Science.gov

Sample records for a2b5 neural progenitor

  1. PAR1 inhibition suppresses the self-renewal and growth of A2B5-defined glioma progenitor cells and their derived gliomas in vivo

    PubMed Central

    Auvergne, R; Wu, C; Connell, A; Au, S; Cornwell, A; Osipovitch, M; Benraiss, A; Dangelmajer, S; Guerrero-Cazares, H; Quinones-Hinojosa, A; Goldman, SA

    2016-01-01

    Glioblastoma (GBM) remains the most common and lethal intracranial tumor. In a comparison of gene expression by A2B5-defined tumor-initiating progenitor cells (TPCs) to glial progenitor cells derived from normal adult human brain, we found that the F2R gene encoding PAR1 was differentially overexpressed by A2B5-sorted TPCs isolated from gliomas at all stages of malignant development. In this study, we asked if PAR1 is causally associated with glioma progression. Lentiviral knockdown of PAR1 inhibited the expansion and self-renewal of human GBM-derived A2B5+ TPCs in vitro, while pharmacological inhibition of PAR 1 similarly slowed both the growth and migration of A2B5+ TPCs in culture. In addition, PAR1 silencing potently suppressed tumor expansion in vivo, and significantly prolonged the survival of mice following intracranial transplantation of human TPCs. These data strongly suggest the importance of PAR1 to the self-renewal and tumorigenicity of A2B5-defined glioma TPCs; as such, the abrogation of PAR1-dependent signaling pathways may prove a promising strategy for gliomas. PMID:26616854

  2. Immunodiagnosis of tumors in vivo using radiolabeled monoclonal antibody A2B5

    SciTech Connect

    Reintgen, D.S.; Shimizu, K.; Coleman, E.; Briner, W.; Kitzmiller, J.; Eisenbarth, G.; Seigler, H.F.

    1983-07-01

    Recently a murine monoclonal antibody (A2B5) has been described that reacts with a membrane associated GQ ganglioside common to peptide secreting normal cells and tumors. In vitro binding data demonstrated the presence of this ganglioside on neurons, adrenal medulla, and pancreatic islets, along with neuroendocrine tumors such as insulinomas, pheochromocytomas, melanomas and neuroblastomas. Negative binding has previously been shown for tissue sections from liver, kidney, colon, lung, stomach, and tumors not derived from the neural crest. Because of the specificity at A2B5 in vitro, this monoclonal antibody was labeled with /sup 131/I for in vivo tumor localization studies. Daily radionuclear scans were obtained in 5 KX rats bearing the radiation induced rat insulinoma with disappearance of the label from the blood pool and concentration in the tumor so that by the fourth day, the only activity present by scan was in the insulinoma. In addition A2B5 also localized to five different human melanoma cells lines grown in nude mice with high tumor/blood levels compared to normal tissues, while no localization is seen in nudes carrying osteosarcomas, colon, bladder, and renal cell carcinomas. In addition antibody A2B5 did not concentrate in any normal tissue though the antigen is present on several. The finding that A2B5 reacts across species lines (mouse, rat, man) lends itself to obvious diagnostic and therapeutic possibilities.

  3. Characterization of Proliferating Neural Progenitors after Spinal Cord Injury in Adult Zebrafish

    PubMed Central

    Hui, Subhra Prakash; Nag, Tapas Chandra; Ghosh, Sukla

    2015-01-01

    Zebrafish can repair their injured brain and spinal cord after injury unlike adult mammalian central nervous system. Any injury to zebrafish spinal cord would lead to increased proliferation and neurogenesis. There are presences of proliferating progenitors from which both neuronal and glial loss can be reversed by appropriately generating new neurons and glia. We have demonstrated the presence of multiple progenitors, which are different types of proliferating populations like Sox2+ neural progenitor, A2B5+ astrocyte/ glial progenitor, NG2+ oligodendrocyte progenitor, radial glia and Schwann cell like progenitor. We analyzed the expression levels of two common markers of dedifferentiation like msx-b and vimentin during regeneration along with some of the pluripotency associated factors to explore the possible role of these two processes. Among the several key factors related to pluripotency, pou5f1 and sox2 are upregulated during regeneration and associated with activation of neural progenitor cells. Uncovering the molecular mechanism for endogenous regeneration of adult zebrafish spinal cord would give us more clues on important targets for future therapeutic approach in mammalian spinal cord repair and regeneration. PMID:26630262

  4. Characterization of Proliferating Neural Progenitors after Spinal Cord Injury in Adult Zebrafish.

    PubMed

    Hui, Subhra Prakash; Nag, Tapas Chandra; Ghosh, Sukla

    2015-01-01

    Zebrafish can repair their injured brain and spinal cord after injury unlike adult mammalian central nervous system. Any injury to zebrafish spinal cord would lead to increased proliferation and neurogenesis. There are presences of proliferating progenitors from which both neuronal and glial loss can be reversed by appropriately generating new neurons and glia. We have demonstrated the presence of multiple progenitors, which are different types of proliferating populations like Sox2+ neural progenitor, A2B5+ astrocyte/ glial progenitor, NG2+ oligodendrocyte progenitor, radial glia and Schwann cell like progenitor. We analyzed the expression levels of two common markers of dedifferentiation like msx-b and vimentin during regeneration along with some of the pluripotency associated factors to explore the possible role of these two processes. Among the several key factors related to pluripotency, pou5f1 and sox2 are upregulated during regeneration and associated with activation of neural progenitor cells. Uncovering the molecular mechanism for endogenous regeneration of adult zebrafish spinal cord would give us more clues on important targets for future therapeutic approach in mammalian spinal cord repair and regeneration.

  5. Immunodiagnosis of tumors in vivo using radiolabeled monoclonal antibody A2B5.

    PubMed

    Reintgen, D S; Shimizu, K; Coleman, E; Briner, W; Kitzmiller, J; Eisenbarth, G; Seigler, H F

    1983-07-01

    Recently a murine monoclonal antibody (A2B5) has been described that reacts with a membrane associated GQ ganglioside common to peptide secreting normal cells and tumors. In vitro binding data demonstrated the presence of this ganglioside on neurons, adrenal medulla, and pancreatic islets, along with neuroendocrine tumors such as insulinomas, pheochromocytomas, melanomas and neuroblastomas. Negative binding has previously been shown for tissue sections from liver, kidney, colon, lung, stomach, and tumors not derived from the neural crest. Because of the specificity at A2B5 in vitro, this monoclonal antibody was labeled with 131I for in vivo tumor localization studies. Daily radionuclear scans were obtained in 5 KX rats bearing the radiation induced rat insulinoma with disappearance of the label from the blood pool and concentration in the tumor so that by the fourth day, the only activity present by scan was in the insulinoma. Tissue-counting data showed tumor/blood ratios (av +/- SE, 1.29 +/- 0.25) of A2B5 activity two to ten times the average activity found in other organs (0.28 +/- 0.05). No tumor concentration of the control nonspecific monoclonal antibody P3X63 was evident (0.27 +/- 0.04). In addition A2B5 also localized to five different human melanoma cells lines grown in nude mice with high tumor/blood levels (1.04 +/- 0.27) compared to normal tissues (0.32 +/- 0.05) (P = .0005), while no localization is seen in nudes carrying osteosarcomas, colon, bladder, and renal cell carcinomas. In addition antibody A2B5 did not concentrate in any normal tissue though the antigen is present on several. The finding that A2B5 reacts across species lines (mouse, rat, man) lends itself to obvious diagnostic and therapeutic possibilities.

  6. A genetic analysis of neural progenitor differentiation.

    PubMed

    Geschwind, D H; Ou, J; Easterday, M C; Dougherty, J D; Jackson, R L; Chen, Z; Antoine, H; Terskikh, A; Weissman, I L; Nelson, S F; Kornblum, H I

    2001-02-01

    Genetic mechanisms regulating CNS progenitor function and differentiation are not well understood. We have used microarrays derived from a representational difference analysis (RDA) subtraction in a heterogeneous stem cell culture system to systematically study the gene expression patterns of CNS progenitors. This analysis identified both known and novel genes enriched in progenitor cultures. In situ hybridization in a subset of clones demonstrated that many of these genes were expressed preferentially in germinal zones, some showing distinct ventricular or subventricular zone labeling. Several genes were also enriched in hematopoietic stem cells, suggesting an overlap of gene expression in neural and hematopoietic progenitors. This combination of methods demonstrates the power of using custom microarrays derived from RDA-subtracted libraries for both gene discovery and gene expression analysis in the central nervous system.

  7. Regulation of the nascent brain vascular network by neural progenitors.

    PubMed

    Santhosh, Devi; Huang, Zhen

    2015-11-01

    Neural progenitors are central players in the development of the brain neural circuitry. They not only produce the diverse neuronal and glial cell types in the brain, but also guide their migration in this process. Recent evidence indicates that neural progenitors also play a critical role in the development of the brain vascular network. At an early stage, neural progenitors have been found to facilitate the ingression of blood vessels from outside the neural tube, through VEGF and canonical Wnt signaling. Subsequently, neural progenitors directly communicate with endothelial cells to stabilize nascent brain vessels, in part through down-regulating Wnt pathway activity. Furthermore, neural progenitors promote nascent brain vessel integrity, through integrin αvβ8-dependent TGFβ signaling. In this review, we will discuss the evidence for, as well as questions that remain, regarding these novel roles of neural progenitors and the underlying mechanisms in their regulation of the nascent brain vascular network.

  8. Transient nuclear Prospero induces neural progenitor quiescence.

    PubMed

    Lai, Sen-Lin; Doe, Chris Q

    2014-10-29

    Stem cells can self-renew, differentiate, or enter quiescence. Understanding how stem cells switch between these states is highly relevant for stem cell-based therapeutics. Drosophila neural progenitors (neuroblasts) have been an excellent model for studying self-renewal and differentiation, but quiescence remains poorly understood. In this study, we show that when neuroblasts enter quiescence, the differentiation factor Prospero is transiently detected in the neuroblast nucleus, followed by the establishment of a unique molecular profile lacking most progenitor and differentiation markers. The pulse of low level nuclear Prospero precedes entry into neuroblast quiescence even when the timing of quiescence is advanced or delayed by changing temporal identity factors. Furthermore, loss of Prospero prevents entry into quiescence, whereas a pulse of low level nuclear Prospero can drive proliferating larval neuroblasts into quiescence. We propose that Prospero levels distinguish three progenitor fates: absent for self-renewal, low for quiescence, and high for differentiation.

  9. Expression of Receptors for Tetanus Toxin and Monoclonal Antibody A2B5 by Pancreatic Islet Cells

    NASA Astrophysics Data System (ADS)

    Eisenbarth, G. S.; Shimizu, K.; Bowring, M. A.; Wells, S.

    1982-08-01

    Studies of the reaction of antibody A2B5 and tetanus toxin with pancreatic islet cells, islet cell tumors, and other human amine precursor uptake and decarboxylation (APUD) tumors are described. By indirect immunofluorescence, antibody A2B5 and tetanus toxin were shown to specifically bind to the plasma membrane of human, rat, chicken, and mouse islet cells. The binding of antibody A2B5 to the cell surface of living islet cells has allowed isolation of these cells from a suspension of pancreatic cells by using a fluorescence-activated cell sorter. In studies designed to determine whether tetanus toxin and antibody A2B5 bound to the same surface antigen, A2B5 and tetanus toxin did not compete for binding to normal islet cells, a human islet cell tumor, or a rat islet cell tumor. In addition to binding to islet cell tumors, antibody A2B5 reacts with frozen sections, isolated cells, and cell lines of neural, neural crest, and APUD origin.

  10. Transient nuclear Prospero induces neural progenitor quiescence

    PubMed Central

    Lai, Sen-Lin; Doe, Chris Q

    2014-01-01

    Stem cells can self-renew, differentiate, or enter quiescence. Understanding how stem cells switch between these states is highly relevant for stem cell-based therapeutics. Drosophila neural progenitors (neuroblasts) have been an excellent model for studying self-renewal and differentiation, but quiescence remains poorly understood. In this study, we show that when neuroblasts enter quiescence, the differentiation factor Prospero is transiently detected in the neuroblast nucleus, followed by the establishment of a unique molecular profile lacking most progenitor and differentiation markers. The pulse of low level nuclear Prospero precedes entry into neuroblast quiescence even when the timing of quiescence is advanced or delayed by changing temporal identity factors. Furthermore, loss of Prospero prevents entry into quiescence, whereas a pulse of low level nuclear Prospero can drive proliferating larval neuroblasts into quiescence. We propose that Prospero levels distinguish three progenitor fates: absent for self-renewal, low for quiescence, and high for differentiation. DOI: http://dx.doi.org/10.7554/eLife.03363.001 PMID:25354199

  11. Skeletal muscle neural progenitor cells exhibit properties of NG2-glia

    PubMed Central

    Birbrair, Alexander; Zhang, Tan; Wang, Zhong-Min; Messi, María Laura; Enikolopov, Grigori N.; Mintz, Akiva; Delbono, Osvaldo

    2013-01-01

    Reversing brain degeneration and trauma lesions will depend on cell therapy. Our previous work identified neural precursor cells derived from the skeletal muscle of Nestin-GFP transgenic mice, but their identity, origin, and potential survival in the brain are only vaguely understood. In this work, we show that Nestin-GFP+ progenitor cells share morphological and molecular markers with NG2-glia, including NG2, PDGFRα, O4, NGF receptor (p75), glutamate receptor-1(AMPA), and A2B5 expression. Although these cells exhibit NG2, they do not express other pericyte markers, such as α-SMA or connexin-43, and do not differentiate into the muscle lineage. Patch-clamp studies displayed outward potassium currents, probably carried through Kir6.1 channels. Given their potential therapeutic application, we compared their abundance in tissues and concluded that skeletal muscle is the richest source of predifferentiated neural precursor cells. We found that these cells migrate toward the neurogenic subventricular zone displaying their typical morphology and nestin-GFP expression two weeks after brain injection. For translational purposes, we sought to identify these neural progenitor cells in wild-type species by developing a DsRed expression vector under Nestin-Intron II control. This approach revealed them in nonhuman primates and aging rodents throughout the lifespan. PMID:22999866

  12. Skeletal muscle neural progenitor cells exhibit properties of NG2-glia.

    PubMed

    Birbrair, Alexander; Zhang, Tan; Wang, Zhong-Min; Messi, María Laura; Enikolopov, Grigori N; Mintz, Akiva; Delbono, Osvaldo

    2013-01-01

    Reversing brain degeneration and trauma lesions will depend on cell therapy. Our previous work identified neural precursor cells derived from the skeletal muscle of Nestin-GFP transgenic mice, but their identity, origin, and potential survival in the brain are only vaguely understood. In this work, we show that Nestin-GFP+ progenitor cells share morphological and molecular markers with NG2-glia, including NG2, PDGFRα, O4, NGF receptor (p75), glutamate receptor-1(AMPA), and A2B5 expression. Although these cells exhibit NG2, they do not express other pericyte markers, such as α-SMA or connexin-43, and do not differentiate into the muscle lineage. Patch-clamp studies displayed outward potassium currents, probably carried through Kir6.1 channels. Given their potential therapeutic application, we compared their abundance in tissues and concluded that skeletal muscle is the richest source of predifferentiated neural precursor cells. We found that these cells migrate toward the neurogenic subventricular zone displaying their typical morphology and nestin-GFP expression two weeks after brain injection. For translational purposes, we sought to identify these neural progenitor cells in wild-type species by developing a DsRed expression vector under Nestin-Intron II control. This approach revealed them in nonhuman primates and aging rodents throughout the lifespan.

  13. A Mechanism for the Inhibition of Neural Progenitor Cell Proliferation by Cocaine

    PubMed Central

    Lee, Chun-Ting; Chen, Jia; Hayashi, Teruo; Tsai, Shang-Yi; Sanchez, Joseph F; Errico, Stacie L; Amable, Rose; Su, Tsung-Ping; Lowe, Ross H; Huestis, Marilyn A; Shen, James; Becker, Kevin G; Geller, Herbert M; Freed, William J

    2008-01-01

    Background Prenatal exposure of the developing brain to cocaine causes morphological and behavioral abnormalities. Recent studies indicate that cocaine-induced proliferation inhibition and/or apoptosis in neural progenitor cells may play a pivotal role in causing these abnormalities. To understand the molecular mechanism through which cocaine inhibits cell proliferation in neural progenitors, we sought to identify the molecules that are responsible for mediating the effect of cocaine on cell cycle regulation. Methods and Findings Microarray analysis followed by quantitative real-time reverse transcription PCR was used to screen cocaine-responsive and cell cycle-related genes in a neural progenitor cell line where cocaine exposure caused a robust anti-proliferative effect by interfering with the G1-to-S transition. Cyclin A2, among genes related to the G1-to-S cell cycle transition, was most strongly down-regulated by cocaine. Down-regulation of cyclin A was also found in cocaine-treated human primary neural and A2B5+ progenitor cells, as well as in rat fetal brains exposed to cocaine in utero. Reversing cyclin A down-regulation by gene transfer counteracted the proliferation inhibition caused by cocaine. Further, we found that cocaine-induced accumulation of reactive oxygen species, which involves N-oxidation of cocaine via cytochrome P450, promotes cyclin A down-regulation by causing an endoplasmic reticulum (ER) stress response, as indicated by increased phosphorylation of eIF2α and expression of ATF4. In the developing rat brain, the P450 inhibitor cimetidine counteracted cocaine-induced inhibition of neural progenitor cell proliferation as well as down-regulation of cyclin A. Conclusions Our results demonstrate that down-regulation of cyclin A underlies cocaine-induced proliferation inhibition in neural progenitors. The down-regulation of cyclin A is initiated by N-oxidative metabolism of cocaine and consequent ER stress. Inhibition of cocaine N

  14. GSK-3 is a master regulator of neural progenitor homeostasis

    PubMed Central

    Kim, Woo-Yang; Wang, Xinshuo; Wu, Yaohong; Doble, Bradley W; Patel, Satish; Woodgett, James R; Snider, William D

    2016-01-01

    The development of the brain requires the exquisite coordination of progenitor proliferation and differentiation to achieve complex circuit assembly. It has been suggested that glycogen synthase kinase 3 (GSK-3) acts as an integrating molecule for multiple proliferation and differentiation signals because of its essential role in the RTK, Wnt and Shh signaling pathways. We created conditional mutations that deleted both the α and β forms of GSK-3 in mouse neural progenitors. GSK-3 deletion resulted in massive hyperproliferation of neural progenitors along the entire neuraxis. Generation of both intermediate neural progenitors and postmitotic neurons was markedly suppressed. These effects were associated with the dysregulation of β-catenin, Sonic Hedgehog, Notch and fibroblast growth factor signaling. Our results indicate that GSK-3 signaling is an essential mediator of homeostatic controls that regulate neural progenitors during mammalian brain development. PMID:19801986

  15. Pannexin 1 regulates postnatal neural stem and progenitor cell proliferation

    PubMed Central

    2012-01-01

    Background Pannexin 1 forms ion and metabolite permeable hexameric channels and is abundantly expressed in the brain. After discovering pannexin 1 expression in postnatal neural stem and progenitor cells we sought to elucidate its functional role in neuronal development. Results We detected pannexin 1 in neural stem and progenitor cells in vitro and in vivo. We manipulated pannexin 1 expression and activity in Neuro2a neuroblastoma cells and primary postnatal neurosphere cultures to demonstrate that pannexin 1 regulates neural stem and progenitor cell proliferation likely through the release of adenosine triphosphate (ATP). Conclusions Permeable to ATP, a potent autocrine/paracine signaling metabolite, pannexin 1 channels are ideally suited to influence the behavior of neural stem and progenitor cells. Here we demonstrate they play a robust role in the regulation of neural stem and progenitor cell proliferation. Endogenous postnatal neural stem and progenitor cells are crucial for normal brain health, and their numbers decline with age. Furthermore, these special cells are highly responsive to neurological injury and disease, and are gaining attention as putative targets for brain repair. Therefore, understanding the fundamental role of pannexin 1 channels in neural stem and progenitor cells is of critical importance for brain health and disease. PMID:22458943

  16. Neural Progenitors Adopt Specific Identities by Directly Repressing All Alternative Progenitor Transcriptional Programs.

    PubMed

    Kutejova, Eva; Sasai, Noriaki; Shah, Ankita; Gouti, Mina; Briscoe, James

    2016-03-21

    In the vertebrate neural tube, a morphogen-induced transcriptional network produces multiple molecularly distinct progenitor domains, each generating different neuronal subtypes. Using an in vitro differentiation system, we defined gene expression signatures of distinct progenitor populations and identified direct gene-regulatory inputs corresponding to locations of specific transcription factor binding. Combined with targeted perturbations of the network, this revealed a mechanism in which a progenitor identity is installed by active repression of the entire transcriptional programs of other neural progenitor fates. In the ventral neural tube, sonic hedgehog (Shh) signaling, together with broadly expressed transcriptional activators, concurrently activates the gene expression programs of several domains. The specific outcome is selected by repressive input provided by Shh-induced transcription factors that act as the key nodes in the network, enabling progenitors to adopt a single definitive identity from several initially permitted options. Together, the data suggest design principles relevant to many developing tissues.

  17. Analysing human neural stem cell ontogeny by consecutive isolation of Notch active neural progenitors.

    PubMed

    Edri, Reuven; Yaffe, Yakey; Ziller, Michael J; Mutukula, Naresh; Volkman, Rotem; David, Eyal; Jacob-Hirsch, Jasmine; Malcov, Hagar; Levy, Carmit; Rechavi, Gideon; Gat-Viks, Irit; Meissner, Alexander; Elkabetz, Yechiel

    2015-03-23

    Decoding heterogeneity of pluripotent stem cell (PSC)-derived neural progeny is fundamental for revealing the origin of diverse progenitors, for defining their lineages, and for identifying fate determinants driving transition through distinct potencies. Here we have prospectively isolated consecutively appearing PSC-derived primary progenitors based on their Notch activation state. We first isolate early neuroepithelial cells and show their broad Notch-dependent developmental and proliferative potential. Neuroepithelial cells further yield successive Notch-dependent functional primary progenitors, from early and midneurogenic radial glia and their derived basal progenitors, to gliogenic radial glia and adult-like neural progenitors, together recapitulating hallmarks of neural stem cell (NSC) ontogeny. Gene expression profiling reveals dynamic stage-specific transcriptional patterns that may link development of distinct progenitor identities through Notch activation. Our observations provide a platform for characterization and manipulation of distinct progenitor cell types amenable for developing streamlined neural lineage specification paradigms for modelling development in health and disease.

  18. Fas Activation Increases Neural progenitor Cell Survival

    PubMed Central

    Knight, Julia C.; Scharf, Eugene L.; Mao-Draayer, Yang

    2015-01-01

    Although there is a sizable amount of research focusing on adult neural progenitor cells (NPCs) as a therapeutic approach for many neurodegenerative diseases, including multiple sclerosis, little is known about the pathways that govern NPC survival and apoptosis. Fas, a member of the death receptor superfamily, plays a well-characterized role in the immune system, but its function in neural stem cells remains uncertain. Our study focuses on the effects of Fas on NPC survival in vitro. Activation of Fas by recombinant Fas ligand (FasL) did not induce apoptosis in murine NPCs in culture. In fact, both an increase in the amount of viable cells and a decrease in apoptotic and dying cells were observed with FasL treatment. Our data indicate that FasL-mediated adult NPC neuroprotection is characterized by a reduction in apoptosis, but not increased proliferation. Further investigation of this effect revealed that the antiapoptotic effects of FasL are mediated by the up-regulation of Birc3, an inhibitor of apoptosis protein (IAP). Conversely, the observed effect is not the result of altered caspase activation or FLIP (Fas-associated death domain-like interleukin-1beta-converting enzyme inhibitory protein) up-regulation, which is known to inhibit caspase-8-mediated cell death in T cells. Our data indicate that murine adult NPCs are resistant to FasL-induced cell death. Activation of Fas increased cell survival by decreasing apoptosis through Birc3 up-regulation. These results describe a novel pathway involved in NPC survival. PMID:19830835

  19. Harmine stimulates proliferation of human neural progenitors

    PubMed Central

    Dakic, Vanja; Maciel, Renata de Moraes; Drummond, Hannah; Nascimento, Juliana M.; Trindade, Pablo

    2016-01-01

    Harmine is the β-carboline alkaloid with the highest concentration in the psychotropic plant decoction Ayahuasca. In rodents, classical antidepressants reverse the symptoms of depression by stimulating neuronal proliferation. It has been shown that Ayahuasca presents antidepressant effects in patients with depressive disorder. In the present study, we investigated the effects of harmine in cell cultures containing human neural progenitor cells (hNPCs, 97% nestin-positive) derived from pluripotent stem cells. After 4 days of treatment, the pool of proliferating hNPCs increased by 71.5%. Harmine has been reported as a potent inhibitor of the dual specificity tyrosine-phosphorylation-regulated kinase (DYRK1A), which regulates cell proliferation and brain development. We tested the effect of analogs of harmine, an inhibitor of DYRK1A (INDY), and an irreversible selective inhibitor of monoamine oxidase (MAO) but not DYRK1A (pargyline). INDY but not pargyline induced proliferation of hNPCs similarly to harmine, suggesting that inhibition of DYRK1A is a possible mechanism to explain harmine effects upon the proliferation of hNPCs. Our findings show that harmine enhances proliferation of hNPCs and suggest that inhibition of DYRK1A may explain its effects upon proliferation in vitro and antidepressant effects in vivo. PMID:27957390

  20. Immunocytochemical characterisation of neural stem-progenitor cells from green terror cichlid Aequidens rivulatus.

    PubMed

    Wen, C M; Chen, M M; Nan, F H; Wang, C S

    2017-01-01

    In this study, cultures of neural stem-progenitor cells (NSPC) from the brain of green terror cichlid Aequidens rivulatus were established and various NSPCs were demonstrated using immunocytochemistry. All of the NSPCs expressed brain lipid-binding protein, dopamine- and cAMP-regulated neuronal phosphoprotein 32 (DARPP-32), oligodendrocyte transcription factor 2, paired box 6 and sex determining region Y-box 2. The intensity and localisation of these proteins, however, varied among the different NSPCs. Despite being intermediate cells, NSPCs can be divided into radial glial cells, oligodendrocyte progenitor cells (OPC) and neuroblasts by expressing the astrocyte marker glial fibrillary acidic protein (GFAP), OPC marker A2B5 and neuronal markers, including acetyl-tubulin, βIII-tubulin, microtubule-associated protein 2 and neurofilament protein. Nevertheless, astrocytes were polymorphic and were the most dominant cells in the NSPC cultures. By using Matrigel, radial glia exhibiting a long GFAP(+) or DARPP-32(+) fibre and neurons exhibiting a significant acetyl-tubulin(+) process were obtained. The results confirmed that NSPCs obtained from A. rivulatus brains can proliferate and differentiate into neurons in vitro. Clonal culture can be useful for further studying the distinct NSPCs.

  1. Neural progenitors, neurogenesis and the evolution of the neocortex.

    PubMed

    Florio, Marta; Huttner, Wieland B

    2014-06-01

    The neocortex is the seat of higher cognitive functions and, in evolutionary terms, is the youngest part of the mammalian brain. Since its origin, the neocortex has expanded in several mammalian lineages, and this is particularly notable in humans. This expansion reflects an increase in the number of neocortical neurons, which is determined during development and primarily reflects the number of neurogenic divisions of distinct classes of neural progenitor cells. Consequently, the evolutionary expansion of the neocortex and the concomitant increase in the numbers of neurons produced during development entail interspecies differences in neural progenitor biology. Here, we review the diversity of neocortical neural progenitors, their interspecies variations and their roles in determining the evolutionary increase in neuron numbers and neocortex size.

  2. NFIX regulates neural progenitor cell differentiation during hippocampal morphogenesis.

    PubMed

    Heng, Yee Hsieh Evelyn; McLeay, Robert C; Harvey, Tracey J; Smith, Aaron G; Barry, Guy; Cato, Kathleen; Plachez, Céline; Little, Erica; Mason, Sharon; Dixon, Chantelle; Gronostajski, Richard M; Bailey, Timothy L; Richards, Linda J; Piper, Michael

    2014-01-01

    Neural progenitor cells have the ability to give rise to neurons and glia in the embryonic, postnatal and adult brain. During development, the program regulating whether these cells divide and self-renew or exit the cell cycle and differentiate is tightly controlled, and imbalances to the normal trajectory of this process can lead to severe functional consequences. However, our understanding of the molecular regulation of these fundamental events remains limited. Moreover, processes underpinning development of the postnatal neurogenic niches within the cortex remain poorly defined. Here, we demonstrate that Nuclear factor one X (NFIX) is expressed by neural progenitor cells within the embryonic hippocampus, and that progenitor cell differentiation is delayed within Nfix(-/-) mice. Moreover, we reveal that the morphology of the dentate gyrus in postnatal Nfix(-/-) mice is abnormal, with fewer subgranular zone neural progenitor cells being generated in the absence of this transcription factor. Mechanistically, we demonstrate that the progenitor cell maintenance factor Sry-related HMG box 9 (SOX9) is upregulated in the hippocampus of Nfix(-/-) mice and demonstrate that NFIX can repress Sox9 promoter-driven transcription. Collectively, our findings demonstrate that NFIX plays a central role in hippocampal morphogenesis, regulating the formation of neuronal and glial populations within this structure.

  3. L1 Retrotransposition in Neural Progenitor Cells.

    PubMed

    Muotri, Alysson R

    2016-01-01

    Long interspersed nucleotide element 1 (LINE-1 or L1) is a family of non-LTR retrotransposons that can replicate and reintegrate into the host genome. L1s have considerably influenced mammalian genome evolution by retrotransposing during germ cell development or early embryogenesis, leading to massive genome expansion. For many years, L1 retrotransposons were viewed as a selfish DNA parasite that had no contribution in somatic cells. Historically, L1s were thought to only retrotranspose during gametogenesis and in neoplastic processes, but recent studies have shown that L1s are extremely active in the mouse, rat, and human neuronal progenitor cells (NPCs). These de novo L1 insertions can impact neuronal transcriptional expression, creating unique transcriptomes of individual neurons, possibly contributing to the uniqueness of the individual cognition and mental disorders in humans.

  4. Methylene blue promotes quiescence of rat neural progenitor cells.

    PubMed

    Xie, Luokun; Choudhury, Gourav R; Wang, Jixian; Park, Yong; Liu, Ran; Yuan, Fang; Zhang, Chun-Li; Yorio, Thomas; Jin, Kunlin; Yang, Shao-Hua

    2014-01-01

    Neural stem cell-based treatment holds a new therapeutic opportunity for neurodegenerative disorders. Here, we investigated the effect of methylene blue on proliferation and differentiation of rat neural progenitor cells (NPCs) both in vitro and in vivo. We found that methylene blue inhibited proliferation and promoted quiescence of NPCs in vitro without affecting committed neuronal differentiation. Consistently, intracerebroventricular infusion of methylene blue significantly inhibited NPC proliferation at the subventricular zone (SVZ). Methylene blue inhibited mTOR signaling along with down-regulation of cyclins in NPCs in vitro and in vivo. In summary, our study indicates that methylene blue may delay NPC senescence through enhancing NPCs quiescence.

  5. Proliferation control in neural stem and progenitor cells

    PubMed Central

    Homem, Catarina CF; Repic, Marko; Knoblich, Juergen A

    2015-01-01

    Neural circuit function can be drastically affected by variations in the number of cells that are produced during development or by a reduction in adult cell number due to disease. Unlike many other organs, the brain is unable to compensate for such changes by increasing cell numbers or altering the size of the cells. For this reason, unique cell cycle and cell growth control mechanisms operate in the developing and adult brain. In Drosophila melanogaster and mammalian neural stem and progenitor cells these mechanisms are intricately coordinated with the developmental age and the nutritional, metabolic and hormonal state of the animal. Defects in neural stem cell proliferation that result in the generation of incorrect cell numbers or defects in neural stem cell differentiation can cause microcephaly or megalencephaly. PMID:26420377

  6. Exfoliated Human Olfactory Neuroepithelium: A Source of Neural Progenitor Cells.

    PubMed

    Jiménez-Vaca, Ana L; Benitez-King, Gloria; Ruiz, Víctor; Ramírez-Rodríguez, Gerardo B; Hernández-de la Cruz, Beatriz; Salamanca-Gómez, Fabio A; González-Márquez, Humberto; Ramírez-Sánchez, Israel; Ortíz-López, Leonardo; Vélez-Del Valle, Cristina; Ordoñez-Razo, Rosa Ma

    2017-04-08

    Neural progenitor cells (NPC) contained in the human adult olfactory neuroepithelium (ONE) possess an undifferentiated state, the capability of self-renewal, the ability to generate neural and glial cells as well as being kept as neurospheres in cell culture conditions. Recently, NPC have been isolated from human or animal models using high-risk surgical methods. Therefore, it was necessary to improve methodologies to obtain and maintain human NPC as well as to achieve better knowledge of brain disorders. In this study, we propose the establishment and characterization of NPC cultures derived from the human olfactory neuroepithelium, using non-invasive procedures. Twenty-two healthy individuals (29.7 ± 4.5 years of age) were subjected to nasal exfoliation. Cells were recovered and kept as neurospheres under serum-free conditions. The neural progenitor origin of these neurospheres was determined by immunocytochemistry and qPCR. Their ability for self-renewal and multipotency was analyzed by clonogenic and differentiation assays, respectively. In the cultures, the ONE cells preserved the phenotype of the neurospheres. The expression levels of Nestin, Musashi, Sox2, and βIII-tubulin demonstrated the neural origin of the neurospheres; 48% of the cells separated could generate neurospheres, determining that they retained their self-renewal capacity. Neurospheres were differentiated in the absence of growth factors (EGF and FGF), and their multipotency ability was maintained as well. We were also able to isolate and grow human neural progenitor cells (neurospheres) through nasal exfoliates (non-invasive method) of the ONE from healthy adults, which is an extremely important contribution for the study of brain disorders and for the development of new therapies.

  7. Requirement for Foxd3 in Maintenance of Neural Crest Progenitors

    PubMed Central

    Teng, Lu; Mundell, Nathan A.; Frist, Audrey Y.; Wang, Qiaohong; Labosky, Patricia A.

    2008-01-01

    Summary Understanding the molecular mechanisms of stem cell maintenance is critical for the ultimate goal of manipulating stem cells for treatment of disease. Foxd3 is required early in mouse embryogenesis; Foxd3−/− embryos fail around the time of implantation, cells of the inner cell mass cannot be maintained in vitro, and blastocyst-derived stem cell lines cannot be established. Here, we report that Foxd3 is required for maintenance of the multipotent mammalian neural crest. Using tissue specific deletion of Foxd3 in the neural crest, we show that Foxd3flox/−; Wnt1-Cre mice die perinatally with a catastrophic loss of neural crest-derived structures. Cranial neural crest tissues are either missing or severely reduced in size, the peripheral nervous system consists of reduced dorsal root ganglia and cranial nerves, and the entire gastrointestinal tract is devoid of neural crest derivatives. These results demonstrate a global role for this transcriptional repressor in all aspects of neural crest maintenance along the anterior-posterior axis, and establish an unprecedented molecular link between multiple divergent progenitor lineages of the mammalian embryo. PMID:18367558

  8. Playing Well with Others: Extrinsic Cues Regulate Neural Progenitor Temporal Identity to Generate Neuronal Diversity.

    PubMed

    Syed, Mubarak Hussain; Mark, Brandon; Doe, Chris Q

    2017-09-09

    During neurogenesis, vertebrate and Drosophila progenitors change over time as they generate a diverse population of neurons and glia. Vertebrate neural progenitors have long been known to use both progenitor-intrinsic and progenitor-extrinsic cues to regulate temporal patterning. In contrast, virtually all temporal patterning mechanisms discovered in Drosophila neural progenitors (neuroblasts) involve progenitor-intrinsic temporal transcription factor cascades. Recent results, however, have revealed several extrinsic pathways that regulate Drosophila neuroblast temporal patterning: nutritional cues regulate the timing of neuroblast proliferation/quiescence and a steroid hormone cue that is required for temporal transcription factor expression. Here, we discuss newly discovered extrinsic cues regulating neural progenitor temporal identity in Drosophila, highlight conserved mechanisms, and raise open questions for the future. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Grhl2 is required in non-neural tissues for neural progenitor survival and forebrain development

    PubMed Central

    Menke, Chelsea; Cionni, Megan; Siggers, Trevor; Bulyk, Martha L.; Beier, David R.; Stottmann, Rolf W.

    2015-01-01

    Grainyhead-like genes are part of a highly conserved gene family that play a number of roles in ectoderm development and maintenance in mammals. Here we identify a novel allele of Grhl2, cleft-face 3 (clft3), in a mouse line recovered from an ENU mutagenesis screen for organogenesis defects. Homozygous clft3 mutants have a number of phenotypes in common with other alleles of Grhl2. We note a significant effect of genetic background on the clft3 phenotype. One of these is a reduction in size of the telencephalon where we find abnormal patterns of neural progenitor mitosis and apoptosis in mutant brains. Interestingly, Grhl2 is not expressed in the developing forebrain, suggesting this is a survival factor for neural progenitors exerting a paracrine effect on the neural tissue from the overlying ectoderm where Grhl2 is highly expressed. PMID:26177923

  10. Neural and oligodendrocyte progenitor cells: transferrin effects on cell proliferation

    PubMed Central

    Silvestroff, Lucas; Franco, Paula Gabriela; Pasquini, Juana María

    2013-01-01

    NSC (neural stem cells)/NPC (neural progenitor cells) are multipotent and self-renew throughout adulthood in the SVZ (subventricular zone) of the mammalian CNS (central nervous system). These cells are considered interesting targets for CNS neurodegenerative disorder cell therapies, and understanding their behaviour in vitro is crucial if they are to be cultured prior to transplantation. We cultured the SVZ tissue belonging to newborn rats under the form of NS (neurospheres) to evaluate the effects of Tf (transferrin) on cell proliferation. The NS were heterogeneous in terms of the NSC/NPC markers GFAP (glial fibrillary acidic protein), Nestin and Sox2 and the OL (oligodendrocyte) progenitor markers NG2 (nerve/glia antigen 2) and PDGFRα (platelet-derived growth factor receptor α). The results of this study indicate that aTf (apoTransferrin) is able to increase cell proliferation of SVZ-derived cells in vitro, and that these effects were mediated at least in part by the TfRc1 (Tf receptor 1). Since OPCs (oligodendrocyte progenitor cells) represent a significant proportion of the proliferating cells in the SVZ-derived primary cultures, we used the immature OL cell line N20.1 to show that Tf was able to augment the proliferation rate of OPC, either by adding aTf to the culture medium or by overexpressing rat Tf in situ. The culture medium supplemented with ferric iron, together with aTf, increased the DNA content, while ferrous iron did not. The present work provides data that could have a potential application in human cell replacement therapies for neurodegenerative disease and/or CNS injury that require the use of in vitro amplified NPCs. PMID:23368675

  11. Neural stem/progenitor cells in Alzheimer's disease.

    PubMed

    Tincer, Gizem; Mashkaryan, Violeta; Bhattarai, Prabesh; Kizil, Caghan

    2016-03-01

    Alzheimer's disease (AD) is the most prevalent neurodegenerative disease and a worldwide health challenge. Different therapeutic approaches are being developed to reverse or slow the loss of affected neurons. Another plausible therapeutic way that may complement the studies is to increase the survival of existing neurons by mobilizing the existing neural stem/progenitor cells (NSPCs) - i.e. "induce their plasticity" - to regenerate lost neurons despite the existing pathology and unfavorable environment. However, there is controversy about how NSPCs are affected by the unfavorable toxic environment during AD. In this review, we will discuss the use of stem cells in neurodegenerative diseases and in particular how NSPCs affect the AD pathology and how neurodegeneration affects NSPCs. In the end of this review, we will discuss how zebrafish as a useful model organism with extensive regenerative ability in the brain might help to address the molecular programs needed for NSPCs to respond to neurodegeneration by enhanced neurogenesis.

  12. Time-lapse live imaging of clonally related neural progenitor cells in the developing zebrafish forebrain.

    PubMed

    Dong, Zhiqiang; Wagle, Mahendra; Guo, Su

    2011-04-06

    Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36 hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

  13. Maternal embryonic leucine zipper kinase (MELK) regulates multipotent neural progenitor proliferation.

    PubMed

    Nakano, Ichiro; Paucar, Andres A; Bajpai, Ruchi; Dougherty, Joseph D; Zewail, Amani; Kelly, Theresa K; Kim, Kevin J; Ou, Jing; Groszer, Matthias; Imura, Tetsuya; Freije, William A; Nelson, Stanley F; Sofroniew, Michael V; Wu, Hong; Liu, Xin; Terskikh, Alexey V; Geschwind, Daniel H; Kornblum, Harley I

    2005-08-01

    Maternal embryonic leucine zipper kinase (MELK) was previously identified in a screen for genes enriched in neural progenitors. Here, we demonstrate expression of MELK by progenitors in developing and adult brain and that MELK serves as a marker for self-renewing multipotent neural progenitors (MNPs) in cultures derived from the developing forebrain and in transgenic mice. Overexpression of MELK enhances (whereas knockdown diminishes) the ability to generate neurospheres from MNPs, indicating a function in self-renewal. MELK down-regulation disrupts the production of neurogenic MNP from glial fibrillary acidic protein (GFAP)-positive progenitors in vitro. MELK expression in MNP is cell cycle regulated and inhibition of MELK expression down-regulates the expression of B-myb, which is shown to also mediate MNP proliferation. These findings indicate that MELK is necessary for proliferation of embryonic and postnatal MNP and suggest that it regulates the transition from GFAP-expressing progenitors to rapid amplifying progenitors in the postnatal brain.

  14. Raman spectroscopy for discrimination of neural progenitor cells and their lineages (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Chen, Keren; Ong, William; Chew, Sing Yian; Liu, Quan

    2017-02-01

    Neurological diseases are one of the leading causes of adult disability and they are estimated to cause more deaths than cancer in the elderly population by 2040. Stem cell therapy has shown great potential in treating neurological diseases. However, before cell therapy can be widely adopted in the long term, a number of challenges need to be addressed, including the fundamental research about cellular development of neural progenitor cells. To facilitate the fundamental research of neural progenitor cells, many methods have been developed to identify neural progenitor cells. Although great progress has been made, there is still lack of an effective method to achieve fast, label-free and noninvasive differentiation of neural progenitor cells and their lineages. As a fast, label-free and noninvasive technique, spontaneous Raman spectroscopy has been conducted to characterize many types of stem cells including neural stem cells. However, to our best knowledge, it has not been studied for the discrimination of neural progenitor cells from specific lineages. Here we report the differentiation of neural progenitor cell from their lineages including astrocytes, oligodendrocytes and neurons using spontaneous Raman spectroscopy. Moreover, we also evaluate the influence of system parameters during spectral acquisition on the quality of measured Raman spectra and the accuracy of classification using the spectra, which yield a set of optimal system parameters facilitating future studies.

  15. Human neural progenitor cells promote photoreceptor survival in retinal explants.

    PubMed

    Englund-Johansson, Ulrica; Mohlin, Camilla; Liljekvist-Soltic, Ingela; Ekström, Per; Johansson, Kjell

    2010-02-01

    Different types of progenitor and stem cells have been shown to provide neuroprotection in animal models of photoreceptor degeneration. The present study was conducted to investigate whether human neural progenitor cells (HNPCs) have neuroprotective properties on retinal explants models with calpain- and caspase-3-dependent photoreceptor cell death. In the first experiments, HNPCs in a feeder layer were co-cultured for 6 days either with postnatal rd1 mouse or normal rat retinas. Retinal histological sections were used to determine outer nuclear layer (ONL) thickness, and to detect the number of photoreceptors with labeling for calpain activity, cleaved caspase-3 and TUNEL. The ONL thickness of co-cultured rat and rd1 retinas was found to be almost 10% and 40% thicker, respectively, compared to controls. Cell counts of calpain activity, cleaved caspase-3 and TUNEL labeled photoreceptors in both models revealed a 30-50% decrease when co-cultured with HNPCs. The results represent significant increases of photoreceptor survival in the co-cultured retinas. In the second experiments, for an identification of putative survival factors, or a combination of them, a growth factor profile was performed on conditioned medium. The relative levels of various growth factors were analyzed by densitometric measurements of growth factor array membranes. Following growth factors were identified as most potential survival factors; granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GMCSF), insulin-like growth factor II (IGF-II), neurotrophic factor 3 (NT-3), placental growth factor (PIGF), transforming growth factors (TGF-beta1 and TGF-beta2) and vascular endothelial growth factor (VEGF-D). HNPCs protect both against calpain- and caspase-3-dependent photoreceptor cell death in the rd1 mouse and against caspase-3-dependent photoreceptor cell death in normal rat retinas in vitro. The protective effect is possibly achieved by a variety of

  16. Genome-wide gene amplification during differentiation of neural progenitor cells in vitro.

    PubMed

    Fischer, Ulrike; Keller, Andreas; Voss, Meike; Backes, Christina; Welter, Cornelius; Meese, Eckart

    2012-01-01

    DNA sequence amplification is a phenomenon that occurs predictably at defined stages during normal development in some organisms. Developmental gene amplification was first described in amphibians during gametogenesis and has not yet been described in humans. To date gene amplification in humans is a hallmark of many tumors. We used array-CGH (comparative genomic hybridization) and FISH (fluorescence in situ hybridization) to discover gene amplifications during in vitro differentiation of human neural progenitor cells. Here we report a complex gene amplification pattern two and five days after induction of differentiation of human neural progenitor cells. We identified several amplified genes in neural progenitor cells that are known to be amplified in malignant tumors. There is also a striking overlap of amplified chromosomal regions between differentiating neural progenitor cells and malignant tumor cells derived from astrocytes. Gene amplifications in normal human cells as physiological process has not been reported yet and may bear resemblance to developmental gene amplifications in amphibians and insects.

  17. Amplification of neural stem cell proliferation by intermediate progenitor cells in Drosophila brain development.

    PubMed

    Bello, Bruno C; Izergina, Natalya; Caussinus, Emmanuel; Reichert, Heinrich

    2008-02-19

    In the mammalian brain, neural stem cells divide asymmetrically and often amplify the number of progeny they generate via symmetrically dividing intermediate progenitors. Here we investigate whether specific neural stem cell-like neuroblasts in the brain of Drosophila might also amplify neuronal proliferation by generating symmetrically dividing intermediate progenitors. Cell lineage-tracing and genetic marker analysis show that remarkably large neuroblast lineages exist in the dorsomedial larval brain of Drosophila. These lineages are generated by brain neuroblasts that divide asymmetrically to self renew but, unlike other brain neuroblasts, do not segregate the differentiating cell fate determinant Prospero to their smaller daughter cells. These daughter cells continue to express neuroblast-specific molecular markers and divide repeatedly to produce neural progeny, demonstrating that they are proliferating intermediate progenitors. The proliferative divisions of these intermediate progenitors have novel cellular and molecular features; they are morphologically symmetrical, but molecularly asymmetrical in that key differentiating cell fate determinants are segregated into only one of the two daughter cells. Our findings provide cellular and molecular evidence for a new mode of neurogenesis in the larval brain of Drosophila that involves the amplification of neuroblast proliferation through intermediate progenitors. This type of neurogenesis bears remarkable similarities to neurogenesis in the mammalian brain, where neural stem cells as primary progenitors amplify the number of progeny they generate through generation of secondary progenitors. This suggests that key aspects of neural stem cell biology might be conserved in brain development of insects and mammals.

  18. G protein-coupled receptor signaling through Gq and JNK negatively regulates neural progenitor cell migration

    PubMed Central

    Mizuno, Norikazu; Kokubu, Hiroshi; Sato, Maiko; Nishimura, Akiyuki; Yamauchi, Junji; Kurose, Hitoshi; Itoh, Hiroshi

    2005-01-01

    In the early development of the central nervous system, neural progenitor cells divide in an asymmetric manner and migrate along the radial glia cells. The radial migration is an important process for the proper lamination of the cerebral cortex. Recently, a new mode of the radial migration was found at the intermediate zone where the neural progenitor cells become multipolar and reduce the migration rate. However, the regulatory signals for the radial migration are unknown. Using the migration assay in vitro, we examined how neural progenitor cell migration is regulated. Neural progenitor cells derived from embryonic mouse telencephalon migrated on laminin-coated dishes. Endothelin (ET)-1 inhibited the neural progenitor cell migration. This ET-1 effect was blocked by BQ788, a specific inhibitor of the ETB receptor, and by the expression of a carboxyl-terminal peptide of Gαq but not Gαi. The expression of constitutively active mutant of Gαq, GαqR183C, inhibited the migration of neural progenitor cells. Moreover, the inhibitory effect of ET-1 was suppressed by the c-Jun N-terminal kinase (JNK) inhibitor SP600125 and the expression of the JNK-binding domain of JNK-interacting protein-1, a specific inhibitor of the JNK pathway. Using the slice culture system of embryonic brain, we demonstrated that ET-1 and the constitutively active mutant of Gαq caused the retention of the neural progenitor cells in the intermediate zone and JNK-binding domain of JNK-interacting protein-1 abrogated the effect of ET-1. These results indicated that G protein-coupled receptor signaling negatively regulates neural progenitor cell migration through Gq and JNK. PMID:16116085

  19. Establishment of Human Neural Progenitor Cells from Human Induced Pluripotent Stem Cells with Diverse Tissue Origins

    PubMed Central

    Fukusumi, Hayato; Shofuda, Tomoko; Bamba, Yohei; Yamamoto, Atsuyo; Kanematsu, Daisuke; Handa, Yukako; Okita, Keisuke; Nakamura, Masaya; Yamanaka, Shinya; Okano, Hideyuki; Kanemura, Yonehiro

    2016-01-01

    Human neural progenitor cells (hNPCs) have previously been generated from limited numbers of human induced pluripotent stem cell (hiPSC) clones. Here, 21 hiPSC clones derived from human dermal fibroblasts, cord blood cells, and peripheral blood mononuclear cells were differentiated using two neural induction methods, an embryoid body (EB) formation-based method and an EB formation method using dual SMAD inhibitors (dSMADi). Our results showed that expandable hNPCs could be generated from hiPSC clones with diverse somatic tissue origins. The established hNPCs exhibited a mid/hindbrain-type neural identity and uniform expression of neural progenitor genes. PMID:27212953

  20. Regulation of adult neural progenitor cell functions by purinergic signaling.

    PubMed

    Tang, Yong; Illes, Peter

    2017-02-01

    Extracellular purines are signaling molecules in the neurogenic niches of the brain and spinal cord, where they activate cell surface purinoceptors at embryonic neural stem cells (NSCs) and adult neural progenitor cells (NPCs). Although mRNA and protein are expressed at NSCs/NPCs for almost all subtypes of the nucleotide-sensitive P2X/P2Y, and the nucleoside-sensitive adenosine receptors, only a few of those have acquired functional significance. ATP is sequentially degraded by ecto-nucleotidases to ADP, AMP, and adenosine with agonistic properties for distinct receptor-classes. Nucleotides/nucleosides facilitate or inhibit NSC/NPC proliferation, migration and differentiation. The most ubiquitous effect of all agonists (especially of ATP and ADP) appears to be the facilitation of cell proliferation, usually through P2Y1Rs and sometimes through P2X7Rs. However, usually P2X7R activation causes necrosis/apoptosis of NPCs. Differentiation can be initiated by P2Y2R-activation or P2X7R-blockade. A key element in the transduction mechanism of either receptor is the increase of the intracellular free Ca(2+) concentration, which may arise due to its release from intracellular storage sites (G protein-coupling; P2Y) or due to its passage through the receptor-channel itself from the extracellular space (ATP-gated ion channel; P2X). Further research is needed to clarify how purinergic signaling controls NSC/NPC fate and how the balance between the quiescent and activated states is established with fine and dynamic regulation. GLIA 2017;65:213-230. © 2016 Wiley Periodicals, Inc.

  1. Facilitating neural stem/progenitor cell niche calibration for neural lineage differentiation by polyelectrolyte multilayer films.

    PubMed

    Lee, I-Chi; Wu, Yu-Chieh

    2014-09-01

    Neural stem/progenitor cells (NSPCs) are a possible candidate for advancing development and lineage control in neural engineering. Differentiated protocols have been developed in this field to generate neural progeny and to establish neural networks. However, continued refinement is required to enhance differentiation specificity and prevent the generation of unwanted cell types. In this study, we fabricated a niche-modulated system to investigate surface effects on NSPC differentiation by the formation of polyelectrolyte multilayer (PEM) films governed by electrostatic interactions of poly-l-glutamine acid as a polyanion and poly-l-lysine as a polycation. The serum- and chemical agent-free system provided a clean and clear platform to observe in isolation the interaction between surface niche and stem cell differentiation. We found that NSPCs were inducible on PEM films of up to eight alternating layers. In addition, neurite outgrowth, neuron percentage, and synaptic function were regulated by layer number and the surface charge of the terminal layer. The average process outgrowth length was over 500μm on PLL/PLGA(n=7.5) only after 3 days of culture. Moreover, the quantity and quality of the differentiated neurons were enhanced as the number of layers increased, especially when the terminal layer was poly-l-lysine. Our results achieve important targets of neural engineering, including long processes, large neural network size, and large amounts of functional neurons. Our methodology for nanoscale control of material deposition can be successfully applied for surface modification, neural niche modulation, and neural engineering applications. Copyright © 2014. Published by Elsevier B.V.

