precursors from breast skin and expanded them in vitro for over ten passages. We showed that 48% of these cells were proliferating after the first passage, while this growth rate decreased after the second passage. We demonstrated that 70% of these cells were nestin-positive after the third passage, while only 17% were neurofilament M-positive after 7 days of differentiation. These neuronal precursors expressed betaIII tubulin, the dendritic marker MAP2 and the presynaptic marker synaptophysin after 7 days of in vitro maturation. They also expressed the postsynaptic marker PSD95 and the late neuronal markers NeuN and neurofilament H after 21 days of differentiation, demonstrating they became terminally differentiated neurons. These markers were still expressed after 50 days of culture. The generation of autologous neurons from an accessible adult human source opens many potential therapeutic applications and has a great potential for the development of experimental studies on normal human neurons.

  9. Heteromeric complexes of heat shock protein 70 (HSP70) family members, including Hsp70B′, in differentiated human neuronal cells

    PubMed Central

    Chow, Ari M.; Mok, Philip; Xiao, Dawn; Khalouei, Sam

    2010-01-01

    Human neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis have been termed “protein misfolding disorders.” Upregulation of heat shock proteins that target misfolded aggregation-prone proteins has been proposed as a potential therapeutic strategy to counter neurodegenerative disorders. The heat shock protein 70 (HSP70) family is well characterized for its cytoprotective effects against cell death and has been implicated in neuroprotection by overexpression studies. HSP70 family members exhibit sequence and structural conservation. The significance of the multiplicity of HSP70 proteins is unknown. In this study, coimmunoprecipitation was employed to determine if association of HSP70 family members occurs, including Hsp70B′ which is present in the human genome but not in mouse and rat. Heteromeric complexes of Hsp70B′, Hsp70, and Hsc70 were detected in differentiated human SH-SY5Y neuronal cells. Hsp70B′ also formed complexes with Hsp40 suggesting a common co-chaperone for HSP70 family members. PMID:20084477

  10. [Astragalus induces human amniotic epithelial cells (WISH) to differentiate toward neurons, inhibits the expression of Notch1 and promotes cell survival].

    PubMed

    Chen, Xu-Dong; Wang, Jian-Guo

    2012-12-25

    The aim of the study was to investigate the effect of astragalus on differentiation of human amniotic epithelial cell line WISH into neurons, the expression of Notch1 gene and cell viability. WISH were randomly divided into astragalus group (4 subgroups), alltransretinoic acid (RA) group and control group. Astragalus group and RA group were induced to differentiate into neurocytes by using chemical inducer RA and astragalus, respectively. The expression of neuron-specific enolase (NSE), microtubule associated protein 2 (MAP-2), Nestin and GFAP of induced cells in three groups were detected using immunocytochemical method. RT- PCR was further used to detect the expression of Oct4, Notch1, Hes1, Nestin and NSE. The cell viability was measured by methyl thiazolyl tetrazolium methods. Under the convert microscope it was observed that WISH cells started to change their shape, and there were several axon or dendrite-like processes out from the cell body induced by astragalus for 24 h or RA for 12 h. The positive cell rates of NSE and MAP-2 in 100 μL/mL astragalus-induced group were less than those in RA-induced group at 48 h (P < 0.05), but higher than those in control group. Cell viability in astragalus group was higher than that of RA group (P < 0.05). While the positive cell rates of Nestin and GFAP in 100 μL/mL astragalus-induced group were higher than those in RA-induced group at 48 h (P < 0.05). The positive cell rates of Nestin in the two induced groups were lower than those in control group. RT-PCR showed that the expressions of Oct4, Notch1 and Hes1 in RA and astragalus (100 μL/mL) groups were less than those in control group, but the expression of NSE was higher than that in control group. These results suggest that astragalus (especially at 100 μL/mL, 48 h) and RA can both induce human amniotic epithelial cell line WISH cells into neuron-like cells, but astragalus induction has a higher cell survival rate than RA induction, and the expression of Notch1

  11. Nutrient restriction during early life reduces cell proliferation in the hippocampus at adulthood but does not impair the neuronal differentiation process of the new generated cells.