  2. CPAP promotes timely cilium disassembly to maintain neural progenitor pool.

    PubMed

    Gabriel, Elke; Wason, Arpit; Ramani, Anand; Gooi, Li Ming; Keller, Patrick; Pozniakovsky, Andrei; Poser, Ina; Noack, Florian; Telugu, Narasimha Swamy; Calegari, Federico; Šarić, Tomo; Hescheler, Jürgen; Hyman, Anthony A; Gottardo, Marco; Callaini, Giuliano; Alkuraya, Fowzan Sami; Gopalakrishnan, Jay

    2016-04-15

    A mutation in the centrosomal-P4.1-associated protein (CPAP) causes Seckel syndrome with microcephaly, which is suggested to arise from a decline in neural progenitor cells (NPCs) during development. However, mechanisms ofNPCs maintenance remain unclear. Here, we report an unexpected role for the cilium inNPCs maintenance and identifyCPAPas a negative regulator of ciliary length independent of its role in centrosome biogenesis. At the onset of cilium disassembly,CPAPprovides a scaffold for the cilium disassembly complex (CDC), which includes Nde1, Aurora A, andOFD1, recruited to the ciliary base for timely cilium disassembly. In contrast, mutatedCPAPfails to localize at the ciliary base associated with inefficientCDCrecruitment, long cilia, retarded cilium disassembly, and delayed cell cycle re-entry leading to premature differentiation of patientiPS-derivedNPCs. AberrantCDCfunction also promotes premature differentiation ofNPCs in SeckeliPS-derived organoids. Thus, our results suggest a role for cilia in microcephaly and its involvement during neurogenesis and brain size control. © 2016 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

  3. Neural stem/progenitor cells in Alzheimer’s disease

    PubMed Central

    Tincer, Gizem; Mashkaryan, Violeta; Bhattarai, Prabesh; Kizil, Caghan

    2016-01-01

    Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease and a worldwide health challenge. Different therapeutic approaches are being developed to reverse or slow the loss of affected neurons. Another plausible therapeutic way that may complement the studies is to increase the survival of existing neurons by mobilizing the existing neural stem/progenitor cells (NSPCs) — i.e. “induce their plasticity” — to regenerate lost neurons despite the existing pathology and unfavorable environment. However, there is controversy about how NSPCs are affected by the unfavorable toxic environment during AD. In this review, we will discuss the use of stem cells in neurodegenerative diseases and in particular how NSPCs affect the AD pathology and how neurodegeneration affects NSPCs. In the end of this review, we will discuss how zebrafish as a useful model organism with extensive regenerative ability in the brain might help to address the molecular programs needed for NSPCs to respond to neurodegeneration by enhanced neurogenesis. PMID:27505014

  4. Neural progenitors, patterning and ecology in neocortical origins

    PubMed Central

    Aboitiz, Francisco; Zamorano, Francisco

    2013-01-01

    The anatomical organization of the mammalian neocortex stands out among vertebrates for its laminar and columnar arrangement, featuring vertically oriented, excitatory pyramidal neurons. The evolutionary origin of this structure is discussed here in relation to the brain organization of other amniotes, i.e., the sauropsids (reptiles and birds). Specifically, we address the developmental modifications that had to take place to generate the neocortex, and to what extent these modifications were shared by other amniote lineages or can be considered unique to mammals. In this article, we propose a hypothesis that combines the control of proliferation in neural progenitor pools with the specification of regional morphogenetic gradients, yielding different anatomical results by virtue of the differential modulation of these processes in each lineage. Thus, there is a highly conserved genetic and developmental battery that becomes modulated in different directions according to specific selective pressures. In the case of early mammals, ecological conditions like nocturnal habits and reproductive strategies are considered to have played a key role in the selection of the particular brain patterning mechanisms that led to the origin of the neocortex. PMID:24273496

  5. Arsenic and fluoride induce neural progenitor cell apoptosis.

    PubMed

    Rocha, R A; Gimeno-Alcañiz, J V; Martín-Ibañez, R; Canals, J M; Vélez, D; Devesa, V

    2011-06-24

    The aim of the present study is to determine the effect of inorganic arsenic (As) and its metabolites on the viability of the neural progenitor cell (NPC) line C17.2, in order to evaluate cellular mechanisms involved in As developmental neurotoxicity. Moreover, we analyzed the effects of the coexposure to As and fluoride (F), a situation to which some populations are commonly exposed. Our results show that NPCs are not susceptible to pentavalent As species [arsenate, monomethylarsonic acid, and dimethylarsinic acid] and F alone. However, the trivalent metabolites of arsenate [arsenite, monomethylarsonous acid, and dimethylarsinous acid] are toxic at concentrations below 1 mg/l, and this susceptibility increases when there is coexposure with F (≥ 5 mg/l). Arsenite triggers apoptosis after 24 h of exposure, whereas monomethylarsonous acid produces necrosis at very short times (2 h). Arsenite leads to an increase in intracellular Ca levels and generation of reactive oxygen species, which may cause a decrease in mitochondrial transmembrane potential, release of cytochrome c, and consequent activation of caspases. A slight activation of calpain also takes place, which might favor activation of the mitochondrial pathway or might activate other pathways. The treatment with some antioxidants such as quercetin and α-tocopherol shows only a partial reduction of the cytotoxicity.

  6. Heterogeneity of neural progenitor cells revealed by enhancers in the nestin gene

    PubMed Central

    Yaworsky, Paul J.; Kappen, Claudia

    2014-01-01

    Using transgenic embryos, we have identified two distinct CNS progenitor cell-specific enhancers, each requiring the cooperation of at least two independent regulatory sites, within the second intron of the rat nestin gene. One enhancer is active throughout the developing CNS while the other is specifically active in the ventral midbrain. These experiments demonstrate that neural progenitor cells in the midbrain constitute a unique subpopulation based upon their ability to activate the midbrain regulatory elements. Our finding of differential enhancer activity from a gene encoding a structural protein reveals a previously unrecognized diversity in neural progenitor cell populations. PMID:9917366

  7. Msx1-Positive Progenitors in the Retinal Ciliary Margin Give Rise to Both Neural and Non-neural Progenies in Mammals.

    PubMed

    Bélanger, Marie-Claude; Robert, Benoit; Cayouette, Michel

    2017-01-23

    In lower vertebrates, stem/progenitor cells located in a peripheral domain of the retina, called the ciliary margin zone (CMZ), cooperate with retinal domain progenitors to build the mature neural retina. In mammals, it is believed that the CMZ lacks neurogenic potential and that the retina develops from one pool of multipotent retinal progenitor cells (RPCs). Here we identify a population of Msx1-expressing progenitors in the mouse CMZ that is both molecularly and functionally distinct from RPCs. Using genetic lineage tracing, we report that Msx1 progenitors have unique developmental properties compared with RPCs. Msx1 lineages contain both neural retina and non-neural ciliary epithelial progenies and overall generate fewer photoreceptors than classical RPC lineages. Furthermore, we show that the endocytic adaptor protein Numb regulates the balance between neural and non-neural fates in Msx1 progenitors. These results uncover a population of CMZ progenitors, distinct from classical RPCs, that also contributes to mammalian retinogenesis.

  8. Light-induced Notch activity controls neurogenic and gliogenic potential of neural progenitors.

    PubMed

    Kim, Kyung-Tai; Song, Mi-Ryoung

    2016-10-28

    Oscillations in Notch signaling are essential for reserving neural progenitors for cellular diversity in developing brains. Thus, steady and prolonged overactivation of Notch signaling is not suitable for generating neurons. To acquire greater temporal control of Notch activity and mimic endogenous oscillating signals, here we adopted a light-inducible transgene system to induce active form of Notch NICD in neural progenitors. Alternating Notch activity saved more progenitors that are prone to produce neurons creating larger number of mixed clones with neurons and progenitors in vitro, compared to groups with no light or continuous light stimulus. Furthermore, more upper layer neurons and astrocytes arose upon intermittent Notch activity, indicating that dynamic Notch activity maintains neural progeny and fine-tune neuron-glia diversity.

  9. Canonical Wnt signaling transiently stimulates proliferation and enhances neurogenesis in neonatal neural progenitor cultures

    SciTech Connect

    Hirsch, Cordula; Campano, Louise M.; Woehrle, Simon; Hecht, Andreas . E-mail: andreas.hecht@mol-med.uni-freiburg.de

    2007-02-01

    Canonical Wnt signaling triggers the formation of heterodimeric transcription factor complexes consisting of {beta}-catenin and T cell factors, and thereby controls the execution of specific genetic programs. During the expansion and neurogenic phases of embryonic neural development canonical Wnt signaling initially controls proliferation of neural progenitor cells, and later neuronal differentiation. Whether Wnt growth factors affect neural progenitor cells postnatally is not known. Therefore, we have analyzed the impact of Wnt signaling on neural progenitors isolated from cerebral cortices of newborn mice. Expression profiling of pathway components revealed that these cells are fully equipped to respond to Wnt signals. However, Wnt pathway activation affected only a subset of neonatal progenitors and elicited a limited increase in proliferation and neuronal differentiation in distinct subsets of cells. Moreover, Wnt pathway activation only transiently stimulated S-phase entry but did not support long-term proliferation of progenitor cultures. The dampened nature of the Wnt response correlates with the predominant expression of inhibitory pathway components and the rapid actuation of negative feedback mechanisms. Interestingly, in differentiating cell cultures activation of canonical Wnt signaling reduced Hes1 and Hes5 expression suggesting that during postnatal neural development, Wnt/{beta}-catenin signaling enhances neurogenesis from progenitor cells by interfering with Notch pathway activity.

  10. Phenotype and Stability of Neural Differentiation of Androgenetic Murine ES Cell-Derived Neural Progenitor Cells

    PubMed Central

    Wolber, Wanja; Ahmad, Ruhel; Choi, Soon Won; Eckardt, Sigrid; McLaughlin, K. John; Schmitt, Jessica; Geis, Christian; Heckmann, Manfred; Sirén, Anna-Leena; Müller, Albrecht M.

    2013-01-01

    Uniparental zygotes with two paternal (androgenetic, AG) or two maternal genomes (gynogenetic, GG) cannot develop into viable offsprings but form blastocysts from which pluripotent embryonic stem (ES) cells can be derived. For most organs, it is unclear whether uniparental ES cells can give rise to stably expandable somatic stem cells that can repair injured tissues. Even if previous reports indicated that the capacity of AG ES cells to differentiate in vitro into pan-neural progenitor cells (pNPCs) and into cells expressing neural markers is similar to biparental [normal fertilized (N)] ES cells, their potential for functional neurogenesis is not known. Here we show that murine AG pNPCs give rise to neuron-like cells, which then generate sodium-driven action potentials while maintaining fidelity of imprinted gene expression. Neural engraftment after intracerebral transplantation was achieved only by late (22 days) AG and N pNPCs with in vitro low colony-forming cell (CFC) capacity. However, persisting CFC formation seen, in particular, in early (13 or 16 days) differentiation cultures of N and AG pNPCs correlated with a high incidence of trigerm layer teratomas. As AG ES cells display functional neurogenesis and in vivo stability similar to N ES cells, they represent a unique model system to study the roles of paternal and maternal genomes on neural development and on the development of imprinting-associated brain diseases. PMID:26858862

  11. Differential Effects of Isoxazole-9 on Neural Stem/Progenitor Cells, Oligodendrocyte Precursor Cells, and Endothelial Progenitor Cells

    PubMed Central

    Maki, Takakuni; Shindo, Akihiro; Osumi, Noriko; Zhao, Jing; Lin, Hong; Holder, Julie C.; Chuang, Tsu Tshen; McNeish, John D.; Arai, Ken; Lo, Eng H.

    2015-01-01

    Adult mammalian brain can be plastic after injury and disease. Therefore, boosting endogenous repair mechanisms would be a useful therapeutic approach for neurological disorders. Isoxazole-9 (Isx-9) has been reported to enhance neurogenesis from neural stem/progenitor cells (NSPCs). However, the effects of Isx-9 on other types of progenitor/precursor cells remain mostly unknown. In this study, we investigated the effects of Isx-9 on the three major populations of progenitor/precursor cells in brain: NSPCs, oligodendrocyte precursor cells (OPCs), and endothelial progenitor cells (EPCs). Cultured primary NSPCs, OPCs, or EPCs were treated with various concentrations of Isx-9 (6.25, 12.5, 25, 50 μM), and their cell numbers were counted in a blinded manner. Isx-9 slightly increased the number of NSPCs and effectively induced neuronal differentiation of NSPCs. However, Isx-9 significantly decreased OPC number in a concentration-dependent manner, suggesting cytotoxicity. Isx-9 did not affect EPC cell number. But in a matrigel assay of angiogenesis, Isx-9 significantly inhibited tube formation in outgrowth endothelial cells derived from EPCs. This potential anti-tube-formation effect of Isx-9 was confirmed in a brain endothelial cell line. Taken together, our data suggest that mechanisms and targets for promoting stem/progenitor cells in the central nervous system may significantly differ between cell types. PMID:26407349

  12. Topological defects control collective dynamics in neural progenitor cell cultures

    NASA Astrophysics Data System (ADS)

    Kawaguchi, Kyogo; Kageyama, Ryoichiro; Sano, Masaki

    2017-04-01

    Cultured stem cells have become a standard platform not only for regenerative medicine and developmental biology but also for biophysical studies. Yet, the characterization of cultured stem cells at the level of morphology and of the macroscopic patterns resulting from cell-to-cell interactions remains largely qualitative. Here we report on the collective dynamics of cultured murine neural progenitor cells (NPCs), which are multipotent stem cells that give rise to cells in the central nervous system. At low densities, NPCs moved randomly in an amoeba-like fashion. However, NPCs at high density elongated and aligned their shapes with one another, gliding at relatively high velocities. Although the direction of motion of individual cells reversed stochastically along the axes of alignment, the cells were capable of forming an aligned pattern up to length scales similar to that of the migratory stream observed in the adult brain. The two-dimensional order of alignment within the culture showed a liquid-crystalline pattern containing interspersed topological defects with winding numbers of +1/2 and -1/2 (half-integer due to the nematic feature that arises from the head-tail symmetry of cell-to-cell interaction). We identified rapid cell accumulation at +1/2 defects and the formation of three-dimensional mounds. Imaging at the single-cell level around the defects allowed us to quantify the velocity field and the evolving cell density; cells not only concentrate at +1/2 defects, but also escape from -1/2 defects. We propose a generic mechanism for the instability in cell density around the defects that arises from the interplay between the anisotropic friction and the active force field.

  13. The influence of electric fields on hippocampal neural progenitor cells.

    PubMed

    Ariza, Carlos Atico; Fleury, Asha T; Tormos, Christian J; Petruk, Vadim; Chawla, Sagar; Oh, Jisun; Sakaguchi, Donald S; Mallapragada, Surya K

    2010-12-01

    The differentiation and proliferation of neural stem/progenitor cells (NPCs) depend on various in vivo environmental factors or cues, which may include an endogenous electrical field (EF), as observed during nervous system development and repair. In this study, we investigate the morphologic, phenotypic, and mitotic alterations of adult hippocampal NPCs that occur when exposed to two EFs of estimated endogenous strengths. NPCs treated with a 437 mV/mm direct current (DC) EF aligned perpendicularly to the EF vector and had a greater tendency to differentiate into neurons, but not into oligodendrocytes or astrocytes, compared to controls. Furthermore, NPC process growth was promoted perpendicularly and inhibited anodally in the 437 mV/mm DC EF. Yet fewer cells were observed in the DC EF, which in part was due to a decrease in cell viability. The other EF applied was a 46 mV/mm alternating current (AC) EF. However, the 46 mV/mm AC EF showed no major differences in alignment or differentiation, compared to control conditions. For both EF treatments, the percent of mitotic cells during the last 14 h of the experiment were statistically similar to controls. Reported here, to our knowledge, is the first evidence of adult NPC differentiation affected in an EF in vitro. Further investigation and application of EFs on stem cells is warranted to elucidate the utility of EFs to control phenotypic behavior. With progress, the use of EFs may be engineered to control differentiation and target the growth of transplanted cells in a stem cell-based therapy to treat nervous system disorders.

  14. Adult retinal pigment epithelium cells express neural progenitor properties and the neuronal precursor protein doublecortin.

    PubMed

    Engelhardt, Maren; Bogdahn, Ulrich; Aigner, Ludwig

    2005-04-08

    The adult mammalian retina is devoid of any detectable neurogenesis. However, different cell types have been suggested to potentially act as neural progenitors in the adult mammalian retina in vitro, such as ciliary body (CB), Muller glia, and retinal pigment epithelium (RPE) cells. In rodents and humans, strong evidence for neural stem or progenitor properties exists only for CB-derived cells, but not for other retinal cell types. Here, we provide a comparative analysis of adult rat CB- and RPE-derived cells suggesting that the two cell types share certain neural progenitor properties in vitro. CB and RPE cells expressed neural progenitor markers such as Nestin, Flk-1, Hes1, and Musashi. They proliferated under adherent and neurosphere conditions and showed limited self-renewal. Moreover, they differentiated into neuronal and glial cells based on the expression of differentiation markers such as the young neuronal marker beta-III tubulin and the glial and progenitor markers GFAP and NG2. Expression of beta-III tubulin was found in cells with neuronal and non-neuronal morphology. A subpopulation of RPE- and CB-derived progenitor cells expressed the neurogenesis-specific protein doublecortin (DCX). Interestingly, DCX expression defined a beta-III tubulin-positive CB and RPE fraction with a distinct neuronal morphology. In summary, the data suggest that RPE cells share with CB cells the potential to de-differentiate into a cell type with neural progenitor-like identity. In addition, DCX expression might define the neuronal-differentiating RPE- and CB-derived progenitor population.

  15. Presenilin-1 regulates neural progenitor cell differentiation in the adult brain

    PubMed Central

    Gadadhar, Archana; Marr, Robert; Lazarov, Orly

    2011-01-01

    Presenilin-1 (PS1) is the catalytic core of the aspartyl protease γ-secretase. Previous genetic studies using germ-line deletion of PS1 and conditional knockout mice demonstrated that PS1 plays an essential role in neuronal differentiation during neural development, but it remained unclear whether PS1 plays a similar role in neurogenesis in the adult brain. Here we show that neural progenitor cells infected with lentiviral vectors expressing short interfering RNA (siRNA) for the exclusive knockdown of PS1 in the neurogenic microenvironments, exhibit a dramatic enhancement of cell differentiation. Infected cells differentiated into neurons, astrocytes and oligodendrocytes, suggesting that multipotentiality of neural progenitor cells is not affected by reduced levels of PS1. Neurosphere cultures treated with γ-secretase inhibitors exhibit a similar phenotype of enhanced cell differentiation, suggesting that PS1 function in neural progenitor cells is γ-secretase-dependent. Neurospheres infected with lentiviral vectors expressing siRNA for the targeting of PS1 differentiated even in the presence of the proliferation factors epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), suggesting that PS1 dominates EFG and bFGF signaling pathways. Reduction of PS1 expression in neural progenitor cells was accompanied by a decrease in epidermal growth factor receptor (EGFR) and β-catenin expression level, suggesting that they are downstream essential transducers of PS1 signaling in adult neural progenitor cells. These findings suggest a physiological role for PS1 in adult neurogenesis, and a potential target for the manipulation of neural progenitor cell differentiation. PMID:21325529

  16. Mir-23a and mir-125b regulate neural stem/progenitor cell proliferation by targeting Musashi1

    PubMed Central

    Gioia, Ubaldo; Di Carlo, Valerio; Caramanica, Pasquale; Toselli, Camilla; Cinquino, Antonella; Marchioni, Marcella; Laneve, Pietro; Biagioni, Stefano; Bozzoni, Irene; Cacci, Emanuele; Caffarelli, Elisa

    2014-01-01

    Musashi1 is an RNA binding protein that controls the neural cell fate, being involved in maintaining neural progenitors in their proliferative state. In particular, its downregulation is needed for triggering early neural differentiation programs. In this study, we profiled microRNA expression during the transition from neural progenitors to differentiated astrocytes and underscored 2 upregulated microRNAs, miR-23a and miR-125b, that sinergically act to restrain Musashi1 expression, thus creating a regulatory module controlling neural progenitor proliferation. PMID:25483045

  17. Mir-23a and mir-125b regulate neural stem/progenitor cell proliferation by targeting Musashi1.

    PubMed

    Gioia, Ubaldo; Di Carlo, Valerio; Caramanica, Pasquale; Toselli, Camilla; Cinquino, Antonella; Marchioni, Marcella; Laneve, Pietro; Biagioni, Stefano; Bozzoni, Irene; Cacci, Emanuele; Caffarelli, Elisa

    2014-01-01

    Musashi1 is an RNA binding protein that controls the neural cell fate, being involved in maintaining neural progenitors in their proliferative state. In particular, its downregulation is needed for triggering early neural differentiation programs. In this study, we profiled microRNA expression during the transition from neural progenitors to differentiated astrocytes and underscored 2 upregulated microRNAs, miR-23a and miR-125b, that sinergically act to restrain Musashi1 expression, thus creating a regulatory module controlling neural progenitor proliferation.

  18. Progenitors of the protochordate ocellus as an evolutionary origin of the neural crest

    PubMed Central

    2013-01-01

    The neural crest represents a highly multipotent population of embryonic stem cells found only in vertebrate embryos. Acquisition of the neural crest during the evolution of vertebrates was a great advantage, providing Chordata animals with the first cellular cartilage, bone, dentition, advanced nervous system and other innovations. Today not much is known about the evolutionary origin of neural crest cells. Here we propose a novel scenario in which the neural crest originates from neuroectodermal progenitors of the pigmented ocelli in Amphioxus-like animals. We suggest that because of changes in photoreception needs, these multipotent progenitors of photoreceptors gained the ability to migrate outside of the central nervous system and subsequently started to give rise to neural, glial and pigmented progeny at the periphery. PMID:23575111

  19. Induction of Excess Centrosomes in Neural Progenitor Cells during the Development of Radiation-Induced Microcephaly

    PubMed Central

    Shimada, Mikio; Matsuzaki, Fumio; Kato, Akihiro; Kobayashi, Junya; Matsumoto, Tomohiro; Komatsu, Kenshi

    2016-01-01

    The embryonic brain is one of the tissues most vulnerable to ionizing radiation. In this study, we showed that ionizing radiation induces apoptosis in the neural progenitors of the mouse cerebral cortex, and that the surviving progenitor cells subsequently develop a considerable amount of supernumerary centrosomes. When mouse embryos at Day 13.5 were exposed to γ-rays, brains sizes were reduced markedly in a dose-dependent manner, and these size reductions persisted until birth. Immunostaining with caspase-3 antibodies showed that apoptosis occurred in 35% and 40% of neural progenitor cells at 4 h after exposure to 1 and 2 Gy, respectively, and this was accompanied by a disruption of the apical layer in which mitotic spindles were positioned in unirradiated mice. At 24 h after 1 Gy irradiation, the apoptotic cells were completely eliminated and proliferation was restored to a level similar to that of unirradiated cells, but numerous spindles were localized outside the apical layer. Similarly, abnormal cytokinesis, which included multipolar division and centrosome clustering, was observed in 19% and 24% of the surviving neural progenitor cells at 48 h after irradiation with 1 and 2 Gy, respectively. Because these cytokinesis aberrations derived from excess centrosomes result in growth delay and mitotic catastrophe-mediated cell elimination, our findings suggest that, in addition to apoptosis at an early stage of radiation exposure, radiation-induced centrosome overduplication could contribute to the depletion of neural progenitors and thereby lead to microcephaly. PMID:27367050

  20. 12-Deoxyphorbols Promote Adult Neurogenesis by Inducing Neural Progenitor Cell Proliferation via PKC Activation.

    PubMed

    Geribaldi-Doldán, Noelia; Flores-Giubi, Eugenia; Murillo-Carretero, Maribel; García-Bernal, Francisco; Carrasco, Manuel; Macías-Sánchez, Antonio J; Domínguez-Riscart, Jesús; Verástegui, Cristina; Hernández-Galán, Rosario; Castro, Carmen

    2015-07-29

    Neuropsychiatric and neurological disorders frequently occur after brain insults associated with neuronal loss. Strategies aimed to facilitate neuronal renewal by promoting neurogenesis constitute a promising therapeutic option to treat neuronal death-associated disorders. In the adult brain, generation of new neurons occurs physiologically throughout the entire life controlled by extracellular molecules coupled to intracellular signaling cascades. Proteins participating in these cascades within neurogenic regions constitute potential pharmacological targets to promote neuronal regeneration of injured areas of the central nervous system. We have performed in vitro and in vivo approaches to determine neural progenitor cell proliferation to understand whether activation of kinases of the protein kinase C family facilitates neurogenesis in the adult brain. We have demonstrated that protein kinase C activation by phorbol-12-myristate-13-acetate induces neural progenitor cell proliferation in vitro. We also show that the nontumorogenic protein kinase C activator prostratin exerts a proliferative effect on neural progenitor cells in vitro. This effect can be reverted by addition of the protein kinase C inhibitor G06850, demonstrating that the effect of prostratin is mediated by protein kinase C activation. Additionally, we show that prostratin treatment in vivo induces proliferation of neural progenitor cells within the dentate gyrus of the hippocampus and the subventricular zone. Finally, we describe a library of diterpenes with a 12-deoxyphorbol structure similar to that of prostratin that induces a stronger effect than prostratin on neural progenitor cell proliferation both in vitro and in vivo. This work suggests that protein kinase C activation is a promising strategy to expand the endogenous neural progenitor cell population to promote neurogenesis and highlights the potential of 12-deoxyphorbols as pharmaceutical agents to facilitate neuronal renewal. © The

  1. 12-Deoxyphorbols Promote Adult Neurogenesis by Inducing Neural Progenitor Cell Proliferation via PKC Activation

    PubMed Central

    Geribaldi-Doldán, Noelia; Flores-Giubi, Eugenia; Murillo-Carretero, Maribel; García-Bernal, Francisco; Carrasco, Manuel; Macías-Sánchez, Antonio J.; Domínguez-Riscart, Jesús; Verástegui, Cristina; Hernández-Galán, Rosario

    2016-01-01

    Background: Neuropsychiatric and neurological disorders frequently occur after brain insults associated with neuronal loss. Strategies aimed to facilitate neuronal renewal by promoting neurogenesis constitute a promising therapeutic option to treat neuronal death-associated disorders. In the adult brain, generation of new neurons occurs physiologically throughout the entire life controlled by extracellular molecules coupled to intracellular signaling cascades. Proteins participating in these cascades within neurogenic regions constitute potential pharmacological targets to promote neuronal regeneration of injured areas of the central nervous system. Methodology: We have performed in vitro and in vivo approaches to determine neural progenitor cell proliferation to understand whether activation of kinases of the protein kinase C family facilitates neurogenesis in the adult brain. Results: We have demonstrated that protein kinase C activation by phorbol-12-myristate-13-acetate induces neural progenitor cell proliferation in vitro. We also show that the nontumorogenic protein kinase C activator prostratin exerts a proliferative effect on neural progenitor cells in vitro. This effect can be reverted by addition of the protein kinase C inhibitor G06850, demonstrating that the effect of prostratin is mediated by protein kinase C activation. Additionally, we show that prostratin treatment in vivo induces proliferation of neural progenitor cells within the dentate gyrus of the hippocampus and the subventricular zone. Finally, we describe a library of diterpenes with a 12-deoxyphorbol structure similar to that of prostratin that induces a stronger effect than prostratin on neural progenitor cell proliferation both in vitro and in vivo. Conclusions: This work suggests that protein kinase C activation is a promising strategy to expand the endogenous neural progenitor cell population to promote neurogenesis and highlights the potential of 12-deoxyphorbols as pharmaceutical

  2. Transplantation of neural progenitors enhances production of endogenous cells in the impaired brain.

    PubMed

    Ben-Shaanan, T L; Ben-Hur, T; Yanai, J

    2008-02-01

    Grafting of neural progenitors has been shown to reverse a wide variety of neurobehavioral defects. While their role of replacing injured cells and restoring damaged circuitries has been shown, it is widely accepted that this cannot be the only mechanism, as therapy can occur even when an insufficient number of transplanted cells are found. We hypothesized that one major mechanism by which transplanted neural progenitors exert their therapeutic effect is by enhancing endogenous cells production. Consequently, in an allographic model of transplantation, prenatally heroin-exposed genetically heterogeneous (HS) mice were made defective in their hippocampal neurobehavioral function by exposing their mothers to heroin (10 mg kg(-1) heroin on gestation days 9-18). Hippocampal damage was confirmed by deficient performance in the Morris maze (P<0.009), and decreased production of endogenous cells in the dentate gyrus by 39% was observed. On postnatal day 35, they received an HS-derived neural progenitors transplant followed by repeated bromodeoxyuridine injections. The transplant returned endogenous cells production to normal levels (P<0.006) and reversed the behavioral defects (P<0.03), despite the fact that only 0.0334% of the transplanted neural progenitors survived and that they differentiated mainly to astrocytes. An immunological study demonstrated the presence of macrophages and T cells as a possible explanation for the paucity of the transplanted cells. This study suggests one mechanism for the therapeutic action of neural progenitors, the enhancement of the production of endogenous cells, pointing to future clinical applications in this direction by use of neural progenitors or by analogous cell-inducing techniques.

  3. [A technique of rhesus monkey neural progenitor cells intravitreal transplant to rats].

    PubMed

    Bian, Hui; Fan, Yao-Dong; Guo, Li-Yun; Yu, Hua-Lin

    2012-02-01

    To investigate a simple and effective intraocular xenotransplant technique of rhesus monkey neural progenitor cells to rats, mechanical injury was induced in the rat's right retina. And the GFP-labeled rhesus monkey neural progenitor cells suspension was slowly injected into the vitreous space of the right injured and left control eye. Confocal image suggested that the xenografted cells survived in both the injured and control eye, meanwhile the cells integrated in the injured right retina. The results demonstrated that intravitreal xenotransplant could be adopted as a simple and reliable method.

  4. NRAGE mediates p38 activation and neural progenitor apoptosis via the bone morphogenetic protein signaling cascade.

    PubMed

    Kendall, Stephen E; Battelli, Chiara; Irwin, Sarah; Mitchell, Jane G; Glackin, Carlotta A; Verdi, Joseph M

    2005-09-01

    Understanding the molecular events that govern neural progenitor lineage commitment, mitotic arrest, and differentiation into functional progeny are germane to our understanding of neocortical development. Members of the family of bone morphogenetic proteins (BMPs) play pivotal roles in regulating neural differentiation and apoptosis during neurogenesis through combined actions involving Smad and TAK1 activation. We demonstrate that BMP signaling is required for the induction of apoptosis of neural progenitors and that NRAGE is a mandatory component of the signaling cascade. NRAGE possesses the ability to bind and function with the TAK1-TAB1-XIAP complex facilitating the activation of p38. Disruption of NRAGE or any other member of the noncanonical signaling cascaded is sufficient to block p38 activation and thus the proapoptotic signals generated through BMP exposure. The function of NRAGE is independent of Smad signaling, but the introduction of a dominant-negative Smad5 also rescues neural progenitor apoptosis, suggesting that both canonical and noncanonical pathways can converge and regulate BMP-mediated apoptosis. Collectively, these results establish NRAGE as an integral component in BMP signaling and clarify its role during neural progenitor development.

  5. NRAGE Mediates p38 Activation and Neural Progenitor Apoptosis via the Bone Morphogenetic Protein Signaling Cascade

    PubMed Central

    Kendall, Stephen E.; Battelli, Chiara; Irwin, Sarah; Mitchell, Jane G.; Glackin, Carlotta A.; Verdi, Joseph M.

    2005-01-01

    Understanding the molecular events that govern neural progenitor lineage commitment, mitotic arrest, and differentiation into functional progeny are germane to our understanding of neocortical development. Members of the family of bone morphogenetic proteins (BMPs) play pivotal roles in regulating neural differentiation and apoptosis during neurogenesis through combined actions involving Smad and TAK1 activation. We demonstrate that BMP signaling is required for the induction of apoptosis of neural progenitors and that NRAGE is a mandatory component of the signaling cascade. NRAGE possesses the ability to bind and function with the TAK1-TAB1-XIAP complex facilitating the activation of p38. Disruption of NRAGE or any other member of the noncanonical signaling cascaded is sufficient to block p38 activation and thus the proapoptotic signals generated through BMP exposure. The function of NRAGE is independent of Smad signaling, but the introduction of a dominant-negative Smad5 also rescues neural progenitor apoptosis, suggesting that both canonical and noncanonical pathways can converge and regulate BMP-mediated apoptosis. Collectively, these results establish NRAGE as an integral component in BMP signaling and clarify its role during neural progenitor development. PMID:16107717

  6. Electroacupuncture Promotes Proliferation of Amplifying Neural Progenitors and Preserves Quiescent Neural Progenitors from Apoptosis to Alleviate Depressive-Like and Anxiety-Like Behaviours

    PubMed Central

    Yang, Liu; Yue, Na; Zhu, Xiaocang; Han, Qiuqin; Li, Bin; Liu, Qiong; Wu, Gencheng; Yu, Jin

    2014-01-01

    The present study was designed to investigate the effects of electroacupuncture (EA) on depressive-like and anxiety-like behaviours and neural progenitors in the hippocampal dentate gyrus (DG) in a chronic unpredictable stress (CUS) rat model of depression. After being exposed to a CUS procedure for 2 weeks, rats were subjected to EA treatment, which was performed on acupoints Du-20 (Bai-Hui) and GB-34 (Yang-Ling-Quan), once every other day for 15 consecutive days (including 8 treatments), with each treatment lasting for 30 min. The behavioural tests (i.e., forced swimming test, elevated plus-maze test, and open-field entries test) revealed that EA alleviated the depressive-like and anxiety-like behaviours of the stressed rats. Immunohistochemical results showed that proliferative cells (BrdU-positive) in the EA group were significantly larger in number compared with the Model group. Further, the results showed that EA significantly promoted the proliferation of amplifying neural progenitors (ANPs) and simultaneously inhibited the apoptosis of quiescent neural progenitors (QNPs). In a word, the mechanism underlying the antidepressant-like effects of EA is associated with enhancement of ANPs proliferation and preserving QNPs from apoptosis. PMID:24719647

  7. Membrane biophysics define neuron and astrocyte progenitors in the neural lineage

    PubMed Central

    Nourse, J.L.; Prieto, J.L.; Dickson, A.R.; Lu, J.; Pathak, M.M.; Tombola, F.; Demetriou, M.; Lee, A.P.; Flanagan, L.A.

    2014-01-01

    Neural stem and progenitor cells (NSPCs) are heterogeneous populations of self-renewing stem cells and more committed progenitors that differentiate into neurons, astrocytes and oligodendrocytes. Accurately identifying and characterizing the different progenitor cells in this lineage has continued to be a challenge for the field. We found previously that populations of NSPCs with more neurogenic progenitors (NPs) can be distinguished from those with more astrogenic progenitors (APs) by their inherent biophysical properties, specifically the electrophysiological property of whole cell membrane capacitance, which we characterized with dielectrophoresis (DEP). Here we hypothesize that inherent electrophysiological properties are sufficient to define NPs and APs and test this by determining whether isolation of cells solely by these properties specifically separates NPs and APs. We found NPs and APs are enriched in distinct fractions after separation by electrophysiological properties using DEP. A single round of DEP isolation provided greater NP enrichment than sorting with PSA-NCAM, which is considered an NP marker. Additionally, cell surface N-linked glycosylation was found to significantly affect cell fate-specific electrophysiological properties, providing a molecular basis for the cell membrane characteristics. Inherent plasma membrane biophysical properties are thus sufficient to define progenitor cells of differing fate potential in the neural lineage, can be used to specifically isolate these cells, and are linked to patterns of glycosylation on the cell surface. PMID:24105912

  8. Differences and similarities between human and chimpanzee neural progenitors during cerebral cortex development

    PubMed Central

    Mora-Bermúdez, Felipe; Badsha, Farhath; Kanton, Sabina; Camp, J Gray; Vernot, Benjamin; Köhler, Kathrin; Voigt, Birger; Okita, Keisuke; Maricic, Tomislav; He, Zhisong; Lachmann, Robert; Pääbo, Svante; Treutlein, Barbara; Huttner, Wieland B

    2016-01-01

    Human neocortex expansion likely contributed to the remarkable cognitive abilities of humans. This expansion is thought to primarily reflect differences in proliferation versus differentiation of neural progenitors during cortical development. Here, we have searched for such differences by analysing cerebral organoids from human and chimpanzees using immunohistofluorescence, live imaging, and single-cell transcriptomics. We find that the cytoarchitecture, cell type composition, and neurogenic gene expression programs of humans and chimpanzees are remarkably similar. Notably, however, live imaging of apical progenitor mitosis uncovered a lengthening of prometaphase-metaphase in humans compared to chimpanzees that is specific to proliferating progenitors and not observed in non-neural cells. Consistent with this, the small set of genes more highly expressed in human apical progenitors points to increased proliferative capacity, and the proportion of neurogenic basal progenitors is lower in humans. These subtle differences in cortical progenitors between humans and chimpanzees may have consequences for human neocortex evolution. DOI: http://dx.doi.org/10.7554/eLife.18683.001 PMID:27669147

  9. ApoE is required for maintenance of the dentate gyrus neural progenitor pool

    PubMed Central

    Yang, Cui-Ping; Gilley, Jennifer A.; Zhang, Gui; Kernie, Steven G.

    2011-01-01

    Many genes regulating adult neurogenesis have been identified and are known to play similar roles during early neuronal development. We recently identified apolipoprotein E (ApoE) as a gene the expression of which is essentially absent in early brain progenitors but becomes markedly upregulated in adult dentate gyrus stem/progenitor cells. Here, we demonstrate that ApoE deficiency impairs adult dentate gyrus development by affecting the neural progenitor pool over time. We utilized ApoE-deficient mice crossed to a nestin-GFP reporter to demonstrate that dentate gyrus progenitor cells proliferate more rapidly at early ages, which is subsequently accompanied by an overall decrease in neural progenitor cell number at later time points. This appears to be secondary to over-proliferation early in life and ultimate depletion of the Type 1 nestin- and GFAP-expressing neural stem cells. We also rescue the proliferation phenotype with an ApoE-expressing retrovirus, demonstrating that ApoE works directly in this regard. These data provide novel insight into late hippocampal development and suggest a possible role for ApoE in neurodegenerative diseases. PMID:21880781

  10. Pannexin 2 Is Expressed by Postnatal Hippocampal Neural Progenitors and Modulates Neuronal Commitment*

    PubMed Central

    Swayne, Leigh Anne; Sorbara, Catherine D.; Bennett, Steffany A. L.

    2010-01-01

    The pannexins (Panx1, -2, and -3) are a mammalian family of putative single membrane channels discovered through homology to invertebrate gap junction-forming proteins, the innexins. Because connexin gap junction proteins are known regulators of neural stem and progenitor cell proliferation, migration, and specification, we asked whether pannexins, specifically Panx2, play a similar role in the postnatal hippocampus. We show that Panx2 protein is differentially expressed by multipotential progenitor cells and mature neurons. Both in vivo and in vitro, Type I and IIa stem-like neural progenitor cells express an S-palmitoylated Panx2 species localizing to Golgi and endoplasmic reticulum membranes. Protein expression is down-regulated during neurogenesis in neuronally committed Type IIb and III progenitor cells and immature neurons. Panx2 is re-expressed by neurons following maturation. Protein expressed by mature neurons is not palmitoylated and localizes to the plasma membrane. To assess the impact of Panx2 on neuronal differentiation, we used short hairpin RNA to suppress Panx2 expression in Neuro2a cells. Knockdown significantly accelerated the rate of neuronal differentiation. Neuritic extension and the expression of antigenic markers of mature neurons occurred earlier in stable lines expressing Panx2 short hairpin RNA than in controls. Together, these findings describe an endogenous post-translational regulation of Panx2, specific to early neural progenitor cells, and demonstrate that this expression plays a role in modulating the timing of their commitment to a neuronal lineage. PMID:20529862

  11. Spatiotemporal analyses of neural lineages after embryonic and postnatal progenitor targeting combining different reporters

    PubMed Central

    Figueres-Oñate, Maria; García-Marqués, Jorge; Pedraza, Maria; De Carlos, Juan Andrés; López-Mascaraque, Laura

    2015-01-01

    Genetic lineage tracing with electroporation is one of the most powerful techniques to target neural progenitor cells and their progeny. However, the spatiotemporal relationship between neural progenitors and their final phenotype remain poorly understood. One critical factor to analyze the cell fate of progeny is reporter integration into the genome of transfected cells. To address this issue, we performed postnatal and in utero co-electroporations of different fluorescent reporters to label, in both cerebral cortex and olfactory bulb, the progeny of subventricular zone neural progenitors. By comparing fluorescent reporter expression in the adult cell progeny, we show a differential expression pattern within the same cell lineage, depending on electroporation stage and cell identity. Further, while neuronal lineages arise from many progenitors in proliferative zones after few divisions, glial lineages come from fewer progenitors that accomplish many cell divisions. Together, these data provide a useful guide to select a strategy to track the cell fate of a specific cell population and to address whether a different proliferative origin might be correlated with functional heterogeneity. PMID:25852461

  12. Notch signaling patterns neurogenic ectoderm and regulates the asymmetric division of neural progenitors in sea urchin embryos.

    PubMed

    Mellott, Dan O; Thisdelle, Jordan; Burke, Robert D

    2017-08-29

    We have examined regulation of neurogenesis by Delta/Notch signaling in sea urchin embryos. At gastrulation neural progenitors enter S-phase coincident with expression of Sp-SoxC. We used a BAC (bacterial artificial chromosome) containing GFP knocked into the Sp-SoxC locus to label neural progenitors. Live imaging and immunolocalizations indicate that Sp-SoxC-expressing cells divide producing pairs of adjacent cells expressing GFP. Over an interval of about 6 h, one cell fragments, undergoes apoptosis, and expresses high levels of activated Caspase3. A Notch reporter indicates that Notch signaling is activated in cells adjacent to cells expressing Sp-SoxC. Inhibition of γ-secretase, injection of Sp-Delta morpholinos, or CRISPR/Cas9-induced mutation of Sp-Delta results in supernumerary neural progenitors and neurons. Interfering with Notch signaling increases neural progenitor recruitment and pairs of neural progenitors. Thus, Notch signaling restricts the number of neural progenitors recruited and regulates the fate of progeny of the asymmetric division. We propose a model in which localized signaling converts ectodermal and ciliary band cells to neural progenitors that divide asymmetrically to produce a neural precursor and an apoptotic cell. © 2017. Published by The Company of Biologists Ltd.

  13. Characterization of Human Neural Progenitor Cell Models for Developmental Neurotoxicity Screening

    EPA Science Inventory

    Current testing methods for developmental neurotoxicity (DNT) make evaluation of the effects of large numbers of chemicals impractical and prohibitively expensive. As such, we are evaluating two different human neural progenitor cell (hNPC) models for their utility in screens for...

  14. Isolation of neural crest derived chromaffin progenitors from adult adrenal medulla.

    PubMed

    Chung, Kuei-Fang; Sicard, Flavie; Vukicevic, Vladimir; Hermann, Andreas; Storch, Alexander; Huttner, Wieland B; Bornstein, Stefan R; Ehrhart-Bornstein, Monika

    2009-10-01

    Chromaffin cells of the adrenal medulla are neural crest-derived cells of the sympathoadrenal lineage. Unlike the closely-related sympathetic neurons, a subpopulation of proliferation-competent cells exists even in the adult. Here, we describe the isolation, expansion, and in vitro characterization of proliferation-competent progenitor cells from the bovine adrenal medulla. Similar to neurospheres, these cells, when prevented from adherence to the culture dish, grew in spheres, which we named chromospheres. These chromospheres were devoid of mRNA specific for smooth muscle cells (MYH11) or endothelial cells (PECAM1). During sphere formation, markers for differentiated chromaffin cells, such as phenylethanolamine-N-methyl transferase, were downregulated while neural progenitor markers nestin, vimentin, musashi 1, and nerve growth factor receptor, as well as markers of neural crest progenitor cells such as Sox1 and Sox9, were upregulated. Clonal analysis and bromo-2'-deoxyuridine-incorporation analysis demonstrated the self-renewing capacity of chromosphere cells. Differentiation protocols using NGF and BMP4 or dexamethasone induced neuronal or endocrine differentiation, respectively. Electrophysiological analyses of neural cells derived from chromospheres revealed functional properties of mature nerve cells, such as tetrodotoxin-sensitive sodium channels and action potentials. Our study provides evidence that proliferation and differentiation competent chromaffin progenitor cells can be isolated from adult adrenal medulla and that these cells might harbor the potential for the treatment of neurodegenerative diseases, such as Parkinson's disease.

  15. Characterization of Human Neural Progenitor Cell Models for Developmental Neurotoxicity Screening

    EPA Science Inventory

    Current testing methods for developmental neurotoxicity (DNT) make evaluation of the effects of large numbers of chemicals impractical and prohibitively expensive. As such, we are evaluating two different human neural progenitor cell (hNPC) models for their utility in screens for...

  16. MicroRNA-9 controls a migratory mechanism in human neural progenitor cells.

    PubMed

    Uchida, Nobuko

    2010-04-02

    MicroRNAs play roles in developmental switching; however, their roles in human neural progenitor cells (hNPCs) is poorly understood. In this issue of Cell Stem Cell, Delaloy et al. (2010) report that proliferation and migration choices in hNPCs are regulated by miR-9.

  17. Scaffolds for Intracerebral Grafting of Neural Progenitor Cells After Cerebral Infarction: A Systematic Review

    PubMed Central

    Cohen, Laura K; Jensen, Matthew B

    2015-01-01

    Context Intracerebral grafting of neural progenitor cells is a promising potential treatment to improve recovery after stroke, but the structural disruption and cavitation of brain tissue that occurs creates an unfavorable environment for graft cell survival. To overcome this obstacle, scaffold materials have been used as extracellular matrix to provide structural support for the transplanted cells. Many materials could potentially be used as scaffolds for this application. Evidence Acquisition We performed a systematic review to determine the available evidence supporting specific scaffolds for neural progenitor cell grafting after stroke. Articles were identified with a MeSH search on PubMed. Relevant references and “related articles” of selected manuscripts were also reviewed. Full original articles published prior to May 2013 presenting unique experimental data describing intracerebral grafting of neural progenitor cells in a scaffold after cerebral infarction were included in our study. All selected articles were reviewed thoroughly by the authors for relevant data. Results We found reports of use of scaffolds composed of polyglycolic acid, poly [lactic-co-glycolic acid] particles (with and without VEGF), hyaluronan-heparin-collagen hydrogel, Matrigel, collagen and extracellular matrix derived from porcine brain and urinary bladder. While multiple beneficial effects were reported, the optimal scaffold is unclear as we found no direct comparisons. Conclusions We conclude that multiple scaffolds appear promising for neural progenitor cell grafting after stroke, but further research is needed to optimize this neurorestorative approach. Thus, we hope to provide a basic understanding of the state of scaffolds for neural progenitor cell grafting after stroke and to encourage further research. Based on the methods of the discussed studies, we propose a standardized set of outcomes that would best be used to evaluate and compare the effectiveness of a given

  18. Low Density Lipoprotein Receptor Related Proteins as Regulators of Neural Stem and Progenitor Cell Function

    PubMed Central

    Landowski, Lila M.; Young, Kaylene M.

    2016-01-01

    The central nervous system (CNS) is a highly organised structure. Many signalling systems work in concert to ensure that neural stem cells are appropriately directed to generate progenitor cells, which in turn mature into functional cell types including projection neurons, interneurons, astrocytes, and oligodendrocytes. Herein we explore the role of the low density lipoprotein (LDL) receptor family, in particular family members LRP1 and LRP2, in regulating the behaviour of neural stem and progenitor cells during development and adulthood. The ability of LRP1 and LRP2 to bind a diverse and extensive range of ligands, regulate ligand endocytosis, recruit nonreceptor tyrosine kinases for direct signal transduction and signal in conjunction with other receptors, enables them to modulate many crucial neural cell functions. PMID:26949399

  19. Characterization of a population of neural progenitor cells in the infant hippocampus

    PubMed Central

    Paine, S M L; Willsher, A R; Nicholson, S L; Sebire, N J; Jacques, T S

    2014-01-01

    Aims Abnormalities of the hippocampus are associated with a range of diseases in children, including epilepsy and sudden death. A population of rod cells in part of the hippocampus, the polymorphic layer of the dentate gyrus, has long been recognized in infants. Previous work suggested that these cells were microglia and that their presence was associated with chronic illness and sudden infant death syndrome. Prompted by the observations that a sensitive immunohistochemical marker of microglia used in diagnostic practice does not typically stain these cells and that the hippocampus is a site of postnatal neurogenesis, we hypothesized that this transient population of cells were not microglia but neural progenitors. Methods Using archived post mortem tissue, we applied a broad panel of antibodies to establish the immunophenotype of these cells in 40 infants dying suddenly of causes that were either explained or remained unexplained, following post mortem investigation. Results The rod cells were consistently negative for the microglial markers CD45, CD68 and HLA-DR. The cells were positive, in varying proportions, for the neural progenitor marker, doublecortin, the neural stem cell marker, nestin and the neural marker, TUJ1. Conclusions These data support our hypothesis that the rod cells of the polymorphic layer of the dentate gyrus in the infant hippocampus are not microglia but a population of neural progenitors. These findings advance our understanding of postnatal neurogenesis in the human hippocampus in health and disease and are of diagnostic importance, allowing reactive microglia to be distinguished from the normal population of neural progenitors. PMID:23742713

  20. Single Cell Analysis Reveals Transcriptional Heterogeneity of Neural Progenitors in the Human Cortex

    PubMed Central

    Johnson, Matthew B.; Wang, Peter P.; Atabay, Kutay D.; Murphy, Elisabeth A.; Doan, Ryan N.; Hecht, Jonathan; Walsh, Christopher A.

    2017-01-01

    The human cerebral cortex depends for its normal development and size on a precisely controlled balance between self-renewal and differentiation of diverse neural progenitor cells. Specialized progenitors that are common in humans, but virtually absent in rodents, called ‘outer radial glia’ (ORG), have been suggested to be crucial to the evolutionary expansion of the human cortex. We combined progenitor subtype-specific sorting with transcriptome-wide RNA-sequencing to identify genes enriched in human ORG, which included targets of the transcription factor Neurogenin and previously uncharacterized, evolutionarily dynamic long noncoding RNAs. We show that activating the Neurogenin pathway in ferret progenitors promotes delamination and outward migration. Finally, single-cell transcriptional profiling in human, ferret, and mouse revealed more cells co-expressing proneural Neurogenin targets in human compared to other species, suggesting greater neuronal lineage commitment and differentiation of self-renewing progenitors. Thus, we find that the abundance of human ORG is paralleled by increased transcriptional heterogeneity of cortical progenitors. PMID:25734491

  1. Migratory neuronal progenitors arise from the neural plate borders in tunicates

    PubMed Central

    Stolfi, Alberto; Ryan, Kerrianne; Meinertzhagen, Ian A.; Christiaen, Lionel

    2015-01-01

    The neural crest is an evolutionary novelty that fostered the emergence of vertebrate anatomical innovations such as the cranium and jaws1. During embryonic development, multipotent neural crest cells are specified at the lateral borders of the neural plate before delaminating, migrating, and differentiating into various cell types. In invertebrate chordates (cephalochordates and tunicates), neural plate border cells express conserved factors such as Msx, Snail, and Pax3/7 and generate melanin-containing pigment cells2-4, a derivative of the neural crest in vertebrates. However, invertebrate neural plate border cells have not been shown to generate homologues of other neural crest derivatives. Thus, proposed models of neural crest evolution postulate vertebrate-specific elaborations on an ancestral neural plate border program, through acquisition of migratory capabilities and the potential to generate multiple cell types5-7. Here we show that a particular neuronal cell type in the tadpole larva of the tunicate Ciona intestinalis, the bipolar tail neuron, shares a set of features with neural crest-derived spinal ganglia neurons in vertebrates. Bipolar tail neuron precursors derive from caudal neural plate border cells, delaminate, and migrate along the paraxial mesoderm on either side of the neural tube, eventually differentiating into afferent neurons that form synaptic contacts with both epidermal sensory cells and motor neurons. We propose that the neural plate borders of the chordate ancestor already produced migratory peripheral neurons and pigment cells, and that the neural crest evolved through the acquisition of a multipotent progenitor regulatory state upstream of multiple, pre-existing neural plate border cell differentiation programs. PMID:26524532

  2. DISP3 promotes proliferation and delays differentiation of neural progenitor cells.

    PubMed

    Zíková, Martina; Konířová, Jana; Ditrychová, Karolína; Corlett, Alicia; Kolář, Michal; Bartůněk, Petr

    2014-11-03

    DISP3 (PTCHD2), a sterol-sensing domain-containing protein, is highly expressed in neural tissue but its role in neural differentiation is unknown. In the present study we used a multipotent cerebellar progenitor cell line, C17.2, to investigate the impact of DISP3 on the proliferation and differentiation of neural precursors. We found that ectopically expressed DISP3 promotes cell proliferation and alters expression of genes that are involved in tumorigenesis. Finally, the differentiation profile of DISP3-expressing cells was altered, as evidenced by delayed expression of neural specific markers and a reduced capacity to undergo neural differentiation. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  3. Organogenesis relies on SoxC transcription factors for the survival of neural and mesenchymal progenitors

    PubMed Central

    Bhattaram, Pallavi; Penzo-Méndez, Alfredo; Sock, Elisabeth; Colmenares, Clemencia; Kaneko, Kotaro J.; Vassilev, Alex; DePamphilis, Melvin L.; Wegner, Michael; Lefebvre, Véronique

    2010-01-01

    During organogenesis, neural and mesenchymal progenitor cells give rise to many cell lineages, but their molecular requirements for self-renewal and lineage decisions are incompletely understood. In this study, we show that their survival critically relies on the redundantly acting SoxC transcription factors Sox4, Sox11 and Sox12. The more SoxC alleles that are deleted in mouse embryos, the more severe and widespread organ hypoplasia is. SoxC triple-null embryos die at midgestation unturned and tiny, with normal patterning and lineage specification, but with massively dying neural and mesenchymal progenitor cells. Specific inactivation of SoxC genes in neural and mesenchymal cells leads to selective apoptosis of these cells, suggesting SoxC cell-autonomous roles. Tead2 functionally interacts with SoxC genes in embryonic development, and is a direct target of SoxC proteins. SoxC genes therefore ensure neural and mesenchymal progenitor cell survival, and function in part by activating this transcriptional mediator of the Hippo signalling pathway. PMID:20596238

  4. Organogenesis relies on SoxC transcription factors for the survival of neural and mesenchymal progenitors.

    PubMed

    Bhattaram, Pallavi; Penzo-Méndez, Alfredo; Sock, Elisabeth; Colmenares, Clemencia; Kaneko, Kotaro J; Vassilev, Alex; Depamphilis, Melvin L; Wegner, Michael; Lefebvre, Véronique

    2010-04-12

    During organogenesis, neural and mesenchymal progenitor cells give rise to many cell lineages, but their molecular requirements for self-renewal and lineage decisions are incompletely understood. In this study, we show that their survival critically relies on the redundantly acting SoxC transcription factors Sox4, Sox11 and Sox12. The more SoxC alleles that are deleted in mouse embryos, the more severe and widespread organ hypoplasia is. SoxC triple-null embryos die at midgestation unturned and tiny, with normal patterning and lineage specification, but with massively dying neural and mesenchymal progenitor cells. Specific inactivation of SoxC genes in neural and mesenchymal cells leads to selective apoptosis of these cells, suggesting SoxC cell-autonomous roles. Tead2 functionally interacts with SoxC genes in embryonic development, and is a direct target of SoxC proteins. SoxC genes therefore ensure neural and mesenchymal progenitor cell survival, and function in part by activating this transcriptional mediator of the Hippo signalling pathway.

  5. Pleiotrophin enhances PDGFB-induced gliomagenesis through increased proliferation of neural progenitor cells.

    PubMed

    Zhang, Lei; Laaniste, Liisi; Jiang, Yiwen; Alafuzoff, Irina; Uhrbom, Lene; Dimberg, Anna

    2016-12-06

    Pleiotrophin (PTN) augments tumor growth by increasing proliferation of tumor cells and promoting vascular abnormalization, but its role in early gliomagenesis has not been evaluated. Through analysis of publically available datasets, we demonstrate that increased PTN mRNA expression is associated with amplification of chromosome 7, identified as one of the earliest steps in glioblastoma development. To elucidate the role of PTN in tumor initiation we employed the RCAS/tv-a model that allows glioma induction by RCAS-virus mediated expression of oncogenes in neural progenitor cells. Intracranial injection of RCAS-PTN did not induce glioma formation when administrated alone, but significantly enhanced RCAS-platelet derived growth factor (PDGF)B-induced gliomagenesis. PTN co-treatment augmented PDGFB-induced Akt activation in neural progenitor cells in vitro, and enhanced neural sphere size associated with increased proliferation. Our data indicates that PTN expression is associated with chromosome 7 gain, and that PTN enhances PDGFB-induced gliomagenesis by stimulating proliferation of neural progenitor cells.

  6. Pleiotrophin enhances PDGFB-induced gliomagenesis through increased proliferation of neural progenitor cells

    PubMed Central

    Zhang, Lei; Laaniste, Liisi; Jiang, Yiwen; Alafuzoff, Irina; Uhrbom, Lene; Dimberg, Anna

    2016-01-01

    Pleiotrophin (PTN) augments tumor growth by increasing proliferation of tumor cells and promoting vascular abnormalization, but its role in early gliomagenesis has not been evaluated. Through analysis of publically available datasets, we demonstrate that increased PTN mRNA expression is associated with amplification of chromosome 7, identified as one of the earliest steps in glioblastoma development. To elucidate the role of PTN in tumor initiation we employed the RCAS/tv-a model that allows glioma induction by RCAS-virus mediated expression of oncogenes in neural progenitor cells. Intracranial injection of RCAS-PTN did not induce glioma formation when administrated alone, but significantly enhanced RCAS-platelet derived growth factor (PDGF)B-induced gliomagenesis. PTN co-treatment augmented PDGFB-induced Akt activation in neural progenitor cells in vitro, and enhanced neural sphere size associated with increased proliferation. Our data indicates that PTN expression is associated with chromosome 7 gain, and that PTN enhances PDGFB-induced gliomagenesis by stimulating proliferation of neural progenitor cells. PMID:27806344

  7. 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.

  8. Function of Mouse Embryonic Stem Cell-Derived Supporting Cells in Neural Progenitor Cell Maturation and Long Term Cxpansion

    PubMed Central

    Guan, Yunqian; Du, Qing-An; Zhu, Wanwan; Zou, Chunlin; Wu, Di; Chen, Ling; Zhang, Yu Alex

    2013-01-01

    Background In the differentiation of mouse embryonic stem (ES) cells into neurons using the 5-stage method, cells in stage 4 are in general used as neural progenitors (NPs) because of their ability to give rise to neurons. The choice of stage 4 raises several questions about neural progenitors such as the type of cell types that are specifically considered to be neural progenitors, the exact time when these progenitors become capable of neurogenesis and whether neurogenesis is an independent and autonomous process or the result of an interaction between NP cells and the surrounding cells. Methodology/Principal Findings In this study, we found that the confluent monolayer cells and neural sphere like cell clusters both appeared in the culture of the first 14 days and the subsequent 6 weeks. However, only the sphere cells are neural progenitors that give rise to neurons and astrocytes. The NP cells require 14 days to mature into neural lineages fully capable of differentiation. We also found that although the confluent monolayer cells do not undergo neurogenesis, they play a crucial role in the growth, differentiation, and apoptosis of the sphere cells, during the first 14 days and long term culture, by secreted factors and direct cell to cell contact. Conclusions/Significance The sphere cells in stage 4 are more committed to developing into neural progenitors than monolayer cells. Interaction between the monolayer cells and sphere cells is important in the development of stage 4 cell characteristics. PMID:23342136

  9. Recent Zika Virus Isolates Induce Premature Differentiation of Neural Progenitors in Human Brain Organoids.

    PubMed

    Gabriel, Elke; Ramani, Anand; Karow, Ulrike; Gottardo, Marco; Natarajan, Karthick; Gooi, Li Ming; Goranci-Buzhala, Gladiola; Krut, Oleg; Peters, Franziska; Nikolic, Milos; Kuivanen, Suvi; Korhonen, Essi; Smura, Teemu; Vapalahti, Olli; Papantonis, Argyris; Schmidt-Chanasit, Jonas; Riparbelli, Maria; Callaini, Giuliano; Krönke, Martin; Utermöhlen, Olaf; Gopalakrishnan, Jay

    2017-03-02

    The recent Zika virus (ZIKV) epidemic is associated with microcephaly in newborns. Although the connection between ZIKV and neurodevelopmental defects is widely recognized, the underlying mechanisms are poorly understood. Here we show that two recently isolated strains of ZIKV, an American strain from an infected fetal brain (FB-GWUH-2016) and a closely-related Asian strain (H/PF/2013), productively infect human iPSC-derived brain organoids. Both of these strains readily target to and replicate in proliferating ventricular zone (VZ) apical progenitors. The main phenotypic effect was premature differentiation of neural progenitors associated with centrosome perturbation, even during early stages of infection, leading to progenitor depletion, disruption of the VZ, impaired neurogenesis, and cortical thinning. The infection pattern and cellular outcome differ from those seen with the extensively passaged ZIKV strain MR766. The structural changes we see after infection with these more recently isolated viral strains closely resemble those seen in ZIKV-associated microcephaly.

  10. Neural differentiation potential of sympathoadrenal progenitors derived from fresh and cryopreserved neonatal porcine adrenal glands.

    PubMed

    Bozhok, G A; Sidorenko, O S; Plaksina, E M; Gurina, T M; Sukach, A N; Kholodnyy, V S; Ustichenko, V D; Bilyavskaya, S B; Bondarenko, T P; Legach, E I

    2016-10-01

    Stem/progenitor cells are thought to have the potential in the treatment of severe neurodegenerative diseases. Recently, sympathoadrenal progenitors expressing specific markers of neural crest derivatives and capable to differentiate into neurons were discovered in adult bovine and human adrenal glands, but there was no reported data on cryopreservation of sympathoadrenal progenitors. The aim of the present study was to examine the neural differentiation potential of sympathoadrenal progenitors derived from fresh and cryopreserved neonatal porcine adrenal glands. Considering impact of various initial state of frozen biomaterial on cell recovery, we carried out a comparative estimation of cryopreservation outcome both for adrenal tissue fragments and isolated primary cells. The estimation consisted of determining cell yield, viability, ability to adhere, proliferate and differentiate in vitro. Cells isolated from the fresh adrenal glands were cultured until confluence. A formation of sympathoadrenal progenitors-embedded spherical cell colonies, whose cells are differentiated then into βIII-tubulin-positive cells with neuron-like morphology, was observed on the monolayer. The colonies were well preserved after cryopreservation of cell culture with a cooling rate of 1 °C/min in the cryoprotectant media containing 5-15% of dimethylsulfoxide. Adrenal tissue fragments were cryopreserved in the presence of 10% dimethylsulfoxide at the cooling rates of 0.3; 1: 5; 40 and > 100 °C/min. Sympathoadrenal progenitors were recovered after cryopreservation with 0.3 °C/min cooling rate but not higher. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Defining a developmental path to neural fate by global expression profiling of mouse embryonic stem cells and adult neural stem/progenitor cells.

    PubMed

    Aiba, Kazuhiro; Sharov, Alexei A; Carter, Mark G; Foroni, Chiara; Vescovi, Angelo L; Ko, Minoru S H

    2006-04-01

    To understand global features of gene expression changes during in vitro neural differentiation, we carried out the microarray analysis of embryonic stem cells (ESCs), embryonal carcinoma cells, and adult neural stem/progenitor (NS) cells. Expression profiling of ESCs during differentiation in monolayer culture revealed three distinct phases: undifferentiated ESCs, primitive ectoderm-like cells, and neural progenitor cells. Principal component (PC) analysis revealed that these cells were aligned on PC1 over the course of 6 days. This PC1 represents approximately 4,000 genes, the expression of which increased with neural commitment/differentiation. Furthermore, NS cells derived from adult brain and their differentiated cells were positioned along this PC axis further away from undifferentiated ESCs than embryonic stem-derived neural progenitors. We suggest that this PC1 defines a path to neural fate, providing a scale for the degree of commitment/differentiation.

  12. Aging neural progenitors lose competence to respond to mitogenic Notch signaling

    PubMed Central

    Farnsworth, Dylan R.; Bayraktar, Omer Ali; Doe, Chris Q.

    2015-01-01

    Drosophila neural stem cells (neuroblasts) are a powerful model system for investigating stem cell self-renewal, specification of temporal identity, and progressive restriction in competence. Notch signaling is a conserved cue that is an important determinant of cell fate in many contexts across animal development; for example mammalian T cell differentiation in the thymus and neuroblast specification in Drosophila are both regulated by Notch signaling. However, Notch also functions as a mitogen, and constitutive Notch signaling potentiates T cell leukemia as well as Drosophila neuroblast tumors. While the role of Notch signaling has been studied in these and other cell types, it remains unclear how stem cells and progenitors change competence to respond to Notch over time. Notch is required in type II neuroblasts for normal development of their transit amplifying progeny, intermediate neural progenitors (INPs). Here we find that aging INPs lose competence to respond to constitutively active Notch signaling. Moreover, we show that reducing the levels of the old INP temporal transcription factor Eyeless/Pax6 allows Notch signaling to promote the de-differentiation of INP progeny into ectopic INPs, thereby creating a proliferative mass of ectopic progenitors in the brain. These findings provide a new system for studying progenitor competence, and identify a novel role for the conserved transcription factor Eyeless/Pax6 in blocking Notch signaling during development. PMID:26585279

  13. Aging Neural Progenitors Lose Competence to Respond to Mitogenic Notch Signaling.

    PubMed

    Farnsworth, Dylan R; Bayraktar, Omer Ali; Doe, Chris Q

    2015-12-07

    Drosophila neural stem cells (neuroblasts) are a powerful model system for investigating stem cell self-renewal, specification of temporal identity, and progressive restriction in competence. Notch signaling is a conserved cue that is an important determinant of cell fate in many contexts across animal development; for example, mammalian T cell differentiation in the thymus and neuroblast specification in Drosophila are both regulated by Notch signaling. However, Notch also functions as a mitogen, and constitutive Notch signaling potentiates T cell leukemia as well as Drosophila neuroblast tumors. While the role of Notch signaling has been studied in these and other cell types, it remains unclear how stem cells and progenitors change competence to respond to Notch over time. Notch is required in type II neuroblasts for normal development of their transit amplifying progeny, intermediate neural progenitors (INPs). Here, we find that aging INPs lose competence to respond to constitutively active Notch signaling. Moreover, we show that reducing the levels of the old INP temporal transcription factor Eyeless/Pax6 allows Notch signaling to promote the de-differentiation of INP progeny into ectopic INPs, thereby creating a proliferative mass of ectopic progenitors in the brain. These findings provide a new system for studying progenitor competence and identify a novel role for the conserved transcription factor Eyeless/Pax6 in blocking Notch signaling during development. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Rapid genetic targeting of pial surface neural progenitors and immature neurons by neonatal electroporation

    PubMed Central

    2012-01-01

    Background Recent findings have indicated the presence of a progenitor domain at the marginal zone/layer 1 of the cerebral cortex, and it has been suggested that these progenitors have neurogenic and gliogenic potential. However, their contribution to the histogenesis of the cortex remains poorly understood due to difficulties associated with genetically manipulating these unique cells in a population-specific manner. Results We have adapted the electroporation technique to target pial surface cells for rapid genetic manipulation at postnatal day 2. In vivo data show that most of these cells proliferate and progressively differentiate into both neuronal and glial subtypes. Furthermore, these cells localize to the superficial layers of the optic tectum and cerebral cortex prior to migration away from the surface. Conclusions We provide a foundation upon which future studies can begin to elucidate the molecular controls governing neural progenitor fate, migration, differentiation, and contribution to cortical and tectal histogenesis. Furthermore, specific genetic targeting of such neural progenitor populations will likely be of future clinical interest. PMID:22776033

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

    NASA Astrophysics Data System (ADS)

    Jose, Anumol; Krishnan, Lissy K.

    2010-06-01

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

  16. Heparin prevents Zika virus induced-cytopathic effects in human neural progenitor cells.

    PubMed

    Ghezzi, Silvia; Cooper, Lynsay; Rubio, Alicia; Pagani, Isabel; Capobianchi, Maria Rosaria; Ippolito, Giuseppe; Pelletier, Julien; Meneghetti, Maria Cecilia Z; Lima, Marcelo A; Skidmore, Mark A; Broccoli, Vania; Yates, Edwin A; Vicenzi, Elisa

    2017-04-01

    The recent Zika virus (ZIKV) outbreak, which mainly affected Brazil and neighbouring states, demonstrated the paucity of information concerning the epidemiology of several flaviruses, but also highlighted the lack of available agents with which to treat such emerging diseases. Here, we show that heparin, a widely used anticoagulant, while exerting a modest inhibitory effect on Zika Virus replication, fully prevents virus-induced cell death of human neural progenitor cells (NPCs).

  17. Transplanted Neural Progenitor Cells from Distinct Sources Migrate Differentially in an Organotypic Model of Brain Injury

    PubMed Central

    Ngalula, Kapinga P.; Cramer, Nathan; Schell, Michael J.; Juliano, Sharon L.

    2015-01-01

    Brain injury is a major cause of long-term disability. The possibility exists for exogenously derived neural progenitor cells to repair damage resulting from brain injury, although more information is needed to successfully implement this promising therapy. To test the ability of neural progenitor cells (NPCs) obtained from rats to repair damaged neocortex, we transplanted neural progenitor cell suspensions into normal and injured slice cultures of the neocortex acquired from rats on postnatal day 0–3. Donor cells from E16 embryos were obtained from either the neocortex, including the ventricular zone (VZ) for excitatory cells, ganglionic eminence (GE) for inhibitory cells or a mixed population of the two. Cells were injected into the ventricular/subventricular zone (VZ/SVZ) or directly into the wounded region. Transplanted cells migrated throughout the cortical plate with GE and mixed population donor cells predominately targeting the upper cortical layers, while neocortically derived NPCs from the VZ/SVZ migrated less extensively. In the injured neocortex, transplanted cells moved predominantly into the wounded area. NPCs derived from the GE tended to be immunoreactive for GABAergic markers while those derived from the neocortex were more strongly immunoreactive for other neuronal markers such as MAP2, TUJ1, or Milli-Mark. Cells transplanted in vitro acquired the electrophysiological characteristics of neurons, including action potential generation and reception of spontaneous synaptic activity. This suggests that transplanted cells differentiate into neurons capable of functionally integrating with the host tissue. Together, our data suggest that transplantation of neural progenitor cells holds great potential as an emerging therapeutic intervention for restoring function lost to brain damage. PMID:26500604

  18. Short term interactions with long term consequences: modulation of chimeric vessels by neural progenitors.

    PubMed

    Williams, Cicely; Rauch, Millicent Ford; Michaud, Michael; Robinson, Rebecca; Xu, Hao; Madri, Joseph; Lavik, Erin

    2012-01-01

    Vessels are a critical and necessary component of most tissues, and there has been substantial research investigating vessel formation and stabilization. Several groups have investigated coculturing endothelial cells with a second cell type to promote formation and stabilization of vessels. Some have noted that long-term vessels derived from implanted cocultures are often chimeric consisting of both host and donor cells. The questions arise as to whether the coculture cell might impact the chimeric nature of the microvessels and can modulate the density of donor cells over time. If long-term engineered microvessels are primarily of host origin, any impairment of the host's angiogenic ability has significant implications for the long-term success of the implant. If one can modulate the host versus donor response, one may be able to overcome a host's angiogenic impairment. Furthermore, if one can modulate the donor contribution, one may be able to engineer microvascular networks to deliver molecules a patient lacks systemically for long times. To investigate the impact of the cocultured cell on the host versus donor contributions of endothelial cells in engineered microvascular networks, we varied the ratio of the neural progenitors to endothelial cells in subcutaneously implanted poly(ethylene glycol)/poly-L-lysine hydrogels. We found that the coculture of neural progenitors with endothelial cells led to the formation of chimeric host-donor vessels, and the ratio of neural progenitors has a significant impact on the long term residence of donor endothelial cells in engineered microvascular networks in vivo even though the neural progenitors are only present transiently in the system. We attribute this to the short term paracrine signaling between the two cell types. This suggests that one can modulate the host versus donor contributions using short-term paracrine signaling which has broad implications for the application of engineered microvascular networks and

  19. Short Term Interactions with Long Term Consequences: Modulation of Chimeric Vessels by Neural Progenitors

    PubMed Central

    Williams, Cicely; Rauch, Millicent Ford; Michaud, Michael; Robinson, Rebecca; Xu, Hao; Madri, Joseph; Lavik, Erin

    2012-01-01

    Vessels are a critical and necessary component of most tissues, and there has been substantial research investigating vessel formation and stabilization. Several groups have investigated coculturing endothelial cells with a second cell type to promote formation and stabilization of vessels. Some have noted that long-term vessels derived from implanted cocultures are often chimeric consisting of both host and donor cells. The questions arise as to whether the coculture cell might impact the chimeric nature of the microvessels and can modulate the density of donor cells over time. If long-term engineered microvessels are primarily of host origin, any impairment of the host's angiogenic ability has significant implications for the long-term success of the implant. If one can modulate the host versus donor response, one may be able to overcome a host's angiogenic impairment. Furthermore, if one can modulate the donor contribution, one may be able to engineer microvascular networks to deliver molecules a patient lacks systemically for long times. To investigate the impact of the cocultured cell on the host versus donor contributions of endothelial cells in engineered microvascular networks, we varied the ratio of the neural progenitors to endothelial cells in subcutaneously implanted poly(ethylene glycol)/poly-L-lysine hydrogels. We found that the coculture of neural progenitors with endothelial cells led to the formation of chimeric host-donor vessels, and the ratio of neural progenitors has a significant impact on the long term residence of donor endothelial cells in engineered microvascular networks in vivo even though the neural progenitors are only present transiently in the system. We attribute this to the short term paracrine signaling between the two cell types. This suggests that one can modulate the host versus donor contributions using short-term paracrine signaling which has broad implications for the application of engineered microvascular networks and

  20. In Vitro and Ex Ovo Culture of Reptilian and Avian Neural Progenitor Cells.

    PubMed

    Yamashita, Wataru; Shimizu, Toyo; Nomura, Tadashi

    2017-01-01

    Reptiles and birds have been highlighted as excellent experimental models for the study of developmental biology; however, due to technical limitations in cellular analysis, dynamics of neural stem/progenitor cells of these animals remain unclear. In this chapter, we introduce the protocols for neurosphere culture and ex ovo embryonic culture of developing reptilian and avian embryos, which are modified from the method originally established for rodent embryos. Applications of these techniques provide powerful strategies for the study of comparative neural development of amniotes.

  1. Effects of Chronic Low-Dose Radiation on Human Neural Progenitor Cells

    NASA Astrophysics Data System (ADS)

    Katsura, Mari; Cyou-Nakamine, Hiromasa; Zen, Qin; Zen, Yang; Nansai, Hiroko; Amagasa, Shota; Kanki, Yasuharu; Inoue, Tsuyoshi; Kaneki, Kiyomi; Taguchi, Akashi; Kobayashi, Mika; Kaji, Toshiyuki; Kodama, Tatsuhiko; Miyagawa, Kiyoshi; Wada, Youichiro; Akimitsu, Nobuyoshi; Sone, Hideko

    2016-01-01

    The effects of chronic low-dose radiation on human health have not been well established. Recent studies have revealed that neural progenitor cells are present not only in the fetal brain but also in the adult brain. Since immature cells are generally more radiosensitive, here we investigated the effects of chronic low-dose radiation on cultured human neural progenitor cells (hNPCs) derived from embryonic stem cells. Radiation at low doses of 31, 124 and 496 mGy per 72 h was administered to hNPCs. The effects were estimated by gene expression profiling with microarray analysis as well as morphological analysis. Gene expression was dose-dependently changed by radiation. By thirty-one mGy of radiation, inflammatory pathways involving interferon signaling and cell junctions were altered. DNA repair and cell adhesion molecules were affected by 124 mGy of radiation while DNA synthesis, apoptosis, metabolism, and neural differentiation were all affected by 496 mGy of radiation. These in vitro results suggest that 496 mGy radiation affects the development of neuronal progenitor cells while altered gene expression was observed at a radiation dose lower than 100 mGy. This study would contribute to the elucidation of the clinical and subclinical phenotypes of impaired neuronal development induced by chronic low-dose radiation.

  2. Effects of Chronic Low-Dose Radiation on Human Neural Progenitor Cells

    PubMed Central

    Katsura, Mari; Cyou-Nakamine, Hiromasa; Zen, Qin; Zen, Yang; Nansai, Hiroko; Amagasa, Shota; Kanki, Yasuharu; Inoue, Tsuyoshi; Kaneki, Kiyomi; Taguchi, Akashi; Kobayashi, Mika; Kaji, Toshiyuki; Kodama, Tatsuhiko; Miyagawa, Kiyoshi; Wada, Youichiro; Akimitsu, Nobuyoshi; Sone, Hideko

    2016-01-01

    The effects of chronic low-dose radiation on human health have not been well established. Recent studies have revealed that neural progenitor cells are present not only in the fetal brain but also in the adult brain. Since immature cells are generally more radiosensitive, here we investigated the effects of chronic low-dose radiation on cultured human neural progenitor cells (hNPCs) derived from embryonic stem cells. Radiation at low doses of 31, 124 and 496 mGy per 72 h was administered to hNPCs. The effects were estimated by gene expression profiling with microarray analysis as well as morphological analysis. Gene expression was dose-dependently changed by radiation. By thirty-one mGy of radiation, inflammatory pathways involving interferon signaling and cell junctions were altered. DNA repair and cell adhesion molecules were affected by 124 mGy of radiation while DNA synthesis, apoptosis, metabolism, and neural differentiation were all affected by 496 mGy of radiation. These in vitro results suggest that 496 mGy radiation affects the development of neuronal progenitor cells while altered gene expression was observed at a radiation dose lower than 100 mGy. This study would contribute to the elucidation of the clinical and subclinical phenotypes of impaired neuronal development induced by chronic low-dose radiation. PMID:26795421

  3. Adult Olfactory Bulb Interneuron Phenotypes Identified by Targeting Embryonic and Postnatal Neural Progenitors

    PubMed Central

    Figueres-Oñate, Maria; López-Mascaraque, Laura

    2016-01-01

    Neurons are generated during embryonic development and in adulthood, although adult neurogenesis is restricted to two main brain regions, the hippocampus and olfactory bulb. The subventricular zone (SVZ) of the lateral ventricles generates neural stem/progenitor cells that continually provide the olfactory bulb (OB) with new granule or periglomerular neurons, cells that arrive from the SVZ via the rostral migratory stream. The continued neurogenesis and the adequate integration of these newly generated interneurons is essential to maintain homeostasis in the olfactory bulb, where the differentiation of these cells into specific neural cell types is strongly influenced by temporal cues. Therefore, identifying the critical features that control the generation of adult OB interneurons at either pre- or post-natal stages is important to understand the dynamic contribution of neural stem cells. Here, we used in utero and neonatal SVZ electroporation along with a transposase-mediated stable integration plasmid, in order to track interneurons and glial lineages in the OB. These plasmids are valuable tools to study the development of OB interneurons from embryonic and post-natal SVZ progenitors. Accordingly, we examined the location and identity of the adult progeny of embryonic and post-natally transfected progenitors by examining neurochemical markers in the adult OB. These data reveal the different cell types in the olfactory bulb that are generated in function of age and different electroporation conditions. PMID:27242400

  4. The influence of immunosuppressive drugs on neural stem/progenitor cell fate in vitro

    SciTech Connect

    Skardelly, Marco; Glien, Anja; Groba, Claudia; Schlichting, Nadine; Kamprad, Manja; Meixensberger, Juergen; Milosevic, Javorina

    2013-12-10

    In allogenic and xenogenic transplantation, adequate immunosuppression plays a major role in graft survival, especially over the long term. The effect of immunosuppressive drugs on neural stem/progenitor cell fate has not been sufficiently explored. The focus of this study is to systematically investigate the effects of the following four different immunotherapeutic strategies on human neural progenitor cell survival/death, proliferation, metabolic activity, differentiation and migration in vitro: (1) cyclosporine A (CsA), a calcineurin inhibitor; (2) everolimus (RAD001), an mTOR-inhibitor; (3) mycophenolic acid (MPA, mycophenolate), an inhibitor of inosine monophosphate dehydrogenase and (4) prednisolone, a steroid. At the minimum effective concentration (MEC), we found a prominent decrease in hNPCs' proliferative capacity (BrdU incorporation), especially for CsA and MPA, and an alteration of the NAD(P)H-dependent metabolic activity. Cell death rate, neurogenesis, gliogenesis and cell migration remained mostly unaffected under these conditions for all four immunosuppressants, except for apoptotic cell death, which was significantly increased by MPA treatment. - Highlights: • Four immunosuppresants (ISs) were tested in human neural progenitor cells in vitro. • Cyclosporine A and mycophenolic acid showed a prominent anti-proliferative activity • Mycophenolic acid exhibited a significant pro-apoptotic effect. • NAD(P)H-dependent metabolic activity was occasionally induced by ISs. • Neuronal differentiation and migration potential remained unaffected by ISs treatment.

  5. Effects of Chronic Low-Dose Radiation on Human Neural Progenitor Cells.

    PubMed

    Katsura, Mari; Cyou-Nakamine, Hiromasa; Zen, Qin; Zen, Yang; Nansai, Hiroko; Amagasa, Shota; Kanki, Yasuharu; Inoue, Tsuyoshi; Kaneki, Kiyomi; Taguchi, Akashi; Kobayashi, Mika; Kaji, Toshiyuki; Kodama, Tatsuhiko; Miyagawa, Kiyoshi; Wada, Youichiro; Akimitsu, Nobuyoshi; Sone, Hideko

    2016-01-22

    The effects of chronic low-dose radiation on human health have not been well established. Recent studies have revealed that neural progenitor cells are present not only in the fetal brain but also in the adult brain. Since immature cells are generally more radiosensitive, here we investigated the effects of chronic low-dose radiation on cultured human neural progenitor cells (hNPCs) derived from embryonic stem cells. Radiation at low doses of 31, 124 and 496 mGy per 72 h was administered to hNPCs. The effects were estimated by gene expression profiling with microarray analysis as well as morphological analysis. Gene expression was dose-dependently changed by radiation. By thirty-one mGy of radiation, inflammatory pathways involving interferon signaling and cell junctions were altered. DNA repair and cell adhesion molecules were affected by 124 mGy of radiation while DNA synthesis, apoptosis, metabolism, and neural differentiation were all affected by 496 mGy of radiation. These in vitro results suggest that 496 mGy radiation affects the development of neuronal progenitor cells while altered gene expression was observed at a radiation dose lower than 100 mGy. This study would contribute to the elucidation of the clinical and subclinical phenotypes of impaired neuronal development induced by chronic low-dose radiation.

  6. Secretin deficiency causes impairment in survival of neural progenitor cells in mice.

    PubMed

    Jukkola, Peter I; Rogers, Justin T; Kaspar, Brian K; Weeber, Edwin J; Nishijima, Ichiko

    2011-03-01

    Hippocampal neurogenesis is the lifelong production of new neurons in the central nervous system (CNS), and affects many physiological and pathophysiological conditions, including neurobehavioral disorders. The early postnatal stage is the most prominent neurogenesis period; however, the functional role of neurogenesis in this developing stage has not been well characterized. To understand the role of hippocampal neurogenesis in the postnatal developing period, we analyzed secretin, a neuropeptide, which is expressed significantly higher in the development stage. Secretin is a pleiotropic neuropeptide hormone that belongs to the secretin/VIP/glucagon peptide family. Although secretin was originally isolated in the gastrointestinal system, it has been found that secretin itself acts as a neuropeptide in the CNS. Here, we report a new function of secretin as a survival factor for neural progenitor cells in the hippocampus. We found that secretin-deficient mice exhibit decreased numbers of BrdU-labeled new neurons and dramatically increased apoptosis of doublecortin-positive neural progenitor cells in the subgranular zone of the dentate gyrus (DG) during the early postnatal period. Furthermore, we found that reduced survival of neural progenitor cells leads to decreased volume of DG, reduced long-term potentiation and impaired spatial learning ability in adults. Our studies demonstrate that secretin has important implications for neurogenesis in postnatal development, and affects neurobehavioral function in the adult mouse.

  7. Structurally Distinct LewisX Glycans Distinguish Subpopulations of Neural Stem/Progenitor Cells*

    PubMed Central

    Hennen, Eva; Czopka, Tim; Faissner, Andreas

    2011-01-01

    There is increasing evidence that the stem and progenitor cell population that builds the central nervous system is very heterogeneous. Stem cell markers with the potential to divide this cell pool into subpopulations with distinct characteristics are sparse. We were looking for new cell type-specific antigens to further subdivide the progenitor pool. Here, we introduce the novel monoclonal antibody clone 5750. We show that it specifically labels cell surfaces of neural stem and progenitor cells. When 5750-expressing cells were isolated by fluorescence-activated cell sorting from embryonic mouse brains, the sorted population showed increased neurosphere forming capacity and multipotency. Neurospheres generated from 5750-positive cells could self-renew and remained multipotent even after prolonged passaging. Carbohydrate binding assays revealed that the 5750 antibody specifically binds to LewisX-related carbohydrates. Interestingly, we found that the LewisX epitope recognized by clone 5750 differs from those detected by other anti-LewisX antibody clones like 487LeX, SSEA-1LeX, and MMALeX. Our data further reveal that individual anti-LewisX clones can be successfully used to label and deplete different subpopulations of neural cells in vivo and in vitro. In conclusion, we present a new tool for the isolation and characterization of neural subpopulations and provide insights into the complexity of cell surface glycosylation. PMID:21385876

  8. USP9X deubiquitylating enzyme maintains RAPTOR protein levels, mTORC1 signalling and proliferation in neural progenitors.

    PubMed

    Bridges, Caitlin R; Tan, Men-Chee; Premarathne, Susitha; Nanayakkara, Devathri; Bellette, Bernadette; Zencak, Dusan; Domingo, Deepti; Gecz, Jozef; Murtaza, Mariyam; Jolly, Lachlan A; Wood, Stephen A

    2017-03-24

    USP9X, is highly expressed in neural progenitors and, essential for neural development in mice. In humans, mutations in USP9X are associated with neurodevelopmental disorders. To understand USP9X's role in neural progenitors, we studied the effects of altering its expression in both the human neural progenitor cell line, ReNcell VM, as well as neural stem and progenitor cells derived from Nestin-cre conditionally deleted Usp9x mice. Decreasing USP9X resulted in ReNcell VM cells arresting in G0 cell cycle phase, with a concomitant decrease in mTORC1 signalling, a major regulator of G0/G1 cell cycle progression. Decreased mTORC1 signalling was also observed in Usp9x-null neurospheres and embryonic mouse brains. Further analyses revealed, (i) the canonical mTORC1 protein, RAPTOR, physically associates with Usp9x in embryonic brains, (ii) RAPTOR protein level is directly proportional to USP9X, in both loss- and gain-of-function experiments in cultured cells and, (iii) USP9X deubiquitlyating activity opposes the proteasomal degradation of RAPTOR. EdU incorporation assays confirmed Usp9x maintains the proliferation of neural progenitors similar to Raptor-null and rapamycin-treated neurospheres. Interestingly, loss of Usp9x increased the number of sphere-forming cells consistent with enhanced neural stem cell self-renewal. To our knowledge, USP9X is the first deubiquitylating enzyme shown to stabilize RAPTOR.

  9. Neural progenitor cell transplants into the developing and mature central nervous system.

    PubMed

    Sakaguchi, D S; Van Hoffelen, S J; Grozdanic, S D; Kwon, Y H; Kardon, R H; Young, M J

    2005-05-01

    When developing cell transplant strategies to repair the diseased or injured central nervous system (CNS), it is essential to consider host-graft interactions and how they may influence the outcome of the transplants. Recent studies have demonstrated that transplanted neural progenitor cells (NPCs) can differentiate and integrate morphologically into developing mammalian retinas. Is the ability to differentiate and to undergo structural integration into the CNS unique to specific progenitor cells, or is this plasticity a function of host environment, or both? To address these issues we have used the developing retina of the Brazilian opossum and have compared the structural integration of brain and retinal progenitor cells transplanted into the eyes at different developmental stages. The Brazilian opossum, Monodelphis domestica, is a small pouchless marsupial native to South America. This animal's lack of a pouch and fetal-like nature at birth circumvents the need for in utero surgical procedures, and thus provides an ideal environment in which to study the interactions between developing host tissues and transplanted NPCs. To test whether NPCs affect visual function we transplanted adult hippocampal progenitor cells (AHPCs) into normal, healthy adult rat eyes and performed noninvasive functional recordings. Monitoring of the retina and optic nerve over time by electroretinography and pupillometry revealed no severe perturbation in visual function in the transplant recipient eyes. Taken together, our findings suggest that the age of the host environment can strongly influence NPC differentiation and that transplantation of neural progenitor cells may be a useful strategy aimed at treating neurodegeneration and pathology of the CNS.

  10. Abnormal development of NG2+PDGFRα+ neural progenitor cells leads to neonatal hydrocephalus in a ciliopathy mouse model

    PubMed Central

    Carter, Calvin S.; Vogel, Timothy W.; Zhang, Qihong; Seo, Seongjin; Swiderski, Ruth E.; Moninger, Thomas O.; Cassell, Martin D.; Thedens, Daniel R.; Keppler-Noreuil, Kim M.; Nopoulos, Peggy; Nishimura, Darryl Y.; Searby, Charles C.; Bugge, Kevin; Sheffield, Val C.

    2012-01-01

    Hydrocephalus is a common neurological disorder leading to expansion of the cerebral ventricles and is associated with significant morbidity and mortality. Most neonatal cases are of unknown etiology and are likely to display complex inheritance involving multiple genes and environmental factors. Identifying molecular mechanisms for neonatal hydrocephalus and developing non-invasive treatment modalities are high priorities. Here we employ a hydrocephalic mouse model of the human ciliopathy Bardet-Biedl Syndrome (BBS) and identify a role for neural progenitors in the pathogenesis of neonatal hydrocephalus. We found that hydrocephalus in this mouse model is caused by aberrant PDGFRα signaling, resulting in increased apoptosis and impaired proliferation of NG2+PDGFRα+ neural progenitors. Targeting this pathway with lithium treatment rescued NG2+PDGFRα+ progenitor cell proliferation in BBS mutant mice, reducing ventricular volume. Our findings demonstrate that neural progenitors are critical in the pathogenesis of neonatal hydrocephalus and we identify novel therapeutic targets for this common neurological disorder. PMID:23160237

  11. Stress, glucocorticoid hormones, and hippocampal neural progenitor cells: implications to mood disorders

    PubMed Central

    Kino, Tomoshige

    2015-01-01

    The hypothalamic-pituitary-adrenal (HPA) axis and its end-effectors glucocorticoid hormones play central roles in the adaptive response to numerous stressors that can be either internal or external. Thus, this system has a strong impact on the brain hippocampus and its major functions, such as cognition, memory as well as behavior, and mood. The hippocampal area of the adult brain contains neural stem cells or more committed neural progenitor cells, which retain throughout the human life the ability of self-renewal and to differentiate into multiple neural cell lineages, such as neurons, astrocytes, and oligodendrocytes. Importantly, these characteristic cells contribute significantly to the above-indicated functions of the hippocampus, while various stressors and glucocorticoids influence proliferation, differentiation, and fate of these cells. This review offers an overview of the current understanding on the interactions between the HPA axis/glucocorticoid stress-responsive system and hippocampal neural progenitor cells by focusing on the actions of glucocorticoids. Also addressed is a further discussion on the implications of such interactions to the pathophysiology of mood disorders. PMID:26347657

  12. Defining properties of neural crest-derived progenitor cells from the apex of human developing tooth.

    PubMed

    Degistirici, Ozer; Jaquiery, Claude; Schönebeck, Bodo; Siemonsmeier, Jürgen; Götz, Werner; Martin, Ivan; Thie, Michael

    2008-02-01

    The connective tissue of the human tooth arises from cells that are derived from the cranial neural crest and, thus, are termed as "ectomesenchymal cells." Here, cells being located in a pad-like tissue adjacent to the apex of the developing tooth, which we designated the third molar pad, were separated by the microexplant technique. When outgrowing from the explant, dental neural crest-derived progenitor cells (dNC-PCs) adhered to plastic, proliferated steadily, and displayed a fibroblast-like morphology. At the mRNA level, dNC-PCs expressed neural crest marker genes like Sox9, Snail1, Snail2, Twist1, Msx2, and Dlx6. Cytofluorometric analysis indicated that cells were positive for CD49d (alpha4 integrin), CD56 (NCAM), and PDGFRalpha, while negative for CD31, CD34, CD45, and STRO-1. dNC-PCs could be differentiated into neurogenic, chondrogenic, and osteogenic lineages and were shown to produce bone matrix in athymic mice. These results demonstrate that human third molar pad possesses neural crest-derived cells that represent multipotent stem/progenitor cells. As a rather large amount of dNC-PCs could be obtained from each single third molar, cells may be used to regenerate a wide range of tissues within the craniofacial region of humans.

  13. Effects of Substrate and Co-Culture on Neural Progenitor Cell Differentiation

    SciTech Connect

    Jones, Erin Boote

    2008-01-01

    In recent years the study of stem and progenitor cells has moved to the forefront of research. Since the isolation of human hematopoietic stem cells in 1988 and the subsequent discovery of a self renewing population of multipotent cells in many tissues, many researchers have envisioned a better understanding of development and potential clinical usage in intractable diseases. Both these goals, however, depend on a solid understanding of the intracellular and extracellular forces that cause stem cells to differentiate to a specific cell fate. Many diseases of large scale cell loss have been suggested as candidates for stem cell based treatments. It is proposed that replacing the function of the damaged or defective cells by specific differentiation of stem or progenitor cells could treat the disease. Before cells can be directed to specific lineages, the mechanisms of differentiation must be better understood. Differentiation in vivo is an intensively complex system that is difficult to study. The goal of this research is to develop further understanding of the effects of soluble and extracellular matrix (ECM) cues on the differentiation of neural progenitor cells with the use of a simplified in vitro culture system. Specific research objectives are to study the differentiation of neural progenitor cells in response to astrocyte conditioned medium and protein substrate composition and concentration. In an effort to reveal the mechanism of the conditioned medium interaction, a test for the presence of a feedback loop between progenitor cells and astrocytes is presented along with an examination of conditioned medium storage temperature, which can reveal enzymatic dependencies. An examination of protein substrate composition and concentration will help to reveal the role of any ECM interactions on differentiation. This thesis is organized into a literature review covering recent advances in use of external modulators of differentiation such as surface coatings, co

  14. Embryonic cerebrospinal fluid in brain development: neural progenitor control.

    PubMed

    Gato, Angel; Alonso, M Isabel; Martín, Cristina; Carnicero, Estela; Moro, José Antonio; De la Mano, Aníbal; Fernández, José M F; Lamus, Francisco; Desmond, Mary E

    2014-08-28

    Due to the effort of several research teams across the world, today we have a solid base of knowledge on the liquid contained in the brain cavities, its composition, and biological roles. Although the cerebrospinal fluid (CSF) is among the most relevant parts of the central nervous system from the physiological point of view, it seems that it is not a permanent and stable entity because its composition and biological properties evolve across life. So, we can talk about different CSFs during the vertebrate life span. In this review, we focus on the CSF in an interesting period, early in vertebrate development before the formation of the choroid plexus. This specific entity is called "embryonic CSF." Based on the structure of the compartment, CSF composition, origin and circulation, and its interaction with neuroepithelial precursor cells (the target cells) we can conclude that embryonic CSF is different from the CSF in later developmental stages and from the adult CSF. This article presents arguments that support the singularity of the embryonic CSF, mainly focusing on its influence on neural precursor behavior during development and in adult life.

  15. Embryonic cerebrospinal fluid in brain development: neural progenitor control

    PubMed Central

    Gato, Angel; Alonso, M. Isabel; Martín, Cristina; Carnicero, Estela; Moro, José Antonio; De la Mano, Aníbal; Fernández, José M. F.; Lamus, Francisco; Desmond, Mary E.

    2014-01-01

    Due to the effort of several research teams across the world, today we have a solid base of knowledge on the liquid contained in the brain cavities, its composition, and biological roles. Although the cerebrospinal fluid (CSF) is among the most relevant parts of the central nervous system from the physiological point of view, it seems that it is not a permanent and stable entity because its composition and biological properties evolve across life. So, we can talk about different CSFs during the vertebrate life span. In this review, we focus on the CSF in an interesting period, early in vertebrate development before the formation of the choroid plexus. This specific entity is called “embryonic CSF.” Based on the structure of the compartment, CSF composition, origin and circulation, and its interaction with neuroepithelial precursor cells (the target cells) we can conclude that embryonic CSF is different from the CSF in later developmental stages and from the adult CSF. This article presents arguments that support the singularity of the embryonic CSF, mainly focusing on its influence on neural precursor behavior during development and in adult life. PMID:25165044

  16. Transient, afferent input-dependent, postnatal niche for neural progenitor cells in the cochlear nucleus

    PubMed Central

    Volkenstein, Stefan; Oshima, Kazuo; Sinkkonen, Saku T.; Corrales, C. Eduardo; Most, Sam P.; Chai, Renjie; Jan, Taha A.; van Amerongen, Renée; Cheng, Alan G.; Heller, Stefan

    2013-01-01

    In the cochlear nucleus (CN), the first central relay of the auditory pathway, the survival of neurons during the first weeks after birth depends on afferent innervation from the cochlea. Although input-dependent neuron survival has been extensively studied in the CN, neurogenesis has not been evaluated as a possible mechanism of postnatal plasticity. Here we show that new neurons are born in the CN during the critical period of postnatal plasticity. Coincidently, we found a population of neural progenitor cells that are controlled by a complex interplay of Wnt, Notch, and TGFβ/BMP signaling, in which low levels of TGFβ/BMP signaling are permissive for progenitor proliferation that is promoted by Wnt and Notch activation. We further show that cells with activated Wnt signaling reside in the CN and that these cells have high propensity for neurosphere formation. Cochlear ablation resulted in diminishment of progenitors and Wnt/β-catenin-active cells, suggesting that the neonatal CN maintains an afferent innervation-dependent population of progenitor cells that display active canonical Wnt signaling. PMID:23940359

  17. Transient, afferent input-dependent, postnatal niche for neural progenitor cells in the cochlear nucleus.

    PubMed

    Volkenstein, Stefan; Oshima, Kazuo; Sinkkonen, Saku T; Corrales, C Eduardo; Most, Sam P; Chai, Renjie; Jan, Taha A; van Amerongen, Renée; Cheng, Alan G; Heller, Stefan

    2013-08-27

    In the cochlear nucleus (CN), the first central relay of the auditory pathway, the survival of neurons during the first weeks after birth depends on afferent innervation from the cochlea. Although input-dependent neuron survival has been extensively studied in the CN, neurogenesis has not been evaluated as a possible mechanism of postnatal plasticity. Here we show that new neurons are born in the CN during the critical period of postnatal plasticity. Coincidently, we found a population of neural progenitor cells that are controlled by a complex interplay of Wnt, Notch, and TGFβ/BMP signaling, in which low levels of TGFβ/BMP signaling are permissive for progenitor proliferation that is promoted by Wnt and Notch activation. We further show that cells with activated Wnt signaling reside in the CN and that these cells have high propensity for neurosphere formation. Cochlear ablation resulted in diminishment of progenitors and Wnt/β-catenin-active cells, suggesting that the neonatal CN maintains an afferent innervation-dependent population of progenitor cells that display active canonical Wnt signaling.

  18. Radial glia and neural progenitors in the adult zebrafish central nervous system.

    PubMed

    Than-Trong, Emmanuel; Bally-Cuif, Laure

    2015-08-01

    The adult central nervous system (CNS) of the zebrafish, owing to its enrichment in constitutive neurogenic niches, is becoming an increasingly used model to address fundamental questions pertaining to adult neural stem cell (NSC) biology, adult neurogenesis and neuronal repair. Studies conducted in several CNS territories (notably the telencephalon, retina, midbrain, cerebellum and spinal cord) highlighted the presence, in these niches, of progenitor cells displaying NSC-like characters. While pointing to radial glial cells (RG) as major long-lasting, constitutively active and/or activatable progenitors in most domains, these studies also revealed a high heterogeneity in the progenitor subtypes used at the top of neurogenic hierarchies, including the persistence of neuroepithelial (NE) progenitors in some areas. Likewise, dissecting the molecular pathways underlying RG maintenance and recruitment under physiological conditions and upon repair in the zebrafish model revealed shared processes but also specific cascades triggering or sustaining reparative NSC recruitment. Together, the zebrafish adult brain reveals an extensive complexity of adult NSC niches, properties and control pathways, which extends existing understanding of adult NSC biology and gives access to novel mechanisms of efficient NSC maintenance and recruitment in an adult vertebrate brain.

  19. BLOS2 negatively regulates Notch signaling during neural and hematopoietic stem and progenitor cell development

    PubMed Central

    Zhou, Wenwen; He, Qiuping; Zhang, Chunxia; He, Xin; Cui, Zongbin; Liu, Feng; Li, Wei

    2016-01-01

    Notch signaling plays a crucial role in controling the proliferation and differentiation of stem and progenitor cells during embryogenesis or organogenesis, but its regulation is incompletely understood. BLOS2, encoded by the Bloc1s2 gene, is a shared subunit of two lysosomal trafficking complexes, biogenesis of lysosome-related organelles complex-1 (BLOC-1) and BLOC-1-related complex (BORC). Bloc1s2−/− mice were embryonic lethal and exhibited defects in cortical development and hematopoiesis. Loss of BLOS2 resulted in elevated Notch signaling, which consequently increased the proliferation of neural progenitor cells and inhibited neuronal differentiation in cortices. Likewise, ablation of bloc1s2 in zebrafish or mice led to increased hematopoietic stem and progenitor cell production in the aorta-gonad-mesonephros region. BLOS2 physically interacted with Notch1 in endo-lysosomal trafficking of Notch1. Our findings suggest that BLOS2 is a novel negative player in regulating Notch signaling through lysosomal trafficking to control multiple stem and progenitor cell homeostasis in vertebrates. DOI: http://dx.doi.org/10.7554/eLife.18108.001 PMID:27719760

  20. The early postnatal nonhuman primate neocortex contains self-renewing multipotent neural progenitor cells.

    PubMed

    Homman-Ludiye, Jihane; Merson, Tobias D; Bourne, James A

    2012-01-01

    The postnatal neocortex has traditionally been considered a non-neurogenic region, under non-pathological conditions. A few studies suggest, however, that a small subpopulation of neural cells born during postnatal life can differentiate into neurons that take up residence within the neocortex, implying that postnatal neurogenesis could occur in this region, albeit at a low level. Evidence to support this hypothesis remains controversial while the source of putative neural progenitors responsible for generating new neurons in the postnatal neocortex is unknown. Here we report the identification of self-renewing multipotent neural progenitor cells (NPCs) derived from the postnatal day 14 (PD14) marmoset monkey primary visual cortex (V1, striate cortex). While neuronal maturation within V1 is well advanced by PD14, we observed cells throughout this region that co-expressed Sox2 and Ki67, defining a population of resident proliferating progenitor cells. When cultured at low density in the presence of epidermal growth factor (EGF) and/or fibroblast growth factor 2 (FGF-2), dissociated V1 tissue gave rise to multipotent neurospheres that exhibited the ability to differentiate into neurons, oligodendrocytes and astrocytes. While the capacity to generate neurones and oligodendrocytes was not observed beyond the third passage, astrocyte-restricted neurospheres could be maintained for up to 6 passages. This study provides the first direct evidence for the existence of multipotent NPCs within the postnatal neocortex of the nonhuman primate. The potential contribution of neocortical NPCs to neural repair following injury raises exciting new possibilities for the field of regenerative medicine.

  1. USP9X Enhances the Polarity and Self-Renewal of Embryonic Stem Cell-derived Neural Progenitors

    PubMed Central

    Jolly, Lachlan A.; Taylor, Verdon

    2009-01-01

    The substrate-specific deubiquitylating enzyme USP9X is a putative “stemness” gene expressed in many progenitor cell populations. To test its function in embryonic stem cell-derived neural progenitor/stem cells, we expressed USP9X from a Nestin promoter. Elevated USP9X levels resulted in two phenomena. First, it produced a dramatically altered cellular architecture wherein the majority (>80%) of neural progenitors was arranged into radial clusters. These progenitors expressed markers of radial glial cells and were highly polarized with adherens junction proteins (N-cadherin, β-catenin, and AF-6) and apical markers (Prominin1, atypical protein kinase C-ζ) as well as Notch, Numb, and USP9X itself, concentrated at the center. The cluster centers were also devoid of nuclei and so resembled the apical end-feet of radial progenitors in the neural tube. Second, USP9X overexpression caused a fivefold increase in the number of radial progenitors and neurons, in the absence of exogenous growth factors. 5-Bromo-2′-deoxyuridine labeling, as well as the examination of the brain lipid-binding protein:βIII-tubulin ratio, indicated that nestin-USP9X enhanced the self-renewal of radial progenitors but did not block their subsequent differentiation to neurons and astrocytes. nestin-USP9X radial progenitors reformed clusters after passage as single cells, whereas control cells did not, suggesting it aids the establishment of polarity. We propose that USP9X-induced polarization of these neural progenitors results in their radial arrangement, which provides an environment conducive for self-renewal. PMID:19176755

  2. Prominin-1 (CD133) and the Cell Biology of Neural Progenitors and Their Progeny.

    PubMed

    Sykes, Alex M; Huttner, Wieland B

    2013-01-01

    Our group discovered prominin-1 in search for markers to study the cell polarity of neural stem and progenitor cells in the developing brain. Over the past 15 years, prominin-1, also called CD133, has not only become a frequently used marker of neural stem cells and neural cancer stem cells, as is in fact the case of somatic (cancer) stem cells in general, but has also been used to understand the symmetric versus asymmetric division of the neural stem cells in the context of their apical-basal polarity. Moreover, studying prominin-1 on neural stem cells has revealed a novel fate of the midbody, that is, midbody release, and key differences in this release between normal stem cells and cancer-derived cells. Other subcellular aspects of neural stem cells, the understanding of which has been promoted by studying prominin-1, pertain to the organization of plasma membrane protrusions and the membrane microdomains they contain. Of particular relevance in this context is the primary cilium of neuroepithelial cells and its transformation into the outer segment of retinal photoreceptor cells, a process in which prominin-1 exerts a vital role.

  3. Requirement for Foxd3 in the maintenance of neural crest progenitors.

    PubMed

    Teng, Lu; Mundell, Nathan A; Frist, Audrey Y; Wang, Qiaohong; Labosky, Patricia A

    2008-05-01

    Understanding the molecular mechanisms of stem cell maintenance is crucial for the ultimate goal of manipulating stem cells for the treatment of disease. Foxd3 is required early in mouse embryogenesis; Foxd3(-/-) embryos fail around the time of implantation, cells of the inner cell mass cannot be maintained in vitro, and blastocyst-derived stem cell lines cannot be established. Here, we report that Foxd3 is required for maintenance of the multipotent mammalian neural crest. Using tissue-specific deletion of Foxd3 in the neural crest, we show that Foxd3(flox/-); Wnt1-Cre mice die perinatally with a catastrophic loss of neural crest-derived structures. Cranial neural crest tissues are either missing or severely reduced in size, the peripheral nervous system consists of reduced dorsal root ganglia and cranial nerves, and the entire gastrointestinal tract is devoid of neural crest derivatives. These results demonstrate a global role for this transcriptional repressor in all aspects of neural crest maintenance along the anterior-posterior axis, and establish an unprecedented molecular link between multiple divergent progenitor lineages of the mammalian embryo.

  4. The mother superior mutation ablates foxd3 activity in neural crest progenitor cells and depletes neural crest derivatives in zebrafish.

    PubMed

    Montero-Balaguer, Mercedes; Lang, Michael R; Sachdev, Sherri Weiss; Knappmeyer, Christiane; Stewart, Rodney A; De La Guardia, Ana; Hatzopoulos, Antonis K; Knapik, Ela W

    2006-12-01

    The zebrafish mutation mother superior (mosm188) leads to a depletion of neural crest (NC) derivatives including the craniofacial cartilage skeleton, the peripheral nervous system (sympathetic neurons, dorsal root ganglia, enteric neurons), and pigment cells. The loss of derivatives is preceded by a reduction in NC-expressed transcription factors, snail1b, sox9b, sox10, and a specific loss of foxd3 expression in NC progenitor cells. We employed genetic linkage analysis and physical mapping to place the mosm188 mutation on zebrafish chromosome 6 in the vicinity of the foxd3 gene. Furthermore, we found that mosm188 does not complement the sym1/foxd3 mutation, indicating that mosm188 resides within the foxd3 locus. Injection of PAC clones containing the foxd3 gene into mosm188 embryos restored foxd3 expression in NC progenitors and suppressed the mosm188 phenotype. However, sequencing the foxd3 transcribed area in mosm188 embryos did not reveal nucleotide changes segregating with the mosm188 phenotype, implying that the mutation most likely resides outside the foxd3-coding region. Based on these findings, we propose that the mosm188 mutation perturbs a NC-specific foxd3 regulatory element. Further analysis of mosm188 mutants and foxd3 morphants revealed that NC cells are initially formed, suggesting that foxd3 function is required to maintain the pool of NC progenitors.

  5. Neural differentiation of novel multipotent progenitor cells from cryopreserved human umbilical cord blood

    SciTech Connect

    Lee, Myoung Woo; Moon, Young Joon; Yang, Mal Sook; Kim, Sun Kyung; Jang, In Keun; Eom, Young-woo; Park, Joon Seong; Kim, Hugh C.; Song, Kye Yong; Park, Soon Cheol; Lim, Hwan Sub; Kim, Young Jin . E-mail: jin@lifecord.co.kr

    2007-06-29

    Umbilical cord blood (UCB) is a rich source of hematopoietic stem cells, with practical and ethical advantages. To date, the presence of other stem cells in UCB remains to be established. We investigated whether other stem cells are present in cryopreserved UCB. Seeded mononuclear cells formed adherent colonized cells in optimized culture conditions. Over a 4- to 6-week culture period, colonized cells gradually developed into adherent mono-layer cells, which exhibited homogeneous fibroblast-like morphology and immunophenotypes, and were highly proliferative. Isolated cells were designated 'multipotent progenitor cells (MPCs)'. Under appropriate conditions for 2 weeks, MPCs differentiated into neural tissue-specific cell types, including neuron, astrocyte, and oligodendrocyte. Differentiated cells presented their respective markers, specifically, NF-L and NSE for neurons, GFAP for astrocytes, and myelin/oligodendrocyte for oligodendrocytes. In this study, we successfully isolated MPCs from cryopreserved UCB, which differentiated into the neural tissue-specific cell types. These findings suggest that cryopreserved human UCB is a useful alternative source of neural progenitor cells, such as MPCs, for experimental and therapeutic applications.