    PubMed

    Matos, R J B; Orozco-Solís, R; Lopes de Souza, S; Manhães-de-Castro, R; Bolaños-Jiménez, F

    2011-11-24

    Maternal malnutrition results in learning deficits and predisposition to anxiety and depression in the offspring that extend into adulthood. At the cellular level, learning and memory rely on the production of new neurons in the dentate gyrus (DG) of the hippocampus, and hippocampal neurogenesis has been associated with the etiology and treatment of depression, but whether adult neurogenesis is affected by malnutrition during early life is not known. To investigate the effects of perinatal undernutrition on neurogenesis at adulthood, pregnant Sprague-Dawley rats were fed either ad libitum (C) or were undernourished by reducing their daily food intake by 50% in relation to the C group during gestation and lactation (FR/FR). At birth, one subset of control pups was cross-fostered to food-restricted dams to constitute a third group of animals that were undernourished during the lactation period only (AdLib/FR). At 90 days of age, pups were injected with bromodeoxyuridine (BrdU) and sacrificed 2 h, 1 week, or 3 weeks later. The number of BrdU-labeled cells in the DG was significantly reduced in the offspring of FR/FR dams in relation to controls at all the time points examined. However, the proportion of new cells exhibiting a neuronal phenotype was higher in FR/FR rats than in controls as revealed by the colabeling at 3 weeks of the BrdU-labeled cells with neuron-specific nuclear protein (NeuN). AdLib/FR animals exhibited also reduced BrdU labeling at 2 h and 1 week. Nevertheless, we found no significant differences at 3 weeks in either the number of BrdU-labeled cells or in the proportion of new neurons between controls and AdLib/FR rats. These results indicate that the decreased number of hippocampal neurons in perinatally undernourished rats is due to the deleterious effects of early nutrient restriction on cell proliferation but not on the neuronal differentiation process of the new generated cells.

  12. Differentiating lower motor neuron syndromes.

    PubMed

    Garg, Nidhi; Park, Susanna B; Vucic, Steve; Yiannikas, Con; Spies, Judy; Howells, James; Huynh, William; Matamala, José M; Krishnan, Arun V; Pollard, John D; Cornblath, David R; Reilly, Mary M; Kiernan, Matthew C

    2017-06-01

    Lower motor neuron (LMN) syndromes typically present with muscle wasting and weakness and may arise from pathology affecting the distal motor nerve up to the level of the anterior horn cell. A variety of hereditary causes are recognised, including spinal muscular atrophy, distal hereditary motor neuropathy and LMN variants of familial motor neuron disease. Recent genetic advances have resulted in the identification of a variety of disease-causing mutations. Immune-mediated disorders, including multifocal motor neuropathy and variants of chronic inflammatory demyelinating polyneuropathy, account for a proportion of LMN presentations and are important to recognise, as effective treatments are available. The present review will outline the spectrum of LMN syndromes that may develop in adulthood and provide a framework for the clinician assessing a patient presenting with predominantly LMN features. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  13. Differentiation of Human Adipose-Derived Stem Cells into Neuron-Like Cells Which Are Compatible with Photocurable Three-Dimensional Scaffolds

    PubMed Central

    Gao, Shane; Zhao, Peng; Lin, Chao; Sun, Yuxi; Wang, Yilei; Zhou, Zhichong; Yang, Danjing; Wang, Xianli; Xu, Hongzhen; Zhou, Fei; Cao, Limei; Zhou, Wei; Ning, Ke

    2014-01-01

    Multipotent human adipose-derived stromal/stem cells (hADSCs) hold a great promise for cell-based therapy for many devastating human diseases, such as spinal cord injury and stroke. If exogenous hADSCs can be cultured in a three-dimensional (3D) scaffold with effective proliferation and differentiation capacity, it will better mimic the in vivo environment, which will have profound impact on the therapeutic application of hADSCs. In this study, a group of elastic-dominant, porous bioscaffolds from photocurable chitosan and gelatin were fabricated and proven to be biocompatible with both hADSCs and hADSC-derived neuron-like cells (hADSC-NLCs) in vitro. The identity of harvested hADSCs was confirmed by their positive immunostaining of mesenchymal stem cell surface markers, CD29, CD44, and CD105, and also positive expression of stem markers, Sox-2, Oct-4, c-Myc, Nanog, and Klf4. Their multipotency was further confirmed by trilineage differentiation of hADSCs toward adipocyte, osteoblast, and chondrocyte. It was found that hADSCs could be conditioned to differentiate into neurons in vitro as determined by immunostaining the markers of Tuj1, MAP2, NeuN, and Synapsin. The hADSCs and hADSC-NLCs were proven to be biocompatible with 3D scaffold, which actually facilitated the proliferation and differentiation of hADSCs in vitro, by MTT assay and their neuronal gene expression profiling. Moreover, hADSC-NLCs, which were mixed with 3D scaffold and transplanted into traumatic