  6. Neural progenitors organize in small-world networks to promote cell proliferation.

    PubMed

    Malmersjö, Seth; Rebellato, Paola; Smedler, Erik; Planert, Henrike; Kanatani, Shigeaki; Liste, Isabel; Nanou, Evanthia; Sunner, Hampus; Abdelhady, Shaimaa; Zhang, Songbai; Andäng, Michael; El Manira, Abdeljabbar; Silberberg, Gilad; Arenas, Ernest; Uhlén, Per

    2013-04-16

    Coherent network activity among assemblies of interconnected cells is essential for diverse functions in the adult brain. However, cellular networks before formations of chemical synapses are poorly understood. Here, embryonic stem cell-derived neural progenitors were found to form networks exhibiting synchronous calcium ion (Ca(2+)) activity that stimulated cell proliferation. Immature neural cells established circuits that propagated electrical signals between neighboring cells, thereby activating voltage-gated Ca(2+) channels that triggered Ca(2+) oscillations. These network circuits were dependent on gap junctions, because blocking prevented electrotonic transmission both in vitro and in vivo. Inhibiting connexin 43 gap junctions abolished network activity, suppressed proliferation, and affected embryonic cortical layer formation. Cross-correlation analysis revealed highly correlated Ca(2+) activities in small-world networks that followed a scale-free topology. Graph theory predicts that such network designs are effective for biological systems. Taken together, these results demonstrate that immature cells in the developing brain organize in small-world networks that critically regulate neural progenitor proliferation.

  7. Optimization of surface-immobilized extracellular matrices for the proliferation of neural progenitor cells derived from induced pluripotent stem cells.

    PubMed

    Komura, Takashi; Kato, Koichi; Konagaya, Shuhei; Nakaji-Hirabayashi, Tadashi; Iwata, Hiroo

    2015-11-01

    Neural progenitor cells derived from induced pluripotent stem cells have been considered as a potential source for cell-transplantation therapy of central nervous disorders. However, efficient methods to expand neural progenitor cells are further required for their clinical applications. In this study, a protein array was fabricated with nine extracellular matrices and used to screen substrates suitable for the expansion of neural progenitor cells derived from mouse induced pluripotent stem cells. The results showed that neural progenitor cells efficiently proliferated on substrates with immobilized laminin-1, laminin-5, or Matrigel. Based on this result, further attempts were made to develop clinically compliant substrates with immobilized polypeptides that mimic laminin-1, one of the most effective extracellular matrices as identified in the array-based screening. We used here recombinant DNA technology to prepare polypeptide containing the globular domain 3 of laminin-1 and immobilized it onto glass-based substrates. Our results showed that neural progenitor cells selectively proliferated on substrate with the immobilized polypeptide while maintaining their differentiated state.

  8. Noncoding RNA in the transcriptional landscape of human neural progenitor cell differentiation

    PubMed Central

    Hecht, Patrick M.; Ballesteros-Yanez, Inmaculada; Grepo, Nicole; Knowles, James A.; Campbell, Daniel B.

    2015-01-01

    Increasing evidence suggests that noncoding RNAs play key roles in cellular processes, particularly in the brain. The present study used RNA sequencing to identify the transcriptional landscape of two human neural progenitor cell lines, SK-N-SH and ReNcell CX, as they differentiate into human cortical projection neurons. Protein coding genes were found to account for 54.8 and 57.0% of expressed genes, respectively, and alignment of RNA sequencing reads revealed that only 25.5–28.1% mapped to exonic regions of the genome. Differential expression analysis in the two cell lines identified altered gene expression in both protein coding and noncoding RNAs as they undergo neural differentiation with 222 differentially expressed genes observed in SK-N-SH cells and 19 differentially expressed genes in ReNcell CX. Interestingly, genes showing differential expression in SK-N-SH cells are enriched in genes implicated in autism spectrum disorder, but not in gene sets related to cancer or Alzheimer's disease. Weighted gene co-expression network analysis (WGCNA) was used to detect modules of co-expressed protein coding and noncoding RNAs in SK-N-SH cells and found four modules to be associated with neural differentiation. These modules contain varying levels of noncoding RNAs ranging from 10.7 to 49.7% with gene ontology suggesting roles in numerous cellular processes important for differentiation. These results indicate that noncoding RNAs are highly expressed in human neural progenitor cells and likely hold key regulatory roles in gene networks underlying neural differentiation and neurodevelopmental disorders. PMID:26557050

  9. Yes-Associated Protein 65 (YAP) Expands Neural Progenitors and Regulates Pax3 Expression in the Neural Plate Border Zone

    PubMed Central

    Gee, Stephen T.; Milgram, Sharon L.; Kramer, Kenneth L.; Conlon, Frank L.; Moody, Sally A.

    2011-01-01

    Yes-associated protein 65 (YAP) contains multiple protein-protein interaction domains and functions as both a transcriptional co-activator and as a scaffolding protein. Mouse embryos lacking YAP did not survive past embryonic day 8.5 and showed signs of defective yolk sac vasculogenesis, chorioallantoic fusion, and anterior-posterior (A-P) axis elongation. Given that the YAP knockout mouse defects might be due in part to nutritional deficiencies, we sought to better characterize a role for YAP during early development using embryos that develop externally. YAP morpholino (MO)-mediated loss-of-function in both frog and fish resulted in incomplete epiboly at gastrulation and impaired axis formation, similar to the mouse phenotype. In frog, germ layer specific genes were expressed, but they were temporally delayed. YAP MO-mediated partial knockdown in frog allowed a shortened axis to form. YAP gain-of-function in Xenopus expanded the progenitor populations in the neural plate (sox2+) and neural plate border zone (pax3+), while inhibiting the expression of later markers of tissues derived from the neural plate border zone (neural crest, pre-placodal ectoderm, hatching gland), as well as epidermis and somitic muscle. YAP directly regulates pax3 expression via association with TEAD1 (N-TEF) at a highly conserved, previously undescribed, TEAD-binding site within the 5′ regulatory region of pax3. Structure/function analyses revealed that the PDZ-binding motif of YAP contributes to the inhibition of epidermal and somitic muscle differentiation, but a complete, intact YAP protein is required for expansion of the neural plate and neural plate border zone progenitor pools. These results provide a thorough analysis of YAP mediated gene expression changes in loss- and gain-of-function experiments. Furthermore, this is the first report to use YAP structure-function analyzes to determine which portion of YAP is involved in specific gene expression changes and the first to show

  10. Yes-associated protein 65 (YAP) expands neural progenitors and regulates Pax3 expression in the neural plate border zone.

    PubMed

    Gee, Stephen T; Milgram, Sharon L; Kramer, Kenneth L; Conlon, Frank L; Moody, Sally A

    2011-01-01

    Yes-associated protein 65 (YAP) contains multiple protein-protein interaction domains and functions as both a transcriptional co-activator and as a scaffolding protein. Mouse embryos lacking YAP did not survive past embryonic day 8.5 and showed signs of defective yolk sac vasculogenesis, chorioallantoic fusion, and anterior-posterior (A-P) axis elongation. Given that the YAP knockout mouse defects might be due in part to nutritional deficiencies, we sought to better characterize a role for YAP during early development using embryos that develop externally. YAP morpholino (MO)-mediated loss-of-function in both frog and fish resulted in incomplete epiboly at gastrulation and impaired axis formation, similar to the mouse phenotype. In frog, germ layer specific genes were expressed, but they were temporally delayed. YAP MO-mediated partial knockdown in frog allowed a shortened axis to form. YAP gain-of-function in Xenopus expanded the progenitor populations in the neural plate (sox2(+)) and neural plate border zone (pax3(+)), while inhibiting the expression of later markers of tissues derived from the neural plate border zone (neural crest, pre-placodal ectoderm, hatching gland), as well as epidermis and somitic muscle. YAP directly regulates pax3 expression via association with TEAD1 (N-TEF) at a highly conserved, previously undescribed, TEAD-binding site within the 5' regulatory region of pax3. Structure/function analyses revealed that the PDZ-binding motif of YAP contributes to the inhibition of epidermal and somitic muscle differentiation, but a complete, intact YAP protein is required for expansion of the neural plate and neural plate border zone progenitor pools. These results provide a thorough analysis of YAP mediated gene expression changes in loss- and gain-of-function experiments. Furthermore, this is the first report to use YAP structure-function analyzes to determine which portion of YAP is involved in specific gene expression changes and the first to show

  11. Effects of acute hypoxia/acidosis on intracellular pH in differentiating neural progenitor cells.

    PubMed

    Nordström, Tommy; Jansson, Linda C; Louhivuori, Lauri M; Akerman, Karl E O

    2012-06-21

    The response of differentiating mouse neural progenitor cells, migrating out from neurospheres, to conditions simulating ischemia (hypoxia and extracellular or intracellular acidosis) was studied. We show here, by using BCECF and single cell imaging to monitor intracellular pH (pH(i)), that two main populations can be distinguished by exposing migrating neural progenitor cells to low extracellular pH or by performing an acidifying ammonium prepulse. The cells dominating at the periphery of the neurosphere culture, which were positive for neuron specific markers MAP-2, calbindin and NeuN had lower initial resting pH(i) and could also easily be further acidified by lowering the extracellular pH. Moreover, in this population, a more profound acidification was seen when the cells were acidified using the ammonium prepulse technique. However, when the cell population was exposed to depolarizing potassium concentrations no alterations in pH(i) took place in this population. In contrast, depolarization caused an increase in pH(i) (by 0.5 pH units) in the cell population closer to the neurosphere body, which region was positive for the radial cell marker (GLAST). This cell population, having higher resting pH(i) (pH 6.9-7.1) also responded to acute hypoxia. During hypoxic treatment the resting pH(i) decreased by 0.1 pH units and recovered rapidly after reoxygenation. Our results show that migrating neural progenitor cells are highly sensitive to extracellular acidosis and that irreversible damage becomes evident at pH 6.2. Moreover, our results show that a response to acidosis clearly distinguishes two individual cell populations probably representing neuronal and radial cells.

  12. CHEMOKINES REGULATE THE MIGRATION OF NEURAL PROGENITORS TO SITES OF NEUROINFLAMMATION

    PubMed Central

    Belmadani, Abdelhak; Tran, Phuong B.; Ren, Dongjun; Miller, Richard J.

    2009-01-01

    Many studies have shown that transplanted or endogenous neural progenitor cells will migrate towards damaged areas of the brain. However, the mechanism underlying this effect is not clear. Here we report that, using hippocampal slice cultures, grafted neural progenitor cells (NPs) migrate towards areas of neuroinflammation, and that chemokines are a major regulator of this process. Migration of NPs was observed after injecting an inflammatory stimulus into the area of the fimbria, and transplanting green fluorescent protein (EGFP)-labeled NPs into the dentate gyrus (DG) of cultured hippocampal slices. 3–7 days following transplantation, EGFP-NPs in control slices showed little tendancy to migrate and had differentiated into neurons and glia. In contrast, in slices injected with inflammatory stimuli, EGFP-NPs migrated towards the site of the injection. NPs in these slices also survived less well. The inflammatory stimuli used were either a combination of the cytokines TNF-α and IFN-γ, the bacterial toxin LPS, the HIV-1 coat protein gp120 or a β-amyloid expressing adenovirus. We showed that these inflammatory stimuli increased the synthesis of numerous chemokines and cytokines by hippocampal slices. When EGFP-NPs from CCR2 ko mice were transplanted into slices they exhibited little migration towards sites of inflammation. Similarly, wild type EGFP-NPs exhibited little migration towards inflammatory sites when transplanted into slices prepared from MCP-1 ko mice. These data indicate that factors secreted by sites of neuroinflammation are attractive to neural progenitors and suggest that chemokines such as MCP-1 play an important role in this process. PMID:16554469

  13. Ror family receptor tyrosine kinases regulate the maintenance of neural progenitor cells in the developing neocortex.

    PubMed

    Endo, Mitsuharu; Doi, Ryosuke; Nishita, Michiru; Minami, Yasuhiro

    2012-04-15

    The Ror family receptor tyrosine kinases (RTKs), Ror1 and Ror2, have been shown to play crucial roles in developmental morphogenesis by acting as receptors or co-receptors to mediate Wnt5a-induced signaling. Although Ror1, Ror2 and Wnt5a are expressed in the developing brain, little is known about their roles in the neural development. Here we show that Ror1, Ror2 and their ligand Wnt5a are highly expressed in neocortical neural progenitor cells (NPCs). Small interfering RNA (siRNA)-mediated suppression of Ror1, Ror2 or Wnt5a in cultured NPCs isolated from embryonic neocortex results in the reduction of βIII-tubulin-positive neurons that are produced from NPCs possibly through the generation of T-box brain 2 (Tbr2)-positive intermediate progenitors. BrdU-labeling experiments further reveal that the proportion of proliferative and neurogenic NPCs, which are positive for neural progenitor cell marker (Pax6) but negative for glial cell marker (glial fibrillary acidic protein; GFAP), is reduced within a few days in culture following knockdown of these molecules, suggesting that Ror1, Ror2 and Wnt5a regulate neurogenesis through the maintenance of NPCs. Moreover, we show that Dishevelled 2 (Dvl2) is involved in Wnt5a-Ror1 and Wnt5a-Ror2 signaling in NPCs, and that suppressed expression of Dvl2 indeed reduces the proportion of proliferative and neurogenic NPCs. Interestingly, suppressed expression of either Ror1 or Ror2 in NPCs in the developing neocortex results in the precocious differentiation of NPCs into neurons, and their forced expression results in delayed differentiation. Collectively, these results indicate that Wnt5a-Ror1 and Wnt5a-Ror2 signaling pathways play roles in maintaining proliferative and neurogenic NPCs during neurogenesis of the developing neocortex.

  14. Activin/Nodal Signaling Supports Retinal Progenitor Specification in a Narrow Time Window during Pluripotent Stem Cell Neuralization

    PubMed Central

    Bertacchi, Michele; Lupo, Giuseppe; Pandolfini, Luca; Casarosa, Simona; D’Onofrio, Mara; Pedersen, Roger A.; Harris, William A.; Cremisi, Federico

    2015-01-01

    Summary Retinal progenitors are initially found in the anterior neural plate region known as the eye field, whereas neighboring areas undertake telencephalic or hypothalamic development. Eye field cells become specified by switching on a network of eye field transcription factors, but the extracellular cues activating this network remain unclear. In this study, we used chemically defined media to induce in vitro differentiation of mouse embryonic stem cells (ESCs) toward eye field fates. Inhibition of Wnt/β-catenin signaling was sufficient to drive ESCs to telencephalic, but not retinal, fates. Instead, retinal progenitors could be generated from competent differentiating mouse ESCs by activation of Activin/Nodal signaling within a narrow temporal window corresponding to the emergence of primitive anterior neural progenitors. Activin also promoted eye field gene expression in differentiating human ESCs. Our results reveal insights into the mechanisms of eye field specification and open new avenues toward the generation of retinal progenitors for translational medicine. PMID:26388287

  15. A comparison of epithelial and neural properties in progenitor cells derived from the adult human ciliary body and brain.

    PubMed

    Moe, Morten C; Kolberg, Rebecca S; Sandberg, Cecilie; Vik-Mo, Einar; Olstorn, Havard; Varghese, Mercy; Langmoen, Iver A; Nicolaissen, Bjørn

    2009-01-01

    Cells isolated from the ciliary body (CB) of the adult human eye possess properties of retinal stem/progenitor cells and can be propagated as spheres in culture. As these cells are isolated from a non-neural epithelium which has neuroepithelial origin, they may have both epithelial and neural lineages. Since it is the properties of neural progenitor cells that are sought after in a future scenario of autotransplantation, we wanted to directly compare human CB spheres with neurospheres derived from the human subventricular zone (SVZ), which is the best characterized neural stem cell niche in the CNS of adults. The CB epithelium was dissected from donor eyes (n = 8). Biopsies from the ventricular wall were harvested during neurosurgery due to epilepsy (n = 7). CB and SVZ tissue were also isolated from Brown Norwegian rats. Dissociated single cells were cultivated in a sphere-promoting medium and passaged every 10-30 days. Fixed spheres were studied by immunohistochemistry, quantitative RT-PCR and scanning/transmission electron microscopy. We found that both CB and SVZ spheres contained a mixed population of cells embedded in extracellular matrix. CB spheres, in contrast to SVZ neurospheres, contained pigmented cells with epithelial morphology that stained for cytokeratins (3/12 + 19), were connected through desmosomes and tight-junctions and produced PEDF. Markers of neural progenitors (nestin, Sox-2, GFAP) were significantly lower expressed in human CB compared to SVZ spheres, and nestin positive cells in the CB spheres also contained pigment. There was higher expression of EGF and TGF-beta receptors in human CB spheres, and a comparative greater activation of the canonical Wnt pathway. These results indicate that adult human CB spheres contain progenitor cells with epithelial properties and limited expression of neural progenitor markers compared to CNS neurospheres. Further studies mapping the regulation between epithelial and neural properties in the adult human

  16. Astrocyte-Secreted Factors Selectively Alter Neural Stem and Progenitor Cell Proliferation in the Fragile X Mouse.

    PubMed

    Sourial, Mary; Doering, Laurie C

    2016-01-01

    An increasing body of evidence indicates that astrocytes contribute to the governance and fine tuning of stem and progenitor cell production during brain development. The effect of astrocyte function in cell production in neurodevelopmental disorders is unknown. We used the Neural Colony Forming Cell assay to determine the effect of astrocyte conditioned media (ACM) on the generation of neurospheres originating from either progenitor cells or functional stem cells in the knock out (KO) Fragile X mouse model. ACM from both normal and Fmr1-KO mice generated higher percentages of smaller neurospheres indicative of restricted proliferation of the progenitor cell population in Fmr1-KO brains. Wild type (WT) neurospheres, but not KO neurospheres, showed enhanced responses to ACM from the Fmr1-KO mice. In particular, Fmr1-KO ACM increased the percentage of large neurospheres generated, representative of spheres produced from neural stem cells. We also used 2D DIGE to initiate identification of the astrocyte-secreted proteins with differential expression between Fmr1-KO and WT cortices and hippocampi. The results further support the critical role of astrocytes in governing neural cell production in brain development and point to significant alterations in neural cell proliferation due to astrocyte secreted factors from the Fragile X brain. • We studied the proliferation of neural stem and progenitor cells in Fragile X.• We examined the role of astrocyte-secreted factors in neural precursor cell biology.• Astrocyte-secreted factors with differential expression in Fragile X identified.

  17. Astrocyte-Secreted Factors Selectively Alter Neural Stem and Progenitor Cell Proliferation in the Fragile X Mouse

    PubMed Central

    Sourial, Mary; Doering, Laurie C.

    2016-01-01

    An increasing body of evidence indicates that astrocytes contribute to the governance and fine tuning of stem and progenitor cell production during brain development. The effect of astrocyte function in cell production in neurodevelopmental disorders is unknown. We used the Neural Colony Forming Cell assay to determine the effect of astrocyte conditioned media (ACM) on the generation of neurospheres originating from either progenitor cells or functional stem cells in the knock out (KO) Fragile X mouse model. ACM from both normal and Fmr1-KO mice generated higher percentages of smaller neurospheres indicative of restricted proliferation of the progenitor cell population in Fmr1-KO brains. Wild type (WT) neurospheres, but not KO neurospheres, showed enhanced responses to ACM from the Fmr1-KO mice. In particular, Fmr1-KO ACM increased the percentage of large neurospheres generated, representative of spheres produced from neural stem cells. We also used 2D DIGE to initiate identification of the astrocyte-secreted proteins with differential expression between Fmr1-KO and WT cortices and hippocampi. The results further support the critical role of astrocytes in governing neural cell production in brain development and point to significant alterations in neural cell proliferation due to astrocyte secreted factors from the Fragile X brain. Highlights: • We studied the proliferation of neural stem and progenitor cells in Fragile X. • We examined the role of astrocyte-secreted factors in neural precursor cell biology. • Astrocyte-secreted factors with differential expression in Fragile X identified. PMID:27242437

  18. Hippocampal adult neurogenesis is maintained by Neil3-dependent repair of oxidative DNA lesions in neural progenitor cells.

    PubMed

    Regnell, Christine Elisabeth; Hildrestrand, Gunn Annette; Sejersted, Yngve; Medin, Tirill; Moldestad, Olve; Rolseth, Veslemøy; Krokeide, Silje Zandstra; Suganthan, Rajikala; Luna, Luisa; Bjørås, Magnar; Bergersen, Linda H

    2012-09-27

    Accumulation of oxidative DNA damage has been proposed as a potential cause of age-related cognitive decline. The major pathway for removal of oxidative DNA base lesions is base excision repair, which is initiated by DNA glycosylases. In mice, Neil3 is the main DNA glycosylase for repair of hydantoin lesions in single-stranded DNA of neural stem/progenitor cells, promoting neurogenesis. Adult neurogenesis is crucial for maintenance of hippocampus-dependent functions involved in behavior. Herein, behavioral studies reveal learning and memory deficits and reduced anxiety-like behavior in Neil3(-/-) mice. Neural stem/progenitor cells from aged Neil3(-/-) mice show impaired proliferative capacity and reduced DNA repair activity. Furthermore, hippocampal neurons in Neil3(-/-) mice display synaptic irregularities. It appears that Neil3-dependent repair of oxidative DNA damage in neural stem/progenitor cells is required for maintenance of adult neurogenesis to counteract the age-associated deterioration of cognitive performance.

  19. Drosophila intermediate neural progenitors produce lineage-dependent related series of diverse neurons

    PubMed Central

    Wang, Yu-Chun; Yang, Jacob S.; Johnston, Rebecca; Ren, Qingzhong; Lee, Ying-Jou; Luan, Haojiang; Brody, Thomas; Odenwald, Ward F.; Lee, Tzumin

    2014-01-01

    Drosophila type II neuroblasts (NBs), like mammalian neural stem cells, deposit neurons through intermediate neural progenitors (INPs) that can each produce a series of neurons. Both type II NBs and INPs exhibit age-dependent expression of various transcription factors, potentially specifying an array of diverse neurons by combinatorial temporal patterning. Not knowing which mature neurons are made by specific INPs, however, conceals the actual variety of neuron types and limits further molecular studies. Here we mapped neurons derived from specific type II NB lineages and found that sibling INPs produced a morphologically similar but temporally regulated series of distinct neuron types. This suggests a common fate diversification program operating within each INP that is modulated by NB age to generate slightly different sets of diverse neurons based on the INP birth order. Analogous mechanisms might underlie the expansion of neuron diversity via INPs in mammalian brain. PMID:24306106

  20. LOXL2 Oxidizes Methylated TAF10 and Controls TFIID-Dependent Genes during Neural Progenitor Differentiation.

    PubMed

    Iturbide, Ane; Pascual-Reguant, Laura; Fargas, Laura; Cebrià, Joan Pau; Alsina, Berta; García de Herreros, Antonio; Peiró, Sandra

    2015-06-04

    Protein function is often regulated and controlled by posttranslational modifications, such as oxidation. Although oxidation has been mainly considered to be uncontrolled and nonenzymatic, many enzymatic oxidations occur on enzyme-selected lysine residues; for instance, LOXL2 oxidizes lysines by converting the ε-amino groups into aldehyde groups. Using an unbiased proteomic approach, we have identified methylated TAF10, a member of the TFIID complex, as a LOXL2 substrate. LOXL2 oxidation of TAF10 induces its release from its promoters, leading to a block in TFIID-dependent gene transcription. In embryonic stem cells, this results in the inactivation of the pluripotency genes and loss of the pluripotent capacity. During zebrafish development, the absence of LOXL2 resulted in the aberrant overexpression of the neural progenitor gene Sox2 and impaired neural differentiation. Thus, lysine oxidation of the transcription factor TAF10 is a controlled protein modification and demonstrates a role for protein oxidation in regulating pluripotency genes.

  1. Fibronectin promotes differentiation of neural crest progenitors endowed with smooth muscle cell potential

    SciTech Connect

    Costa-Silva, Bruno; Coelho da Costa, Meline; Melo, Fernanda Rosene; Neves, Cynara Mendes; Alvarez-Silva, Marcio; Calloni, Giordano Wosgrau; Trentin, Andrea Goncalves

    2009-04-01

    The neural crest (NC) is a model system used to investigate multipotency during vertebrate development. Environmental factors control NC cell fate decisions. Despite the well-known influence of extracellular matrix molecules in NC cell migration, the issue of whether they also influence NC cell differentiation has not been addressed at the single cell level. By analyzing mass and clonal cultures of mouse cephalic and quail trunk NC cells, we show for the first time that fibronectin (FN) promotes differentiation into the smooth muscle cell phenotype without affecting differentiation into glia, neurons, and melanocytes. Time course analysis indicated that the FN-induced effect was not related to massive cell death or proliferation of smooth muscle cells. Finally, by comparing clonal cultures of quail trunk NC cells grown on FN and collagen type IV (CLIV), we found that FN strongly increased both NC cell survival and the proportion of unipotent and oligopotent NC progenitors endowed with smooth muscle potential. In contrast, melanocytic progenitors were prominent in clonogenic NC cells grown on CLIV. Taken together, these results show that FN promotes NC cell differentiation along the smooth muscle lineage, and therefore plays an important role in fate decisions of NC progenitor cells.

  2. Developmentally-regulated subnuclear genome reorganization restricts neural progenitor competence in Drosophila

    PubMed Central

    Kohwi, Minoree; Lupton, Joshua R.; Lai, Sen-Lin; Miller, Michael R.

    2013-01-01

    Stem/progenitor cells often generate distinct cell types in a stereotyped birth order, and over time lose competence to specify earlier-born fates by unknown mechanisms. In Drosophila, the Hunchback transcription factor acts in neural progenitors (neuroblasts) to specify early-born neurons, in part by indirectly inducing the neuronal transcription of its target genes, including the hunchback gene. We used in vivo immuno-DNA FISH and found that the hunchback gene moves to the neuroblast nuclear periphery, a repressive subnuclear compartment, precisely when competence to specify early-born fate is lost, and several hours and cell divisions following termination of its transcription. hunchback movement to the lamina correlated with downregulation of the neuroblast nuclear protein, Distal antenna (Dan). Either prolonging Dan expression or disrupting lamina interfered with hunchback repositioning and extended neuroblast competence. We propose that neuroblasts undergo a developmentally-regulated subnuclear genome reorganization to permanently silence Hunchback target genes that results in loss of progenitor competence. PMID:23332748

  3. Curcumin attenuates harmful effects of arsenic on neural stem/progenitor cells

    PubMed Central

    Jahanbazi Jahan-Abad, Ali; Morteza-zadeh, Parastoo; Sahab Negah, Sajad; Gorji, Ali

    2017-01-01

    Objective: Arsenic, an environmental pollutant, decreases neuronal migration as well as cellular maturation and inhibits the proliferation of neural progenitor cells. Curcumin has been described as an antioxidant and neuroprotective agent with strong therapeutic potential in some neurological disorders. Human adipose-derived stem cells (hADSCs), a source of multipotent stem cells, can self-renew and differentiate into neural cells. The aim of the present study was to investigate the preventive effect of curcumin against arsenic toxic effects on the viability, telomerase activity, and apoptosis of neural stem/progenitor cells (NSPCs) derived from hADSCs. Materials and Methods: The characteristics of human adipose tissue were identified by immunocytochemistry for surface markers namely, CD105, CD73, and CD90. Using neurosphere assay, hADSCs were differentiated into neuronal cells. To characterize neural cells, expression of nestin, SOX2, MAP2, and GFAP were assessed by immunocytochemistry. Cytotoxicity and viability of NSPCs were evaluated by MTT assay. Reactive oxygen species (ROS) generated by arsenic exposure, were measured and caspase 3/7 activity and caspase-3 processing as well as the telomerase activity were determined. Results: The isolated hADSCs positively expressed CD105, CD73, and CD90. Nestin, Sox2, GFAP, and MAP2 were expressed in the neurospheres derived from hADSCs. Curcumin/arsenic co-treatment significantly increased telomerase activity of NSPCs compared to arsenic group. Furthermore, curcumin significantly reduced arsenic-induced apoptosis (via inactivation of caspases) as well as arsenic-associated ROS generation. Conclusion: Our findings revealed that curcumin has the potential to prevent harmful effects of arsenic on neurogenesis. PMID:28884087

  4. 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.

  5. p38 MAP kinase mediates nitric oxide-induced apoptosis of neural progenitor cells.

    PubMed

    Cheng, A; Chan, S L; Milhavet, O; Wang, S; Mattson, M P

    2001-11-16

    Neural progenitor cells (NPC) can proliferate, differentiate into neurons or glial cells, or undergo a form of programmed cell death called apoptosis. Although death of NPC occurs during development of the nervous system and in the adult, the underlying mechanisms are unknown. Here we show that nitric oxide (NO) can induce death of C17.2 NPC by a mechanism requiring activation of p38 MAP kinase, poly(ADP-ribose) polymerase, and caspase-3. Nitric oxide causes release of cytochrome c from mitochondria, and Bcl-2 protects the neural progenitor cells against nitric oxide-induced death, consistent with a pivotal role for mitochondrial changes in controlling the cell death process. Inhibition of p38 MAP kinase by SB203580 abolished NO-induced cell death, cytochrome c release, and activation of caspase-3, indicating that p38 activation serves as an upstream mediator in the cell death process. The anti-apoptotic protein Bcl-2 protected NPC against nitric oxide-induced apoptosis and suppressed activation of p38 MAP kinase. The ability of nitric oxide to trigger death of NPC by a mechanism involving p38 MAP kinase suggests that this diffusible gas may regulate NPC fate in physiological and pathological settings in which NO is produced.

  6. SUMOylation of DISC1: a potential role in neural progenitor proliferation in the developing cortex.

    PubMed

    Tankou, Stephanie; Ishii, Kazuhiro; Elliott, Christina; Yalla, Krishna C; Day, Jon P; Furukori, Keiko; Kubo, Ken-Ichiro; Brandon, Nicholas J; Tang, Qiyi; Hayward, Gary; Nakajima, Kazunori; Houslay, Miles D; Kamiya, Atsushi; Baillie, George; Ishizuka, Koko; Sawa, Akira

    2016-05-01

    DISC1 is a multifunctional, intracellular scaffold protein. At the cellular level, DISC1 plays a pivotal role in neural progenitor proliferation, migration, and synaptic maturation. Perturbation of the biological pathways involving DISC1 is known to lead to behavioral changes in rodents, which supports a clinical report of a Scottish pedigree in which the majority of family members with disruption of the DISC1 gene manifest depression, schizophrenia, and related mental conditions. The discrepancy of modest evidence in genetics but strong biological support for the role of DISC1 in mental conditions suggests a working hypothesis that regulation of DISC1 at the protein level, such as posttranslational modification, may play a role in the pathology of mental conditions. In this study, we report the SUMOylation of DISC1. This posttranslational modification occurs on lysine residues where small ubiquitin-related modifier (SUMO) and its homologs are conjugated to a large number of cellular proteins, which in turn regulates their subcellular distribution and protein stability. By using in silico, biochemical, and cell biological approaches, we now demonstrate that human DISC1 is SUMOylated at one specific lysine 643 (K643). We also show that this residue is crucial for proper neural progenitor proliferation in the developing cortex.

  7. Using human neural crest-derived progenitor cells to investigate osteogenesis: an in vitro study.

    PubMed

    Degistirici, Ozer; Grabellus, Florian; Irsen, Stephan; Schmid, Kurt Werner; Thie, Michael

    2010-04-01

    Human tooth contains a distinct population of neural crest-derived progenitor cells (dNC-PCs) which are known to give rise to specialized daughter cells of an osteogenic lineage. We hypothesised that dNC-PCs could develop into neural crest-derived bone in a self-propagating and extracorporal culture system. Thus, we examined the three-dimensional structure obtained from osteogenic-stimulated dNC-PCs by morphological, biochemical and spectroscopic methods. After the onset of stimulation, cells formed a multilayer with outer cells covering the surface and inner cells secreting a hyaline matrix. With prolonged culture, multilayers contracted and formed a three-dimensional construct which subsequently converted to a calcified mass. Differentiation of progenitor cells was associated with apoptosis. Cell types which survived were smooth muscle actin-positive cells and bone-like cells. The expression of osteoblastic markers and the secretion of a collagenous matrix indicate that the bone cells had acquired their functional phenotype. Furthermore, these cells produced and secreted membrane-bound vesicles into the newly forming matrix. Consequently, an early biomineralized extracellular matrix was found with calcium phosphate deposits being associated with the newly formed collagen matrix framework. The molar calcium-phosphorus-ratio of the mineralized collagen indicated that amorphous calcium phosphate was present within this matrix. The data suggest that stimulated cultures of dNC-PCs are able to recapitulate some processes of the early phase of osteogenesis.

  8. Splicing factor TRA2B is required for neural progenitor survival

    PubMed Central

    Roberts, Jacqueline M; Ennajdaoui, Hanane; Edmondson, Carina; Wirth, Brunhilde; Sanford, Jeremy; Chen, Bin

    2013-01-01

    Alternative splicing of pre-mRNAs can rapidly regulate the expression of large groups of proteins. The RNA binding protein TRA2B (SFRS10) plays well-established roles in developmentally regulated alternative splicing during Drosophila sexual differentiation. TRA2B is also essential for mammalian embryogenesis and is implicated in numerous human diseases. Precise regulation of alternative splicing is critical to the development and function of the central nervous system; however the requirements for specific splicing factors in neurogenesis are poorly understood. In this study we focus on the role of TRA2B in mammalian brain development. We show that, during murine cortical neurogenesis, TRA2B is expressed in both neural progenitors and cortical projection neurons. Using cortex-specific Tra2b mutant mice, we find that TRA2B depletion results in apoptosis of the neural progenitor cells as well as disorganization of the cortical plate. Thus, TRA2B is essential for proper development of the cerebral cortex. PMID:23818142

  9. Acute neonatal glucocorticoid exposure produces selective and rapid cerebellar neural progenitor cell apoptotic death.

    PubMed

    Noguchi, K K; Walls, K C; Wozniak, D F; Olney, J W; Roth, K A; Farber, N B

    2008-10-01

    There has been a growing controversy regarding the continued use of glucocorticoid therapy to treat respiratory dysfunction associated with prematurity, as mounting clinical evidence has shown neonatal exposure produces permanent neuromotor and cognitive deficits. Here we report that, during a selective neonatal window of vulnerability, a single glucocorticoid injection in the mouse produces rapid and selective apoptotic cell death of the proliferating neural progenitor cells in the cerebellar external granule layer and permanent reductions in neuronal cell counts of their progeny, the cerebellar internal granule layer neurons. Our estimates suggest that this mouse window of vulnerability would correspond in the human to a period extending from approximately 20 weeks gestation to 6.5 weeks after birth. This death pathway is critically regulated by the proapoptotic Bcl-2 family member Puma and is independent of p53 expression. These rodent data indicate that there exists a previously unknown window of vulnerability during which a single glucocorticoid exposure at clinically relevant doses can produce neural progenitor cell apoptosis and permanent cerebellar pathology that may be responsible for some of the iatrogenically induced neurodevelopmental abnormalities seen in children exposed to this drug. This vulnerability may be related to the physiological role of glucocorticoids in regulating programmed cell death in the mammalian cerebellum.

  10. Morphine Modulates Adult Neurogenesis and Contextual Memory by Impeding the Maturation of Neural Progenitors

    PubMed Central

    Zhang, Yue; Xu, Chi; Zheng, Hui; Loh, Horace H.; Law, Ping-Yee

    2016-01-01

    The regulation of adult neurogenesis by opiates has been implicated in modulating different addiction cycles. At which neurogenesis stage opiates exert their action remains unresolved. We attempt to define the temporal window of morphine’s inhibition effect on adult neurogenesis by using the POMC-EGFP mouse model, in which newborn granular cells (GCs) can be visualized between days 3–28 post-mitotic. The POMC-EGFP mice were trained under the 3-chambers conditioned place preference (CPP) paradigm with either saline or morphine. We observed after 4 days of CPP training with saline, the number of EGFP-labeled newborn GCs in sub-granular zone (SGZ) hippocampus significantly increased compared to mice injected with saline in their homecage. CPP training with morphine significantly decreased the number of EGFP-labeled GCs, whereas no significant difference in the number of EGFP-labeled GCs was observed with the homecage mice injected with the same dose of morphine. Using cell-type selective markers, we observed that morphine reduced the number of late stage progenitors and immature neurons such as Doublecortin (DCX) and βIII Tubulin (TuJ1) positive cells in the SGZ but did not reduce the number of early progenitors such as Nestin, SOX2, or neurogenic differentiation-1 (NeuroD1) positive cells. Analysis of co-localization between different cell markers shows that morphine reduced the number of adult-born GCs by interfering with differentiation of early progenitors, but not by inducing apoptosis. In addition, when NeuroD1 was over-expressed in DG by stereotaxic injection of lentivirus, it rescued the loss of immature neurons and prolonged the extinction of morphine-trained CPP. These results suggest that under the condition of CPP training paradigm, morphine affects the transition of neural progenitor/stem cells to immature neurons via a mechanism involving NeuroD1. PMID:27078155

  11. Morphine Modulates Adult Neurogenesis and Contextual Memory by Impeding the Maturation of Neural Progenitors.

    PubMed

    Zhang, Yue; Xu, Chi; Zheng, Hui; Loh, Horace H; Law, Ping-Yee

    2016-01-01

    The regulation of adult neurogenesis by opiates has been implicated in modulating different addiction cycles. At which neurogenesis stage opiates exert their action remains unresolved. We attempt to define the temporal window of morphine's inhibition effect on adult neurogenesis by using the POMC-EGFP mouse model, in which newborn granular cells (GCs) can be visualized between days 3-28 post-mitotic. The POMC-EGFP mice were trained under the 3-chambers conditioned place preference (CPP) paradigm with either saline or morphine. We observed after 4 days of CPP training with saline, the number of EGFP-labeled newborn GCs in sub-granular zone (SGZ) hippocampus significantly increased compared to mice injected with saline in their homecage. CPP training with morphine significantly decreased the number of EGFP-labeled GCs, whereas no significant difference in the number of EGFP-labeled GCs was observed with the homecage mice injected with the same dose of morphine. Using cell-type selective markers, we observed that morphine reduced the number of late stage progenitors and immature neurons such as Doublecortin (DCX) and βIII Tubulin (TuJ1) positive cells in the SGZ but did not reduce the number of early progenitors such as Nestin, SOX2, or neurogenic differentiation-1 (NeuroD1) positive cells. Analysis of co-localization between different cell markers shows that morphine reduced the number of adult-born GCs by interfering with differentiation of early progenitors, but not by inducing apoptosis. In addition, when NeuroD1 was over-expressed in DG by stereotaxic injection of lentivirus, it rescued the loss of immature neurons and prolonged the extinction of morphine-trained CPP. These results suggest that under the condition of CPP training paradigm, morphine affects the transition of neural progenitor/stem cells to immature neurons via a mechanism involving NeuroD1.

  12. Neural Progenitor Cells Promote Axonal Growth and Alter Axonal mRNA Localization in Adult Neurons

    PubMed Central

    Merianda, Tanuja T.; Jin, Ying

    2017-01-01

    Abstract The inhibitory environment of the spinal cord and the intrinsic properties of neurons prevent regeneration of axons following CNS injury. However, both ascending and descending axons of the injured spinal cord have been shown to regenerate into grafts of embryonic neural progenitor cells (NPCs). Previous studies have shown that grafts composed of glial-restricted progenitors (GRPs) and neural-restricted progenitors (NRPs) can provide a permissive microenvironment for axon growth. We have used cocultures of adult rat dorsal root ganglion (DRG) neurons together with NPCs, which have shown significant enhancement of axon growth by embryonic rat GRP and GRPs/NRPs, both in coculture conditions and when DRGs are exposed to conditioned medium from the NPC cultures. This growth-promoting effect of NPC-conditioned medium was also seen in injury-conditioned neurons. DRGs cocultured with GRPs/NRPs showed altered expression of regeneration-associated genes at transcriptional and post-transcriptional levels. We found that levels of GAP-43 mRNA increased in DRG cell bodies and axons. However, hepcidin antimicrobial peptide (HAMP) mRNA decreased in the cell bodies of DRGs cocultured with GRPs/NRPs, which is distinct from the increase in cell body HAMP mRNA levels seen in DRGs after injury conditioning. Endogenous GAP-43 and β-actin mRNAs as well as reporter RNAs carrying axonally localizing 3'UTRs of these transcripts showed significantly increased levels in distal axons in the DRGs cocultured with GRPs/NRPs. These results indicate that axon growth promoted by NPCs is associated not only with enhanced transcription of growth-associated genes but also can increase localization of some mRNAs into growing axons. PMID:28197547

  13. Neural Progenitor Cells Promote Axonal Growth and Alter Axonal mRNA Localization in Adult Neurons.

    PubMed

    Merianda, Tanuja T; Jin, Ying; Kalinski, Ashley L; Sahoo, Pabitra K; Fischer, Itzhak; Twiss, Jeffery L

    2017-01-01

    The inhibitory environment of the spinal cord and the intrinsic properties of neurons prevent regeneration of axons following CNS injury. However, both ascending and descending axons of the injured spinal cord have been shown to regenerate into grafts of embryonic neural progenitor cells (NPCs). Previous studies have shown that grafts composed of glial-restricted progenitors (GRPs) and neural-restricted progenitors (NRPs) can provide a permissive microenvironment for axon growth. We have used cocultures of adult rat dorsal root ganglion (DRG) neurons together with NPCs, which have shown significant enhancement of axon growth by embryonic rat GRP and GRPs/NRPs, both in coculture conditions and when DRGs are exposed to conditioned medium from the NPC cultures. This growth-promoting effect of NPC-conditioned medium was also seen in injury-conditioned neurons. DRGs cocultured with GRPs/NRPs showed altered expression of regeneration-associated genes at transcriptional and post-transcriptional levels. We found that levels of GAP-43 mRNA increased in DRG cell bodies and axons. However, hepcidin antimicrobial peptide (HAMP) mRNA decreased in the cell bodies of DRGs cocultured with GRPs/NRPs, which is distinct from the increase in cell body HAMP mRNA levels seen in DRGs after injury conditioning. Endogenous GAP-43 and β-actin mRNAs as well as reporter RNAs carrying axonally localizing 3'UTRs of these transcripts showed significantly increased levels in distal axons in the DRGs cocultured with GRPs/NRPs. These results indicate that axon growth promoted by NPCs is associated not only with enhanced transcription of growth-associated genes but also can increase localization of some mRNAs into growing axons.

  14. Application of human persistent fetal vasculature neural progenitors for transplantation in the inner retina.

    PubMed

    Chen, Guochun; Ma, Jie; Shatos, Marie A; Chen, Huihui; Cyr, Desirée; Lashkari, Kameran

    2012-01-01

    Persistent fetal vasculature (PFV) is a potentially serious developmental anomaly in human eyes, which results from a failure of the primary vitreous and the hyaloid vascular systems to regress during development. Recent findings from our laboratory indicate that fibrovascular membranes harvested from subjects with PFV contain neural progenitor cells (herein called NPPFV cells). Our studies on successful isolation, culture, and characterization of NPPFV cells have shown that they highly express neuronal progenitor markers (nestin, Pax6, and Ki67) as well as retinal neuronal markers (β-III-tubulin and Brn3a). In the presence of retinoic acid and neurotrophins, these cells acquire a neural morphological appearance in vitro, including a round soma and extensive neurites, and express mature neuronal markers (β-III-tubulin and NF200). Further experiments, including real-time qRT-PCR to quantify characteristic gene expression profiles of these cells and Ca(2+) imaging to evaluate the response to stimulation with different neurotransmitters, indicate that NPPFV cells may resemble a more advanced stage of retinal development and show more differentiation toward inner retinal neurons rather than photoreceptors. To explore the potential of inner retinal transplantation, NPPFV cells were transplanted intravitreally into the eyes of adult C57BL/6 mice. Engrafted NPPFV cells survived well in the intraocular environment in presence of rapamycin and some cells migrated into the inner nuclear layer of the retina 1 week posttransplantation. Three weeks after transplantation, NPPFV cells were observed to migrate and integrate in the inner retina. In response to daily intraperitoneal injections of retinoic acid, a portion of transplanted NPPFV cells exhibited retinal ganglion cell-like morphology and expressed mature neuronal markers (β-III-tubulin and synaptophysin). These findings indicate that fibrovascular membranes from human PFV harbor a population of neuronal progenitors

  15. β-chemokine production by neural and glial progenitor cells is enhanced by HIV-1 Tat: Effects on microglial migration

    PubMed Central

    Hahn, Yun Kyung; Vo, Phu; Fitting, Sylvia; Block, Michelle L.; Hauser, Kurt F.; Knapp, Pamela E.

    2010-01-01

    HIV-1 neuropathology results from collective effects of viral proteins and inflammatory mediators on several cell types. Significant damage is mediated indirectly through inflammatory conditions promulgated by glial cells, including microglia that are productively infected by HIV-1, and astroglia. Neural and glial progenitors exist in both developing and adult brains. To determine whether progenitors are targets of HIV-1, a multi-plex assay was performed to assess chemokine/cytokine expression after treatment with viral proteins Tat or gp120. In the initial screen, ten analytes were basally released by murine striatal progenitors. The beta-chemokines CCL5/RANTES, CCL3/MIP-1α, and CCL4/MIP-1β were increased by 12 h exposure to HIV-1 Tat. Secreted factors from Tat-treated progenitors were chemoattractive towards microglia, an effect blocked by 2D7 anti-CCR5 antibody pretreatment. Tat and opiates have interactive effects on astroglial chemokine secretion, but this interaction did not occur in progenitors. gp120 did not affect chemokine/cytokine release, although both CCR5 and CXCR4, which serve as gp120 co-receptors, were detected in progenitors. We postulate that chemokine production by progenitors may be a normal, adaptive process that encourages immune inspection of newly generated cells. Pathogens such as HIV might usurp this function to create a maladaptive state, especially during development or regeneration, when progenitors are numerous. PMID:20403075

  16. FatJ acts via the Hippo mediator Yap1 to restrict the size of neural progenitor cell pools

    PubMed Central

    Van Hateren, Nick J.; Das, Raman M.; Hautbergue, Guillaume M.; Borycki, Anne-Gaëlle; Placzek, Marysia; Wilson, Stuart A.

    2011-01-01

    The size, composition and functioning of the spinal cord is likely to depend on appropriate numbers of progenitor and differentiated cells of a particular class, but little is known about how cell numbers are controlled in specific cell cohorts along the dorsoventral axis of the neural tube. Here, we show that FatJ cadherin, identified in a large-scale RNA interference (RNAi) screen of cadherin genes expressed in the neural tube, is localised to progenitors in intermediate regions of the neural tube. Loss of function of FatJ promotes an increase in dp4-vp1 progenitors and a concomitant increase in differentiated Lim1+/Lim2+ neurons. Our studies reveal that FatJ mediates its action via the Hippo pathway mediator Yap1: loss of downstream Hippo components can rescue the defect caused by loss of FatJ. Together, our data demonstrate that RNAi screens are feasible in the chick embryonic neural tube, and show that FatJ acts through the Hippo pathway to regulate cell numbers in specific subsets of neural progenitor pools and their differentiated progeny. PMID:21521736

  17. Adult neurogenesis and repair of the adult CNS with neural progenitors, precursors, and stem cells.

    PubMed

    Emsley, Jason G; Mitchell, Bartley D; Kempermann, Gerd; Macklis, Jeffrey D

    2005-04-01

    Recent work in neuroscience has shown that the adult central nervous system contains neural progenitors, precursors, and stem cells that are capable of generating new neurons, astrocytes, and oligodendrocytes. While challenging previous dogma that no new neurons are born in the adult mammalian CNS, these findings bring with them future possibilities for the development of novel neural repair strategies. The purpose of this review is to present current knowledge about constitutively occurring adult mammalian neurogenesis, to highlight the critical differences between "neurogenic" and "non-neurogenic" regions in the adult brain, and to describe the cardinal features of two well-described neurogenic regions-the subventricular zone/olfactory bulb system, and the dentate gyrus of the hippocampus. We also provide an overview of currently used models for studying neural precursors in vitro, mention some precursor transplantation models, and emphasize that, in this rapidly growing field of neuroscience, one must take caution with respect to a variety of methodological considerations for studying neural precursor cells both in vitro and in vivo. The possibility of repairing neural circuitry by manipulating neurogenesis is an intriguing one, and, therefore, we also review recent efforts to understand the conditions under which neurogenesis can be induced in non-neurogenic regions of the adult CNS. This work aims toward molecular and cellular manipulation of endogenous neural precursors in situ, without transplantation. We conclude this review with a discussion of what the function might be of newly generated neurons in the adult brain and provide a summary of current thinking about the consequences of disturbed adult neurogenesis and the reaction of neurogenic regions to disease.

  18. Adult neurogenesis and cellular brain repair with neural progenitors, precursors and stem cells.

    PubMed

    Sohur, U Shivraj; Emsley, Jason G; Mitchell, Bartley D; Macklis, Jeffrey D

    2006-09-29

    Recent work in neuroscience has shown that the adult central nervous system (CNS) contains neural progenitors, precursors and stem cells that are capable of generating new neurons, astrocytes and oligodendrocytes. While challenging the previous dogma that no new neurons are born in the adult mammalian CNS, these findings bring with them the future possibilities for development of novel neural repair strategies. The purpose of this review is to present the current knowledge about constitutively occurring adult mammalian neurogenesis, highlight the critical differences between 'neurogenic' and 'non-neurogenic' regions in the adult brain, and describe the cardinal features of two well-described neurogenic regions-the subventricular zone/olfactory bulb system and the dentate gyrus of the hippocampus. We also provide an overview of presently used models for studying neural precursors in vitro, mention some precursor transplantation models and emphasize that, in this rapidly growing field of neuroscience, one must be cautious with respect to a variety of methodological considerations for studying neural precursor cells both in vitro and in vivo. The possibility of repairing neural circuitry by manipulating neurogenesis is an intriguing one, and, therefore, we also review recent efforts to understand the conditions under which neurogenesis can be induced in non-neurogenic regions of the adult CNS. This work aims towards molecular and cellular manipulation of endogenous neural precursors in situ, without transplantation. We conclude this review with a discussion of what might be the function of newly generated neurons in the adult brain, and provide a summary of present thinking about the consequences of disturbed adult neurogenesis and the reaction of neurogenic regions to disease.

  19. Adult neurogenesis and cellular brain repair with neural progenitors, precursors and stem cells

    PubMed Central

    Shivraj Sohur, U; Emsley, Jason G; Mitchell, Bartley D; Macklis, Jeffrey D

    2006-01-01

    Recent work in neuroscience has shown that the adult central nervous system (CNS) contains neural progenitors, precursors and stem cells that are capable of generating new neurons, astrocytes and oligodendrocytes. While challenging the previous dogma that no new neurons are born in the adult mammalian CNS, these findings bring with them the future possibilities for development of novel neural repair strategies. The purpose of this review is to present the current knowledge about constitutively occurring adult mammalian neurogenesis, highlight the critical differences between ‘neurogenic’ and ‘non-neurogenic’ regions in the adult brain, and describe the cardinal features of two well-described neurogenic regions—the subventricular zone/olfactory bulb system and the dentate gyrus of the hippocampus. We also provide an overview of presently used models for studying neural precursors in vitro, mention some precursor transplantation models and emphasize that, in this rapidly growing field of neuroscience, one must be cautious with respect to a variety of methodological considerations for studying neural precursor cells both in vitro and in vivo. The possibility of repairing neural circuitry by manipulating neurogenesis is an intriguing one, and, therefore, we also review recent efforts to understand the conditions under which neurogenesis can be induced in non-neurogenic regions of the adult CNS. This work aims towards molecular and cellular manipulation of endogenous neural precursors in situ, without transplantation. We conclude this review with a discussion of what might be the function of newly generated neurons in the adult brain, and provide a summary of present thinking about the consequences of disturbed adult neurogenesis and the reaction of neurogenic regions to disease. PMID:16939970

  20. Brief Report: Robo1 Regulates the Migration of Human Subventricular Zone Neural Progenitor Cells During Development.

    PubMed

    Guerrero-Cazares, Hugo; Lavell, Emily; Chen, Linda; Schiapparelli, Paula; Lara-Velazquez, Montserrat; Capilla-Gonzalez, Vivian; Clements, Anna Christina; Drummond, Gabrielle; Noiman, Liron; Thaler, Katrina; Burke, Anne; Quiñones-Hinojosa, Alfredo

    2017-07-01

    Human neural progenitor cell (NPC) migration within the subventricular zone (SVZ) of the lateral ganglionic eminence is an active process throughout early brain development. The migration of human NPCs from the SVZ to the olfactory bulb during fetal stages resembles what occurs in adult rodents. As the human brain develops during infancy, this migratory stream is drastically reduced in cell number and becomes barely evident in adults. The mechanisms regulating human NPC migration are unknown. The Slit-Robo signaling pathway has been defined as a chemorepulsive cue involved in axon guidance and neuroblast migration in rodents. Slit and Robo proteins expressed in the rodent brain help guide neuroblast migration from the SVZ through the rostral migratory stream to the olfactory bulb. Here, we present the first study on the role that Slit and Robo proteins play in human-derived fetal neural progenitor cell migration (hfNPC). We describe that Robo1 and Robo2 isoforms are expressed in the human fetal SVZ. Furthermore, we demonstrate that Slit2 is able to induce a chemorepellent effect on the migration of hfNPCs derived from the human fetal SVZ. In addition, when Robo1 expression is inhibited, hfNPCs are unable to migrate to the olfactory bulb of mice when injected in the anterior SVZ. Our findings indicate that the migration of human NPCs from the SVZ is partially regulated by the Slit-Robo axis. This pathway could be regulated to direct the migration of NPCs in human endogenous neural cell therapy. Stem Cells 2017;35:1860-1865. © 2017 AlphaMed Press.

  1. Quantitative changes in gene transcription during induction of differentiation in porcine neural progenitor cells

    PubMed Central

    Yang, Jing; Gu, Ping; Menges, Steven

    2012-01-01

    Purpose Differentiation of neural stem/progenitor cells involves changes in the gene expression of these cells. Less clear is the extent to which incremental changes occur and the time course of such changes, particularly in non-rodents. Methods Using porcine genome microarrays, we analyzed changes in the expression of 23,256 genes in porcine neural progenitor cells (pNPCs) subject to two established differentiation protocols. In addition, we performed sequential quantitative assessment of a defined transcription profile consisting of 15 progenitor- and lineage-associated genes following exposure to the same treatment protocols, to examine the temporal dynamics of phenotypic changes following induction of differentiation. Immunocytochemistry was also used to examine the expression of seven of these phenotypically important genes at the protein level. Initial primary isolates were passaged four times in proliferation medium containing 20 ng/ml epidermal growth factor (EGF) and 20 ng/ml basic fibroblast growth factor (bFGF) before differentiation was induced. Differentiation was induced by medium without EGF or bFGF and containing either 10 ng/ml ciliary neurotrophic factor or 10% fetal bovine serum (FBS). Cultures were fed every two days and harvested on days 0, 1, 3, and 5 for quantitative real-time PCR. Results The microarray results illustrated and contrasted the global shifts in the porcine transcriptome associated with both treatment conditions. PCR confirmed dramatic upregulation of transcripts for myelin basic protein (up to 88 fold), claudin 11 (up to 32 fold), glial fibrillary acidic protein (GFAP; up to 26 fold), together with notable (>twofold) increases in message for microtubule associated protein 2 (MAP2) and C-X-C chemokine receptor type 4 (CXCR4), Janus kinase 1 (Jak1), signal transducer and activator of transcription 1 (STAT1), and signal transducer and activator of transcription 3 (STAT3). Transcripts for nestin and Krüppel-like factor 4 (KLF4

  2. Preclinical Analysis of Fetal Human Mesencephalic Neural Progenitor Cell Lines: Characterization and Safety In Vitro and In Vivo.

    PubMed

    Moon, Jisook; Schwarz, Sigrid C; Lee, Hyun-Seob; Kang, Jun Mo; Lee, Young-Eun; Kim, Bona; Sung, Mi-Young; Höglinger, Günter; Wegner, Florian; Kim, Jin Su; Chung, Hyung-Min; Chang, Sung Woon; Cha, Kwang Yul; Kim, Kwang-Soo; Schwarz, Johannes

    2016-09-02

    : We have developed a good manufacturing practice for long-term cultivation of fetal human midbrain-derived neural progenitor cells. The generation of human dopaminergic neurons may serve as a tool of either restorative cell therapies or cellular models, particularly as a reference for phenotyping region-specific human neural stem cell lines such as human embryonic stem cells and human inducible pluripotent stem cells. We cultivated 3 different midbrain neural progenitor lines at 10, 12, and 14 weeks of gestation for more than a year and characterized them in great detail, as well as in comparison with Lund mesencephalic cells. The whole cultivation process of tissue preparation, cultivation, and cryopreservation was developed using strict serum-free conditions and standardized operating protocols under clean-room conditions. Long-term-cultivated midbrain-derived neural progenitor cells retained stemness, midbrain fate specificity, and floorplate markers. The potential to differentiate into authentic A9-specific dopaminergic neurons was markedly elevated after prolonged expansion, resulting in large quantities of functional dopaminergic neurons without genetic modification. In restorative cell therapeutic approaches, midbrain-derived neural progenitor cells reversed impaired motor function in rodents, survived well, and did not exhibit tumor formation in immunodeficient nude mice in the short or long term (8 and 30 weeks, respectively). We conclude that midbrain-derived neural progenitor cells are a promising source for human dopaminergic neurons and suitable for long-term expansion under good manufacturing practice, thus opening the avenue for restorative clinical applications or robust cellular models such as high-content or high-throughput screening.

  3. Preclinical Analysis of Fetal Human Mesencephalic Neural Progenitor Cell Lines: Characterization and Safety In Vitro and In Vivo.

    PubMed

    Moon, Jisook; Schwarz, Sigrid C; Lee, Hyun-Seob; Kang, Jun Mo; Lee, Young-Eun; Kim, Bona; Sung, Mi-Young; Höglinger, Günter; Wegner, Florian; Kim, Jin Su; Chung, Hyung-Min; Chang, Sung Woon; Cha, Kwang Yul; Kim, Kwang-Soo; Schwarz, Johannes

    2017-02-01

    We have developed a good manufacturing practice for long-term cultivation of fetal human midbrain-derived neural progenitor cells. The generation of human dopaminergic neurons may serve as a tool of either restorative cell therapies or cellular models, particularly as a reference for phenotyping region-specific human neural stem cell lines such as human embryonic stem cells and human inducible pluripotent stem cells. We cultivated 3 different midbrain neural progenitor lines at 10, 12, and 14 weeks of gestation for more than a year and characterized them in great detail, as well as in comparison with Lund mesencephalic cells. The whole cultivation process of tissue preparation, cultivation, and cryopreservation was developed using strict serum-free conditions and standardized operating protocols under clean-room conditions. Long-term-cultivated midbrain-derived neural progenitor cells retained stemness, midbrain fate specificity, and floorplate markers. The potential to differentiate into authentic A9-specific dopaminergic neurons was markedly elevated after prolonged expansion, resulting in large quantities of functional dopaminergic neurons without genetic modification. In restorative cell therapeutic approaches, midbrain-derived neural progenitor cells reversed impaired motor function in rodents, survived well, and did not exhibit tumor formation in immunodeficient nude mice in the short or long term (8 and 30 weeks, respectively). We conclude that midbrain-derived neural progenitor cells are a promising source for human dopaminergic neurons and suitable for long-term expansion under good manufacturing practice, thus opening the avenue for restorative clinical applications or robust cellular models such as high-content or high-throughput screening. Stem Cells Translational Medicine 2017;6:576-588.

  4. EVALUATION OF HUMAN NEURAL PROGENITOR CELLS FOR DEVELOPMENTAL NEUROTOXICITY SCREENING: TIME COURSE OF EFFECTS ON CELL PROLIFERATION AND VIABILITY.

    EPA Science Inventory

    Current testing methods for developmental neurotoxicity (DNT) make evaluation of the effects of large numbers of chemicals impractical and prohibitively expensive. As such, we are evaluating human neural progenitor cells (NPCs) as a screen for DNT. ReNcell CX (ReN CX) cells are a...

  5. Tumor suppressor Nf2 limits expansion of the neural progenitor pool by inhibiting Yap/Taz transcriptional coactivators

    PubMed Central

    Lavado, Alfonso; He, Yu; Paré, Joshua; Neale, Geoffrey; Olson, Eric N.; Giovannini, Marco; Cao, Xinwei

    2013-01-01

    Brain development requires a precise balance between expansion of the neural progenitor pool and the production of postmitotic neurons and glia. Disruption of this equilibrium results in a myriad of structural abnormalities and disorders of the nervous system. The molecular mechanism that restricts neural progenitor expansion is poorly understood. Here we show that the tumor suppressor neurofibromatosis 2 (Nf2; merlin) limits the expansion of neural progenitor cells (NPCs) in the mammalian dorsal telencephalon. Nf2 is localized at the apical region of NPCs. In the absence of Nf2, NPCs of the cortical hem, hippocampal primordium and neocortical primordium overexpand, while production of Cajal-Retzius cells and hippocampal neurons decreases, resulting in severe malformation of the hippocampus in adult mice. We further show that Nf2 functions by inhibiting the Yap/Taz transcriptional coactivators, probably through a mechanism that is distinct from the canonical Hippo pathway. Overexpressing human YAP in NPCs causes a hippocampal malformation phenotype that closely resembles that of Nf2 mutants and, importantly, deleting Yap in the Nf2 mutant background largely restores hippocampal development. Our studies uncover Nf2 as an important inhibitor of neural progenitor expansion and establish Yap/Taz as key downstream effectors of Nf2 during brain development. PMID:23863479

  6. The postischemic environment differentially impacts teratoma or tumor formation after transplantation of human embryonic stem cell-derived neural progenitors.

    PubMed

    Seminatore, Christine; Polentes, Jerome; Ellman, Ditte; Kozubenko, Nataliya; Itier, Valerie; Tine, Samir; Tritschler, Laurent; Brenot, Marion; Guidou, Emmanuelle; Blondeau, Johanna; Lhuillier, Mickael; Bugi, Aurore; Aubry, Laetitia; Jendelova, Pavla; Sykova, Eva; Perrier, Anselme L; Finsen, Bente; Onteniente, Brigitte

    2010-01-01

    Risk of tumorigenesis is a major obstacle to human embryonic and induced pluripotent stem cell therapy. Likely linked to the stage of differentiation of the cells at the time of implantation, formation of teratoma/tumors can also be influenced by factors released by the host tissue. We have analyzed the relative effects of the stage of differentiation and the postischemic environment on the formation of adverse structures by transplanted human embryonic stem cell-derived neural progenitors. Four differentiation stages were identified on the basis of quantitative polymerase chain reaction expression of pluripotency, proliferation, and differentiation markers. Neural progenitors were transplanted at these 4 stages into rats with no, small, or large middle cerebral artery occlusion lesions. The fate of each transplant was compared with their pretransplantation status 1 to 4 months posttransplantation. The influence of the postischemic environment was limited to graft survival and occurrence of nonneuroectodermal structures after transplantation of very immature neural progenitors. Both effects were lost with differentiation. We identified a particular stage of differentiation characterized in vitro by a rebound of proliferative activity that produced highly proliferative grafts susceptible to threaten surrounding host tissues. The effects of the ischemic environment on the formation of teratoma by transplanted human embryonic stem cell-derived neural progenitors are limited to early differentiation stages that will likely not be used for stem cell therapy. In contrast, hyperproliferation observed at later stages of differentiation corresponds to an intrinsic activity that should be monitored to avoid tumorigenesis.

  7. EVALUATION OF HUMAN NEURAL PROGENITOR CELLS FOR DEVELOPMENTAL NEUROTOXICITY SCREENING: TIME COURSE OF EFFECTS ON CELL PROLIFERATION AND VIABILITY.

    EPA Science Inventory

    Current testing methods for developmental neurotoxicity (DNT) make evaluation of the effects of large numbers of chemicals impractical and prohibitively expensive. As such, we are evaluating human neural progenitor cells (NPCs) as a screen for DNT. ReNcell CX (ReN CX) cells are a...

  8. ERK-dependent and -independent pathways trigger human neural progenitor cell migration

    SciTech Connect

    Moors, Michaela . E-mail: moors@uni-duesseldorf.de; Cline, Jason E. . E-mail: jason.cline@uni-duesseldorf.de; Abel, Josef . E-mail: josef.abel@uni-duesseldorf.de; Fritsche, Ellen . E-mail: ellen.fritsche@uni-duesseldorf.de

    2007-05-15

    Besides differentiation and apoptosis, cell migration is a basic process in brain development in which neural cells migrate several centimeters within the developing brain before reaching their proper positions and forming the right connections. For identifying signaling events that control neural migration and are therefore potential targets of chemicals to disturb normal brain development, we developed a human neurosphere-based migration assay based on normal human neural progenitor (NHNP) cells, in which the distance is measured that cells wander over time. Applying this assay, we investigated the role of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) in the regulation of NHNP cell migration. Exposure to model substances like ethanol or phorbol 12-myristate 13-acetate (PMA) revealed a correlation between ERK1/2 activation and cell migration. The participation of phospho-(P-) ERK1/2 was confirmed by exposure of the cells to the MEK inhibitor PD98059, which directly prohibits ERK1/2 phosphorylation and inhibited cell migration. We identified protein kinase C (PKC) and epidermal growth factor receptor (EGFR) as upstream signaling kinases governing ERK1/2 activation, thereby controlling NHNP cell migration. Additionally, treatments with src kinase inhibitors led to a diminished cell migration without affecting ERK1/2 phosphorylation. Based on these results, we postulate that migration of NHNP cells is controlled via ERK1/2-dependent and -independent pathways.

  9. In vitro expansion of fetal neural progenitors as adherent cell lines.

    PubMed

    Pollard, Steven M

    2013-01-01

    In vitro studies of neural progenitors isolated from the developing mouse have provided important insights into intrinsic and extrinsic pathways that control their behavior. However, use of primary cultures or neurospheres established from fetal tissues in cell population-based assays can be compromised by cellular heterogeneity. A complementary approach that addresses this issue is the establishment of adherent clonal neural stem (NS) cell lines. Here I describe protocols and troubleshooting advice for establishing adherent NS cell lines from the mouse fetal forebrain. NS cells grow as pure cultures in defined serum-free conditions as adherent monolayers and are therefore amenable to chemical/genetic screens, biochemical studies, and population-based analysis of gene expression or transcriptional regulation (e.g. RNA-Seq and ChIP-Seq). NS cell lines therefore represent a tractable cellular model system to explore the molecular and cellular biology of neural stem cell self-renewal and differentiation. Similar protocols can be extended to rat and human embryos, as well as human brain tumors.

  10. Mesenchymal stem cells prime proliferating adult neural progenitors toward an oligodendrocyte fate.

    PubMed

    Steffenhagen, Carolin; Dechant, Franz-Xaver; Oberbauer, Eleni; Furtner, Tanja; Weidner, Norbert; Küry, Patrick; Aigner, Ludwig; Rivera, Francisco J

    2012-07-20

    Oligodendrogenesis encompasses lineage specification of neural progenitor cells (NPCs) and differentiation into oligodendrocytes that ultimately culminates in the myelination of central nervous system axons. Each individual process must be tightly regulated by extracellular and cell-intrinsic mechanisms, whose identities are barely understood. We had previously demonstrated that soluble factors derived from rat mesenchymal stem cells (MSCs) induce oligodendrogenesis in differentiating adult NPCs under differentiation conditions. However, since lineage specification predominantly occurs in proliferating progenitors and not necessarily during early differentiation, we investigated if soluble factors derived from MSCs are able to prime NPCs to the oligodendroglial fate already under proliferation conditions. Therefore, we analyzed the effects of a 3 weeks stimulation of adult NPCs under proliferation conditions with conditioned media derived from MSCs (MSC-CM) in terms of cell morphology, proliferation, cell-specific marker expression profile, response to growth factor withdrawal (GFW), cell-lineage restriction, and expression of glial fate determinants. While MSC-CM did not affect the proliferation rate of NPCs, it boosted the formation of 2', 3'-cyclic-nucleotide-3'-phosphodieesterase (CNPase)- and myelin basic protein-expressing oligodendrocytes after GFW, even when cells were exposed to an astrogenic milieu. Moreover, it reinforced the proper development of oligodendrocytes, since it ensured a sustained expression of the functional marker CNPase. Finally, the presence of MSC-CM reduced the anti-oligodendrogenic determinant Id2 in proliferating NPCs, thus increasing the relative proportion of the pro-oligodendrogenic factor Olig2 expression. In summary, MSCs prime proliferating progenitors and, thus, reinforce cell fate choice and accelerate differentiation toward the oligodendrocyte lineage. The present findings underscore the potential use of MSCs in cell

  11. iPS-derived neural progenitor cells from PPMS patients reveal defect in myelin injury response.

    PubMed

    Nicaise, Alexandra M; Banda, Erin; Guzzo, Rosa M; Russomanno, Kristen; Castro-Borrero, Wanda; Willis, Cory M; Johnson, Kasey M; Lo, Albert C; Crocker, Stephen J

    2017-02-01

    Primary progressive multiple sclerosis (PPMS) is a chronic demyelinating disease of the central nervous system (CNS) currently lacking any effective treatment. Promoting endogenous brain repair offers a potential strategy to halt and possibly restore neurologic function in PPMS. To understand how the microenvironment within white matter lesions plays a role in repair we have focused on neural progenitor cells (NPCs) since these are found in lesions in PPMS and have been found to influence oligodendrocyte progenitor cell maturation (OPCs). To better understand the cellular nature of NPCs in PPMS we developed iPS cells from blood samples of PPMS patients and age matched non-disease spouse or blood relative controls. Using these iPS cell lines we determined that the NPCs from PPMS cases provided no neuroprotection against active CNS demyelination compared to NPCs from control iPS lines which were capable of completely preventing injury. Conditioned media (CM) from PPMS NPCs provides no protection to OPCs and prevents maturation of OPCs into oligodendrocytes in vitro. We also found that CM from PPMS iPS NPCs elicited patient-specific differences in the response to compounds that should foster oligodendrocyte (OL) maturation. Together, these data establish a new model for understanding the nature of myelination defects in PPMS which may lead to novel targeted approaches for preventing demyelination in these patients. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Characterization of Voltage-Gated Potassium Channels in Human Neural Progenitor Cells

    PubMed Central

    Schaarschmidt, Grit; Wegner, Florian; Schwarz, Sigrid C.; Schmidt, Hartmut; Schwarz, Johannes

    2009-01-01

    Background Voltage-gated potassium (Kv) channels are among the earliest ion channels to appear during brain development, suggesting a functional requirement for progenitor cell proliferation and/or differentiation. We tested this hypothesis, using human neural progenitor cells (hNPCs) as a model system. Methodology/Principal Findings In proliferating hNPCs a broad spectrum of Kv channel subtypes was identified using quantitative real-time PCR with a predominant expression of the A-type channel Kv4.2. In whole-cell patch-clamp recordings Kv currents were separated into a large transient component characteristic for fast-inactivating A-type potassium channels (IA) and a small, sustained component produced by delayed-rectifying channels (IK). During differentiation the expression of IA as well as A-type channel transcripts dramatically decreased, while IK producing delayed-rectifiers were upregulated. Both Kv currents were differentially inhibited by selective neurotoxins like phrixotoxin-1 and α-dendrotoxin as well as by antagonists like 4-aminopyridine, ammoniumchloride, tetraethylammonium chloride and quinidine. In viability and proliferation assays chronic inhibition of the A-type currents severely disturbed the cell cycle and precluded proper hNPC proliferation, while the blockade of delayed-rectifiers by α-dendrotoxin increased proliferation. Conclusions/Significance These findings suggest that A-type potassium currents are essential for proper proliferation of immature multipotent hNPCs. PMID:19584922

  13. Ischemia-induced neural stem/progenitor cells in the pia mater following cortical infarction.

    PubMed

    Nakagomi, Takayuki; Molnár, Zoltán; Nakano-Doi, Akiko; Taguchi, Akihiko; Saino, Orie; Kubo, Shuji; Clausen, Martijn; Yoshikawa, Hiroo; Nakagomi, Nami; Matsuyama, Tomohiro

    2011-12-01

    Increasing evidence shows that neural stem/progenitor cells (NSPCs) can be activated in the nonconventional neurogenic zones such as the cortex following ischemic stroke. However, the precise origin, identity, and subtypes of the ischemia-induced NSPCs (iNSPCs), which can contribute to cortical neurogenesis, is currently still unclear. In our present study, using an adult mouse cortical infarction model, we found that the leptomeninges (pia mater), which is widely distributed within and closely associated with blood vessels as microvascular pericytes/perivascular cells throughout central nervous system (CNS), have NSPC activity in response to ischemia and can generate neurons. These observations indicate that microvascular pericytes residing near blood vessels that are distributed from the leptomeninges to the cortex are potential sources of iNSPCs for neurogenesis following cortical infarction. In addition, our results propose a novel concept that the leptomeninges, which cover the entire brain, have an important role in CNS restoration following brain injury such as stroke.

  14. Aging Neural Progenitor Cells Have Decreased Mitochondrial Content and Lower Oxidative Metabolism*

    PubMed Central

    Stoll, Elizabeth A.; Cheung, Willy; Mikheev, Andrei M.; Sweet, Ian R.; Bielas, Jason H.; Zhang, Jing; Rostomily, Robert C.; Horner, Philip J.

    2011-01-01

    Although neurogenesis occurs in discrete areas of the adult mammalian brain, neural progenitor cells (NPCs) produce fewer new neurons with age. To characterize the molecular changes that occur during aging, we performed a proteomic comparison between primary-cultured NPCs from the young adult and aged mouse forebrain. This analysis yielded changes in proteins necessary for cellular metabolism. Mitochondrial quantity and oxygen consumption rates decrease with aging, although mitochondrial DNA in aged NPCs does not have increased mutation rates. In addition, aged cells are resistant to the mitochondrial inhibitor rotenone and proliferate in response to lowered oxygen conditions. These results demonstrate that aging NPCs display an altered metabolic phenotype, characterized by a coordinated shift in protein expression, subcellular structure, and metabolic physiology. PMID:21900249

  15. Molecular signatures associated with ZIKV exposure in human cortical neural progenitors

    PubMed Central

    Zhang, Feiran; Hammack, Christy; Ogden, Sarah C.; Cheng, Yichen; Lee, Emily M.; Wen, Zhexing; Qian, Xuyu; Nguyen, Ha Nam; Li, Yujing; Yao, Bing; Xu, Miao; Xu, Tianlei; Chen, Li; Wang, Zhiqin; Feng, Hao; Huang, Wei-Kai; Yoon, Ki-jun; Shan, Chao; Huang, Luoxiu; Qin, Zhaohui; Christian, Kimberly M.; Shi, Pei-Yong; Xu, Mingjiang; Xia, Menghang; Zheng, Wei; Wu, Hao; Song, Hongjun; Tang, Hengli; Ming, Guo-Li; Jin, Peng

    2016-01-01

    Zika virus (ZIKV) infection causes microcephaly and has been linked to other brain abnormalities. How ZIKV impairs brain development and function is unclear. Here we systematically profiled transcriptomes of human neural progenitor cells exposed to Asian ZIKVC, African ZIKVM, and dengue virus (DENV). In contrast to the robust global transcriptome changes induced by DENV, ZIKV has a more selective and larger impact on expression of genes involved in DNA replication and repair. While overall expression profiles are similar, ZIKVC, but not ZIKVM, induces upregulation of viral response genes and TP53. P53 inhibitors can block the apoptosis induced by both ZIKVC and ZIKVM in hNPCs, with higher potency against ZIKVC-induced apoptosis. Our analyses reveal virus- and strain-specific molecular signatures associated with ZIKV infection. These datasets will help to investigate ZIKV-host interactions and identify neurovirulence determinants of ZIKV. PMID:27580721

  16. Aging neural progenitor cells have decreased mitochondrial content and lower oxidative metabolism.

    PubMed

    Stoll, Elizabeth A; Cheung, Willy; Mikheev, Andrei M; Sweet, Ian R; Bielas, Jason H; Zhang, Jing; Rostomily, Robert C; Horner, Philip J

    2011-11-04

    Although neurogenesis occurs in discrete areas of the adult mammalian brain, neural progenitor cells (NPCs) produce fewer new neurons with age. To characterize the molecular changes that occur during aging, we performed a proteomic comparison between primary-cultured NPCs from the young adult and aged mouse forebrain. This analysis yielded changes in proteins necessary for cellular metabolism. Mitochondrial quantity and oxygen consumption rates decrease with aging, although mitochondrial DNA in aged NPCs does not have increased mutation rates. In addition, aged cells are resistant to the mitochondrial inhibitor rotenone and proliferate in response to lowered oxygen conditions. These results demonstrate that aging NPCs display an altered metabolic phenotype, characterized by a coordinated shift in protein expression, subcellular structure, and metabolic physiology.

  17. NEPRO: a novel Notch effector for maintenance of neural progenitor cells in the neocortex.

    PubMed

    Saito, Tetsuichiro

    2012-01-01

    The Notch pathway is essential for maintaining neural progenitor cells (NPCs) in the developing brain. Activation of the pathway is sufficient to maintain NPCs, whereas loss-of-function mutations in the critical components of the pathway cause precocious neuronal differentiation and NPC depletion. Hairy and Enhancer of split (Hes)-type transcription factors have long been thought to be the only Notch effectors for the maintenance of NPCs. Recently, a novel nuclear protein, Nepro, has been identified as another critical effector of Notch. The Notch pathway is bifurcated into Nepro and Hes-type proteins in the early development of the neocortex. The combination of Nepro and Hes-type proteins is necessary and sufficient for maintaining NPCs downstream of Notch.

  18. Measuring Process Dynamics and Nuclear Migration for Clones of Neural Progenitor Cells

    PubMed Central

    De La Hoz, Edgar Cardenas; Winter, Mark R.; Apostolopoulou, Maria; Temple, Sally

    2016-01-01

    Neural stem and progenitor cells (NPCs) generate processes that extend from the cell body in a dynamic manner. The NPC nucleus migrates along these processes with patterns believed to be tightly coupled to mechanisms of cell cycle regulation and cell fate determination. Here, we describe a new segmentation and tracking approach that allows NPC processes and nuclei to be reliably tracked across multiple rounds of cell division in phase-contrast microscopy images. Results are presented for mouse adult and embryonic NPCs from hundreds of clones, or lineage trees, containing tens of thousands of cells and millions of segmentations. New visualization approaches allow the NPC nuclear and process features to be effectively visualized for an entire clone. Significant differences in process and nuclear dynamics were found among type A and type C adult NPCs, and also between embryonic NPCs cultured from the anterior and posterior cerebral cortex. PMID:27878138

  19. Measuring Process Dynamics and Nuclear Migration for Clones of Neural Progenitor Cells.

    PubMed

    De La Hoz, Edgar Cardenas; Winter, Mark R; Apostolopoulou, Maria; Temple, Sally; Cohen, Andrew R

    2016-10-01

    Neural stem and progenitor cells (NPCs) generate processes that extend from the cell body in a dynamic manner. The NPC nucleus migrates along these processes with patterns believed to be tightly coupled to mechanisms of cell cycle regulation and cell fate determination. Here, we describe a new segmentation and tracking approach that allows NPC processes and nuclei to be reliably tracked across multiple rounds of cell division in phase-contrast microscopy images. Results are presented for mouse adult and embryonic NPCs from hundreds of clones, or lineage trees, containing tens of thousands of cells and millions of segmentations. New visualization approaches allow the NPC nuclear and process features to be effectively visualized for an entire clone. Significant differences in process and nuclear dynamics were found among type A and type C adult NPCs, and also between embryonic NPCs cultured from the anterior and posterior cerebral cortex.

  20. The Drosophila Sp8 transcription factor Buttonhead prevents premature differentiation of intermediate neural progenitors.

    PubMed

    Xie, Yonggang; Li, Xiaosu; Zhang, Xian; Mei, Shaolin; Li, Hongyu; Urso, Andreacarola; Zhu, Sijun

    2014-10-06

    Intermediate neural progenitor cells (INPs) need to avoid differentiation and cell cycle exit while maintaining restricted developmental potential, but mechanisms preventing differentiation and cell cycle exit of INPs are not well understood. In this study, we report that the Drosophila homolog of mammalian Sp8 transcription factor Buttonhead (Btd) prevents premature differentiation and cell cycle exit of INPs in Drosophila larval type II neuroblast (NB) lineages. We show that the loss of Btd leads to elimination of mature INPs due to premature differentiation of INPs into terminally dividing ganglion mother cells. We provide evidence to demonstrate that Btd prevents the premature differentiation by suppressing the expression of the homeodomain protein Prospero in immature INPs. We further show that Btd functions cooperatively with the Ets transcription factor Pointed P1 to promote the generation of INPs. Thus, our work reveals a critical mechanism that prevents premature differentiation and cell cycle exit of Drosophila INPs.

  1. Landscape of monoallelic DNA accessibility in mouse embryonic stem cells and neural progenitor cells

    PubMed Central

    Xu, Jin; Carter, Ava C; Gendrel, Anne-Valerie; Attia, Mikael; Loftus, Joshua; Greenleaf, William J; Tibshirani, Robert; Heard, Edith; Chang, Howard Y

    2017-01-01

    We developed an allele-specific assay for transposase-accessible chromatin with high-throughput sequencing (ATAC–seq) to genotype and profile active regulatory DNA across the genome. Using a mouse hybrid F1 system, we found that monoallelic DNA accessibility across autosomes was pervasive, developmentally programmed and composed of several patterns. Genetically determined accessibility was enriched at distal enhancers, but random monoallelically accessible (RAMA) elements were enriched at promoters and may act as gatekeepers of monoallelic mRNA expression. Allelic choice at RAMA elements was stable across cell generations and bookmarked through mitosis. RAMA elements in neural progenitor cells were biallelically accessible in embryonic stem cells but premarked with bivalent histone modifications; one allele was silenced during differentiation. Quantitative analysis indicated that allelic choice at the majority of RAMA elements is consistent with a stochastic process; however, up to 30% of RAMA elements may deviate from the expected pattern, suggesting a regulated or counting mechanism. PMID:28112738

  2. Neural stem/progenitor cell properties of glial cells in the adult mouse auditory nerve

    PubMed Central

    Lang, Hainan; Xing, Yazhi; Brown, LaShardai N.; Samuvel, Devadoss J.; Panganiban, Clarisse H.; Havens, Luke T.; Balasubramanian, Sundaravadivel; Wegner, Michael; Krug, Edward L.; Barth, Jeremy L.

    2015-01-01

    The auditory nerve is the primary conveyor of hearing information from sensory hair cells to the brain. It has been believed that loss of the auditory nerve is irreversible in the adult mammalian ear, resulting in sensorineural hearing loss. We examined the regenerative potential of the auditory nerve in a mouse model of auditory neuropathy. Following neuronal degeneration, quiescent glial cells converted to an activated state showing a decrease in nuclear chromatin condensation, altered histone deacetylase expression and up-regulation of numerous genes associated with neurogenesis or development. Neurosphere formation assays showed that adult auditory nerves contain neural stem/progenitor cells (NSPs) that were within a Sox2-positive glial population. Production of neurospheres from auditory nerve cells was stimulated by acute neuronal injury and hypoxic conditioning. These results demonstrate that a subset of glial cells in the adult auditory nerve exhibit several characteristics of NSPs and are therefore potential targets for promoting auditory nerve regeneration. PMID:26307538

  3. Effects of addictive drugs on adult neural stem/progenitor cells

    PubMed Central

    Xu, Chi; Loh, Horace H.; Law, Ping-Yee

    2015-01-01

    Neural stem/progenitor cells (NSPCs) undergo a series of developmental processes before giving rise to newborn neurons, astrocytes and oligodendrocytes in adult neurogenesis. During the past decade, the role of NSPCs has been highlighted by studies on adult neurogenesis modulated by addictive drugs. It has been proven that these drugs regulate the proliferation, differentiation and survival of adult NSPCs in different manners, which results in the varying consequences of adult neurogenesis. The effects of addictive drugs on NSPCs are exerted via a variety of different mechanisms and pathways, which interact with one another and contribute to the complexity of NSPC regulation. Here, we review the effects of different addictive drugs on NSPCs, and the related experimental methods and paradigms. We also discuss the current understanding of major signaling molecules, especially the putative common mechanisms, underlying such effects. Finally, we review the future directions of research in this area. PMID:26468052

  4. Selective Differentiation of Neural Progenitor Cells by High-Epitope Density Nanofibers

    NASA Astrophysics Data System (ADS)

    Silva, Gabriel A.; Czeisler, Catherine; Niece, Krista L.; Beniash, Elia; Harrington, Daniel A.; Kessler, John A.; Stupp, Samuel I.

    2004-02-01

    Neural progenitor cells were encapsulated in vitro within a three-dimensional network of nanofibers formed by self-assembly of peptide amphiphile molecules. The self-assembly is triggered by mixing cell suspensions in media with dilute aqueous solutions of the molecules, and cells survive the growth of the nanofibers around them. These nanofibers were designed to present to cells the neurite-promoting laminin epitope IKVAV at nearly van der Waals density. Relative to laminin or soluble peptide, the artificial nanofiber scaffold induced very rapid differentiation of cells into neurons, while discouraging the development of astrocytes. This rapid selective differentiation is linked to the amplification of bioactive epitope presentation to cells by the nanofibers.

  5. Mechanisms of brain evolution: regulation of neural progenitor cell diversity and cell cycle length.

    PubMed

    Borrell, Victor; Calegari, Federico

    2014-09-01

    In the last few years, several studies have revisited long-held assumptions in the field of brain development and evolution providing us with a fundamentally new vision on the mechanisms controlling its size and shape, hence function. Among these studies, some described hitherto unforeseeable subtypes of neural progenitors while others reinterpreted long-known observations about their cell cycle in alternative new ways. Most remarkably, this knowledge combined has allowed the generation of mammalian model organisms in which brain size and folding has been selectively increased giving us the means to understand the mechanisms underlying the evolution of the most complex and sophisticated organ. Here we review the key findings made in this area and make a few conjectures about their evolutionary meaning including the likelihood of Martians conquering our planet. Copyright © 2014 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  6. Transplantation of enteric neural stem/progenitor cells into the irradiated young mouse hippocampus.

    PubMed

    Osman, Ahmed M; Zhou, Kai; Zhu, Changlian; Blomgren, Klas

    2014-01-01

    Radiotherapy is an effective treatment for brain tumors but often results in cognitive deficits in survivors. Transplantation of embryonic or brain-derived neural stem/progenitor cells (BNSPCs) ameliorated cognitive impairment after irradiation (IR) in animal models. However, such an approach in patients requires a clinically relevant source of cells. We show for the first time the utilization of enteric neural stem/progenitor cells (ENSPCs) from the postnatal intestinal wall as a source of autologous cells for brain repair after injury caused by IR. Cells were isolated from the intestinal wall and propagated in vitro for 1 week. Differentiation assays showed that ENSPCs are multipotent and generated neurons, astrocytes, and myofibroblasts. To investigate whether ENSPCs can be used in vivo, postnatal day 9 mice were subjected to a single moderate irradiation dose (6 or 8 Gy). Twelve days later, mice received an intrahippocampal injection of syngeneic ENSPCs. Four weeks after transplantation, 0.5% and 1% of grafted ENSPCs were detected in the dentate gyrus of sham and irradiated animals, respectively, and only 0.1% was detected after 16 weeks. Grafted ENSPCs remained undifferentiated but failed to restore IR-induced loss of BNSPCs and the subsequent impaired growth of the dentate gyrus. We observed microglia activation, astrogliosis, and loss of granule neurons associated with grafted ENSPC clusters. Transplantation of ENSPCs did not ameliorate IR-induced impaired learning and memory. In summary, while autologous ENSPC grafting to the brain worked technically, even in the absence of immunosuppression, the protocols need to be modified to improve survival and integration.

  7. S-phase duration is the main target of cell cycle regulation in neural progenitors of developing ferret neocortex.

    PubMed

    Turrero García, Miguel; Chang, YoonJeung; Arai, Yoko; Huttner, Wieland B

    2016-02-15

    The evolutionary expansion of the neocortex primarily reflects increases in abundance and proliferative capacity of cortical progenitors and in the length of the neurogenic period during development. Cell cycle parameters of neocortical progenitors are an important determinant of cortical development. The ferret (Mustela putorius furo), a gyrencephalic mammal, has gained increasing importance as a model for studying corticogenesis. Here, we have studied the abundance, proliferation, and cell cycle parameters of different neural progenitor types, defined by their differential expression of the transcription factors Pax6 and Tbr2, in the various germinal zones of developing ferret neocortex. We focused our analyses on postnatal day 1, a late stage of cortical neurogenesis when upper-layer neurons are produced. Based on cumulative 5-ethynyl-2'-deoxyuridine (EdU) labeling as well as Ki67 and proliferating cell nuclear antigen (PCNA) immunofluorescence, we determined the duration of the various cell cycle phases of the different neocortical progenitor subpopulations. Ferret neocortical progenitors were found to exhibit longer cell cycles than those of rodents and little variation in the duration of G1 among distinct progenitor types, also in contrast to rodents. Remarkably, the main difference in cell cycle parameters among the various progenitor types was the duration of S-phase, which became shorter as progenitors progressively changed transcription factor expression from patterns characteristic of self-renewal to those of neuron production. Hence, S-phase duration emerges as major target of cell cycle regulation in cortical progenitors of this gyrencephalic mammal. © 2015 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.

  8. HIV-1 alters neural and glial progenitor cell dynamics in the central nervous system: coordinated response to opiates during maturation.

    PubMed

    Hahn, Yun Kyung; Podhaizer, Elizabeth M; Hauser, Kurt F; Knapp, Pamela E

    2012-12-01

    HIV-associated neurocognitive disorders (HANDs) are common sequelae of human immunodeficiency virus (HIV) infection, even when viral titers are well controlled by antiretroviral therapy. Evidence in patients and animal models suggests that neurologic deficits are increased during chronic opiate exposure. We have hypothesized that central nervous system (CNS) progenitor cells in both adult and developing CNS are affected by HIV infection and that opiates exacerbate these effects. To examine this question, neural progenitors were exposed to HIV-1 Tat(1-86) in the developing brain of inducible transgenic mice and in vitro. We examined whether Tat affected the proliferation or balance of progenitor populations expressing nestin, Sox2, and Olig2. Disease relevance was further tested by exposing human-derived progenitors to supernatant from HIV-1 infected monocytes. Studies concentrated on striatum, a region preferentially targeted by HIV and opiates. Results were similar among experimental paradigms. Tat or HIV exposure reduced the proliferation of undifferentiated (Sox2(+)) progenitors and oligodendroglial (Olig2(+)) progenitors. Coexposure to morphine exacerbated the effects of Tat or HIV-1(SF162) supernatant, but partially reversed HIV-1(IIIB) supernatant effects. Populations of Sox2(+) and Olig2(+) cells were also reduced by Tat exposure, although progenitor survival was unaffected. In rare instances, p24 immunolabeling was detected in viable human progenitors by confocal imaging. The vulnerability of progenitors is likely to distort the dynamic balance among neuron/glial populations as the brain matures, perhaps contributing to reports that neurologic disease is especially prevalent in pediatric HIV patients. Pediatric disease is atypical in developed regions but remains a serious concern in resource-limited areas where infection occurs commonly at birth and through breast feeding.

  9. HIV-1 Alters Neural and Glial Progenitor Cell Dynamics in the CNS: Coordinated Response to Opiates during Maturation

    PubMed Central

    Hahn, Yun Kyung; Podhaizer, Elizabeth M.; Hauser, Kurt F.; Knapp, Pamela E.

    2014-01-01

    HIV-associated neurocognitive disorders (HAND) are common sequelae of HIV infection, even when viral titers are well controlled by anti-retroviral therapy. Evidence in patients and animal models suggests that neurologic deficits are increased during chronic opiate exposure. We have hypothesized that CNS progenitor cells in both adult and developing CNS are affected by HIV infection, and that opiates exacerbate these effects. To examine this question, neural progenitors were exposed to HIV-1 Tat1-86 in the developing brain of inducible transgenic mice and in vitro. We examined whether Tat affected the proliferation or balance of progenitor populations expressing nestin, Sox2, and Olig2. Disease relevance was further tested by exposing human-derived progenitors to supernatant from HIV-1 infected monocytes. Studies concentrated on striatum, a region preferentially targeted by HIV and opiates. Results were similar among experimental paradigms. Tat or HIV exposure reduced the proliferation of undifferentiated (Sox2+) progenitors and oligodendroglial (Olig2+) progenitors. Co-exposure to morphine exacerbated the effects of Tat or HIV-1SF162 supernatant, but partially reversed HIV-1IIIB supernatant effects. Populations of Sox2+ and Olig2+ cells were also reduced by Tat exposure, although progenitor survival was unaffected. In rare instances, p24 immunolabeling was detected in viable human progenitors by confocal imaging. The vulnerability of progenitors is likely to distort the dynamic balance among neuron/glial populations as the brain matures, perhaps contributing to reports that neurologic disease is especially prevalent in pediatric HIV patients. Pediatric disease is atypical in developed regions, but remains a serious concern in resource-limited areas where infection occurs commonly at birth and through breast-feeding. PMID:22865725

  10. Environmental factors unveil dormant developmental capacities in multipotent progenitors of the trunk neural crest.

    PubMed

    Coelho-Aguiar, Juliana M; Le Douarin, Nicole M; Dupin, Elisabeth

    2013-12-01

    The neural crest (NC), an ectoderm-derived structure of the vertebrate embryo, gives rise to the melanocytes, most of the peripheral nervous system and the craniofacial mesenchymal tissues (i.e., connective, bone, cartilage and fat cells). In the trunk of Amniotes, no mesenchymal tissues are derived from the NC. In certain in vitro conditions however, avian and murine trunk NC cells (TNCCs) displayed a limited mesenchymal differentiation capacity. Whether this capacity originates from committed precursors or from multipotent TNCCs was unknown. Here, we further investigated the potential of TNCCs to develop into mesenchymal cell types in vitro. We found that, in fact, quail TNCCs exhibit a high ability to differentiate into myofibroblasts, chondrocytes, lipid-laden adipocytes and mineralizing osteoblasts. In single cell cultures, both mesenchymal and neural cell types coexisted in TNCC clonal progeny: 78% of single cells yielded osteoblasts together with glial cells and neurons; moreover, TNCCs generated heterogenous clones with adipocytes, myofibroblasts, melanocytes and/or glial cells. Therefore, alike cephalic NCCs, early migratory TNCCs comprised multipotent progenitors able to generate both mesenchymal and melanocytic/neural derivatives, suggesting a continuum in NC developmental potentials along the neural axis. The skeletogenic capacity of the TNC, which was present in the exoskeletal armor of the extinct basal forms of Vertebrates and which persisted in the distal fin rays of extant teleost fish, thus did not totally disappear during vertebrate evolution. Mesenchymal potentials of the TNC, although not fulfilled during development, are still present in a dormant state in Amniotes and can be disclosed in in vitro culture. Whether these potentials are not expressed in vivo due to the presence of inhibitory cues or to the lack of permissive factors in the trunk environment remains to be understood. © 2013 Published by Elsevier Inc.

  11. The Cellular Prion Protein Controls Notch Signaling in Neural Stem/Progenitor Cells.

    PubMed

    Martin-Lannerée, Séverine; Halliez, Sophie; Hirsch, Théo Z; Hernandez-Rapp, Julia; Passet, Bruno; Tomkiewicz, Céline; Villa-Diaz, Ana; Torres, Juan-Maria; Launay, Jean-Marie; Béringue, Vincent; Vilotte, Jean-Luc; Mouillet-Richard, Sophie

    2017-03-01

    The prion protein is infamous for its involvement in a group of neurodegenerative diseases known as Transmissible Spongiform Encephalopathies. In the longstanding quest to decipher the physiological function of its cellular isoform, PrP(C) , the discovery of its participation to the self-renewal of hematopoietic and neural stem cells has cast a new spotlight on its potential role in stem cell biology. However, still little is known on the cellular and molecular mechanisms at play. Here, by combining in vitro and in vivo murine models of PrP(C) depletion, we establish that PrP(C) deficiency severely affects the Notch pathway, which plays a major role in neural stem cell maintenance. We document that the absence of PrP(C) in a neuroepithelial cell line or in primary neurospheres is associated with drastically reduced expression of Notch ligands and receptors, resulting in decreased levels of Notch target genes. Similar alterations of the Notch pathway are recovered in the neuroepithelium of Prnp(-/-) embryos during a developmental window encompassing neural tube closure. In addition, in line with Notch defects, our data show that the absence of PrP(C) results in altered expression of Nestin and Olig2 as well as N-cadherin distribution. We further provide evidence that PrP(C) controls the expression of the epidermal growth factor receptor (EGFR) downstream from Notch. Finally, we unveil a negative feedback action of EGFR on both Notch and PrP(C) . As a whole, our study delineates a molecular scenario through which PrP(C) takes part to the self-renewal of neural stem and progenitor cells. Stem Cells 2017;35:754-765.

  12. Inhibition of glycogen synthase kinase-3 (GSK3) promotes the neural differentiation of full-term amniotic fluid-derived stem cells towards neural progenitor cells.

    PubMed

    Gao, Liyang; Zhao, Mingyan; Ye, Wei; Huang, Jinzhi; Chu, Jiaqi; Yan, Shouquan; Wang, Chaojun; Zeng, Rong

    2016-08-01

    The amniotic fluid has a heterogeneous population of cells. Some human amniotic fluid-derived stem (hAFS) cells have been shown to harbor the potential to differentiate into neural cells. However, the neural differentiation efficiency of hAFS cells remains low. In this study, we isolated CD117-positive hAFS cells from amniotic fluid and then examined the pluripotency of these cells through the formation of embryoid bodies (EBs). Additionally, we induced the neural differentiation of these cells using neuroectodermal medium. This study revealed that the GSK3-beta inhibitor SB216763 was able to stimulate the proliferation of CD117-positive hAFS cells without influencing their undifferentiated state. Moreover, SB216763 can efficiently promote the neural differentiation of CD117-positive hAFS cells towards neural progenitor cells in the presence of DMEM/F12 and N2 supplement. These findings provide an easy and low-cost method to maintain the proliferation of hAFS cells, as well as induce an efficacious generation of neural progenitor cells from hAFS cells. Such induction of the neural commitment of hAFS cells may provide an option for the treatment of neurodegenerative diseases by hAFS cells-based therapies.

  13. Lin28 promotes the proliferative capacity of neural progenitor cells in brain development

    PubMed Central

    Yang, Mei; Yang, Si-Lu; Herrlinger, Stephanie; Liang, Chen; Dzieciatkowska, Monika; Hansen, Kirk C.; Desai, Ridham; Nagy, Andras; Niswander, Lee; Moss, Eric G.; Chen, Jian-Fu

    2015-01-01

    Neural progenitor cells (NPCs) have distinct proliferation capacities at different stages of brain development. Lin28 is an RNA-binding protein with two homologs in mice: Lin28a and Lin28b. Here we show that Lin28a/b are enriched in early NPCs and their expression declines during neural differentiation. Lin28a single-knockout mice show reduced NPC proliferation, enhanced cell cycle exit and a smaller brain, whereas mice lacking both Lin28a alleles and one Lin28b allele display similar but more severe phenotypes. Ectopic expression of Lin28a in mice results in increased NPC proliferation, NPC numbers and brain size. Mechanistically, Lin28a physically and functionally interacts with Imp1 (Igf2bp1) and regulates Igf2-mTOR signaling. The function of Lin28a/b in NPCs could be attributed, at least in part, to the regulation of their mRNA targets that encode Igf1r and Hmga2. Thus, Lin28a and Lin28b have overlapping functions in temporally regulating NPC proliferation during early brain development. PMID:25922525

  14. Lin28 promotes the proliferative capacity of neural progenitor cells in brain development.

    PubMed

    Yang, Mei; Yang, Si-Lu; Herrlinger, Stephanie; Liang, Chen; Dzieciatkowska, Monika; Hansen, Kirk C; Desai, Ridham; Nagy, Andras; Niswander, Lee; Moss, Eric G; Chen, Jian-Fu

    2015-05-01

    Neural progenitor cells (NPCs) have distinct proliferation capacities at different stages of brain development. Lin28 is an RNA-binding protein with two homologs in mice: Lin28a and Lin28b. Here we show that Lin28a/b are enriched in early NPCs and their expression declines during neural differentiation. Lin28a single-knockout mice show reduced NPC proliferation, enhanced cell cycle exit and a smaller brain, whereas mice lacking both Lin28a alleles and one Lin28b allele display similar but more severe phenotypes. Ectopic expression of Lin28a in mice results in increased NPC proliferation, NPC numbers and brain size. Mechanistically, Lin28a physically and functionally interacts with Imp1 (Igf2bp1) and regulates Igf2-mTOR signaling. The function of Lin28a/b in NPCs could be attributed, at least in part, to the regulation of their mRNA targets that encode Igf1r and Hmga2. Thus, Lin28a and Lin28b have overlapping functions in temporally regulating NPC proliferation during early brain development. © 2015. Published by The Company of Biologists Ltd.

  15. SDF-1/CXCR4 Signaling Maintains Stemness Signature in Mouse Neural Stem/Progenitor Cells

    PubMed Central

    Ling, Thai-Yen; Lin, Hsing-Yu; Liou, Jeffrey Tsai-Jui; Liu, Fei-Chih; Chen, I-Chun; Lee, Sue-Wei; Hsu, Yu

    2017-01-01

    SDF-1 and its primary receptor, CXCR4, are highly expressed in the embryonic central nervous system (CNS) and play a crucial role in brain architecture. Loss of SDF-1/CXCR4 signaling causes abnormal development of neural stem/progenitor cells (NSCs/NPCs) in the cerebellum, hippocampus, and cortex. However, the mechanism of SDF-1/CXCR4 axis in NSCs/NPCs regulation remains unknown. In this study, we found that elimination of SDF-1/CXCR4 transduction caused NSCs/NPCs to lose their stemness characteristics and to encounter neurogenic differentiation. Moreover, Notch and RE1 silencing transcription factor (REST) both play an essential role in NSCs/NPCs maintenance and neuronal differentiation and were dramatically downregulated following SDF-1/CXCR4 cascade inhibition. Finally, we demonstrated that the expression of achaete-scute homolog 1 (Ascl1), a proneural gene, and p27, an antiproliferative gene, were significantly increased after genetic elimination of SDF-1 alleles. Our results support that the loss of functional SDF-1/CXCR4 signaling pathway in NSCs/NPCs induces exit of cell cycle and promotes premature neural differentiation. PMID:28408934

  16. Survivin Improves Reprogramming Efficiency of Human Neural Progenitors by Single Molecule OCT4

    PubMed Central

    Liu, Yinan; Feng, Ruopeng; Wang, Caiyun; Jiang, Sibo; Zhang, Xiaoyan

    2016-01-01

    Induced pluripotent stem (iPS) cells have been generated from human somatic cells by ectopic expression of four Yamanaka factors. Here, we report that Survivin, an apoptosis inhibitor, can enhance iPS cells generation from human neural progenitor cells (NPCs) together with one factor OCT4 (1F-OCT4-Survivin). Compared with 1F-OCT4, Survivin accelerates the process of reprogramming from human NPCs. The neurocyte-originated induced pluripotent stem (NiPS) cells generated from 1F-OCT4-Survivin resemble human embryonic stem (hES) cells in morphology, surface markers, global gene expression profiling, and epigenetic status. Survivin keeps high expression in both iPS and ES cells. During the process of NiPS cell to neural cell differentiation, the expression of Survivin is rapidly decreased in protein level. The mechanism of Survivin promotion of reprogramming efficiency from NPCs may be associated with stabilization of β-catenin in WNT signaling pathway. This hypothesis is supported by experiments of RT-PCR, chromatin immune-precipitation, and Western blot in human ES cells. Our results showed overexpression of Survivin could improve the efficiency of reprogramming from NPCs to iPS cells by one factor OCT4 through stabilization of the key molecule, β-catenin. PMID:27974895

  17. Defining the role of oxygen tension in human neural progenitor fate.

    PubMed

    Xie, Yuan; Zhang, Jin; Lin, Ying; Gaeta, Xavier; Meng, Xiangzhi; Wisidagama, Dona R R; Cinkornpumin, Jessica; Koehler, Carla M; Malone, Cindy S; Teitell, Michael A; Lowry, William E

    2014-11-11

    Hypoxia augments human embryonic stem cell (hESC) self-renewal via hypoxia-inducible factor 2α-activated OCT4 transcription. Hypoxia also increases the efficiency of reprogramming differentiated cells to a pluripotent-like state. Combined, these findings suggest that low O2 tension would impair the purposeful differentiation of pluripotent stem cells. Here, we show that low O2 tension and hypoxia-inducible factor (HIF) activity instead promote appropriate hESC differentiation. Through gain- and loss-of-function studies, we implicate O2 tension as a modifier of a key cell fate decision, namely whether neural progenitors differentiate toward neurons or glia. Furthermore, our data show that even transient changes in O2 concentration can affect cell fate through HIF by regulating the activity of MYC, a regulator of LIN28/let-7 that is critical for fate decisions in the neural lineage. We also identify key small molecules that can take advantage of this pathway to quickly and efficiently promote the development of mature cell types.

  18. Inducible expression of noggin selectively expands neural progenitors in the adult SVZ.

    PubMed

    Morell, M; Tsan, Yao-chang; O'Shea, K Sue

    2015-01-01

    Multipotent, self-renewing stem cells are present throughout the developing nervous system remaining in discrete regions of the adult brain. In the subventricular zone (SVZ) signaling molecules, including the bone morphogenetic proteins and their secreted inhibitor, noggin appear to play a critical role in controlling neural stem cell (NSC) behavior. To examine the function of this signaling pathway in the intact nervous system, we developed a transgenic mouse model in which noggin expression can be induced specifically in NSC via a nestin-driven reverse tetracycline-controlled transactivator (rtTA). In adult animals, the induction of noggin expression promotes the proliferation of neural progenitors in the SVZ, and shifts the differentiation of B cells (NSC) from mature astrocytes to transit amplifying C cells and oligodendrocyte precursor cells without depleting the NSC population. Noggin expression significantly increases neuronal and oligodendrocyte differentiation both in vivo and in vitro when NSCs are grown as neurospheres. These results demonstrate that noggin/BMP interactions tightly control cell fate in the SVZ. Copyright © 2014. Published by Elsevier B.V.

  19. SDF-1/CXCR4 Signaling Maintains Stemness Signature in Mouse Neural Stem/Progenitor Cells.

    PubMed

    Ho, Shih-Yin; Ling, Thai-Yen; Lin, Hsing-Yu; Liou, Jeffrey Tsai-Jui; Liu, Fei-Chih; Chen, I-Chun; Lee, Sue-Wei; Hsu, Yu; Lai, Dar-Ming; Liou, Horng-Huei

    2017-01-01

    SDF-1 and its primary receptor, CXCR4, are highly expressed in the embryonic central nervous system (CNS) and play a crucial role in brain architecture. Loss of SDF-1/CXCR4 signaling causes abnormal development of neural stem/progenitor cells (NSCs/NPCs) in the cerebellum, hippocampus, and cortex. However, the mechanism of SDF-1/CXCR4 axis in NSCs/NPCs regulation remains unknown. In this study, we found that elimination of SDF-1/CXCR4 transduction caused NSCs/NPCs to lose their stemness characteristics and to encounter neurogenic differentiation. Moreover, Notch and RE1 silencing transcription factor (REST) both play an essential role in NSCs/NPCs maintenance and neuronal differentiation and were dramatically downregulated following SDF-1/CXCR4 cascade inhibition. Finally, we demonstrated that the expression of achaete-scute homolog 1 (Ascl1), a proneural gene, and p27, an antiproliferative gene, were significantly increased after genetic elimination of SDF-1 alleles. Our results support that the loss of functional SDF-1/CXCR4 signaling pathway in NSCs/NPCs induces exit of cell cycle and promotes premature neural differentiation.

  20. Astrocytic Calcium Waves Signal Brain Injury to Neural Stem and Progenitor Cells.

    PubMed

    Kraft, Anna; Jubal, Eduardo Rosales; von Laer, Ruth; Döring, Claudia; Rocha, Adriana; Grebbin, Moyo; Zenke, Martin; Kettenmann, Helmut; Stroh, Albrecht; Momma, Stefan

    2017-03-14

    Brain injuries, such as stroke or trauma, induce neural stem cells in the subventricular zone (SVZ) to a neurogenic response. Very little is known about the molecular cues that signal tissue damage, even over large distances, to the SVZ. Based on our analysis of gene expression patterns in the SVZ, 48 hr after an ischemic lesion caused by middle cerebral artery occlusion, we hypothesized that the presence of an injury might be transmitted by an astrocytic traveling calcium wave rather than by diffusible factors or hypoxia. Using a newly established in vitro system we show that calcium waves induced in an astrocytic monolayer spread to neural stem and progenitor cells and increase their self-renewal as well as migratory behavior. These changes are due to an upregulation of the Notch signaling pathway. This introduces the concept of propagating astrocytic calcium waves transmitting brain injury signals over long distances. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  1. Mitochondrial gene replacement in human pluripotent stem cell-derived neural progenitors.

    PubMed

    Iyer, S; Xiao, E; Alsayegh, K; Eroshenko, N; Riggs, M J; Bennett, J P; Rao, R R

    2012-05-01

    Human pluripotent stem cell-derived neural progenitor (hNP) cells are an excellent resource for understanding early neural development and neurodegenerative disorders. Given that many neurodegenerative disorders can be correlated with defects in the mitochondrial genome, optimal utilization of hNP cells requires an ability to manipulate and monitor changes in the mitochondria. Here, we describe a novel approach that uses recombinant human mitochondrial transcription factor A (rhTFAM) protein to transfect and express a pathogenic mitochondrial genome (mtDNA) carrying the G11778A mutation associated with Leber's hereditary optic neuropathy (LHON) disease, into dideoxycytidine (ddC)-treated hNPs. Treatment with ddC reduced endogenous mtDNA and gene expression, without loss of hNP phenotypic markers. Entry of G11778A mtDNA complexed with the rhTFAM was observed in mitochondria of ddC-hNPs. Expression of the pathogenic RNA was confirmed by restriction enzyme analysis of the SfaN1-digested cDNA. On the basis of the expression of neuron-specific class III beta-tubulin, neuronal differentiation occurred. Our results show for the first time that pathogenic mtDNA can be introduced and expressed into hNPs without loss of phenotype or neuronal differentiation potential. This mitochondrial gene replacement technology allows for creation of in vitro stem cell-based models useful for understanding neuronal development and treatment of neurodegenerative disorders.

  2. Bradykinin promotes neuron-generating division of neural progenitor cells through ERK activation.

    PubMed

    Pillat, Micheli M; Lameu, Claudiana; Trujillo, Cleber A; Glaser, Talita; Cappellari, Angélica R; Negraes, Priscilla D; Battastini, Ana M O; Schwindt, Telma T; Muotri, Alysson R; Ulrich, Henning

    2016-09-15

    During brain development, cells proliferate, migrate and differentiate in highly accurate patterns. In this context, published results indicate that bradykinin functions in neural fate determination, favoring neurogenesis and migration. However, mechanisms underlying bradykinin function are yet to be explored. Our findings indicate a previously unidentified role for bradykinin action in inducing neuron-generating division in vitro and in vivo, given that bradykinin lengthened the G1-phase of the neural progenitor cells (NPC) cycle and increased TIS21 (also known as PC3 and BTG2) expression in hippocampus from newborn mice. This role, triggered by activation of the kinin-B2 receptor, was conditioned by ERK1/2 activation. Moreover, immunohistochemistry analysis of hippocampal dentate gyrus showed that the percentage of Ki67(+) cells markedly increased in bradykinin-treated mice, and ERK1/2 inhibition affected this neurogenic response. The progress of neurogenesis depended on sustained ERK phosphorylation and resulted in ERK1/2 translocation to the nucleus in NPCs and PC12 cells, changing expression of genes such as Hes1 and Ngn2 (also known as Neurog2). In agreement with the function of ERK in integrating signaling pathways, effects of bradykinin in stimulating neurogenesis were reversed following removal of protein kinase C (PKC)-mediated sustained phosphorylation. © 2016. Published by The Company of Biologists Ltd.

  3. beta-Chemokine production by neural and glial progenitor cells is enhanced by HIV-1 Tat: effects on microglial migration.

    PubMed

    Hahn, Yun Kyung; Vo, Phu; Fitting, Sylvia; Block, Michelle L; Hauser, Kurt F; Knapp, Pamela E

    2010-07-01

    Human immunodeficiency virus (HIV)-1 neuropathology results from collective effects of viral proteins and inflammatory mediators on several cell types. Significant damage is mediated indirectly through inflammatory conditions promulgated by glial cells, including microglia that are productively infected by HIV-1, and astroglia. Neural and glial progenitors exist in both developing and adult brains. To determine whether progenitors are targets of HIV-1, a multi-plex assay was performed to assess chemokine/cytokine expression after treatment with viral proteins transactivator of transcription (Tat) or glycoprotein 120 (gp120). In the initial screen, ten analytes were basally released by murine striatal progenitors. The beta-chemokines CCL5/regulated upon activation, normal T cell expressed and secreted, CCL3/macrophage inflammatory protein-1alpha, and CCL4/macrophage inflammatory protein-1beta were increased by 12-h exposure to HIV-1 Tat. Secreted factors from Tat-treated progenitors were chemoattractive towards microglia, an effect blocked by 2D7 anti-CCR5 antibody pre-treatment. Tat and opiates have interactive effects on astroglial chemokine secretion, but this interaction did not occur in progenitors. gp120 did not affect chemokine/cytokine release, although both CCR5 and CXCR4, which serve as gp120 co-receptors, were detected in progenitors. We postulate that chemokine production by progenitors may be a normal, adaptive process that encourages immune inspection of newly generated cells. Pathogens such as HIV might usurp this function to create a maladaptive state, especially during development or regeneration, when progenitors are numerous.

  4. Integration of signals along orthogonal axes of the vertebrate neural tube controls progenitor competence and increases cell diversity.

    PubMed

    Sasai, Noriaki; Kutejova, Eva; Briscoe, James

    2014-07-01

    A relatively small number of signals are responsible for the variety and pattern of cell types generated in developing embryos. In part this is achieved by exploiting differences in the concentration or duration of signaling to increase cellular diversity. In addition, however, changes in cellular competence-temporal shifts in the response of cells to a signal-contribute to the array of cell types generated. Here we investigate how these two mechanisms are combined in the vertebrate neural tube to increase the range of cell types and deliver spatial control over their location. We provide evidence that FGF signaling emanating from the posterior of the embryo controls a change in competence of neural progenitors to Shh and BMP, the two morphogens that are responsible for patterning the ventral and dorsal regions of the neural tube, respectively. Newly generated neural progenitors are exposed to FGF signaling, and this maintains the expression of the Nk1-class transcription factor Nkx1.2. Ventrally, this acts in combination with the Shh-induced transcription factor FoxA2 to specify floor plate cells and dorsally in combination with BMP signaling to induce neural crest cells. As development progresses, the intersection of FGF with BMP and Shh signals is interrupted by axis elongation, resulting in the loss of Nkx1.2 expression and allowing the induction of ventral and dorsal interneuron progenitors by Shh and BMP signaling to supervene. Hence a similar mechanism increases cell type diversity at both dorsal and ventral poles of the neural tube. Together these data reveal that tissue morphogenesis produces changes in the coincidence of signals acting along orthogonal axes of the neural tube and this is used to define spatial and temporal transitions in the competence of cells to interpret morphogen signaling.

  5. Inactivation of Geminin in neural crest cells affects the generation and maintenance of enteric progenitor cells, leading to enteric aganglionosis.

    PubMed

    Stathopoulou, Athanasia; Natarajan, Dipa; Nikolopoulou, Pinelopi; Patmanidi, Alexandra L; Lygerou, Zoi; Pachnis, Vassilis; Taraviras, Stavros

    2016-01-15

    Neural crest cells comprise a multipotent, migratory cell population that generates a diverse array of cell and tissue types, during vertebrate development. Enteric Nervous System controls the function of the gastrointestinal tract and is mainly derived from the vagal and sacral neural crest cells. Deregulation on self-renewal and differentiation of the enteric neural crest cells is evident in enteric nervous system disorders, such as Hirschsprung disease, characterized by the absence of ganglia in a variable length of the distal bowel. Here we show that Geminin is essential for Enteric Nervous System generation as mice that lacked Geminin expression specifically in neural crest cells revealed decreased generation of vagal neural crest cells, and enteric neural crest cells (ENCCs). Geminin-deficient ENCCs showed increased apoptosis and decreased cell proliferation during the early stages of gut colonization. Furthermore, decreased number of committed ENCCs in vivo and the decreased self-renewal capacity of enteric progenitor cells in vitro, resulted in almost total aganglionosis resembling a severe case of Hirschsprung disease. Our results suggest that Geminin is an important regulator of self-renewal and survival of enteric nervous system progenitor cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Intermediate progenitors are increased by lengthening of the cell cycle through calcium signaling and p53 expression in human neural progenitors

    PubMed Central

    García-García, Elisa; Pino-Barrio, María José; López-Medina, Laura; Martínez-Serrano, Alberto

    2012-01-01

    During development, neurons can be generated directly from a multipotent progenitor or indirectly through an intermediate progenitor (IP). This last mode of division amplifies the progeny of neurons. The mechanisms governing the generation and behavior of IPs are not well understood. In this work, we demonstrate that the lengthening of the cell cycle enhances the generation of neurons in a human neural progenitor cell system in vitro and also the generation and expansion of IPs. These IPs are insulinoma-associated 1 (Insm1)+/BTG family member 2 (Btg2)−, which suggests an increase in a self-amplifying IP population. Later the cultures express neurogenin 2 (Ngn2) and become neurogenic. The signaling responsible for this cell cycle modulation is investigated. It is found that the release of calcium from the endoplasmic reticulum to the cytosol in response to B cell lymphoma-extra large overexpression or ATP addition lengths the cell cycle and increases the number of IPs and, in turn, the final neuron outcome. Moreover, data suggest that the p53–p21 pathway is responsible for the changes in cell cycle. In agreement with this, increased p53 levels are necessary for a calcium-induced increase in neurons. Our findings contribute to understand how calcium signaling can modulate cell cycle length during neurogenesis. PMID:22323293

  7. Clinical safety of intrathecal administration of mesenchymal stromal cell-derived neural progenitors in multiple sclerosis.

    PubMed

    Harris, Violaine K; Vyshkina, Tamara; Sadiq, Saud A

    2016-12-01

    There is a critical unmet need to develop regenerative therapies for the demyelinating disease multiple sclerosis (MS). We previously characterized the immunoregulatory and trophic properties of neural progenitors derived from bone marrow mesenchymal stromal cells (MSC-NPs) and established that cells derived from MS and non-MS patients alike were therapeutically viable. In an experimental model of MS, intrathecal MSC-NP injection resulted in disease amelioration with decreased T-cell infiltration, and less severe lesion pathology associated with recruitment of resident progenitors to inflammatory sites. In this pilot feasibility study, we investigated safety and dosing of intrathecal MSC-NP therapy in six patients with MS. Patients with progressive MS and advanced disability who were refractory to all other conventional MS treatments were enrolled in the study. For each dose, MSC-NP cells were cultured from autologous MSCs and tested for quality control before intrathecal administration. Patients were evaluated for adverse events and neurological status to assess safety of the treatment. Six patients with progressive MS were treated with between 2 and 5 intrathecal injections of escalating doses of autologous MSC-NPs and were followed an average of 7.4 years after initial injection. There were no safety concerns noted, no serious adverse events, and the multiple dosing regimen was well tolerated. Four of the six patients showed a measurable clinical improvement following MSC-NP treatment. This pilot study supports preliminary first-in-human safety and tolerability of autologous MSC-NP treatment for MS. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  8. Neural stem/progenitor cells are activated during tail regeneration in the leopard gecko (Eublepharis macularius).

    PubMed

    Gilbert, Eab; Vickaryous, M K

    2017-10-04

    As for many lizards, the leopard gecko (Eublepharis macularius) can self-detach its tail to avoid predation and then regenerate a replacement. The replacement tail includes a regenerated spinal cord with a simple morphology: an ependymal layer surrounded by nerve tracts. We hypothesized that cells within the ependymal layer of the original spinal cord include populations of neural stem/progenitor cells (NSPCs) that contribute to the regenerated spinal cord. Prior to tail loss, we performed a bromodeoxyuridine pulse-chase experiment and found that a subset of ependymal layer cells (ELCs) were label-retaining after a 140-day chase period. Next, we conducted a detailed spatiotemporal characterization of these cells before, during, and after tail regeneration. Our findings show that SOX2, a hallmark protein of NSPCs, is constitutively expressed by virtually all ELCs before, during, and after regeneration. We also found that during regeneration, ELCs express an expanded panel of NSPC and lineage-restricted progenitor cell markers, including MSI-1, SOX9 and TUJ1. Using electron microscopy, we determined that multiciliated, uniciliated, and biciliated cells are present, although the latter was only observed in regenerated spinal cords. Our results demonstrate that cells within the ependymal layer of the original, regenerating and fully regenerate spinal cord represent a heterogeneous population. These include radial glia comparable to Type E and Type B cells, and a neuronal-like population of cerebrospinal fluid-contacting cells. We propose that spinal cord regeneration in geckos represents a truncation of the restorative trajectory observed in some urodeles and teleosts, resulting in the formation of a structurally distinct replacement. This article is protected by copyright. All rights reserved. © 2017 Wiley Periodicals, Inc.

  9. Characterization of calcium responses and electrical activity in differentiating mouse neural progenitor cells in vitro.

    PubMed

    de Groot, Martje W G D M; Dingemans, Milou M L; Rus, Katinka H; de Groot, Aart; Westerink, Remco H S

    2014-02-01

    In vitro methods for developmental neurotoxicity (DNT) testing have the potential to reduce animal use and increase insight into cellular and molecular mechanisms underlying chemical-induced alterations in the development of functional neuronal networks. Mouse neural progenitor cells (mNPCs) differentiate into nervous system-specific cell types and have proven valuable to detect DNT using biochemical and morphological techniques. We therefore investigated a number of functional neuronal parameters in primary mNPCs to explore their applicability for neurophysiological in vitro DNT testing. Immunocytochemistry confirmed that mNPCs express neuronal, glial, and progenitor markers at various differentiation durations (1, 7, 14, and 21 days). Because intracellular calcium ([Ca(2+)]i) plays an essential role in neuronal development and function, we measured stimulus-evoked changes in [Ca(2+)]i at these differentiation durations using the Ca(2+)-responsive dye Fura-2. Increases in [Ca(2+)]i (averages ranging from 65 to 226 nM) were evoked by depolarization, ATP, l-glutamic acid, acetylcholine, and dopamine (up to 87%, 57%, 93%, 28%, and 37% responding cells, respectively) and to a lesser extent by serotonin and gamma-aminobutyric acid (both up to 10% responding cells). Notably, the changes in percentage of responsive cells and their response amplitudes over time indicate changes in the expression and functionality of the respective neurotransmitter receptors and related calcium signaling pathways during in vitro differentiation. The development of functional intercellular signaling pathways was confirmed using multielectrode arrays, demonstrating that mNPCs develop electrical activity within 1-2 weeks of differentiation (55% active wells at 14 days of differentiation; mean spike rate of 1.16 spikes/s/electrode). The combined data demonstrate that mNPCs develop functional neuronal characteristics in vitro, making it a promising model to study chemical-induced effects on the

  10. Multipotent caudal neural progenitors derived from human pluripotent stem cells that give rise to lineages of the central and peripheral nervous system.

    PubMed

    Denham, Mark; Hasegawa, Kouichi; Menheniott, Trevelyan; Rollo, Ben; Zhang, Dongcheng; Hough, Shelley; Alshawaf, Abdullah; Febbraro, Fabia; Ighaniyan, Samiramis; Leung, Jessie; Elliott, David A; Newgreen, Donald F; Pera, Martin F; Dottori, Mirella

    2015-06-01

    The caudal neural plate is a distinct region of the embryo that gives rise to major progenitor lineages of the developing central and peripheral nervous system, including neural crest and floor plate cells. We show that dual inhibition of the glycogen synthase kinase 3β and activin/nodal pathways by small molecules differentiate human pluripotent stem cells (hPSCs) directly into a preneuroepithelial progenitor population we named "caudal neural progenitors" (CNPs). CNPs coexpress caudal neural plate and mesoderm markers, and, share high similarities to embryonic caudal neural plate cells in their lineage differentiation potential. Exposure of CNPs to BMP2/4, sonic hedgehog, or FGF2 signaling efficiently directs their fate to neural crest/roof plate cells, floor plate cells, and caudally specified neuroepithelial cells, respectively. Neural crest derived from CNPs differentiated to neural crest derivatives and demonstrated extensive migratory properties in vivo. Importantly, we also determined the key extrinsic factors specifying CNPs from human embryonic stem cell include FGF8, canonical WNT, and IGF1. Our studies are the first to identify a multipotent neural progenitor derived from hPSCs, that is the precursor for major neural lineages of the embryonic caudal neural tube.

  11. Optimizing Culture Medium Composition to Improve Oligodendrocyte Progenitor Cell Yields In Vitro from Subventricular Zone-Derived Neural Progenitor Cell Neurospheres

    PubMed Central

    Franco, Paula G.; Pasquini, Juana M.; Silvestroff, Lucas

    2015-01-01

    Neural Stem and Progenitor Cells (NSC/NPC) are gathering tangible recognition for their uses in cell therapy and cell replacement therapies for human disease, as well as a model system to continue research on overall neural developmental processes in vitro. The Subventricular Zone is one of the largest NSC/NPC niches in the developing mammalian Central Nervous System, and persists through to adulthood. Oligodendrocyte progenitor cell (OPC) enriched cultures are usefull tools for in vitro studies as well as for cell replacement therapies for treating demyelination diseases. We used Subventricular Zone-derived NSC/NPC primary cultures from newborn mice and compared the effects of different growth factor combinations on cell proliferation and OPC yield. The Platelet Derived Growth Factor-AA and BB homodimers had a positive and significant impact on OPC generation. Furthermore, heparin addition to the culture media contributed to further increase overall culture yields. The OPC generated by this protocol were able to mature into Myelin Basic Protein-expressing cells and to interact with neurons in an in vitro co-culture system. As a whole, we describe an optimized in vitro method for increasing OPC. PMID:25837625

  12. Optimizing culture medium composition to improve oligodendrocyte progenitor cell yields in vitro from subventricular zone-derived neural progenitor cell neurospheres.

    PubMed

    Franco, Paula G; Pasquini, Juana M; Silvestroff, Lucas

    2015-01-01

    Neural Stem and Progenitor Cells (NSC/NPC) are gathering tangible recognition for their uses in cell therapy and cell replacement therapies for human disease, as well as a model system to continue research on overall neural developmental processes in vitro. The Subventricular Zone is one of the largest NSC/NPC niches in the developing mammalian Central Nervous System, and persists through to adulthood. Oligodendrocyte progenitor cell (OPC) enriched cultures are usefull tools for in vitro studies as well as for cell replacement therapies for treating demyelination diseases. We used Subventricular Zone-derived NSC/NPC primary cultures from newborn mice and compared the effects of different growth factor combinations on cell proliferation and OPC yield. The Platelet Derived Growth Factor-AA and BB homodimers had a positive and significant impact on OPC generation. Furthermore, heparin addition to the culture media contributed to further increase overall culture yields. The OPC generated by this protocol were able to mature into Myelin Basic Protein-expressing cells and to interact with neurons in an in vitro co-culture system. As a whole, we describe an optimized in vitro method for increasing OPC.

  13. Neural and glial progenitor transplantation as a neuroprotective strategy for Amyotrophic Lateral Sclerosis (ALS).

    PubMed

    Haidet-Phillips, Amanda M; Maragakis, Nicholas J

    2015-12-02

    ALS is a neurodegenerative disease with a prevalence rate of up to 7.4/100,000 and the overall risk of developing ALS over a lifetime is 1:400. Most patients die from respiratory failure following a course of progressive weakness. To date, only one traditional pharmaceutical agent-riluzole, has been shown to afford a benefit on survival but numerous pharmaceutical interventions have been studied in preclinical models of ALS without subsequent translation to patient efficacy. Despite the relative selectivity of motor neuron cell death, animal and tissue culture models of familial ALS suggest that non-neuronal cells significantly contribute to neuronal dysfunction and death. Early efforts to transplant stem cells had focused on motor neuron replacement. More practically for this aggressive neurodegenerative disease, recent studies, preclinical efforts, and early clinical trials have focused on the transplantation of neural stem cells, mesenchymal stem cells, or glial progenitors. Using transgenic mouse or rat models of ALS, a number of studies have shown neuroprotection through a variety of different mechanisms that have included neurotrophic factor secretion, glutamate transporter regulation, and modulation of neuroinflammation, among others. However, given that cell replacement could involve a number of biologically relevant factors, identifying the key pathway(s) that may contribute to neuroprotection remains a challenge. Nevertheless, given the abundant data supporting the interplay between non-neuronal cell types and motor neuron disease propagation, the replacement of disease-carrying host cells by normal cells may be sufficient to confer neuroprotection. Key preclinical issues that currently are being addressed include the most appropriate methods and routes for delivery of cells to disease-relevant regions of the neuraxis, cell survival and migration, and tracking the cells following transplantation. Central to the initial development of stem cell

  14. Neural stem/progenitor cell transplantation for spinal cord injury treatment; A systematic review and meta-analysis.

    PubMed

    Yousefifard, M; Rahimi-Movaghar, V; Nasirinezhad, F; Baikpour, M; Safari, S; Saadat, S; Moghadas Jafari, A; Asady, H; Razavi Tousi, S M T; Hosseini, M

    2016-05-13

    Despite the vast improvements of cell therapy in spinal cord injury treatment, no optimum protocol has been developed for application of neural stem/progenitor cells. In this regard, the present meta-analysis showed that the efficacy of the neural stem/progenitor cell (NSPC) transplantation depends mainly on injury model, intervention phase, transplanted cell count, immunosuppressive use, and probably stem cell source. Improved functional recovery post NSPC transplantation was found to be higher in transection and contusion models. Moreover, NSPC transplantation in acute phase of spinal injury was found to have better functional recovery. Higher doses (>3×10(6)cell/kg) were also shown to be optimum for transplantation, but immunosuppressive agent administration negatively affected the motor function recovery. Scaffold use in NSPC transplantation could also effectively raise functional recovery.

  15. Assessing the efficacy of endoscopic office olfactory biopsy sites to produce neural progenitor cell cultures for the study of neuropsychiatric disorders.

    PubMed

    Wrobel, Bozena B; Mazza, Jill M; Evgrafov, Oleg V; Knowles, James A

    2013-02-01

    The olfactory region is capable of continuous neurogenesis. Situated on the cribriform plate and segments of the superior septum and both superior and middle turbinates, it is accessible through office-based biopsy and can be used to generate neural progenitor cells to study molecular abnormalities associated with neuropsychiatric disorders. The purpose of the study was to evaluate the efficacy of the endoscopic office olfactory biopsy from middle turbinate and superior-posterior septum to produce the neural progenitor cells. Endoscopic office-based biopsy samples were collected and cultured neuronal cells derived from olfactory neuroepithelium (CNON) were established from 40 healthy individuals and 40 schizophrenia patients. All patients underwent biopsies of both the middle turbinate and the superior-posterior septum. Specific culture conditions promoted the growth of neural progenitor cells from these biopsy sites. CNON cultures were established from such outgrowing neuronal cells. The study was institutional review board (IRB)-approved and informed consent was obtained. Cultures were successfully developed from 98.8% of participants. No complications were observed. The single, unsuccessful specimen failed to grow any cell types due to tissue mishandling. Overall, we have observed no significant difference in the effectiveness of biopsy from middle turbinate and superior-posterior septum to produce neural progenitor cells. The middle turbinate biopsies contain viable neural progenitor cells capable of generating neuronal cell cultures. Thus technically more simple biopsy of the middle turbinate can be used to propagate neural progenitor cells. © 2013 ARS-AAOA, LLC.

  16. Changes in metabolic proteins in ex vivo rat retina during glutamate-induced neural progenitor cell induction.

    PubMed

    Tokuda, Kazuhiro; Kuramitsu, Yasuhiro; Baron, Byron; Kitagawa, Takao; Tokuda, Nobuko; Kobayashi, Masaaki; Kimura, Kazuhiro; Sonoda, Koh-Hei; Nakamura, Kazuyuki

    2016-08-01

    Understanding how energy metabolism and related proteins influence neural progenitor cells in adult tissues is critical for developing new strategies in clinical tissue regeneration therapy. We have recently reported that a subtoxic concentration of glutamate-induced neural progenitor cells in the mature ex vivo rat retina. We herein explore changes in the metabolic pathways during the process. We firstly observed an increase in lactate and lactate dehydrogenase concentration in the glutamate-treated retina. We then investigated the levels of glycolytic enzymes and confirmed significant upregulation of pyruvate kinase M type (PKM), especially PKM2, enolase, phosphoglycerate mutase 1 (PGAM1), and inosine-5'-monophosphate dehydrogenase (IMPDH1) in the glutamate-treated retina compared to the untreated retina. An analysis of the subcellular localization of PKM2 revealed nuclear translocation in the treated retina, which has been reported to regulate cell cycle proliferation and glycolytic enzymes. Our findings indicate that the mature rat retina undergoes an increase in aerobic glycolysis. PKM2, both in the cytoplasm and in the nucleus, may thus play an important role during neural progenitor cell induction, as it does in other proliferating cells.

  17. Characterization of Apoptosis Signaling Cascades During the Differentiation Process of Human Neural ReNcell VM Progenitor Cells In Vitro.

    PubMed

    Jaeger, Alexandra; Fröhlich, Michael; Klum, Susanne; Lantow, Margareta; Viergutz, Torsten; Weiss, Dieter G; Kriehuber, Ralf

    2015-11-01

    Apoptosis is an essential physiological process accompanying the development of the central nervous system and human neurogenesis. However, the time scale and the underlying molecular mechanisms are yet poorly understood. Due to this fact, we investigated the functionality and general inducibility of apoptosis in the human neural ReNcell VM progenitor cell line during differentiation and also after exposure to staurosporine (STS) and ultraviolet B (UVB) irradiation. Transmission light microscopy, flow cytometry, and Western-/Immunoblot analysis were performed to compare proliferating and differentiating, in addition to STS- and UVB-treated cells. In particular, from 24 to 72 h post-initiation of differentiation, G0/G1 cell cycle arrest, increased loss of apoptotic cells, activation of pro-apoptotic BAX, Caspase-3, and cleavage of its substrate PARP were observed during cell differentiation and, to a higher extent, after treatment with STS and UVB. We conclude that redundant or defective cells are eliminated by apoptosis, while otherwise fully differentiated cells were less responsive to apoptosis induction by STS than proliferating cells, likely as a result of reduced APAF-1 expression, and increased levels of BCL-2. These data provide the evidence that apoptotic mechanisms in the neural ReNcell VM progenitor cell line are not only functional, but also inducible by external stimuli like growth factor withdrawal or treatment with STS and UVB, which marks this cell line as a suitable model to investigate apoptosis signaling pathways in respect to the differentiation processes of human neural progenitor cells in vitro.

  18. The Interleukin 3 Gene (IL3) Contributes to Human Brain Volume Variation by Regulating Proliferation and Survival of Neural Progenitors

    PubMed Central

    Huang, Liang; Nho, Kwangsik; Deng, Min; Chen, Qiang; Weinberger, Daniel R.; Vasquez, Alejandro Arias; Rijpkema, Mark; Mattay, Venkata S.; Saykin, Andrew J.; Shen, Li; Fernández, Guillén; Franke, Barbara; Chen, Jing-chun; Chen, Xiang-ning; Wang, Jin-kai; Xiao, Xiao; Qi, Xue-bin; Xiang, Kun; Peng, Ying-Mei; Cao, Xiang-yu; Li, Yi; Shi, Xiao-dong; Gan, Lin; Su, Bing

    2012-01-01

    One of the most significant evolutionary changes underlying the highly developed cognitive abilities of humans is the greatly enlarged brain volume. In addition to being far greater than in most other species, the volume of the human brain exhibits extensive variation and distinct sexual dimorphism in the general population. However, little is known about the genetic mechanisms underlying normal variation as well as the observed sex difference in human brain volume. Here we show that interleukin-3 (IL3) is strongly associated with brain volume variation in four genetically divergent populations. We identified a sequence polymorphism (rs31480) in the IL3 promoter which alters the expression of IL3 by affecting the binding affinity of transcription factor SP1. Further analysis indicated that IL3 and its receptors are continuously expressed in the developing mouse brain, reaching highest levels at postnatal day 1–4. Furthermore, we found IL3 receptor alpha (IL3RA) was mainly expressed in neural progenitors and neurons, and IL3 could promote proliferation and survival of the neural progenitors. The expression level of IL3 thus played pivotal roles in the expansion and maintenance of the neural progenitor pool and the number of surviving neurons. Moreover, we found that IL3 activated both estrogen receptors, but estrogen didn’t directly regulate the expression of IL3. Our results demonstrate that genetic variation in the IL3 promoter regulates human brain volume and reveals novel roles of IL3 in regulating brain development. PMID:23226269

  19. Disruption of neural progenitors along the ventricular and subventricular zones in periventricular heterotopia

    PubMed Central

    Ferland, Russell J.; Batiz, Luis Federico; Neal, Jason; Lian, Gewei; Bundock, Elizabeth; Lu, Jie; Hsiao, Yi-Chun; Diamond, Rachel; Mei, Davide; Banham, Alison H.; Brown, Philip J.; Vanderburg, Charles R.; Joseph, Jeffrey; Hecht, Jonathan L.; Folkerth, Rebecca; Guerrini, Renzo; Walsh, Christopher A.; Rodriguez, Esteban M.; Sheen, Volney L.

    2009-01-01

    Periventricular heterotopia (PH) is a disorder characterized by neuronal nodules, ectopically positioned along the lateral ventricles of the cerebral cortex. Mutations in either of two human genes, Filamin A (FLNA) or ADP-ribosylation factor guanine exchange factor 2 (ARFGEF2), cause PH (Fox et al. in ‘Mutations in filamin 1 prevent migration of cerebral cortical neurons in human periventricular heterotopia'. Neuron, 21, 1315–1325, 1998; Sheen et al. in ‘Mutations in ARFGEF2 implicate vesicle trafficking in neural progenitor proliferation and migration in the human cerebral cortex'. Nat. Genet., 36, 69–76, 2004). Recent studies have shown that mutations in mitogen-activated protein kinase kinase kinase-4 (Mekk4), an indirect interactor with FlnA, also lead to periventricular nodule formation in mice (Sarkisian et al. in ‘MEKK4 signaling regulates filamin expression and neuronal migration'. Neuron, 52, 789–801, 2006). Here we show that neurons in post-mortem human PH brains migrated appropriately into the cortex, that periventricular nodules were primarily composed of later-born neurons, and that the neuroependyma was disrupted in all PH cases. As studied in the mouse, loss of FlnA or Big2 function in neural precursors impaired neuronal migration from the germinal zone, disrupted cell adhesion and compromised neuroepithelial integrity. Finally, the hydrocephalus with hop gait (hyh) mouse, which harbors a mutation in Napa [encoding N-ethylmaleimide-sensitive factor attachment protein alpha (α-SNAP)], also develops a progressive denudation of the neuroepithelium, leading to periventicular nodule formation. Previous studies have shown that Arfgef2 and Napa direct vesicle trafficking and fusion, whereas FlnA associates dynamically with the Golgi membranes during budding and trafficking of transport vesicles. Our current findings suggest that PH formation arises from a final common pathway involving disruption of vesicle trafficking, leading to impaired cell

  20. Geminin loss causes neural tube defects through disrupted progenitor specification and neuronal differentiation.

    PubMed

    Patterson, Ethan S; Waller, Laura E; Kroll, Kristen L

    2014-09-01

    Geminin is a nucleoprotein that can directly bind chromatin regulatory complexes to modulate gene expression during development. Geminin knockout mouse embryos are preimplantation lethal by the 32-cell stage, precluding in vivo study of Geminin's role in neural development. Therefore, here we used a conditional Geminin allele in combination with several Cre-driver lines to define an essential role for Geminin during mammalian neural tube (NT) formation and patterning. Geminin was required in the NT within a critical developmental time window (embryonic day 8.5-10.5), when NT patterning and closure occurs. Geminin excision at these stages resulted in strongly diminished expression of genes that mark and promote dorsal NT identities and decreased differentiation of ventral motor neurons, resulting in completely penetrant NT defects, while excision after embryonic day 10.5 did not result in NT defects. When Geminin was deleted specifically in the spinal NT, both NT defects and axial skeleton defects were observed, but neither defect occurred when Geminin was excised in paraxial mesenchyme, indicating a tissue autonomous requirement for Geminin in developing neuroectoderm. Despite a potential role for Geminin in cell cycle control, we found no evidence of proliferation defects or altered apoptosis. Comparisons of gene expression in the NT of Geminin mutant versus wild-type siblings at embryonic day 10.5 revealed decreased expression of key regulators of neurogenesis, including neurogenic bHLH transcription factors and dorsal interneuron progenitor markers. Together, these data demonstrate a requirement for Geminin for NT patterning and neuronal differentiation during mammalian neurulation in vivo.

  1. Gene expression profiling of human neural progenitor cells following the serum-induced astrocyte differentiation.

    PubMed

    Obayashi, Shinya; Tabunoki, Hiroko; Kim, Seung U; Satoh, Jun-ichi

    2009-05-01

    Neural stem cells (NSC) with self-renewal and multipotent properties could provide an ideal cell source for transplantation to treat spinal cord injury, stroke, and neurodegenerative diseases. However, the majority of transplanted NSC and neural progenitor cells (NPC) differentiate into astrocytes in vivo under pathological environments in the central nervous system, which potentially cause reactive gliosis. Because the serum is a potent inducer of astrocyte differentiation of rodent NPC in culture, we studied the effect of the serum on gene expression profile of cultured human NPC to identify the gene signature of astrocyte differentiation of human NPC. Human NPC spheres maintained in the serum-free culture medium were exposed to 10% fetal bovine serum (FBS) for 72 h, and processed for analyzing on a Whole Human Genome Microarray of 41,000 genes, and the microarray data were validated by real-time RT-PCR. The serum elevated the levels of expression of 45 genes, including ID1, ID2, ID3, CTGF, TGFA, METRN, GFAP, CRYAB and CSPG3, whereas it reduced the expression of 23 genes, such as DLL1, DLL3, PDGFRA, SOX4, CSPG4, GAS1 and HES5. Thus, the serum-induced astrocyte differentiation of human NPC is characterized by a counteraction of ID family genes on Delta family genes. Coimmunoprecipitation analysis identified ID1 as a direct binding partner of a proneural basic helix-loop-helix (bHLH) transcription factor MASH1. Luciferase assay indicated that activation of the DLL1 promoter by MASH1 was counteracted by ID1. Bone morphogenetic protein 4 (BMP4) elevated the levels of ID1 and GFAP expression in NPC under the serum-free culture conditions. Because the serum contains BMP4, these results suggest that the serum factor(s), most probably BMP4, induces astrocyte differentiation by upregulating the expression of ID family genes that repress the proneural bHLH protein-mediated Delta expression in human NPC.

  2. Cultivation of human neural progenitor cells in a 3-dimensional self-assembling peptide hydrogel.

    PubMed

    Liedmann, Andrea; Rolfs, Arndt; Frech, Moritz J

    2012-01-11

    The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool. Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells. Here we used PuraMatrix (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types. PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free. Recently we have studied the influence of the PM-concentration on the formation of the scaffold. In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by βIII-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable

  3. Characterization and Molecular Profiling of PSEN1 Familial Alzheimer's Disease iPSC-Derived Neural Progenitors

    PubMed Central

    Sproul, Andrew A.; Jacob, Samson; Pre, Deborah; Kim, Soong Ho; Nestor, Michael W.; Navarro-Sobrino, Miriam; Santa-Maria, Ismael; Zimmer, Matthew; Aubry, Soline; Steele, John W.; Kahler, David J.; Dranovsky, Alex; Arancio, Ottavio; Crary, John F.; Gandy, Sam; Noggle, Scott A.

    2014-01-01

    Presenilin 1 (PSEN1) encodes the catalytic subunit of γ-secretase, and PSEN1 mutations are the most common cause of early onset familial Alzheimer's disease (FAD). In order to elucidate pathways downstream of PSEN1, we characterized neural progenitor cells (NPCs) derived from FAD mutant PSEN1 subjects. Thus, we generated induced pluripotent stem cells (iPSCs) from affected and unaffected individuals from two families carrying PSEN1 mutations. PSEN1 mutant fibroblasts, and NPCs produced greater ratios of Aβ42 to Aβ40 relative to their control counterparts, with the elevated ratio even more apparent in PSEN1 NPCs than in fibroblasts. Molecular profiling identified 14 genes differentially-regulated in PSEN1 NPCs relative to control NPCs. Five of these targets showed differential expression in late onset AD/Intermediate AD pathology brains. Therefore, in our PSEN1 iPSC model, we have reconstituted an essential feature in the molecular pathogenesis of FAD, increased generation of Aβ42/40, and have characterized novel expression changes. PMID:24416243

  4. Intraspinal transplantation of GABAergic neural progenitors attenuates neuropathic pain in rats: a pharmacologic and neurophysiological evaluation

    PubMed Central

    Jergova, Stanislava; Hentall, Ian D.; Gajavelli, Shyam; Varghese, Mathew S.; Sagen, Jacqueline

    2012-01-01

    Dysfunctional γ-aminobutyric acid (GABA)-ergic inhibitory neurotransmission is hypothesized to underlie chronic neuropathic pain. Intraspinal transplantation of GABAergic neural progenitor cells (NPCs) may reduce neuropathic pain by restoring dorsal horn inhibition. Rat NPCs pre-differentiated to a GABAergic phenotype were transplanted into the dorsal horn of rats with unilateral chronic constriction injury (CCI) of the sciatic nerve. GABA signaling in antinociceptive effects of NPC grafts was tested with the GABAA receptor antagonist bicuculline (BIC), GABAB receptor antagonist CGP35348 (CGP) and GABA reuptake inhibitor SKF 89976A (SKF). NPC-treated animals showed decreased hyperalgesia and allodynia 1-3 week post-transplantation; vehicle-injected CCI rats continued displaying pain behaviors. Intrathecal application of BIC or CGP attenuated the antinociceptive effects of the NPC transplants while SKF injection induced analgesia in control rats. Electrophysiological recordings in NPC treated rats showed reduced responses of wide dynamic range (WDR) neurons to peripheral stimulation compared to controls. A spinal application of BIC or CGP increased wind-up response and post-discharges of WDR neurons in NPC treated animals. Results suggest that transplantation of GABAergic NPCs attenuate pain behaviors and reduce exaggerated dorsal horn neuronal firing induced by CCI. The effects of GABA receptor inhibitors suggest participation of continuously released GABA in the grafted animals. PMID:22193109

  5. Reduced CYFIP1 in Human Neural Progenitors Results in Dysregulation of Schizophrenia and Epilepsy Gene Networks.

    PubMed

    Nebel, Rebecca A; Zhao, Dejian; Pedrosa, Erika; Kirschen, Jill; Lachman, Herbert M; Zheng, Deyou; Abrahams, Brett S

    2016-01-01

    Deletions encompassing the BP1-2 region at 15q11.2 increase schizophrenia and epilepsy risk, but only some carriers have either disorder. To investigate the role of CYFIP1, a gene within the region, we performed knockdown experiments in human neural progenitors derived from donors with 2 copies of each gene at the BP1-2 locus. RNA-seq and cellular assays determined that knockdown of CYFIP1 compromised cytoskeletal remodeling. FMRP targets and postsynaptic density genes, each implicated in schizophrenia, were significantly overrepresented among differentially expressed genes (DEGs). Schizophrenia and/or epilepsy genes, but not those associated with randomly selected disorders, were likewise significantly overrepresented. Mirroring the variable expressivity seen in deletion carriers, marked between-line differences were observed for dysregulation of disease genes. Finally, a subset of DEGs showed a striking similarity to known epilepsy genes and represents novel disease candidates. Results support a role for CYFIP1 in disease and demonstrate that disease-related biological signatures are apparent prior to neuronal differentiation.

  6. Epigallocatechin gallate (EGCG) inhibits adhesion and migration of neural progenitor cells in vitro.

    PubMed

    Barenys, Marta; Gassmann, Kathrin; Baksmeier, Christine; Heinz, Sabrina; Reverte, Ingrid; Schmuck, Martin; Temme, Thomas; Bendt, Farina; Zschauer, Tim-Christian; Rockel, Thomas Dino; Unfried, Klaus; Wätjen, Wim; Sundaram, Sivaraj Mohana; Heuer, Heike; Colomina, Maria Teresa; Fritsche, Ellen

    2017-02-01

    Food supplements based on herbal products are widely used during pregnancy as part of a self-care approach. The idea that such supplements are safe and healthy is deeply seated in the general population, although they do not underlie the same strict safety regulations than medical drugs. We aimed to characterize the neurodevelopmental effects of the green tea catechin epigallocatechin gallate (EGCG), which is now commercialized as high-dose food supplement. We used the "Neurosphere Assay" to study the effects and unravel underlying molecular mechanisms of EGCG treatment on human and rat neural progenitor cells (NPCs) development in vitro. EGCG alters human and rat NPC development in vitro. It disturbs migration distance, migration pattern, and nuclear density of NPCs growing as neurospheres. These functional impairments are initiated by EGCG binding to the extracellular matrix glycoprotein laminin, preventing its binding to β1-integrin subunits, thereby prohibiting cell adhesion and resulting in altered glia alignment and decreased number of migrating young neurons. Our data raise a concern on the intake of high-dose EGCG food supplements during pregnancy and highlight the need of an in vivo characterization of the effects of high-dose EGCG exposure during neurodevelopment.

  7. Low oxygen alters mitochondrial function and response to oxidative stress in human neural progenitor cells

    PubMed Central

    Lages, Yury M.; Nascimento, Juliana M.; Lemos, Gabriela A.; Galina, Antonio; Castilho, Leda R.

    2015-01-01

    Oxygen concentration should be carefully regulated in all living tissues, beginning at the early embryonic stages. Unbalances in oxygen regulation can lead to cell death and disease. However, to date, few studies have investigated the consequences of variations in oxygen levels for fetal-like cells. Therefore, in the present work, human neural progenitor cells (NPCs) derived from pluripotent stem cells grown in 3% oxygen (v/v) were compared with NPCs cultured in 21% (v/v) oxygen. Low oxygen concentrations altered the mitochondrial content and oxidative functions of the cells, which led to improved ATP production, while reducing generation of reactive oxygen species (ROS). NPCs cultured in both conditions showed no differences in proliferation and glucose metabolism. Furthermore, antioxidant enzymatic activity was not altered in NPCs cultured in 3% oxygen under normal conditions, however, when exposed to external agents known to induce oxidative stress, greater susceptibility to DNA damage was observed. Our findings indicate that the management of oxygen levels should be considered for in vitro models of neuronal development and drug screening. PMID:26713239

  8. Common mechanisms linking connexin43 to neural progenitor cell migration and glioma invasion.

    PubMed

    Naus, Christian C; Aftab, Qurratulain; Sin, Wun Chey

    2016-02-01

    Cell migration is critical for cell differentiation, tissue formation and organ development. Several mechanisms come to play in the process of cell migration, orchestrating changes in cell polarity, adhesion, process extension and motility. Recent findings have shown that gap junctions, and specifically connexin43 (Cx43), can play a significant role in these processes, impacting adhesion and cytoskeletal rearrangements. Thus Cx43 within a cell regulates its motility and migration via intracellular signaling. Furthermore, Cx43 in the host cells can impact the degree of cellular migration through that tissue. Similarities in these connexin-based processes account for both neural progenitor migration in the developing brain, and for glioma cell invasion in the mature brain. In both cases, Cx43 in the tissue ("soil") in which cells ("seeds") exist facilitates their migration and, for glioma cells, tissue invasion. Cx43 mediates these effects through channel- and non-channel-dependent mechanisms which have similarities in both paradigms of cell migration. This provides insight into developmental processes and pathological situations, as well as possible therapeutic approaches regarding specific functional domains of gap junction proteins. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

  9. CXCL12-mediated murine neural progenitor cell movement requires PI3Kβ activation.

    PubMed

    Holgado, Borja L; Martínez-Muñoz, Laura; Sánchez-Alcañiz, Juan Antonio; Lucas, Pilar; Pérez-García, Vicente; Pérez, Gema; Rodríguez-Frade, José Miguel; Nieto, Marta; Marín, Oscar; Carrasco, Yolanda R; Carrera, Ana C; Alvarez-Dolado, Manuel; Mellado, Mario

    2013-08-01

    The migratory route of neural progenitor/precursor cells (NPC) has a central role in central nervous system development. Although the role of the chemokine CXCL12 in NPC migration has been described, the intracellular signaling cascade involved remains largely unclear. Here we studied the molecular mechanisms that promote murine NPC migration in response to CXCL12, in vitro and ex vivo. Migration was highly dependent on signaling by the CXCL12 receptor, CXCR4. Although the JAK/STAT pathway was activated following CXCL12 stimulation of NPC, JAK activity was not necessary for NPC migration in vitro. Whereas CXCL12 activated the PI3K catalytic subunits p110α and p110β in NPC, only p110β participated in CXCL12-mediated NPC migration. Ex vivo experiments using organotypic slice cultures showed that p110β blockade impaired NPC exit from the medial ganglionic eminence. In vivo experiments using in utero electroporation nonetheless showed that p110β is dispensable for radial migration of pyramidal neurons. We conclude that PI3K p110β is activated in NPC in response to CXCL12, and its activity is necessary for immature interneuron migration to the cerebral cortex.

  10. The Drosophila Sp8 transcription factor Buttonhead prevents premature differentiation of intermediate neural progenitors

    PubMed Central

    Xie, Yonggang; Li, Xiaosu; Zhang, Xian; Mei, Shaolin; Li, Hongyu; Urso, Andreacarola; Zhu, Sijun

    2014-01-01

    Intermediate neural progenitor cells (INPs) need to avoid differentiation and cell cycle exit while maintaining restricted developmental potential, but mechanisms preventing differentiation and cell cycle exit of INPs are not well understood. In this study, we report that the Drosophila homolog of mammalian Sp8 transcription factor Buttonhead (Btd) prevents premature differentiation and cell cycle exit of INPs in Drosophila larval type II neuroblast (NB) lineages. We show that the loss of Btd leads to elimination of mature INPs due to premature differentiation of INPs into terminally dividing ganglion mother cells. We provide evidence to demonstrate that Btd prevents the premature differentiation by suppressing the expression of the homeodomain protein Prospero in immature INPs. We further show that Btd functions cooperatively with the Ets transcription factor Pointed P1 to promote the generation of INPs. Thus, our work reveals a critical mechanism that prevents premature differentiation and cell cycle exit of Drosophila INPs. DOI: http://dx.doi.org/10.7554/eLife.03596.001 PMID:25285448

  11. Fragile x mental retardation protein regulates proliferation and differentiation of adult neural stem/progenitor cells.

    PubMed

    Luo, Yuping; Shan, Ge; Guo, Weixiang; Smrt, Richard D; Johnson, Eric B; Li, Xuekun; Pfeiffer, Rebecca L; Szulwach, Keith E; Duan, Ranhui; Barkho, Basam Z; Li, Wendi; Liu, Changmei; Jin, Peng; Zhao, Xinyu

    2010-04-08

    Fragile X syndrome (FXS), the most common form of inherited mental retardation, is caused by the loss of functional fragile X mental retardation protein (FMRP). FMRP is an RNA-binding protein that can regulate the translation of specific mRNAs. Adult neurogenesis, a process considered important for neuroplasticity and memory, is regulated at multiple molecular levels. In this study, we investigated whether Fmrp deficiency affects adult neurogenesis. We show that in a mouse model of fragile X syndrome, adult neurogenesis is indeed altered. The loss of Fmrp increases the proliferation and alters the fate specification of adult neural progenitor/stem cells (aNPCs). We demonstrate that Fmrp regulates the protein expression of several components critical for aNPC function, including CDK4 and GSK3beta. Dysregulation of GSK3beta led to reduced Wnt signaling pathway activity, which altered the expression of neurogenin1 and the fate specification of aNPCs. These data unveil a novel regulatory role for Fmrp and translational regulation in adult neurogenesis.

  12. Lentiviral vector-mediated genetic modification of human neural progenitor cells for ex vivo gene therapy.

    PubMed

    Capowski, Elizabeth E; Schneider, Bernard L; Ebert, Allison D; Seehus, Corey R; Szulc, Jolanta; Zufferey, Romain; Aebischer, Patrick; Svendsen, Clive N

    2007-07-30

    Human neural progenitor cells (hNPC) hold great potential as an ex vivo system for delivery of therapeutic proteins to the central nervous system. When cultured as aggregates, termed neurospheres, hNPC are capable of significant in vitro expansion. In the current study, we present a robust method for lentiviral vector-mediated gene delivery into hNPC that maintains the differentiation and proliferative properties of neurosphere cultures while minimizing the amount of viral vector used and controlling the number of insertion sites per population. This method results in long-term, stable expression even after differentiation of the hNPC to neurons and astrocytes and allows for generation of equivalent transgenic populations of hNPC. In addition, the in vitro analysis presented predicts the behavior of transgenic lines in vivo when transplanted into a rodent model of Parkinson's disease. The methods presented provide a powerful tool for assessing the impact of factors such as promoter systems or different transgenes on the therapeutic utility of these cells.

  13. Zika virus infection induces mitosis abnormalities and apoptotic cell death of human neural progenitor cells

    PubMed Central

    Souza, Bruno S. F.; Sampaio, Gabriela L. A.; Pereira, Ciro S.; Campos, Gubio S.; Sardi, Silvia I.; Freitas, Luiz A. R.; Figueira, Claudio P.; Paredes, Bruno D.; Nonaka, Carolina K. V.; Azevedo, Carine M.; Rocha, Vinicius P. C.; Bandeira, Antonio C.; Mendez-Otero, Rosalia; dos Santos, Ricardo Ribeiro; Soares, Milena B. P.

    2016-01-01

    Zika virus (ZIKV) infection has been associated with severe complications both in the developing and adult nervous system. To investigate the deleterious effects of ZIKV infection, we used human neural progenitor cells (NPC), derived from induced pluripotent stem cells (iPSC). We found that NPC are highly susceptible to ZIKV and the infection results in cell death. ZIKV infection led to a marked reduction in cell proliferation, ultrastructural alterations and induction of autophagy. Induction of apoptosis of Sox2+ cells was demonstrated by activation of caspases 3/7, 8 and 9, and by ultrastructural and flow cytometry analyses. ZIKV-induced death of Sox2+ cells was prevented by incubation with the pan-caspase inhibitor, Z-VAD-FMK. By confocal microscopy analysis we found an increased number of cells with supernumerary centrosomes. Live imaging showed a significant increase in mitosis abnormalities, including multipolar spindle, chromosome laggards, micronuclei and death of progeny after cell division. FISH analysis for chromosomes 12 and 17 showed increased frequency of aneuploidy, such as monosomy, trisomy and polyploidy. Our study reinforces the link between ZIKV and abnormalities in the developing human brain, including microcephaly. PMID:28008958

  14. CXCR4/CXCR7 molecular involvement in neuronal and neural progenitor migration: focus in CNS repair.

    PubMed

    Merino, José Joaquín; Bellver-Landete, Victor; Oset-Gasque, María Jesús; Cubelos, Beatriz

    2015-01-01

    In the adult brain, neural progenitor cells (NPCs) reside in the subventricular zone (SVZ) of the lateral ventricles, the dentate gyrus and the olfactory bulb. Following CNS insult, NPCs from the SVZ can migrate along the rostral migratory stream (RMS), a migration of NPCs that is directed by proinflammatory cytokines. Cells expressing CXCR4 follow a homing signal that ultimately leads to neuronal integration and CNS repair, although such molecules can also promote NPC quiescence. The ligand, SDF1 alpha (or CXCL12) is one of the chemokines secreted at sites of injury that it is known to attract NSC-derived neuroblasts, cells that express CXCR4. In function of its concentration, CXCL12 can induce different responses, promoting NPC migration at low concentrations while favoring cell adhesion via EGF and the alpha 6 integrin at high CXCL12 concentrations. However, the preclinical effectiveness of chemokines and their relationship with NPC mobilization requires further study, particularly with respect to CNS repair. NPC migration may also be affected by the release of cytokines or chemokines induced by local inflammation, through autocrine or paracrine mechanisms, as well as through erythropoietin (EPO) or nitric oxide (NO) release. CXCL12 activity requires G-coupled proteins and the availability of its ligand may be modulated by its binding to CXCR7, for which it shows a stronger affinity than for CXCR4.

  15. Astaxanthin Improves Stem Cell Potency via an Increase in the Proliferation of Neural Progenitor Cells

    PubMed Central

    Kim, Jeong-Hwan; Nam, Soo-Wan; Kim, Byung-Woo; Choi, Woobong; Lee, Jong-Hwan; Kim, Wun-Jae; Choi, Yung-Hyun

    2010-01-01

    The present study was designed to investigate the question of whether or not astaxanthin improves stem cell potency via an increase in proliferation of neural progenitor cells (NPCs). Treatment with astaxanthin significantly increased proliferation and colony formation of NPCs. For identification of possible activated signaling molecules involved in active cell proliferation occurring after astaxanthin treatment, total protein levels of several proliferation-related proteins, and expression levels of proliferation-related transcription factors, were assessed in NPCs. In Western blot analysis, astaxanthin induced significant activation of phosphatidylinositol 3-kinase (PI3K) and its downstream mediators in a time-dependent manner. Results of RT-PCR analysis showed upregulation of proliferation-related transcription factors and stemness genes. To estimate the relevance of PI3K and mitogen-activated protein, or extracellular signal-regulated kinase kinase (MEK) signaling pathways in cell growth of astaxanthin-treated NPCs, inhibition assays were performed with LY294002, a specific inhibitor of PI3K, and PD98059, a specific inhibitor of MEK, respectively. These results clearly showed that astaxanthin induces proliferation of NPCs via activation of the PI3K and MEK signaling pathways and improves stem cell potency via stemness acting signals. PMID:21614195

  16. Effects of neural progenitor cell transplantation in children with severe cerebral palsy.

    PubMed

    Luan, Zuo; Liu, Weipeng; Qu, Suqing; Du, Kan; He, Sheng; Wang, Zhaoyan; Yang, Yinxiang; Wang, Caiying; Gong, Xiaojun

    2012-01-01

    Cerebral palsy (CP) is a chronic nervous system disease that severely damages the physical and developmental health of children. Traditional treatment brings about only improvement of mild to moderate CP, but severe CP still lacks effective interventions. To explore safety and efficacy of using neural progenitor cells (NPCs) to treat CP in children, we performed NPC transplantation in 45 patients with severe CP by injecting NPCs derived from aborted fetal tissue into the lateral ventricle. Gross motor function measures (GMFM), the Peabody Developmental Motor Scale-Fine Motor (PDMS-FM) test, and a unified survey questionnaire designed specifically for children with CP were used to evaluate neurological function of the patients. Motor development was significantly accelerated within the first month after cell transplantation, but the rate of improvement gradually slowed to preoperative levels. However, after 1 year, the developmental level in each functional sphere (gross motor, fine motor, and cognition) of the treatment group was significantly higher compared to the control group. No delayed complications of this therapy were noted. These results suggest that NPC transplantation is a safe and effective therapeutic method for treating children with severe CP.

  17. [Treatment of cerebral palsy with transplantation of human neural progenitor cells].

    PubMed

    Liu, Wei-Peng; Qu, Su-Qing; Luan, Zuo; Gong, Xiao-Jun; Wang, Zhao-Yan

    2012-10-01

    To study the clinical efficacy of transplantation of human neural progenitor cells (hNPCs) in the treatment of severe cerebral palsy (CP) in children. Forty-five children with CP were voluntarily accepted transplantation of hNPCs. The cells obtained from the forebrain of 10 to 12-week-fetus were cultured and amplified into hNPCs. Then the hNPCs were injected into the cerebral ventricle of the patients with the supersonic guidance. Dyssomnia, irritability and muscular tension were improved in one patient 3 days after transplantation. The clinical improvements were observed in the majority of the patients 1 month after transplantation. The therapeutic effects slowed down 3 to 6 months after transplantation. One year after transplantation the gross and fine motor skills and the congnition ability in the transplantation group were considerably surpassed to those in the control group. No delayed severe complications were observed after transplantation. No tumorigenesis was noted 5 years after transplantation. The transplantation of hNPCs as a novel therapy is effective and safe for severe CP. Many investigations are needed to evaluate the effect of the therapy.

  18. Endogenous neural stem/progenitor cells stabilize the cortical microenvironment after traumatic brain injury.

    PubMed

    Dixon, Kirsty J; Theus, Michelle H; Nelersa, Claudiu M; Mier, Jose; Travieso, Lissette G; Yu, Tzong-Shiue; Kernie, Steven G; Liebl, Daniel J

    2015-06-01

    Although a myriad of pathological responses contribute to traumatic brain injury (TBI), cerebral dysfunction has been closely linked to cell death mechanisms. A number of therapeutic strategies have been studied in an attempt to minimize or ameliorate tissue damage; however, few studies have evaluated the inherent protective capacity of the brain. Endogenous neural stem/progenitor cells (NSPCs) reside in distinct brain regions and have been shown to respond to tissue damage by migrating to regions of injury. Until now, it remained unknown whether these cells have the capacity to promote endogenous repair. We ablated NSPCs in the subventricular zone to examine their contribution to the injury microenvironment after controlled cortical impact (CCI) injury. Studies were performed in transgenic mice expressing the herpes simplex virus thymidine kinase gene under the control of the nestin(δ) promoter exposed to CCI injury. Two weeks after CCI injury, mice deficient in NSPCs had reduced neuronal survival in the perilesional cortex and fewer Iba-1-positive and glial fibrillary acidic protein-positive glial cells but increased glial hypertrophy at the injury site. These findings suggest that the presence of NSPCs play a supportive role in the cortex to promote neuronal survival and glial cell expansion after TBI injury, which corresponds with improvements in motor function. We conclude that enhancing this endogenous response may have acute protective roles after TBI.

  19. LXR agonists promote the proliferation of neural progenitor cells through MEK-ERK pathway.

    PubMed

    Wang, Jing-Zhong; Fang, Yan; Ji, Wei-Dong; Xu, Hui

    2017-01-29

    The liver X receptors (LXRs) are transcriptional regulators of lipid homeostasis and may be critical for neurodegeneration and neurogenesis in vivo. However, it remains largely unknown about the role of LXRs and its agonists in the in vitro proliferation of neural progenitor cells (NPCs). Here we revealed for the first time that LXRs were markedly expressed in mouse NPCs and were critical for the in vitro proliferation. LXR agonists GW3965 and LXR623 promoted the proliferation of wildtype NPCs, but not NPCs from LXR double-knockout mice. Mechanistically, phosphorylation of MEK1/2 and ERK1/2 in NPCs was enhanced upon LXR agonist treatment, while abrogation of MEK/ERK phosphorylation by the inhibitors PD98059 and U0126 impaired the proliferation of wildtype NPCs in the presence or absence of LXR agonists. Collectively, our findings suggest that LXR agonists GW3965 and LXR623 can stimulate the NPC proliferation in LXR- and MEK/ERK-dependent manner. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. 3D Normal Human Neural Progenitor Tissue-Like Assemblies: A Model of Persistent VZV Infection

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas J.

    2013-01-01

    Varicella-zoster virus (VZV) is a neurotropic human alphaherpesvirus that causes varicella upon primary infection, establishes latency in multiple ganglionic neurons, and can reactivate to cause zoster. Live attenuated VZV vaccines are available; however, they can also establish latent infections and reactivate. Studies of VZV latency have been limited to the analyses of human ganglia removed at autopsy, as the virus is strictly a human pathogen. Recently, terminally differentiated human neurons have received much attention as a means to study the interaction between VZV and human neurons; however, the short life-span of these cells in culture has limited their application. Herein, we describe the construction of a model of normal human neural progenitor cells (NHNP) in tissue-like assemblies (TLAs), which can be successfully maintained for at least 180 days in three-dimensional (3D) culture, and exhibit an expression profile similar to that of human trigeminal ganglia. Infection of NHNP TLAs with cell-free VZV resulted in a persistent infection that was maintained for three months, during which the virus genome remained stable. Immediate-early, early and late VZV genes were transcribed, and low-levels of infectious VZV were recurrently detected in the culture supernatant. Our data suggest that NHNP TLAs are an effective system to investigate long-term interactions of VZV with complex assemblies of human neuronal cells.

  1. Rapid Ngn2-induction of excitatory neurons from hiPSC-derived neural progenitor cells

    PubMed Central

    Ho, Seok-Man; Hartley, Brigham J.; Julia, TCW; Beaumont, Michael; Stafford, Khalifa; Slesinger, Paul A.; Brennand, Kristen J.

    2015-01-01

    Since the discovery of somatic reprogramming, human induced pluripotent stem cells (hiPSCs) have been exploited to model a variety of neurological and psychiatric disorders. Because hiPSCs represent an almost limitless source of patient-derived neurons that retain the genetic variations thought to contribute to disease etiology, they have been heralded as a patient-specific platform for high throughput drug screening. However, the utility of current protocols for generating neurons from hiPSCs remains limited by protracted differentiation timelines and heterogeneity of the neuronal phenotypes produced. Neuronal induction via the forced expression of exogenous transcription factors rapidly induces defined populations of functional neurons from fibroblasts and hiPSCs. Here, we describe an adapted protocol that accelerates maturation of functional excitatory neurons from hiPSC-derived neural progenitor cells (NPCs) via lentiviral transduction of Neurogenin 2 (using both mNgn2 and hNGN2). This methodology, relying upon a robust and scalable starting population of hiPSC NPCs, should be readily amenable to scaling for hiPSC-based high-throughput drug screening. PMID:26626326

  2. Methylglyoxal Causes Cell Death in Neural Progenitor Cells and Impairs Adult Hippocampal Neurogenesis.

    PubMed

    Chun, Hye Jeong; Lee, Yujeong; Kim, Ah Hyun; Lee, Jaewon

    2016-04-01

    Methylglyoxal (MG) is formed during normal metabolism by processes like glycolysis, lipid peroxidation, and threonine catabolism, and its accumulation is associated with various degenerative diseases, such as diabetes and arterial atherogenesis. Furthermore, MG has also been reported to have toxic effects on hippocampal neurons. However, these effects have not been studied in the context of neurogenesis. Here, we report that MG adversely affects hippocampal neurogenesis and induces neural progenitor cell (NPC) death. MG significantly reduced C17.2 NPC proliferation, and high concentration of MG (500 μM) induced cell death and elevated oxidative stress. Further, MG was found to activate the ERK signaling pathway, indicating elevated stress response. To determine the effects of MG in vivo, mice were administrated with vehicle or MG (0.5 or 1 % in drinking water) for 4 weeks. The numbers of BrdU-positive cells in hippocampi were significantly lower in MG-treated mice, indicating impaired neurogenesis, but MG did not induce neuronal damage or glial activations. Interestingly, MG reduced memory retention when administered to mice at 1 % but not at 0.5 %. In addition, the levels of hippocampal BDNF and synaptophysin were significantly lower in the hippocampi of mice treated with MG at 1 %. Collectively, our findings suggest MG could be harmful to NPCs and to hippocampal neurogenesis.

  3. Loss of Brap Results in Premature G1/S Phase Transition and Impeded Neural Progenitor Differentiation.

    PubMed

    Lanctot, Alison A; Guo, Yan; Le, Yicong; Edens, Brittany M; Nowakowski, Richard S; Feng, Yuanyi

    2017-08-01

    Cells initiate fate decisions during G1 phase by converting extracellular signals into distinctive cell cycle kinetics. The DNA replication timing is determined in G1 phase; lengthened G1 and hastened S phases correlate with increased neurogenic propensity of neural progenitor cells (NPCs), although the underlying molecular control remains elusive. Here, we report that proper G1 phase completion in NPCs requires Brap, a Ras-Erk signaling modulator with ubiquitin E3 ligase activity. We identified Skp2 and Skp2-associated SCF ubiquitin ligase as a key target of Brap-mediated polyubiquitination. Loss of Brap resulted in elevated Skp2, which increased p27(Kip1) destruction, leading to G1 phase truncation and premature S phase entry. The aberrantly executed G1 in Brap-mutant NPCs, followed by hindered S phase progression and increased G2 phase arrest, which together prolonged the cell cycle, impeded neuronal differentiation and culminated in microcephaly. These findings demonstrate that neuronal differentiation is potentiated during G1 phase by Brap-directed cascade of events in cell signaling and protein turnover. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  4. Endogenous Neural Stem/Progenitor Cells Stabilize the Cortical Microenvironment after Traumatic Brain Injury

    PubMed Central

    Dixon, Kirsty J.; Theus, Michelle H.; Nelersa, Claudiu M.; Mier, Jose; Travieso, Lissette G.; Yu, Tzong-Shiue; Kernie, Steven G.

    2015-01-01

    Abstract Although a myriad of pathological responses contribute to traumatic brain injury (TBI), cerebral dysfunction has been closely linked to cell death mechanisms. A number of therapeutic strategies have been studied in an attempt to minimize or ameliorate tissue damage; however, few studies have evaluated the inherent protective capacity of the brain. Endogenous neural stem/progenitor cells (NSPCs) reside in distinct brain regions and have been shown to respond to tissue damage by migrating to regions of injury. Until now, it remained unknown whether these cells have the capacity to promote endogenous repair. We ablated NSPCs in the subventricular zone to examine their contribution to the injury microenvironment after controlled cortical impact (CCI) injury. Studies were performed in transgenic mice expressing the herpes simplex virus thymidine kinase gene under the control of the nestinδ promoter exposed to CCI injury. Two weeks after CCI injury, mice deficient in NSPCs had reduced neuronal survival in the perilesional cortex and fewer Iba-1-positive and glial fibrillary acidic protein-positive glial cells but increased glial hypertrophy at the injury site. These findings suggest that the presence of NSPCs play a supportive role in the cortex to promote neuronal survival and glial cell expansion after TBI injury, which corresponds with improvements in motor function. We conclude that enhancing this endogenous response may have acute protective roles after TBI. PMID:25290253

  5. The Lysine Acetyltransferase Activator Brpf1 Governs Dentate Gyrus Development through Neural Stem Cells and Progenitors

    PubMed Central

    You, Linya; Yan, Kezhi; Zhou, Jinfeng; Zhao, Hong; Bertos, Nicholas R.; Park, Morag; Wang, Edwin; Yang, Xiang-Jiao

    2015-01-01

    Lysine acetylation has recently emerged as an important post-translational modification in diverse organisms, but relatively little is known about its roles in mammalian development and stem cells. Bromodomain- and PHD finger-containing protein 1 (BRPF1) is a multidomain histone binder and a master activator of three lysine acetyltransferases, MOZ, MORF and HBO1, which are also known as KAT6A, KAT6B and KAT7, respectively. While the MOZ and MORF genes are rearranged in leukemia, the MORF gene is also mutated in prostate and other cancers and in four genetic disorders with intellectual disability. Here we show that forebrain-specific inactivation of the mouse Brpf1 gene causes hypoplasia in the dentate gyrus, including underdevelopment of the suprapyramidal blade and complete loss of the infrapyramidal blade. We trace the developmental origin to compromised Sox2+ neural stem cells and Tbr2+ intermediate neuronal progenitors. We further demonstrate that Brpf1 loss deregulates neuronal migration, cell cycle progression and transcriptional control, thereby causing abnormal morphogenesis of the hippocampus. These results link histone binding and acetylation control to hippocampus development and identify an important epigenetic regulator for patterning the dentate gyrus, a brain structure critical for learning, memory and adult neurogenesis. PMID:25757017

  6. BET bromodomain inhibition promotes neurogenesis while inhibiting gliogenesis in neural progenitor cells.

    PubMed

    Li, Jingjun; Ma, Jing; Meng, Guofeng; Lin, Hong; Wu, Sharon; Wang, Jamie; Luo, Jie; Xu, Xiaohong; Tough, David; Lindon, Matthew; Rioja, Inmaculada; Zhao, Jing; Mei, Hongkang; Prinjha, Rab; Zhong, Zhong

    2016-09-01

    Neural stem cells and progenitor cells (NPCs) are increasingly appreciated to hold great promise for regenerative medicine to treat CNS injuries and neurodegenerative diseases. However, evidence for effective stimulation of neuronal production from endogenous or transplanted NPCs for neuron replacement with small molecules remains limited. To identify novel chemical entities/targets for neurogenesis, we had established a NPC phenotypic screen assay and validated it using known small-molecule neurogenesis inducers. Through screening small molecule libraries with annotated targets, we identified BET bromodomain inhibition as a novel mechanism for enhancing neurogenesis. BET bromodomain proteins, Brd2, Brd3, and Brd4 were found to be downregulated in NPCs upon differentiation, while their levels remain unaltered in proliferating NPCs. Consistent with the pharmacological study using bromodomain selective inhibitor (+)-JQ-1, knockdown of each BET protein resulted in an increase in the number of neurons with simultaneous reduction in both astrocytes and oligodendrocytes. Gene expression profiling analysis demonstrated that BET bromodomain inhibition induced a broad but specific transcription program enhancing directed differentiation of NPCs into neurons while suppressing cell cycle progression and gliogenesis. Together, these results highlight a crucial role of BET proteins as epigenetic regulators in NPC development and suggest a therapeutic potential of BET inhibitors in treating brain injuries and neurodegenerative diseases. Copyright © 2016 Helmholtz Zentrum München. Published by Elsevier B.V. All rights reserved.

  7. Differential Expression of CXCL12 and CXCR4 During Human Fetal Neural Progenitor Cell Differentiation

    PubMed Central

    Peng, Hui; Kolb, Ryan; Kennedy, J. E.

    2007-01-01

    Stromal cell-derived factor 1 alpha (SDF-1α, CXCL12) and its receptor CXCR4 play an important role in the central nervous system (CNS) development and adulthood by mediating cell migration, enhancing precursor cell proliferation, assisting in neuronal circuit formation, and possibly regulating migration during repair. The expression pattern of CXCR4 and CXCL12 during neurogenesis has not been thoroughly elucidated. In this study, we investigated the expression of CXCL12 and CXCR4 during neural progenitor cells (NPC) differentiation by microarray analysis and reverse transcriptase-polymerase chain reaction (RT-PCR) using human fetal NPC as a model system. The production of CXCL12 was measured by enzyme-linked immunosorbent assay (ELISA). CXCR4 expression was determined by florescence-activated cell sorting (FACS) analysis, immunocytochemical staining, and CXCR4-mediated inhibition of cyclic AMP (cAMP) accumulation. Our data demonstrated that CXCR4 expression is significantly upregulated when NPC are differentiated into neuronal precursors, whereas CXCL12 is upregulated when differentiated into astrocytes. We also provide evidence that CXCR4 localization changes as neurons mature. In neuronal precursors, CXCR4 is localized in both neuronal processes and the cell body, whereas in mature neurons, it is primarily expressed on axons and dendrites. This differential expression of CXCR4 and CXCL12 may be important for the temporal regulation of neuronal migration and circuit formation during development and possibly in adult neurogenesis and repair. PMID:18040858

  8. Injury-induced neurogenesis: consideration of resident microglia as supportive of neural progenitor cells

    PubMed Central

    McPherson, Christopher A.; Kraft, Andrew D.; Harry, G. Jean

    2010-01-01

    The induction of neurogenesis in the adult subgranular zone (SGZ) by injury is often accompanied by changes in the extracellular environment that can have significant impacts on neural progenitor cells (NPC). We examined the induction of neurogenesis in the SGZ at 72 hrs following an injection of the hippocampal toxicant, trimethyltin (TMT; 2mg/kg, ip) inducing apoptosis in dentate granule neurons. BrdU+ incorporation during the active period of neuronal death indicated NPC proliferation and migration of newly generated cells into the granule cell layer (GCL). BrdU+ cells were transiently in contact with process bearing microglia within the inner SGZ layer. Contact with GFAP+ astrocyte processes occurred once cells were within the GCL. A small percentage of the BrdU+ cells within the SGZ region showed immunoreactivity for tumor necrosis factor (TNF) p75 receptor (TNFp75R). In mice deficient for TNFp75R, TMT injection produced an equivalent level of dentate granule cell death however; BrdU+ cells were localized at the SGZ as compared to the presence of cells within the GCL in the WT mice dosed with TMT. These data suggest that cells generated by NPCs in the SGZ induced with a focal lesion to the dentate granule neurons of adolescent mice maintain the capacity to utilize the neuroinflammation and microglia responses within their environment for migration into the GCL. PMID:20524106

  9. Mifepristone-inducible transgene expression in neural progenitor cells in vitro and in vivo

    PubMed Central

    Hjelm, BE; Grunseich, C; Gowing, G; Avalos, P; Tian, J; Shelley, BC; Mooney, M; Narwani, K; Shi, Y; Svendsen, CN; Wolfe, JH; Fischbeck, KH; Pierson, TM

    2016-01-01

    Numerous gene and cell therapy strategies are being developed for the treatment of neurodegenerative disorders. Many of these strategies use constitutive expression of therapeutic transgenic proteins, and although functional in animal models of disease, this method is less likely to provide adequate flexibility for delivering therapy to humans. Ligand-inducible gene expression systems may be more appropriate for these conditions, especially within the central nervous system (CNS). Mifepristone’s ability to cross the blood–brain barrier makes it an especially attractive ligand for this purpose. We describe the production of a mifepristone-inducible vector system for regulated expression of transgenes within the CNS. Our inducible system used a lentivirus-based vector platform for the ex vivo production of mifepristone-inducible murine neural progenitor cells that express our transgenes of interest. These cells were processed through a series of selection steps to ensure that the cells exhibited appropriate transgene expression in a dose-dependent and temporally controlled manner with minimal background activity. Inducible cells were then transplanted into the brains of rodents, where they exhibited appropriate mifepristone-inducible expression. These studies detail a strategy for regulated expression in the CNS for use in the development of safe and efficient gene therapy for neurological disorders. PMID:26863047

  10. Mifepristone-inducible transgene expression in neural progenitor cells in vitro and in vivo.

    PubMed

    Hjelm, B E; Grunseich, C; Gowing, G; Avalos, P; Tian, J; Shelley, B C; Mooney, M; Narwani, K; Shi, Y; Svendsen, C N; Wolfe, J H; Fischbeck, K H; Pierson, T M

    2016-05-01

    Numerous gene and cell therapy strategies are being developed for the treatment of neurodegenerative disorders. Many of these strategies use constitutive expression of therapeutic transgenic proteins, and although functional in animal models of disease, this method is less likely to provide adequate flexibility for delivering therapy to humans. Ligand-inducible gene expression systems may be more appropriate for these conditions, especially within the central nervous system (CNS). Mifepristone's ability to cross the blood-brain barrier makes it an especially attractive ligand for this purpose. We describe the production of a mifepristone-inducible vector system for regulated expression of transgenes within the CNS. Our inducible system used a lentivirus-based vector platform for the ex vivo production of mifepristone-inducible murine neural progenitor cells that express our transgenes of interest. These cells were processed through a series of selection steps to ensure that the cells exhibited appropriate transgene expression in a dose-dependent and temporally controlled manner with minimal background activity. Inducible cells were then transplanted into the brains of rodents, where they exhibited appropriate mifepristone-inducible expression. These studies detail a strategy for regulated expression in the CNS for use in the development of safe and efficient gene therapy for neurological disorders.

  11. The effect of interferon-{beta} on mouse neural progenitor cell survival and differentiation

    SciTech Connect

    Hirsch, Marek; Knight, Julia; Tobita, Mari; Soltys, John; Panitch, Hillel; Mao-Draayer, Yang

    2009-10-16

    Interferon-{beta} (IFN-{beta}) is a mainstay therapy for relapse-remitting multiple sclerosis (MS). However, the direct effects of IFN-{beta} on the central nervous system (CNS) are not well understood. To determine whether IFN-{beta} has direct neuroprotective effects on CNS cells, we treated adult mouse neural progenitor cells (NPCs) in vitro with IFN-{beta} and examined the effects on proliferation, apoptosis, and differentiation. We found that mouse NPCs express high levels of IFN{alpha}/{beta} receptor (IFNAR). In response to IFN-{beta} treatment, no effect was observed on differentiation or proliferation. However, IFN-{beta} treated mouse NPCs demonstrated decreased apoptosis upon growth factor withdrawal. Pathway-specific polymerase chain reaction (PCR) arrays demonstrated that IFN-{beta} treatment upregulated the STAT 1 and 2 signaling pathway, as well as GFRA2, NOD1, Caspases 1 and 12, and TNFSF10. These results suggest that IFN-{beta} can directly affect NPC survival, possibly playing a neuroprotective role in the CNS by modulating neurotrophic factors.

  12. Patient-specific models of microglia-mediated engulfment of synapses and neural progenitors

    PubMed Central

    Sellgren, C M; Sheridan, S D; Gracias, J; Xuan, D; Fu, T; Perlis, R H

    2017-01-01

    Engulfment of synapses and neural progenitor cells (NPCs) by microglia is critical for the development and maintenance of proper brain circuitry, and has been implicated in neurodevelopmental as well as neurodegenerative disease etiology. We have developed and validated models of these mechanisms by reprogramming microglia-like cells from peripheral blood mononuclear cells, and combining them with NPCs and neurons derived from induced pluripotent stem cells to create patient-specific cellular models of complement-dependent synaptic pruning and elimination of NPCs. The resulting microglia-like cells express appropriate markers and function as primary human microglia, while patient-matched macrophages differ markedly. As a demonstration of disease-relevant application, we studied the role of C4, recently implicated in schizophrenia, in engulfment of synaptic structures by human microglia. The ability to create complete patient-specific cellular models of critical microglial functions utilizing samples taken during a single clinical visit will extend the ability to model central nervous system disease while facilitating high-throughput screening. PMID:27956744

  13. Neurotoxic effect of 2,5-hexanedione on neural progenitor cells and hippocampal neurogenesis.

    PubMed

    Kim, Min-Sun; Park, Hee Ra; Park, Mikyung; Kim, So Jung; Kwon, Mugil; Yu, Byung Pal; Chung, Hae Young; Kim, Hyung Sik; Kwack, Seung Jun; Kang, Tae Seok; Kim, Seung Hee; Lee, Jaewon

    2009-06-16

    2,5-Hexanedione (HD), a metabolite of n-hexane, causes central and peripheral neuropathy leading to motor neuron deficits. Although chronic exposure to n-hexane is known to cause gradual sensorimotor neuropathy, there are no reports on the effects of low doses of HD on neurogenesis in the central nervous system. In the current study, we explored HD toxicity in murine neural progenitor cells (NPC), primary neuronal culture and young adult mice. HD (500 nM approximately 50 microM) dose-dependently suppressed NPC proliferation and cell viability, and also increased the production of reactive oxygen species (ROS). HD (10 or 50 mg/kg for 2 weeks) inhibited hippocampal neuronal and NPC proliferation in 6-week-old male ICR mice, as measured by BrdU incorporation in the dentate gyrus, indicating HD impaired hippocampal neurogenesis. In addition, elevated microglial activation was observed in the hippocampal CA3 region and lateral ventricles of HD-treated mice. Lastly, HD dose-dependently decreased the viability of primary cultured neurons. Based on biochemical and histochemical evidence from both cell culture and HD-treated animals, the neurotoxic mechanisms by which HD inhibits NPC proliferation and hippocampal neurogenesis may relate to its ability to elicit an increased generation of deleterious ROS.

  14. Simple SPION Incubation as an Efficient Intracellular Labeling Method for Tracking Neural Progenitor Cells Using MRI

    PubMed Central

    D. M., Jayaseema; Lai, Jiann-Shiun; Hueng, Dueng-Yuan; Chang, Chen

    2013-01-01

    Cellular magnetic resonance imaging (MRI) has been well-established for tracking neural progenitor cells (NPC). Superparamagnetic iron oxide nanoparticles (SPIONs) approved for clinical application are the most common agents used for labeling. Conventionally, transfection agents (TAs) were added with SPIONs to facilitate cell labeling because SPIONs in the native unmodified form were deemed inefficient for intracellular labeling. However, compelling evidence also shows that simple SPION incubation is not invariably ineffective. The labeling efficiency can be improved by prolonged incubation and elevated iron doses. The goal of the present study was to establish simple SPION incubation as an efficient intracellular labeling method. To this end, NPCs derived from the neonatal subventricular zone were incubated with SPIONs (Feridex®) and then evaluated in vitro with regard to the labeling efficiency and biological functions. The results showed that, following 48 hours of incubation at 75 µg/ml, nearly all NPCs exhibited visible SPION intake. Evidence from light microscopy, electron microscopy, chemical analysis, and magnetic resonance imaging confirmed the effectiveness of the labeling. Additionally, biological assays showed that the labeled NPCs exhibited unaffected viability, oxidative stress, apoptosis and differentiation. In the demonstrated in vivo cellular MRI experiment, the hypointensities representing the SPION labeled NPCs remained observable throughout the entire tracking period. The findings indicate that simple SPION incubation without the addition of TAs is an efficient intracellular magnetic labeling method. This simple approach may be considered as an alternative approach to the mainstream labeling method that involves the use of TAs. PMID:23468856

  15. Simple SPION incubation as an efficient intracellular labeling method for tracking neural progenitor cells using MRI.

    PubMed

    Chen, Chiao-Chi V; Ku, Min-Chi; D M, Jayaseema; Lai, Jiann-Shiun; Hueng, Dueng-Yuan; Chang, Chen

    2013-01-01

    Cellular magnetic resonance imaging (MRI) has been well-established for tracking neural progenitor cells (NPC). Superparamagnetic iron oxide nanoparticles (SPIONs) approved for clinical application are the most common agents used for labeling. Conventionally, transfection agents (TAs) were added with SPIONs to facilitate cell labeling because SPIONs in the native unmodified form were deemed inefficient for intracellular labeling. However, compelling evidence also shows that simple SPION incubation is not invariably ineffective. The labeling efficiency can be improved by prolonged incubation and elevated iron doses. The goal of the present study was to establish simple SPION incubation as an efficient intracellular labeling method. To this end, NPCs derived from the neonatal subventricular zone were incubated with SPIONs (Feridex®) and then evaluated in vitro with regard to the labeling efficiency and biological functions. The results showed that, following 48 hours of incubation at 75 µg/ml, nearly all NPCs exhibited visible SPION intake. Evidence from light microscopy, electron microscopy, chemical analysis, and magnetic resonance imaging confirmed the effectiveness of the labeling. Additionally, biological assays showed that the labeled NPCs exhibited unaffected viability, oxidative stress, apoptosis and differentiation. In the demonstrated in vivo cellular MRI experiment, the hypointensities representing the SPION labeled NPCs remained observable throughout the entire tracking period. The findings indicate that simple SPION incubation without the addition of TAs is an efficient intracellular magnetic labeling method. This simple approach may be considered as an alternative approach to the mainstream labeling method that involves the use of TAs.

  16. Permeability transition pore-mediated mitochondrial superoxide flashes regulate cortical neural progenitor differentiation.

    PubMed

    Hou, Yan; Mattson, Mark P; Cheng, Aiwu

    2013-01-01

    In the process of neurogenesis, neural progenitor cells (NPCs) cease dividing and differentiate into postmitotic neurons that grow dendrites and an axon, become excitable, and establish synapses with other neurons. Mitochondrial biogenesis and aerobic metabolism provide energy substrates required to support the differentiation, growth and synaptic activity of neurons. Mitochondria may also serve signaling functions and, in this regard, it was recently reported that mitochondria can generate rapid bursts of superoxide (superoxide flashes), the frequency of which changes in response to environmental conditions and signals including oxygen levels and Ca(2+) fluxes. Here we show that the frequency of mitochondrial superoxide flashes increases as embryonic cerebral cortical neurons differentiate from NPCs, and provide evidence that the superoxide flashes serve a signaling function that is critical for the differentiation process. The superoxide flashes are mediated by mitochondrial permeability transition pore (mPTP) opening, and pharmacological inhibition of the mPTP suppresses neuronal differentiation. Moreover, superoxide flashes and neuronal differentiation are inhibited by scavenging of mitochondrial superoxide. Conversely, manipulations that increase superoxide flash frequency accelerate neuronal differentiation. Our findings reveal a regulatory role for mitochondrial superoxide flashes, mediated by mPTP opening, in neuronal differentiation.

  17. Lesion-induced increase in survival and migration of human neural progenitor cells releasing GDNF

    PubMed Central

    Behrstock, Soshana; Ebert, Allison D.; Klein, Sandra; Schmitt, Melanie; Moore, Jeannette M.; Svendsen, Clive N.

    2009-01-01

    The use of human neural progenitor cells (hNPC) has been proposed to provide neuronal replacement or astrocytes delivering growth factors for brain disorders such as Parkinson’s and Huntington’s disease. Success in such studies likely requires migration from the site of transplantation and integration into host tissue in the face of ongoing damage. In the current study, hNPC modified to release glial cell line derived neurotrophic factor (hNPCGDNF) were transplanted into either intact or lesioned animals. GDNF release itself had no effect on the survival, migration or differentiation of the cells. The most robust migration and survival was found using a direct lesion of striatum (Huntington’s model) with indirect lesions of the dopamine system (Parkinson’s model) or intact animals showing successively less migration and survival. No lesion affected differentiation patterns. We conclude that the type of brain injury dictates migration and integration of hNPC which has important consequences when considering transplantation of these cells as a therapy for neurodegenerative diseases. PMID:19044202

  18. Activation of neural progenitor cells in human eyes with proliferative vitreoretinopathy.

    PubMed

    Johnsen, Erik O; Frøen, Rebecca C; Albert, Réka; Omdal, Bente K; Sarang, Zsolt; Berta, András; Nicolaissen, Bjørn; Petrovski, Goran; Moe, Morten C

    2012-05-01

    In addition to the ability for self-renewal and functional differentiation, neural stem/progenitor cells (NSCs) can respond to CNS injuries by targeted migration. In lower vertebrates, retinal injury is known to activate NSCs in the ciliary marginal zone (CMZ). Cells expressing markers of NSCs are also present in the ciliary body epithelium (CE) and in Müller glia in the peripheral retina (PR) of the adult human eye. However, these cells seem to be quiescent in the adult human eye and recent reports have shown that CE cells have limited properties of NSCs. In order to further clarify whether NSCs exist in the adult human eye, we tested whether NSC-like cells could be activated in eyes with proliferative vitreoretinopathy (PVR). The PR and CE were studied for NSC-associated markers in human enucleated control eyes and eyes with confirmed PVR, as well as in a mouse model of PVR. Furthermore, cells isolated from vitreous samples obtained during vitrectomies for retinal detachment were directly fixed or cultured in a stem cell-promoting medium and compared to cells cultured from the post-mortem retina and CE. In situ characterization of the normal eyes revealed robust expression of markers present in NSCs (Nestin, Sox2, Pax6) only around peripheral cysts of the proximal pars plana region and the PR, the latter population also staining for the glial marker GFAP. Although there were higher numbers of dividing cells in the CE of PVR eyes than in controls, we did not detect NSC-associated markers in the CE except around the proximal pars plana cysts. In the mice PVR eyes, Nestin activation was also found in the CE. In human PVR eyes, proliferation of both non-glial and glial cells co-staining NSC-associated markers was evident around the ora serrata region. Spheres formed in 7/10 vitreous samples from patients with PVR compared to 2/15 samples from patients with no known PVR, and expressed glial - and NSC-associated markers both after direct fixation and repetitive

  19. P2X7 receptors at adult neural progenitor cells of the mouse subventricular zone.

    PubMed

    Messemer, Nanette; Kunert, Christin; Grohmann, Marcus; Sobottka, Helga; Nieber, Karen; Zimmermann, Herbert; Franke, Heike; Nörenberg, Wolfgang; Straub, Isabelle; Schaefer, Michael; Riedel, Thomas; Illes, Peter; Rubini, Patrizia

    2013-10-01

    Neurogenesis requires the balance between the proliferation of newly formed progenitor cells and subsequent death of surplus cells. RT-PCR and immunocytochemistry demonstrated the presence of P2X7 receptor mRNA and immunoreactivity in cultured neural progenitor cells (NPCs) prepared from the adult mouse subventricular zone (SVZ). Whole-cell patch-clamp recordings showed a marked potentiation of the inward current responses both to ATP and the prototypic P2X7 receptor agonist dibenzoyl-ATP (Bz-ATP) at low Ca(2+) and zero Mg(2+) concentrations in the bath medium. The Bz-ATP-induced currents reversed their polarity near 0 mV; in NPCs prepared from P2X7(-/-) mice, Bz-ATP failed to elicit membrane currents. The general P2X/P2Y receptor antagonist PPADS and the P2X7 selective antagonists Brilliant Blue G and A-438079 strongly depressed the effect of Bz-ATP. Long-lasting application of Bz-ATP induced an initial current, which slowly increased to a steady-state response. In combination with the determination of YO-PRO uptake, these experiments suggest the dilation of a receptor-channel and/or the recruitment of a dye-uptake pathway. Ca(2+)-imaging by means of Fura-2 revealed that in a Mg(2+)-deficient bath medium Bz-ATP causes [Ca(2+)](i) transients fully depending on the presence of external Ca(2+). The MTT test indicated a concentration-dependent decrease in cell viability by Bz-ATP treatment. Correspondingly, Bz-ATP led to an increase in active caspase 3 immunoreactivity, indicating a P2X7-controlled apoptosis. In acute SVZ brain slices of transgenic Tg(nestin/EGFP) mice, patch-clamp recordings identified P2X7 receptors at NPCs with pharmacological properties identical to those of their cultured counterparts. We suggest that the apoptotic/necrotic P2X7 receptors at NPCs may be of particular relevance during pathological conditions which lead to increased ATP release and thus could counterbalance the ensuing excessive cell proliferation.

  20. Humanized neuronal chimeric mouse brain generated by neonatally engrafted human iPSC-derived primitive neural progenitor cells

    PubMed Central

    Chen, Chen

    2016-01-01

    The creation of a humanized chimeric mouse nervous system permits the study of human neural development and disease pathogenesis using human cells in vivo. Humanized glial chimeric mice with the brain and spinal cord being colonized by human glial cells have been successfully generated. However, generation of humanized chimeric mouse brains repopulated by human neurons to possess a high degree of chimerism have not been well studied. Here we created humanized neuronal chimeric mouse brains by neonatally engrafting the distinct and highly neurogenic human induced pluripotent stem cell (hiPSC)–derived rosette-type primitive neural progenitors. These neural progenitors predominantly differentiate to neurons, which disperse widely throughout the mouse brain with infiltration of the cerebral cortex and hippocampus at 6 and 13 months after transplantation. Building upon the hiPSC technology, we propose that this potentially unique humanized neuronal chimeric mouse model will provide profound opportunities to define the structure, function, and plasticity of neural networks containing human neurons derived from a broad variety of neurological disorders. PMID:27882348

  1. Prolonged Mitosis of Neural Progenitors Alters Cell Fate in the Developing Brain.

    PubMed

    Pilaz, Louis-Jan; McMahon, John J; Miller, Emily E; Lennox, Ashley L; Suzuki, Aussie; Salmon, Edward; Silver, Debra L

    2016-01-06

    Embryonic neocortical development depends on balanced production of progenitors and neurons. Genetic mutations disrupting progenitor mitosis frequently impair neurogenesis; however, the link between altered mitosis and cell fate remains poorly understood. Here we demonstrate that prolonged mitosis of radial glial progenitors directly alters neuronal fate specification and progeny viability. Live imaging of progenitors from a neurogenesis mutant, Magoh(+/-), reveals that mitotic delay significantly correlates with preferential production of neurons instead of progenitors, as well as apoptotic progeny. Independently, two pharmacological approaches reveal a causal relationship between mitotic delay and progeny fate. As mitotic duration increases, progenitors produce substantially more apoptotic progeny or neurons. We show that apoptosis, but not differentiation, is p53 dependent, demonstrating that these are distinct outcomes of mitotic delay. Together our findings reveal that prolonged mitosis is sufficient to alter fates of radial glia progeny and define a new paradigm to understand how mitosis perturbations underlie brain size disorders such as microcephaly.

  2. Multipotent Caudal Neural Progenitors Derived from Human Pluripotent Stem Cells That Give Rise to Lineages of the Central and Peripheral Nervous System

    PubMed Central

    Hasegawa, Kouichi; Menheniott, Trevelyan; Rollo, Ben; Zhang, Dongcheng; Hough, Shelley; Alshawaf, Abdullah; Febbraro, Fabia; Ighaniyan, Samiramis; Leung, Jessie; Elliott, David A.; Newgreen, Donald F.; Pera, Martin F.

    2015-01-01

    Abstract The caudal neural plate is a distinct region of the embryo that gives rise to major progenitor lineages of the developing central and peripheral nervous system, including neural crest and floor plate cells. We show that dual inhibition of the glycogen synthase kinase 3β and activin/nodal pathways by small molecules differentiate human pluripotent stem cells (hPSCs) directly into a preneuroepithelial progenitor population we named “caudal neural progenitors” (CNPs). CNPs coexpress caudal neural plate and mesoderm markers, and, share high similarities to embryonic caudal neural plate cells in their lineage differentiation potential. Exposure of CNPs to BMP2/4, sonic hedgehog, or FGF2 signaling efficiently directs their fate to neural crest/roof plate cells, floor plate cells, and caudally specified neuroepithelial cells, respectively. Neural crest derived from CNPs differentiated to neural crest derivatives and demonstrated extensive migratory properties in vivo. Importantly, we also determined the key extrinsic factors specifying CNPs from human embryonic stem cell include FGF8, canonical WNT, and IGF1. Our studies are the first to identify a multipotent neural progenitor derived from hPSCs, that is the precursor for major neural lineages of the embryonic caudal neural tube. Stem Cells 2015;33:1759–1770 PMID:25753817

  3. Endothelial progenitor cells and neural progenitor cells synergistically protect cerebral endothelial cells from Hypoxia/reoxygenation-induced injury via activating the PI3K/Akt pathway.

    PubMed

    Wang, Jinju; Chen, Yusen; Yang, Yi; Xiao, Xiang; Chen, Shuzhen; Zhang, Cheng; Jacobs, Bradley; Zhao, Bin; Bihl, Ji; Chen, Yanfang

    2016-02-03

    Protection of cerebral endothelial cells (ECs) from hypoxia/reoxygenation (H/R)-induced injury is an important strategy for treating ischemic stroke. In this study, we investigated whether co-culture with endothelial progenitor cells (EPCs) and neural progenitor cells (NPCs) synergistically protects cerebral ECs against H/R injury and the underlying mechanism. EPCs and NPCs were respectively generated from inducible pluripotent stem cells. Human brain ECs were used to produce an in vitro H/R-injury model. Data showed: 1) Co-culture with EPCs and NPCs synergistically inhibited H/R-induced reactive oxygen species (ROS) over-production, apoptosis, and improved the angiogenic and barrier functions (tube formation and permeability) in H/R-injured ECs. 2) Co-culture with NPCs up-regulated the expression of vascular endothelial growth factor receptor 2 (VEGFR2). 3) Co-culture with EPCs and NPCs complementarily increased vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF) levels in conditioned medium, and synergistically up-regulated the expression of p-Akt/Akt and p-Flk1/VEGFR2 in H/R-injured ECs. 4) Those effects could be decreased or abolished by inhibition of both VEGFR2 and tyrosine kinase B (TrkB) or phosphatidylinositol-3-kinase (PI3K). Our data demonstrate that EPCs and NPCs synergistically protect cerebral ECs from H/R-injury, via activating the PI3K/Akt pathway which mainly depends on VEGF and BDNF paracrine.

  4. S100B and APP Promote a Gliocentric Shift and Impaired Neurogenesis in Down Syndrome Neural Progenitors

    PubMed Central

    Lu, Jie; Esposito, Giuseppe; Scuderi, Caterina; Steardo, Luca; Delli-Bovi, Laurent C.; Hecht, Jonathan L.; Dickinson, Bryan C.; Chang, Christopher J.; Mori, Takashi; Sheen, Volney

    2011-01-01

    Down syndrome (DS) is a developmental disorder associated with mental retardation (MR) and early onset Alzheimer's disease (AD). These CNS phenotypes are attributed to ongoing neuronal degeneration due to constitutive overexpression of chromosome 21 (HSA21) genes. We have previously shown that HSA21 associated S100B contributes to oxidative stress and apoptosis in DS human neural progenitors (HNPs). Here we show that DS HNPs isolated from fetal frontal cortex demonstrate not only disturbances in redox states within the mitochondria and increased levels of progenitor cell death but also transition to more gliocentric progenitor phenotypes with a consequent reduction in neuronogenesis. HSA21 associated S100B and amyloid precursor protein (APP) levels are simultaneously increased within DS HNPs, their secretions are synergistically enhanced in a paracrine fashion, and overexpressions of these proteins disrupt mitochondrial membrane potentials and redox states. HNPs show greater susceptibility to these proteins as compared to neurons, leading to cell death. Ongoing inflammation through APP and S100B overexpression further promotes a gliocentric HNPs phenotype. Thus, the loss in neuronal numbers seen in DS is not merely due to increased HNPs cell death and neurodegeneration, but also a fundamental gliocentric shift in the progenitor pool that impairs neuronal production. PMID:21779383

  5. S100B and APP promote a gliocentric shift and impaired neurogenesis in Down syndrome neural progenitors.

    PubMed

    Lu, Jie; Esposito, Giuseppe; Scuderi, Caterina; Steardo, Luca; Delli-Bovi, Laurent C; Hecht, Jonathan L; Dickinson, Bryan C; Chang, Christopher J; Mori, Takashi; Sheen, Volney

    2011-01-01

    Down syndrome (DS) is a developmental disorder associated with mental retardation (MR) and early onset Alzheimer's disease (AD). These CNS phenotypes are attributed to ongoing neuronal degeneration due to constitutive overexpression of chromosome 21 (HSA21) genes. We have previously shown that HSA21 associated S100B contributes to oxidative stress and apoptosis in DS human neural progenitors (HNPs). Here we show that DS HNPs isolated from fetal frontal cortex demonstrate not only disturbances in redox states within the mitochondria and increased levels of progenitor cell death but also transition to more gliocentric progenitor phenotypes with a consequent reduction in neuronogenesis. HSA21 associated S100B and amyloid precursor protein (APP) levels are simultaneously increased within DS HNPs, their secretions are synergistically enhanced in a paracrine fashion, and overexpressions of these proteins disrupt mitochondrial membrane potentials and redox states. HNPs show greater susceptibility to these proteins as compared to neurons, leading to cell death. Ongoing inflammation through APP and S100B overexpression further promotes a gliocentric HNPs phenotype. Thus, the loss in neuronal numbers seen in DS is not merely due to increased HNPs cell death and neurodegeneration, but also a fundamental gliocentric shift in the progenitor pool that impairs neuronal production.

  6. Inhibition of autophagy prevents irradiation-induced neural stem and progenitor cell death in the juvenile mouse brain.

    PubMed

    Wang, Yafeng; Zhou, Kai; Li, Tao; Xu, Yiran; Xie, Cuicui; Sun, Yanyan; Zhang, Yaodong; Rodriguez, Juan; Blomgren, Klas; Zhu, Changlian

    2017-03-23

    Radiotherapy is an effective tool in the treatment of malignant brain tumors. However, damage to brain stem and progenitor cells constitutes a major problem and is associated with long-term side effects. Autophagy has been shown to be involved in cell death, and the purpose of this study was to evaluate the effect of autophagy inhibition on neural stem and progenitor cell death in the juvenile brain. Ten-day-old selective Atg7 knockout (KO) mice and wild-type (WT) littermates were subjected to a single 6Gy dose of whole-brain irradiation. Cell death and proliferation as well as microglia activation and inflammation were evaluated in the dentate gyrus of the hippocampus and in the cerebellum at 6 h after irradiation. We found that cell death was reduced in Atg7 KO compared with WT mice at 6 h after irradiation. The number of activated microglia increased significantly in both the dentate gyrus and the cerebellum of WT mice after irradiation, but the increase was lower in the Atg7 KO mice. The levels of proinflammatory cytokines and chemokines decreased, especially in the cerebellum, in the Atg7 KO group. These results suggest that autophagy might be a potential target for preventing radiotherapy-induced neural stem and progenitor cell death and its associated long-term side effects.

  7. Brain injury expands the numbers of neural stem cells and progenitors in the SVZ by enhancing their responsiveness to EGF

    PubMed Central

    Alagappan, Dhivyaa; Lazzarino, Deborah A; Felling, Ryan J; Balan, Murugabaskar; Kotenko, Sergei V; Levison, Steven W

    2009-01-01

    There is an increase in the numbers of neural precursors in the SVZ (subventricular zone) after moderate ischaemic injuries, but the extent of stem cell expansion and the resultant cell regeneration is modest. Therefore our studies have focused on understanding the signals that regulate these processes towards achieving a more robust amplification of the stem/progenitor cell pool. The goal of the present study was to evaluate the role of the EGFR [EGF (epidermal growth factor) receptor] in the regenerative response of the neonatal SVZ to hypoxic/ischaemic injury. We show that injury recruits quiescent cells in the SVZ to proliferate, that they divide more rapidly and that there is increased EGFR expression on both putative stem cells and progenitors. With the amplification of the precursors in the SVZ after injury there is enhanced sensitivity to EGF, but not to FGF (fibroblast growth factor)-2. EGF-dependent SVZ precursor expansion, as measured using the neurosphere assay, is lost when the EGFR is pharmacologically inhibited, and forced expression of a constitutively active EGFR is sufficient to recapitulate the exaggerated proliferation of the neural stem/progenitors that is induced by hypoxic/ischaemic brain injury. Cumulatively, our results reveal that increased EGFR signalling precedes that increase in the abundance of the putative neural stem cells and our studies implicate the EGFR as a key regulator of the expansion of SVZ precursors in response to brain injury. Thus modulating EGFR signalling represents a potential target for therapies to enhance brain repair from endogenous neural precursors following hypoxic/ischaemic and other brain injuries. PMID:19570028

  8. Multipotent neural stem cells generate glial cells of the central complex through transit amplifying intermediate progenitors in Drosophila brain development.

    PubMed

    Viktorin, Gudrun; Riebli, Nadia; Popkova, Anna; Giangrande, Angela; Reichert, Heinrich

    2011-08-15

    The neural stem cells that give rise to the neural lineages of the brain can generate their progeny directly or through transit amplifying intermediate neural progenitor cells (INPs). The INP-producing neural stem cells in Drosophila are called type II neuroblasts, and their neural progeny innervate the central complex, a prominent integrative brain center. Here we use genetic lineage tracing and clonal analysis to show that the INPs of these type II neuroblast lineages give rise to glial cells as well as neurons during postembryonic brain development. Our data indicate that two main types of INP lineages are generated, namely mixed neuronal/glial lineages and neuronal lineages. Genetic loss-of-function and gain-of-function experiments show that the gcm gene is necessary and sufficient for gliogenesis in these lineages. The INP-derived glial cells, like the INP-derived neuronal cells, make major contributions to the central complex. In postembryonic development, these INP-derived glial cells surround the entire developing central complex neuropile, and once the major compartments of the central complex are formed, they also delimit each of these compartments. During this process, the number of these glial cells in the central complex is increased markedly through local proliferation based on glial cell mitosis. Taken together, these findings uncover a novel and complex form of neurogliogenesis in Drosophila involving transit amplifying intermediate progenitors. Moreover, they indicate that type II neuroblasts are remarkably multipotent neural stem cells that can generate both the neuronal and the glial progeny that make major contributions to one and the same complex brain structure.

  9. Optimal Poly(l-lysine) Grafting Density in Hydrogels for Promoting Neural Progenitor Cell Functions

    PubMed Central

    Cai, Lei; Lu, Jie; Sheen, Volney; Wang, Shanfeng

    2012-01-01

    Recently we have developed a photo-polymerizable poly(l-lysine) (PLL) that can be covalently incorporated into poly(ethylene glycol) diacrylate (PEGDA) hydrogels to improve their bioactivity by providing positive charges. To explore the potential of these PLL-grafted PEGDA hydrogels as a cell delivery vehicle and luminal filler in nerve guidance conduits for peripheral and central nerve regeneration, we varied the amount of pendent PLL chains in the hydrogels by photo-crosslinking PEGDA with weight compositions of PLL (ϕPLL) of 0, 1%, 2%, 3%, and 5%. We further investigated the effect of PLL grafting density on E14 mouse neural progenitor cell (NPC) behavior including cell viability, attachment, proliferation, differentiation, and gene expression. The amount of actually grafted PLL and charge densities were characterized, showing a proportional increase with the feed composition ϕPLL. NPC viability in 3D hydrogels was significantly improved in a PLL grafting density-dependent manner at days 7 and 14 post-encapsulation. Similarly, NPC attachment and proliferation were promoted on the PLL-grafted hydrogels with increasing ϕPLL up to 2%. More intriguingly, NPC lineage commitment was dramatically altered by the amount of grafted PLL chains in the hydrogels. NPC differentiation demonstrated a parabolic or non-monotonic dependence on ϕPLL, resulting in cells mostly differentiated toward mature neurons with extensive neurite formation and astrocytes rather than oligodendrocytes on the PLL-grafted hydrogels with ϕPLL of 2%, whereas the neutral hydrogels and PLL-grafted hydrogels with higher ϕPLL of 5% support NPC differentiation less. Gene expression of lineage markers further illustrated this trend, indicating that PLL-grafted hydrogels with an optimal ϕPLL of 2% could be a promising cell carrier that promoted NPC functions for treatment of nerve injuries. PMID:22533450

  10. A radiotherapy technique to limit dose to neural progenitor cell niches without compromising tumor coverage

    PubMed Central

    Redmond, Kristin J.; Achanta, Pragathi; Grossman, Stuart A.; Armour, Michael; Reyes, Juvenal; Kleinberg, Lawrence; Tryggestad, Erik; Quinones-Hinojosa, Alfredo

    2015-01-01

    Radiation therapy (RT) for brain tumors is associated with neurocognitive toxicity which may be a result of damage to neural progenitor cells (NPCs). We present a novel technique to limit the radiation dose to NPC without compromising tumor coverage. A study was performed in mice to examine the rationale and another was conducted in humans to determine its feasibility. C57BL/6 mice received localized radiation using a dedicated animal irradiation system with on-board CT imaging with either: (1) Radiation which spared NPC containing regions; (2) Radiation which did not spare these niches; or (3) Sham irradiation. Mice were sacrificed 24 h later and the brains were processed for immunohistochemical Ki-67 staining. For the human component of the study, 33 patients with primary brain tumors were evaluated. Two intensity modulated radiotherapy (IMRT) plans were retrospectively compared: a standard clinical plan and a plan which spares NPC regions while maintaining the same dose coverage of the tumor. The change in radiation dose to the contralateral NPC-containing regions was recorded. In the mouse model, non-NPC-sparing radiation treatment resulted in a significant decrease in the number of Ki67+ cells in dentate gyrus (DG) (P = 0.008) and subventricular zone (SVZ) (P = 0.005) compared to NPC-sparing radiation treatment. In NPC-sparing clinical plans, NPC regions received significantly lower radiation dose with no clinically relevant changes in tumor coverage. This novel radiation technique should significantly reduce radiation doses to NPC containing regions of the brain which may reduce neurocognitive deficits following RT for brain tumors. PMID:21327710

  11. Store-Operated CRAC Channels Regulate Gene Expression and Proliferation in Neural Progenitor Cells

    PubMed Central

    Somasundaram, Agila; Shum, Andrew K.; McBride, Helen J.; Kessler, John A.; Feske, Stefan; Miller, Richard J.

    2014-01-01

    Calcium signals regulate many critical processes during vertebrate brain development including neurogenesis, neurotransmitter specification, and axonal outgrowth. However, the identity of the ion channels mediating Ca2+ signaling in the developing nervous system is not well defined. Here, we report that embryonic and adult mouse neural stem/progenitor cells (NSCs/NPCs) exhibit store-operated Ca2+ entry (SOCE) mediated by Ca2+ release-activated Ca2+ (CRAC) channels. SOCE in NPCs was blocked by the CRAC channel inhibitors La3+, BTP2, and 2-APB and Western blots revealed the presence of the canonical CRAC channel proteins STIM1 and Orai1. Knock down of STIM1 or Orai1 significantly diminished SOCE in NPCs, and SOCE was lost in NPCs from transgenic mice lacking Orai1 or STIM1 and in knock-in mice expressing the loss-of-function Orai1 mutant, R93W. Therefore, STIM1 and Orai1 make essential contributions to SOCE in NPCs. SOCE in NPCs was activated by epidermal growth factor and acetylcholine, the latter occurring through muscarinic receptors. Activation of SOCE stimulated gene transcription through calcineurin/NFAT (nuclear factor of activated T cells) signaling through a mechanism consistent with local Ca2+ signaling by Ca2+ microdomains near CRAC channels. Importantly, suppression or deletion of STIM1 and Orai1 expression significantly attenuated proliferation of embryonic and adult NPCs cultured as neurospheres and, in vivo, in the subventricular zone of adult mice. These findings show that CRAC channels serve as a major route of Ca2+ entry in NPCs and regulate key effector functions including gene expression and proliferation, indicating that CRAC channels are important regulators of mammalian neurogenesis. PMID:24990931

  12. Effect of neurosphere size on the growth rate of human neural stem/progenitor cells.

    PubMed

    Mori, Hideki; Ninomiya, Kazuaki; Kino-oka, Masahiro; Shofuda, Tomoko; Islam, Mohammed Omedul; Yamasaki, Mami; Okano, Hideyuki; Taya, Masahito; Kanemura, Yonehiro

    2006-12-01

    Neural stem/progenitor cells (NSPCs) proliferate as aggregates in vitro, but the mechanism of aggregation is not fully understood. Here, we report that aggregation promotes the proliferation of NSPCs. We found that the proliferation rate was linear and depended on the size of the aggregate; that is, the population doubling time of the NSPCs gradually decreased as the diameter approached 250 micro m and flattened to a nearly constant value beyond this diameter. Given this finding, and with the intent of enhancing the efficiency of human NSPC expansion, we induced the NSPCs to form aggregates close to 250 micro m in diameter quickly by culturing them in plates with U-bottomed wells. The NSPCs formed aggregates effectively in the U-bottomed wells, with cell numbers approximately 1.5 times greater than those in the aggregates that formed spontaneously in flat-bottomed wells. In addition, this effect of aggregation involved cell-cell signaling molecules of the Notch1 pathway. In the U-bottomed wells, Hes1 and Hes5, which are target genes of the Notch signal, were expressed at higher levels than in the control, flat-bottomed wells. The amount of cleaved Notch1 was also higher in the cells cultured in the U-bottomed wells. The addition of gamma-secretase inhibitor, which blocks Notch signaling, suppressed cell proliferation in the U-bottomed wells. These results suggest that the three-dimensional architecture of NSPC aggregates would create a microenvironment that promotes the proliferation of human NSPCs.

  13. Risk assessment for the combinational effects of food color additives: neural progenitor cells and hippocampal neurogenesis.

    PubMed

    Park, Mikyung; Park, Hee Ra; Kim, So Jung; Kim, Min-Sun; Kong, Kyoung Hye; Kim, Hyun Soo; Gong, Ein Ji; Kim, Mi Eun; Kim, Hyung Sik; Lee, Byung Mu; Lee, Jaewon

    2009-01-01

    In 2006, the Korea Food and Drug Administration reported that combinations of dietary colors such as allura red AC (R40), tartrazine (Y4), sunset yellow FCF (Y5), amaranth (R2), and brilliant blue FCF (B1) are widely used in food manufacturing. Although individual tar food colors are controlled based on acceptable daily intake (ADI), there is no apparent information available for how combinations of these additives affect food safety. In the current study, the potencies of single and combination use of R40, Y4, Y5, R2, and B1 were examined on neural progenitor cell (NPC) toxicity, a biomarker for developmental stage, and neurogenesis, indicative of adult central nervous system (CNS) functions. R40 and R2 reduced NPC proliferation and viability in mouse multipotent NPC, in the developing CNS model. Among several combinations tested in mouse model, combination of Y4 and B1 at 1000-fold higher than average daily intake in Korea significantly decreased numbers of newly generated cells in adult mouse hippocampus, indicating potent adverse actions on hippocampal neurogenesis. However, other combinations including R40 and R2 did not affect adult hippocampal neurogenesis in the dentate gyrus. Evidence indicates that single and combination use of most tar food colors may be safe with respect to risk using developmental NPC and adult hippocampal neurogenesis. However, the response to excessively high dose combination of Y4 and B1 is suggestive of synergistic effects to suppress proliferation of NPC in adult hippocampus. Data indicated that combinations of tar colors may adversely affect both developmental and adult hippocampal neurogenesis; thus, further extensive studies are required to assess the safety of these additive combinations.

  14. Diffusible Factors Secreted by Glioblastoma and Medulloblastoma Cells Induce Oxidative Stress in Bystander Neural Stem Progenitors

    PubMed Central

    Sharma, Neha; Colangelo, Nicholas W.; de Toledo, Sonia M.

    2016-01-01

    Harmful effects that alter the homeostasis of neural stem or progenitor cells (NSPs) can affect regenerative processes in the central nervous system. We investigated the effect of soluble factors secreted by control or 137Cs-γ-irradiated glioblastoma or medulloblastoma cells on redox-modulated endpoints in recipient human NSPs. Growth medium harvested from the nonirradiated brain tumor cells, following 24 h of growth, induced prominent oxidative stress in recipient NSPs as judged by overall increases in mitochondrial superoxide radical levels (p < .001), activation of c-jun N-terminal kinase, and decrease in the active form of FoxO3a. The induced oxidative stress was associated with phosphorylation of p53 on serine 15, a marker of DNA damage, induction of the cyclin-cyclin dependent kinase inhibitors p21Waf1 and p27Kip1, and perturbations in cell cycle progression (p < .001). These changes were also associated with increased apoptosis as determined by enhanced annexin V staining (p < .001) and caspase 8 activation (p < .05) and altered expression of critical regulators of self-renewal, proliferation, and differentiation. Exposure of the tumor cells to radiation only slightly altered the induced oxidative changes in the bystander NSPs, except for medium from irradiated medulloblastoma cells that was more potent at inducing apoptosis in the NSPs than medium from nonirradiated cells (p < .001). The elucidation of such stressful bystander effects provides avenues to understand the biochemical events underlying the development or exacerbation of degenerative outcomes associated with brain cancers. It is also relevant to tissue culture protocols whereby growth medium conditioned by tumor cells is often used to support the growth of stem cells. PMID:27511909

  15. In utero gene therapy rescues microcephaly caused by Pqbp1-hypofunction in neural stem progenitor cells

    PubMed Central

    Ito, H; Shiwaku, H; Yoshida, C; Homma, H; Luo, H; Chen, X; Fujita, K; Musante, L; Fischer, U; Frints, S G M; Romano, C; Ikeuchi, Y; Shimamura, T; Imoto, S; Miyano, S; Muramatsu, S-i; Kawauchi, T; Hoshino, M; Sudol, M; Arumughan, A; Wanker, E E; Rich, T; Schwartz, C; Matsuzaki, F; Bonni, A; Kalscheuer, V M; Okazawa, H

    2015-01-01

    Human mutations in PQBP1, a molecule involved in transcription and splicing, result in a reduced but architecturally normal brain. Examination of a conditional Pqbp1-knockout (cKO) mouse with microcephaly failed to reveal either abnormal centrosomes or mitotic spindles, increased neurogenesis from the neural stem progenitor cell (NSPC) pool or increased cell death in vivo. Instead, we observed an increase in the length of the cell cycle, particularly for the M phase in NSPCs. Corresponding to the developmental expression of Pqbp1, the stem cell pool in vivo was decreased at E10 and remained at a low level during neurogenesis (E15) in Pqbp1-cKO mice. The expression profiles of NSPCs derived from the cKO mouse revealed significant changes in gene groups that control the M phase, including anaphase-promoting complex genes, via aberrant transcription and RNA splicing. Exogenous Apc4, a hub protein in the network of affected genes, recovered the cell cycle, proliferation, and cell phenotypes of NSPCs caused by Pqbp1-cKO. These data reveal a mechanism of brain size control based on the simple reduction of the NSPC pool by cell cycle time elongation. Finally, we demonstrated that in utero gene therapy for Pqbp1-cKO mice by intraperitoneal injection of the PQBP1-AAV vector at E10 successfully rescued microcephaly with preserved cortical structures and improved behavioral abnormalities in Pqbp1-cKO mice, opening a new strategy for treating this intractable developmental disorder. PMID:25070536

  16. Concentration-dependent effect of nerve growth factor on cell fate determination of neural progenitors.

    PubMed

    Zhang, Lei; Jiang, Hui; Hu, Zhengqing

    2011-10-01

    Stem cell-based spiral ganglion neuron (SGN) replacement therapy has been proposed to be a promising strategy to restore hearing either via replacing degenerated neurons or by improving the efficacy of cochlear implants which rely on functional neurons. However, lack of suitable donor cells and low survival rate of implanted cells are the major obstacles to successful implementation of therapeutic transplantation. The present study investigated the potential of mouse inner ear statoacoustic ganglion (SAG)-derived neural progenitors (NPs) to differentiate toward SGN-like glutamatergic cells and the influence to cell survival and differentiation when nerve growth factor (NGF) was supplied. We found that SAG-NPs could form neurospheres, proliferate, and differentiate into cells expressing neuronal protein neurofilament and β-III tubulin. NGF affected the cell fate of SAG-NP in a concentration-dependent manner in vitro. Low concentration of NGF (2-5 ng/mL) promoted cell proliferation. Medium concentration of NGF (20-40 ng/mL) stimulated cells to differentiate into bi-polar SGN-like cells expressing glutamatergic proteins. High concentration of NGF (100 ng/mL) could rescue cells from induced apoptosis. In the in vivo study, NGF (100 ng/mL) dramatically enhanced SAG-NP survival rate after implantation into adult mammalian inner ear. This finding raises the possibility to further induce these NPs to differentiate into SGN-like neurons in future in vivo study. In conclusion, given the capability of proliferation and differentiation into SGN-like cells with the supplement of NGF in vitro, SAG-NPs can serve as donor cells in stem cell-based SGN replacement therapy. NGF improved the survival of SAG-NPs not only in vitro but also in vivo.

  17. Intravenous injection of neural progenitor cells improved depression-like behavior after cerebral ischemia

    PubMed Central

    Moriyama, Y; Takagi, N; Tanonaka, K

    2011-01-01

    Poststroke depression (PSD) occurs in approximately one-third of stroke survivors and is one of the serious sequelae of stroke. The onset of PSD causes delayed functional recovery by rehabilitation and also increases cognitive impairment. However, appropriate strategies for the therapy against ischemia-induced depression-like behaviors still remain to be developed. Such behaviors have been associated with a reduced level of brain-derived neurotrophic factor (BDNF). In addition, accumulating evidence indicates the ability of stem cells to improve cerebral ischemia-induced brain injuries. However, it remains to be clarified as to the effect of neural progenitor cells (NPCs) on PSD and the association between BDNF level and PSD. Using NPCs, we investigated the effect of intravenous injection of NPCs on PSD. We showed that injection of NPCs improved ischemia-induced depression-like behaviors in the forced-swimming test and sucrose preference test without having any effect on the viable area between vehicle- and NPC-injected ischemic rats. The injection of NPCs prevented the decrease in the level of BDNF in the ipsilateral hemisphere. The levels of phosphorylated CREB, ERK and Akt, which have been implicated in events downstream of BDNF signaling, were also decreased after cerebral ischemia. NPC injection inhibited these decreases in the phosphorylation of CREB and ERK, but not that of Akt. Our findings provide evidence that injection of NPCs may have therapeutic potential for the improvement of depression-like behaviors after cerebral ischemia and that these effects might be associated with restoring BDNF-ERK-CREB signaling. PMID:22832603

  18. Resveratrol Inhibits the Proliferation of Neural Progenitor Cells and Hippocampal Neurogenesis*

    PubMed Central

    Park, Hee Ra; Kong, Kyoung Hye; Yu, Byung Pal; Mattson, Mark P.; Lee, Jaewon

    2012-01-01

    Resveratrol is a phytoalexin and natural phenol that is present at relatively high concentrations in peanuts and red grapes and wine. Based upon studies of yeast and invertebrate models, it has been proposed that ingestion of resveratrol may also have anti-aging actions in mammals including humans. It has been suggested that resveratrol exerts its beneficial effects on health by activating the same cellular signaling pathways that are activated by dietary energy restriction (DR). Some studies have reported therapeutic actions of resveratrol in animal models of metabolic and neurodegenerative disorders. However, the effects of resveratrol on cell, tissue and organ function in healthy subjects are largely unknown. In the present study, we evaluated the potential effects of resveratrol on the proliferation and survival of neural progenitor cells (NPCs) in culture, and in the hippocampus of healthy young adult mice. Resveratrol reduced the proliferation of cultured mouse multi-potent NPCs, and activated AMP-activated protein kinase (AMPK), in a concentration-dependent manner. Administration of resveratrol to mice (1–10 mg/kg) resulted in activation of AMPK, and reduced the proliferation and survival of NPCs in the dentate gyrus of the hippocampus. Resveratrol down-regulated the levels of the phosphorylated form of cyclic AMP response element-binding protein (pCREB) and brain-derived neurotrophic factor (BDNF) in the hippocampus. Finally, resveratrol-treated mice exhibited deficits in hippocampus-dependent spatial learning and memory. Our findings suggest that resveratrol, unlike DR, adversely affects hippocampal neurogenesis and cognitive function by a mechanism involving activation of AMPK and suppression of CREB and BDNF signaling. PMID:23105098

  19. The effect of cannabichromene on adult neural stem/progenitor cells.

    PubMed

    Shinjyo, Noriko; Di Marzo, Vincenzo

    2013-11-01

    Apart from the psychotropic compound Δ(9)-tetrahydrocannabinol (THC), evidence suggests that other non-psychotropic phytocannabinoids are also of potential clinical use. This study aimed at elucidating the effect of major non-THC phytocannabinoids on the fate of adult neural stem progenitor cells (NSPCs), which are an essential component of brain function in health as well as in pathology. We tested three compounds: cannabidiol, cannabigerol, and cannabichromene (CBC), and found that CBC has a positive effect on the viability of mouse NSPCs during differentiation in vitro. The expression of NSPC and astrocyte markers nestin and Glial fibrillary acidic protein (GFAP), respectively, was up- and down-regulated, respectively. CBC stimulated ERK1/2 phosphorylation; however, this effect had a slower onset in comparison to typical MAPK stimulation. A MEK inhibitor, U0126, antagonized the up-regulation of nestin but not the down-regulation of GFAP. Based on a previous report, we studied the potential involvement of the adenosine A1 receptor in the effect of CBC on these cells and found that the selective adenosine A1 receptor antagonist, DPCPX, counteracted both ERK1/2 phosphorylation and up-regulation of nestin by CBC, indicating that also adenosine is involved in these effects of CBC, but possibly not in CBC inhibitory effect on GFAP expression. Next, we measured ATP levels as an equilibrium marker of adenosine and found higher ATP levels during differentiation of NSPCs in the presence of CBC. Taken together, our results suggest that CBC raises the viability of NSPCs while inhibiting their differentiation into astroglia, possibly through up-regulation of ATP and adenosine signalling. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. Inhibition of neurosphere formation in neural stem/progenitor cells by acrylamide.

    PubMed

    Chen, Jong-Hang; Lee, Don-Ching; Chen, Mei-Shu; Ko, Ying-Chin; Chiu, Ing-Ming

    2015-01-01

    Previous studies showed that transplantation of cultured neural stem/progenitor cells (NSPCs) could improve functional recovery for various neurological diseases. This study aims to develop a stem cell-based model for predictive toxicology of development in the neurological system after acrylamide exposure. Treatment of mouse (KT98/F1B-GFP) and human (U-1240 MG/F1B-GFP) NSPCs with 0.5 mM acrylamide resulted in the inhibition of neurosphere formation (definition of self-renewal ability in NSPCs), but not inhibition of cell proliferation. Apoptosis and differentiation of KT98 (a precursor of KT98/F1B-GFP) and KT98/F1B-GFP are not observed in acrylamide-treated neurospheres. Analysis of secondary neurosphere formation and differentiation of neurons and glia illustrated that acrylamide-treated KT98 and KT98/F1B-GFP neurospheres retain the NSPC properties, such as self-renewal and differentiation capacity. Correlation of acrylamide-inhibited neurosphere formation with cell-cell adhesion was observed in mouse NSPCs by live cell image analysis and the presence of acrylamide. Protein expression levels of cell adhesion molecules [neural cell adhesion molecule (NCAM) and N-cadherin] and extracellular signal-regulated kinases (ERK) in acrylamide-treated KT98/F1B-GFP and U-1240 MG/F1B-GFP neurospheres demonstrated that NCAM decreased and phospho-ERK (pERK) increased, whereas expression of N-cadherin remained unchanged. Analysis of AKT (protein kinase B, PKB)/β-catenin pathway showed decrease in phospho-AKT (p-AKT) and cyclin D1 expression in acrylamide-treated neurospheres of KT98/F1B-GFP. Furthermore, PD98059, an ERK phosphorylation inhibitor, attenuated acrylamide-induced ERK phosphorylation, indicating that pERK contributed to the cell proliferation, but not in neurosphere formation in mouse NSPCs. Coimmunoprecipitation results of KT98/F1B-GFP cell lysates showed that the complex of NCAM and fibroblast growth factor receptor 1 (FGFR1) is present in the neurosphere, and the

  1. Heads-up: new roles for the fragile X mental retardation protein in neural stem and progenitor cells.

    PubMed

    Callan, Matthew A; Zarnescu, Daniela C

    2011-06-01

    Fragile X syndrome (FXS) is the most common form of inherited mental retardation and is caused by the loss of function for Fragile X Mental Retardation Protein (FMRP), a selective RNA-binding protein with a demonstrated role in the localized translation of target mRNAs at synapses. Several recent studies provide compelling evidence for a new role of FMRP in the development of the nervous system, during neurogenesis. Using a multi-faceted approach and a variety of model systems ranging from cultured neurospheres and progenitor cells to in vivo Drosophila and mouse models these reports indicate that FMRP is required for neural stem and progenitor cell proliferation, differentiation, survival, as well as regulation of gene expression. Here we compare and contrast these recent reports and discuss the implications of FMRP's new role in embryonic and adult neurogenesis, including the development of novel therapeutic approaches to FXS and related neurological disorders such as autism.

  2. The APC/C cofactor Cdh1 prevents replicative stress and p53-dependent cell death in neural progenitors.

    PubMed

    Eguren, Manuel; Porlan, Eva; Manchado, Eusebio; García-Higuera, Irene; Cañamero, Marta; Fariñas, Isabel; Malumbres, Marcos

    2013-01-01

    The E3-ubiquitin ligase APC/C-Cdh1 is essential for endoreduplication but its relevance in the mammalian mitotic cell cycle is still unclear. Here we show that genetic ablation of Cdh1 in the developing nervous system results in hypoplastic brain and hydrocephalus. These defects correlate with enhanced levels of Cdh1 substrates and increased entry into the S phase in neural progenitors. However, cell division is prevented in the absence of Cdh1 due to hyperactivation of cyclin-dependent kinases, replicative stress, induction of p53, G2 arrest and apoptotic death of these progenitor cells. Concomitant ablation of p53 rescues apoptosis but not replicative stress, resulting in the presence of damaged neurons throughout the adult brain. These data indicate that the inactivation of Cdh1 in vivo results in replicative stress, cell cycle arrest and cell death, supporting recent therapeutic proposals aimed to inhibit the APC/C in tumours.

  3. A targeted secretome profiling by multiplexed immunoassay revealed that secreted chemokine ligand 2 (MCP-1/CCL2) affects neural differentiation in mesencephalic neural progenitor cells.

    PubMed

    Colucci-D'Amato, Luca; Cicatiello, Anna Emilia; Reccia, Mafalda Giovanna; Volpicelli, Floriana; Severino, Valeria; Russo, Rosita; Sandomenico, Annamaria; Doti, Nunzianna; D'Esposito, Vittoria; Formisano, Pietro; Chambery, Angela

    2015-02-01

    Chemokines and cytokines, primarily known for their roles in the immune and inflammatory response, have also been identified as key components of the neurogenic niche where they are involved in the modulation of neural stem cell proliferation and differentiation. However, a complete understanding of the functional role played in neural differentiation and a comprehensive profiling of these secreted molecules are lacking. By exploiting the multiplexing capability of magnetic bead-based immunoassays, we have investigated the changes of the expression levels of a set of chemokines and cytokines released from the pluripotent neural cell line mes-c-myc A1 following its differentiation from a proliferating phenotype (A1P) toward a neural (A1D) phenotype. We found a subset of molecules exclusively released from A1P, whereas others were differentially detected in A1P and A1D conditioned media. Among them, we identified monocyte chemoattractant protein-1/chemokine ligand 2 (MCP-1/CCL2) as a proneurogenic factor able to affect neuronal differentiation of A1 cells as well as of neuroblasts from primary cultures and to induce the elongation and/or formation of neuritic processes. Altogether, data are suggestive of a main role played by the CCL2/CCR2 signaling pathway and in general of the network of secreted cytokines/chemokines in the differentiation of neural progenitor cells toward a neural fate.

  4. Low-Dose Methylmercury-Induced Apoptosis and Mitochondrial DNA Mutation in Human Embryonic Neural Progenitor Cells

    PubMed Central

    Yan, Mengling; Zhao, Lina; Wu, Qing; Wu, Chunhua

    2016-01-01

    Methylmercury (MeHg) is a long-lasting organic pollutant primarily found in the aquatic environment. The developing brain is particularly sensitive to MeHg due to reduced proliferation of neural stem cell. Although several mechanisms of MeHg-induced apoptosis have been defined in culture models, it remains unclear whether mitochondrial DNA (mtDNA) mutation is involved in the toxic effect of MeHg, especially in the neural progenitor cells. In the present study, the ReNcell CX cell, a human neural progenitor cells (hNPCs) line, was exposed to nanomolar concentrations of MeHg (≤50 nM). We found that MeHg altered mitochondrial metabolic function and induced apoptosis. In addition, we observed that MeHg induced ROS production in a dose-dependent manner in hNPCs cells, which was associated with significantly increased expressions of ND1, Cytb, and ATP6. To elucidate the mechanism underlying MeHg toxicity on mitochondrial function, we examined the ATP content and mitochondrial membrane potential in MeHg-treated hNPCs. Our study showed that MeHg exposure led to decreased ATP content and reduced mitochondrial membrane potential, which failed to match the expansion in mtDNA copy number, suggesting impaired mtDNA. Collectively, these results demonstrated that MeHg induced toxicity in hNPCs through altering mitochondrial function and inducing oxidative damage to mtDNA. PMID:27525052

  5. Huntingtin Is Required for Neural But Not Cardiac/Pancreatic Progenitor Differentiation of Mouse Embryonic Stem Cells In vitro

    PubMed Central

    Yu, Man Shan; Tanese, Naoko

    2017-01-01

    Mutation in the huntingtin (HTT) gene causes Huntington’s disease (HD). It is an autosomal dominant trinucleotide-repeat expansion disease in which CAG repeat sequence expands to >35. This results in the production of mutant HTT protein with an increased stretch of glutamines near the N-terminus. The wild type HTT gene encodes a 350 kD protein whose function remains elusive. Mutant HTT protein has been implicated in transcription, axonal transport, cytoskeletal structure/function, signal transduction, and autophagy. HD is characterized by the appearance of nuclear inclusions and degeneration of the striatum. Although HTT protein is expressed early in embryos, most patients develop symptoms in mid-life. It is also unclear why the ubiquitously expressed mutant HTT specifically causes striatal atrophy. Wild type Htt is essential for development as Htt knockout mice die at day E7.5. Increasing evidence suggests mutant Htt may alter neurogenesis and development of striatal neurons resulting in neuronal loss. Using a mouse embryonic stem cell model, we examined the role of Htt in neural differentiation. We found cells lacking Htt inefficient in generating neural stem cells. In contrast differentiation into progenitors of mesoderm and endoderm lineages was not affected. The data suggests Htt is essential for neural but not cardiac/pancreatic progenitor differentiation of embryonic stem cells in vitro. PMID:28270748

  6. HDAC inhibition amplifies gap junction communication in neural progenitors: Potential for cell-mediated enzyme prodrug therapy

    SciTech Connect

    Khan, Zahidul . E-mail: Zahidul.Khan@ki.se; Akhtar, Monira; Asklund, Thomas; Juliusson, Bengt . E-mail: Tomas.Ekstrom@ki.se

    2007-08-01

    Enzyme prodrug therapy using neural progenitor cells (NPCs) as delivery vehicles has been applied in animal models of gliomas and relies on gap junction communication (GJC) between delivery and target cells. This study investigated the effects of histone deacetylase (HDAC) inhibitors on GJC for the purpose of facilitating transfer of therapeutic molecules from recombinant NPCs. We studied a novel immortalized midbrain cell line, NGC-407 of embryonic human origin having neural precursor characteristics, as a potential delivery vehicle. The expression of gap junction protein connexin 43 (C x 43) was analyzed by western blot and immunocytochemistry. While C x 43 levels were decreased in untreated differentiating NGC-407 cells, the HDAC inhibitor 4-phenylbutyrate (4-PB) increased C x 43 expression along with increased membranous deposition in both proliferating and differentiating cells. Simultaneously, Ser 279/282-phosphorylated form of C x 43 was declined in both culture conditions by 4-PB. The 4-PB effect in NGC-407 cells was verified by using HNSC.100 human neural progenitors and Trichostatin A. Improved functional GJC is of imperative importance for therapeutic strategies involving intercellular transport of low molecular-weight compounds. We show here an enhancement by 4-PB, of the functional GJC among NGC-407 cells, as well as between NGC-407 and human glioma cells, as indicated by increased fluorescent dye transfer.

  7. Running rescues defective adult neurogenesis by shortening the length of the cell cycle of neural stem and progenitor cells.

    PubMed

    Farioli-Vecchioli, Stefano; Mattera, Andrea; Micheli, Laura; Ceccarelli, Manuela; Leonardi, Luca; Saraulli, Daniele; Costanzi, Marco; Cestari, Vincenzo; Rouault, Jean-Pierre; Tirone, Felice

    2014-07-01

    Physical exercise increases the generation of new neurons in adult neurogenesis. However, only few studies have investigated the beneficial effects of physical exercise in paradigms of impaired neurogenesis. Here, we demonstrate that running fully reverses the deficient adult neurogenesis within the hippocampus and subventricular zone of the lateral ventricle, observed in mice lacking the antiproliferative gene Btg1. We also evaluated for the first time how running influences the cell cycle kinetics of stem and precursor subpopulations of wild-type and Btg1-null mice, using a new method to determine the cell cycle length. Our data show that in wild-type mice running leads to a cell cycle shortening only of NeuroD1-positive progenitor cells. In contrast, in Btg1-null mice, physical exercise fully reactivates the defective hippocampal neurogenesis, by shortening the S-phase length and the overall cell cycle duration of both neural stem (glial fibrillary acidic protein(+) and Sox2(+)) and progenitor (NeuroD1(+)) cells. These events are sufficient and necessary to reactivate the hyperproliferation observed in Btg1-null early-postnatal mice and to expand the pool of adult neural stem and progenitor cells. Such a sustained increase of cell proliferation in Btg1-null mice after running provides a long-lasting increment of proliferation, differentiation, and production of newborn neurons, which rescues the impaired pattern separation previously identified in Btg1-null mice. This study shows that running positively affects the cell cycle kinetics of specific subpopulations of newly generated neurons and suggests that the plasticity of neural stem cells without cell cycle inhibitory control is reactivated by running, with implications for the long-term modulation of neurogenesis. © 2014 AlphaMed Press.

  8. Differing Lectin Binding Profiles among Human Embryonic Stem Cells and Derivatives Aid in the Isolation of Neural Progenitor Cells

    PubMed Central

    Dodla, Mahesh C.; Young, Amber; Venable, Alison; Hasneen, Kowser; Rao, Raj R.; Machacek, David W.; Stice, Steven L.

    2011-01-01

    Human embryonic stem cells (hESCs) and their differentiated progeny allow for investigation of important changes/events during normal embryonic development. Currently most of the research is focused on proteinacous changes occurring as a result of differentiation of stem cells and little is known about changes in cell surface glycosylation patterns. Identification of cell lineage specific glycans can help in understanding their role in maintenance, proliferation and differentiation. Furthermore, these glycans can serve as markers for isolation of homogenous populations of cells. Using a panel of eight biotinylated lectins, the glycan expression of hESCs, hESCs-derived human neural progenitors (hNP) cells, and hESCs-derived mesenchymal progenitor (hMP) cells was investigated. Our goal was to identify glycans that are unique for hNP cells and use the corresponding lectins for cell isolation. Flow cytometry and immunocytochemistry were used to determine expression and localization of glycans, respectively, in each cell type. These results show that the glycan expression changes upon differentiation of hESCs and is different for neural and mesenchymal lineage. For example, binding of PHA-L lectin is low in hESCs (14±4.4%) but significantly higher in differentiated hNP cells (99±0.4%) and hMP cells (90±3%). Three lectins: VVA, DBA and LTL have low binding in hESCs and hMP cells, but significantly higher binding in hNP cells. Finally, VVA lectin binding was used to isolate hNP cells from a mixed population of hESCs, hNP cells and hMP cells. This is the first report that compares glycan expression across these human stem cell lineages and identifies significant differences. Also, this is the first study that uses VVA lectin for isolation for human neural progenitor cells. PMID:21850265

  9. 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.

  10. Differentiation of human CD146-positive endometrial stem cells to adipogenic-, osteogenic-, neural progenitor-, and glial-like cells.

    PubMed

    Fayazi, Mehri; Salehnia, Mojdeh; Ziaei, Saeideh

    2015-04-01

    The aim of this study was to investigate the potential differentiation of CD146(+) endometrial stem cells to several lineages. Endometrial stromal cells were cultured using Dulbecco's modified Eagle's medium/Hams F-12 (DMEM/F-12) and were passaged every 7-10 d when cultures reached 80-100% of confluency. The immunophenotypes of single endometrial cells were analyzed using flow cytometry at fourth passage. Then the CD146(+) cells were sorted using magnetic-activated cell sorting, and they were cultured and analyzed for in vitro differentiation to several lineages. Detection of adipocyte- and osteocyte-like cells were assessed by oil red O and alizarin red staining, respectively. For detection of neural progenitor and oligodendrocyte-like cells, the cells were immunostained by neurofilament 68 and oligo2, respectively. The rates of CD90, CD105, CD146, CD31, CD34, and CD9 of cultured endometrial cells were 94.98 ± 3%, 95.77 ± 2.5%, 27.61 ± 2%, 0.79 ± 0.05%, 1.43 ± 0.1%, and 1.01 ± 0.06%, respectively. CD146(+) cells were isolated to high purity. CD146(+)-differentiated cells to adipogenic cell with typical lipid-rich vacuoles and osteogenic cells were observed and confirmed their mesenchymal origin. They also differentiated into neural progenitor and glial differentiation by retinoic acid, basic fibroblast growth factor, and epidermal growth factor signaling molecules, respectively, and confirmed by neurofilament 68 and oligo2 immunocytochemistry. The efficiency of differentiation to neural progenitor and oligodendrocyte-like cells was 90 ± 3.4% and 79 ± 2.8%, respectively. This study showed that CD146(+) cells from human endometrium after in vitro cultivation can differentiate into adipogenic-, osteogenic-, neural progenitor-, and glial-like cells. They may provide available alternative source of stem cells for future cell-based therapies and tissue engineering applications.

  11. 2-Bromopalmitate impairs neural stem/progenitor cell proliferation, promotes cell apoptosis and induces malformation in zebrafish embryonic brain.

    PubMed

    Wang, Chen; Chen, Xueran; Shi, Wei; Wang, Fen; Du, Zhaoxia; Li, Xian; Yao, Yao; Liu, Tong; Shao, Tong; Li, Gang; Hao, Aijun

    2015-01-01

    2-Bromopalmitate (2BP) is a widely used palmitoylation inhibitor. Besides, it has been reported that 2BP can inhibit T-cell activation, making it a potential immunosuppressor for the treatment of autoimmune diseases. Although the important roles of palmitoylation in a neural system have been noted during the past decades, the effect of 2BP on neural development is still not very clear. In this study, we demonstrated that 25 μM-100 μM 2BP exposure caused apparent neural malformation in the presumptive brains of zebrafish embryos at 14 hpf. Further studies implied that the mRNA quantities and distributions of neural stem/progenitor cell (NSPC) markers (neurog1, sox2, and sox3) in the affected regions of 50 μM 2BP treated embryos significantly decreased. In addition, we found that 2BP impaired the NSPC proliferation at 10 hpf and 14 hpf as well as promoted cell apoptosis at 14 hpf, consistent with which the interference with FGF/ERK signaling pathway was also detected. For the first time, this study provided information about the toxicity and teratogenicity of 2BP for neural development in vivo. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Up-regulation of DRP-3 long isoform during the induction of neural progenitor cells by glutamate treatment in the ex vivo rat retina

    SciTech Connect

    Tokuda, Kazuhiro; Kuramitsu, Yasuhiro; Byron, Baron; Kitagawa, Takao; Tokuda, Nobuko; Kobayashi, Daiki; Nagayama, Megumi; Araki, Norie; Sonoda, Koh-Hei; Nakamura, Kazuyuki

    2015-08-07

    Glutamate has been shown to induce neural progenitor cells in the adult vertebrate retina. However, protein dynamics during progenitor cell induction by glutamate are not fully understood. To identify specific proteins involved in the process, we employed two-dimensional electrophoresis-based proteomics on glutamate untreated and treated retinal ex vivo sections. Rat retinal tissues were incubated with 1 mM glutamate for 1 h, followed by incubation in glutamate-free media for a total of 24 h. Consistent with prior reports, it was found that mitotic cells appeared in the outer nuclear layer without any histological damage. Immunohistological evaluations and immunoblotting confirmed the emergence of neuronal progenitor cells in the mature retina treated with glutamate. Proteomic analysis revealed the up-regulation of dihydropyrimidinase-related protein 3 (DRP-3), DRP-2 and stress-induced-phosphoprotein 1 (STIP1) during neural progenitor cell induction by glutamate. Moreover, mRNA expression of DRP-3, especially, its long isoform, robustly increased in the treated retina compared to that in the untreated retina. These results may indicate that glutamate induces neural progenitor cells in the mature rat retina by up-regulating the proteins which mediate cell mitosis and neurite growth. - Highlights: • Glutamate induced neuronal progenitor cells in the mature rat retina. • Proteomic analysis revealed the up-regulation of DRP-3, DRP-2 and STIP1. • mRNA expression of DRP-3, especially, its long isoform, robustly increased.

  13. Effect of enzymatic and mechanical methods of dissociation on neural progenitor cells derived from induced pluripotent stem cells

    PubMed Central

    Jager, Lindsey D.; Canda, Claire-Marie A.; Hall, Crystal A.; Heilingoetter, Cassandra L.; Huynh, Joann; Kwok, Susanna S.; Kwon, Jin H.; Richie, Jacob R.; Jensen, Matthew B.

    2015-01-01

    Purpose To determine the most effective method of dissociating neural stem and progenitor cells into a single-cell suspension. Materials/methods Induced pluripotent stem cells were differentiated toward the neural fate for 4 weeks before clusters were subjected to enzymatic (Accutase, trypsin, TrypLE, dispase, or DNase I) or mechanical (trituration with pipettes of varying size) or combined dissociation. Images of cells were analyzed for cluster size using ImageJ. Results Cells treated with the enzymes Accutase, TrypLE, or trypsin/EDTA, these enzymes followed by trituration, or a combination one of these enzymes followed by incubation with another enzyme, including DNase I, were more likely to be dissociated into a single-cell suspension. Conclusions Cells treated with enzymes or combinations of methods were more likely to be dissociated into a single-cell suspension. PMID:26523795

  14. Effect of enzymatic and mechanical methods of dissociation on neural progenitor cells derived from induced pluripotent stem cells.

    PubMed

    Jager, Lindsey D; Canda, Claire-Marie A; Hall, Crystal A; Heilingoetter, Cassandra L; Huynh, Joann; Kwok, Susanna S; Kwon, Jin H; Richie, Jacob R; Jensen, Matthew B

    2016-03-01

    To determine the most effective method of dissociating neural stem and progenitor cells into a single-cell suspension. Induced pluripotent stem cells were differentiated toward the neural fate for 4 weeks before clusters were subjected to enzymatic (Accutase, trypsin, TrypLE, dispase, or DNase I) or mechanical (trituration with pipettes of varying size) or combined dissociation. Images of cells were analyzed for cluster size using ImageJ. Cells treated with the enzymes Accutase, TrypLE, or trypsin/EDTA, these enzymes followed by trituration, or a combination one of these enzymes followed by incubation with another enzyme, including DNase I, were more likely to be dissociated into a single-cell suspension. Cells treated with enzymes or combinations of methods were more likely to be dissociated into a single-cell suspension. Copyright © 2015 Medical University of Bialystok. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  15. A Notch-Gli2 axis sustains Hedgehog responsiveness of neural progenitors and Müller glia.

    PubMed

    Ringuette, Randy; Atkins, Michael; Lagali, Pamela S; Bassett, Erin A; Campbell, Charles; Mazerolle, Chantal; Mears, Alan J; Picketts, David J; Wallace, Valerie A

    2016-03-01

    Neurogenesis is regulated by the dynamic and coordinated activity of several extracellular signalling pathways, but the basis for crosstalk between these pathways remains poorly understood. Here we investigated regulatory interactions between two pathways that are each required for neural progenitor cell maintenance in the postnatal retina; Hedgehog (Hh) and Notch signalling. Both pathways are activated in progenitor cells in the postnatal retina based on the co-expression of fluorescent pathway reporter transgenes at the single cell level. Disrupting Notch signalling, genetically or pharmacologically, induces a rapid downregulation of all three Gli proteins and inhibits Hh-induced proliferation. Ectopic Notch activation, while not sufficient to promote Hh signalling or proliferation, increases Gli2 protein. We show that Notch regulation of Gli2 in Müller glia renders these cells competent to proliferate in response to Hh. These data suggest that Notch signalling converges on Gli2 to prime postnatal retinal progenitor cells and Müller glia to proliferate in response to Hh.

  16. MicroRNA-130b targets Fmr1 and regulates embryonic neural progenitor cell proliferation and differentiation

    SciTech Connect

    Gong, Xi; Zhang, Kunshan; Wang, Yanlu; Wang, Junbang; Cui, Yaru; Li, Siguang; Luo, Yuping

    2013-10-04

    Highlights: •We found that the 3′ UTR of the Fmr1 mRNA is a target of miR-130b. •MiR-130b suppresses the expression of Fmr1 in mouse embryonic stem cell. •MiR-130b alters the proliferation of mouse embryonic stem cell. •MiR-130b alters fate specification of mouse embryonic stem cell. -- Abstract: Fragile X syndrome, one of the most common forms of inherited mental retardation, is caused by expansion of the CGG repeat in the 5′-untranslated region of the X-linked Fmr1 gene, which results in transcriptional silencing and loss of expression of its encoded protein FMRP. The loss of FMRP increases proliferation and alters fate specification in adult neural progenitor cells (aNPCs). However, little is known about Fmr1 mRNA regulation at the transcriptional and post-transcriptional levels. In the present study, we report that miR-130b regulated Fmr1 expression by directly targeting its 3′-untranslated region (3′ UTR). Up-regulation of miR-130b in mouse embryonic neural progenitor cells (eNPCs) decreased Fmr1 expression, markedly increased eNPC proliferation and altered the differentiation tendency of eNPCs, suggesting that antagonizing miR-130b may be a new therapeutic entry point for treating Fragile X syndrome.

  17. SBE6: a novel long-range enhancer involved in driving sonic hedgehog expression in neural progenitor cells.

    PubMed

    Benabdallah, Nezha S; Gautier, Philippe; Hekimoglu-Balkan, Betul; Lettice, Laura A; Bhatia, Shipra; Bickmore, Wendy A

    2016-11-01

    The expression of genes with key roles in development is under very tight spatial and temporal control, mediated by enhancers. A classic example of this is the sonic hedgehog gene (Shh), which plays a pivotal role in the proliferation, differentiation and survival of neural progenitor cells both in vivo and in vitro. Shh expression in the brain is tightly controlled by several known enhancers that have been identified through genetic, genomic and functional assays. Using chromatin profiling during the differentiation of embryonic stem cells to neural progenitor cells, here we report the identification of a novel long-range enhancer for Shh-Shh-brain-enhancer-6 (SBE6)-that is located 100 kb upstream of Shh and that is required for the proper induction of Shh expression during this differentiation programme. This element is capable of driving expression in the vertebrate brain. Our study illustrates how a chromatin-focused approach, coupled to in vivo testing, can be used to identify new cell-type specific cis-regulatory elements, and points to yet further complexity in the control of Shh expression during embryonic brain development.

  18. SBE6: a novel long-range enhancer involved in driving sonic hedgehog expression in neural progenitor cells

    PubMed Central

    Benabdallah, Nezha S.; Gautier, Philippe; Hekimoglu-Balkan, Betul; Lettice, Laura A.; Bhatia, Shipra

    2016-01-01

    The expression of genes with key roles in development is under very tight spatial and temporal control, mediated by enhancers. A classic example of this is the sonic hedgehog gene (Shh), which plays a pivotal role in the proliferation, differentiation and survival of neural progenitor cells both in vivo and in vitro. Shh expression in the brain is tightly controlled by several known enhancers that have been identified through genetic, genomic and functional assays. Using chromatin profiling during the differentiation of embryonic stem cells to neural progenitor cells, here we report the identification of a novel long-range enhancer for Shh—Shh-brain-enhancer-6 (SBE6)—that is located 100 kb upstream of Shh and that is required for the proper induction of Shh expression during this differentiation programme. This element is capable of driving expression in the vertebrate brain. Our study illustrates how a chromatin-focused approach, coupled to in vivo testing, can be used to identify new cell-type specific cis-regulatory elements, and points to yet further complexity in the control of Shh expression during embryonic brain development. PMID:27852806

  19. Induction of Neural Progenitor-Like Cells from Human Fibroblasts via a Genetic Material-Free Approach

    PubMed Central

    Mirakhori, Fahimeh; Zeynali, Bahman; Rassouli, Hassan; Shahbazi, Ebrahim; Hashemizadeh, Shiva; Kiani, Sahar; Salekdeh, Ghasem Hosseini; Baharvand, Hossein

    2015-01-01

    Background A number of studies generated induced neural progenitor cells (iNPCs) from human fibroblasts by viral delivering defined transcription factors. However, the potential risks associated with gene delivery systems have limited their clinical use. We propose it would be safer to induce neural progenitor-like cells from human adult fibroblasts via a direct non-genetic alternative approach. Methodology/Principal Findings Here, we have reported that seven rounds of TAT-SOX2 protein transduction in a defined chemical cocktail under a 3D sphere culture gradually morphed fibroblasts into neuroepithelial-like colonies. We were able to expand these cells for up to 20 passages. These cells could give rise to cells that expressed neurons and glia cell markers both in vitro and in vivo. Conclusions/Significance These results show that our approach is beneficial for the genetic material-free generation of iNPCs from human fibroblasts where small chemical molecules can provide a valuable, viable strategy to boost and improve induction in a 3D sphere culture. PMID:26266943

  20. Cell cycle and lineage progression of neural progenitors in the ventricular-subventricular zones of adult mice

    PubMed Central

    Ponti, Giovanna; Obernier, Kirsten; Guinto, Cristina; Jose, Lingu; Bonfanti, Luca; Alvarez-Buylla, Arturo

    2013-01-01

    Proliferating neural stem cells and intermediate progenitors persist in the ventricular-subventricular zone (V-SVZ) of the adult mammalian brain. This extensive germinal layer in the walls of the lateral ventricles is the site of birth of different types of interneurons destined for the olfactory bulb. The cell cycle dynamics of stem cells (B1 cells), intermediate progenitors (C cells), and neuroblasts (A cells) in the V-SVZ and the number of times these cells divide remain unknown. Using whole mounts of the walls of the lateral ventricles of adult mice and three cell cycle analysis methods using thymidine analogs, we determined the proliferation dynamics of B1, C, and A cells in vivo. Achaete-scute complex homolog (Ascl)1+ C cells were heterogeneous with a cell cycle length (TC) of 18–25 h and a long S phase length (TS) of 14–17 h. After C cells, Doublecortin+ A cells were the second-most common dividing cell type in the V-SVZ and had a TC of 18 h and TS of 9 h. Human glial fibrillary acidic protein (hGFAP)::GFP+ B1 cells had a surprisingly short Tc of 17–18 h and a TS of 4 h. Progenitor population analysis suggests that following the initial division of B1 cells, C cells divide three times and A cells once, possibly twice. These data provide essential information on the dynamics of adult progenitor cell proliferation in the V-SVZ and how large numbers of new neurons continue to be produced in the adult mammalian brain. PMID:23431204

  1. LIF is Essential for SVZ Neural Stem Cell and Progenitor Homeostasis as Revealed by a Novel Flow Cytometric Analysis

    PubMed Central

    Buono, Krista D.; Vadlamuri, Daimler; Gan, Qiong; Levison, Steven W.

    2013-01-01

    Stem cells rely on extracellular signals produced by the niche, which dictate their ability to self-renew, expand and differentiate. It is essential to have sensitive and reproducible methods of either quantifying or isolating these stem cells and progenitors to understand their intrinsic properties and how extrinsic signals regulate their development. However, stem cells are difficult to distinguish from multipotential progenitors, which may look and act like them. Here we define a 4-color flow cytometry panel using CD133, LeX, CD140a, NG2 to define an NSC as well as 4 classes of multipotential progenitors and 3 classes of bipotential progenitors, several of which have not been previously described. We performed gain and loss of function studies for LIF and show a depletion of NSCs, a subset of multipotential neural precursors and immature oligodendrocytes in LIF null mice. Gain of function studies showed that LIF increased the abundance of these precursors. Our studies also show that these NPs have differential requirements for LIF and CNTF and for EGF, FGF-2 and PDGF for their propagation in vitro. Surprisingly, the related cytokine, CNTF was less potent than LIF in increasing the NSCs and more potent than LIF in increasing the PDGF responsive multipotential precursors. Finally, we show that LIF increases the expression of the core transcription factors: Klf4, Fbx15, Nanog, Sox2 and c-Myc. Altogether our FACS analyses reveal that the neonatal SVZ is far more heterogeneous than previously suspected and our studies provide new insights into the signals and mechanisms that regulate their self-renewal and proliferation. PMID:23258129

  2. Hypoxic-preconditioning enhances the regenerative capacity of neural stem/progenitors in subventricular zone of newborn piglet brain.

    PubMed

    Ara, Jahan; De Montpellier, Sybille

    2013-09-01

    Perinatal hypoxia-ischemia (HI) results in brain injury, whereas mild hypoxic episodes result in preconditioning, which can significantly reduce the vulnerability of the brain to subsequent severe hypoxia-ischemia. Hypoxic-preconditioning (PC) has been shown to enhance cell survival and differentiation of progenitor cells in the central nervous system (CNS). The purpose of this study was to determine whether pretreatment with PC prior to HI stimulates subventricular zone (SVZ) proliferation and neurogenesis in newborn piglets. One-day-old piglets were subjected to PC (8% O2/92% N2) for 3h and 24h later were exposed to HI produced by combination of hypoxia (5% FiO2) for a pre-defined period of 30min and ischemia induced by a period of 10min of hypotension. Here we demonstrate that SVZ derived neural stem/progenitor cells (NSPs) from PC, HI and PC+HI piglets proliferated as neurospheres, expressed neural progenitor and neurodevelopmental markers, and that greater proportion of the spheres generated are multipotential. Neurosphere assay revealed that preconditioning pretreatment increased the number of NSP-derived neurospheres in SVZ following HI compared to normoxic and HI controls. NSPs from preconditioned SVZ generated twice as many neurons and astrocytes in vitro. Injections with 5-Bromo-2-deoxyuridine (BrdU) after PC revealed a robust proliferative response within the SVZ that continued for one week. PC also increased neurogenesis in vivo, doublecortin positive cells with migratory profiles were observed streaming from the SVZ to striatum and neocortex. These findings show that the induction of proliferation and neurogenesis by PC might be a positive adaptation for an efficient repair and plasticity in the event of a hypoxic-ischemic insult.

  3. Seizure induces activation of multiple subtypes of neural progenitors and growth factors in hippocampus with neuronal maturation confined to dentate gyrus

    SciTech Connect

    Indulekha, Chandrasekharan L.; Sanalkumar, Rajendran; Thekkuveettil, Anoopkumar; James, Jackson

    2010-03-19

    Adult hippocampal neurogenesis is altered in response to different physiological and pathological stimuli. GFAP{sup +ve}/nestin{sup +ve} radial glial like Type-1 progenitors are considered to be the resident stem cell population in adult hippocampus. During neurogenesis these Type-1 progenitors matures to GFAP{sup -ve}/nestin{sup +ve} Type-2 progenitors and then to Type-3 neuroblasts and finally differentiates into granule cell neurons. In our study, using pilocarpine-induced seizure model, we showed that seizure initiated activation of multiple progenitors in the entire hippocampal area such as DG, CA1 and CA3. Seizure induction resulted in activation of two subtypes of Type-1 progenitors, Type-1a (GFAP{sup +ve}/nestin{sup +ve}/BrdU{sup +ve}) and Type-1b (GFAP{sup +ve}/nestin{sup +ve}/BrdU{sup -ve}). We showed that majority of Type-1b progenitors were undergoing only a transition from a state of dormancy to activated form immediately after seizures rather than proliferating, whereas Type-1a showed maximum proliferation by 3 days post-seizure induction. Type-2 (GFAP{sup -ve}/nestin{sup +ve}/BrdU{sup +ve}) progenitors were few compared to Type-1. Type-3 (DCX{sup +ve}) progenitors showed increased expression of immature neurons only in DG region by 3 days after seizure induction indicating maturation of progenitors happens only in microenvironment of DG even though progenitors are activated in CA1 and CA3 regions of hippocampus. Also parallel increase in growth factors expression after seizure induction suggests that microenvironmental niche has a profound effect on stimulation of adult neural progenitors.

  4. Sphere-Derived Multipotent Progenitor Cells Obtained From Human Oral Mucosa Are Enriched in Neural Crest Cells.

    PubMed

    Abe, Shigehiro; Yamaguchi, Satoshi; Sato, Yutaka; Harada, Kiyoshi

    2016-01-01

    isolation of neural crest progenitor cells from oral mucosa using this system. Human OMSCs form spheres that exhibit self-renewal capabilities and multipotency, and are enriched with neural crest-derived cells. These oral mucosa sphere-forming cells can generate ectopic bone tissue in vivo. Therefore, the results of the present study show that the sphere culture system can be applied, without the need for complex isolation techniques, to produce multipotent spheres with the properties of neural crest stem cells. Furthermore, a convenient strategy is demonstrated for the isolation and culture of human OMSCs that could have clinical applications. ©AlphaMed Press.

  5. Fat1 interacts with Fat4 to regulate neural tube closure, neural progenitor proliferation and apical constriction during mouse brain development.

    PubMed

    Badouel, Caroline; Zander, Mark A; Liscio, Nicole; Bagherie-Lachidan, Mazdak; Sopko, Richelle; Coyaud, Etienne; Raught, Brian; Miller, Freda D; McNeill, Helen

    2015-08-15

    Mammalian brain development requires coordination between neural precursor proliferation, differentiation and cellular organization to create the intricate neuronal networks of the adult brain. Here, we examined the role of the atypical cadherins Fat1 and Fat4 in this process. We show that mutation of Fat1 in mouse embryos causes defects in cranial neural tube closure, accompanied by an increase in the proliferation of cortical precursors and altered apical junctions, with perturbations in apical constriction and actin accumulation. Similarly, knockdown of Fat1 in cortical precursors by in utero electroporation leads to overproliferation of radial glial precursors. Fat1 interacts genetically with the related cadherin Fat4 to regulate these processes. Proteomic analysis reveals that Fat1 and Fat4 bind different sets of actin-regulating and junctional proteins. In vitro data suggest that Fat1 and Fat4 form cis-heterodimers, providing a mechanism for bringing together their diverse interactors. We propose a model in which Fat1 and Fat4 binding coordinates distinct pathways at apical junctions to regulate neural progenitor proliferation, neural tube closure and apical constriction.

  6. Fat1 interacts with Fat4 to regulate neural tube closure, neural progenitor proliferation and apical constriction during mouse brain development

    PubMed Central

    Badouel, Caroline; Zander, Mark A.; Liscio, Nicole; Bagherie-Lachidan, Mazdak; Sopko, Richelle; Coyaud, Etienne; Raught, Brian; Miller, Freda D.; McNeill, Helen

    2015-01-01

    Mammalian brain development requires coordination between neural precursor proliferation, differentiation and cellular organization to create the intricate neuronal networks of the adult brain. Here, we examined the role of the atypical cadherins Fat1 and Fat4 in this process. We show that mutation of Fat1 in mouse embryos causes defects in cranial neural tube closure, accompanied by an increase in the proliferation of cortical precursors and altered apical junctions, with perturbations in apical constriction and actin accumulation. Similarly, knockdown of Fat1 in cortical precursors by in utero electroporation leads to overproliferation of radial glial precursors. Fat1 interacts genetically with the related cadherin Fat4 to regulate these processes. Proteomic analysis reveals that Fat1 and Fat4 bind different sets of actin-regulating and junctional proteins. In vitro data suggest that Fat1 and Fat4 form cis-heterodimers, providing a mechanism for bringing together their diverse interactors. We propose a model in which Fat1 and Fat4 binding coordinates distinct pathways at apical junctions to regulate neural progenitor proliferation, neural tube closure and apical constriction. PMID:26209645

  7. Cell-cycle-independent transitions in temporal identity of mammalian neural progenitor cells.

    PubMed

    Okamoto, Mayumi; Miyata, Takaki; Konno, Daijiro; Ueda, Hiroki R; Kasukawa, Takeya; Hashimoto, Mitsuhiro; Matsuzaki, Fumio; Kawaguchi, Ayano

    2016-04-20

    During cerebral development, many types of neurons are sequentially generated by self-renewing progenitor cells called apical progenitors (APs). Temporal changes in AP identity are thought to be responsible for neuronal diversity; however, the mechanisms underlying such changes remain largely unknown. Here we perform single-cell transcriptome analysis of individual progenitors at different developmental stages, and identify a subset of genes whose expression changes over time but is independent of differentiation status. Surprisingly, the pattern of changes in the expression of such temporal-axis genes in APs is unaffected by cell-cycle arrest. Consistent with this, transient cell-cycle arrest of APs in vivo does not prevent descendant neurons from acquiring their correct laminar fates. Analysis of cultured APs reveals that transitions in AP gene expression are driven by both cell-intrinsic and -extrinsic mechanisms. These results suggest that the timing mechanisms controlling AP temporal identity function independently of cell-cycle progression and Notch activation mode.

  8. Delayed Rectifier and A-Type Potassium Channels Associated with Kv 2.1 and Kv 4.3 Expression in Embryonic Rat Neural Progenitor Cells

    PubMed Central

    Smith, Dean O.; Rosenheimer, Julie L.; Kalil, Ronald E.

    2008-01-01

    Background Because of the importance of voltage-activated K+ channels during embryonic development and in cell proliferation, we present here the first description of these channels in E15 rat embryonic neural progenitor cells derived from the subventricular zone (SVZ). Activation, inactivation, and single-channel conductance properties of recorded progenitor cells were compared with those obtained by others when these Kv gene products were expressed in oocytes. Methodology/Principal Findings Neural progenitor cells derived from the subventricular zone of E15 embryonic rats were cultured under conditions that did not promote differentiation. Immunocytochemical and Western blot assays for nestin expression indicated that almost all of the cells available for recording expressed this intermediate filament protein, which is generally accepted as a marker for uncommitted embryonic neural progenitor cells. However, a very small numbers of the cells expressed GFAP, a marker for astrocytes, O4, a marker for immature oligodendrocytes, and βIII-tubulin, a marker for neurons. Using immunocytochemistry and Western blots, we detected consistently the expression of Kv2.1, and 4.3. In whole-cell mode, we recorded two outward currents, a delayed rectifier and an A-type current. Conclusions/Significance We conclude that Kv2.1, and 4.3 are expressed in E15 SVZ neural progenitor cells, and we propose that they may be associated with the delayed-rectifier and the A-type currents, respectively, that we recorded. These results demonstrate the early expression of delayed rectifier and A-type K+ currents and channels in embryonic neural progenitor cells prior to the differentiation of these cells. PMID:18270591

  9. Up-regulation of DRP-3 long isoform during the induction of neural progenitor cells by glutamate treatment in the ex vivo rat retina.

    PubMed

    Tokuda, Kazuhiro; Kuramitsu, Yasuhiro; Byron, Baron; Kitagawa, Takao; Tokuda, Nobuko; Kobayashi, Daiki; Nagayama, Megumi; Araki, Norie; Sonoda, Koh-Hei; Nakamura, Kazuyuki

    2015-08-07

    Glutamate has been shown to induce neural progenitor cells in the adult vertebrate retina. However, protein dynamics during progenitor cell induction by glutamate are not fully understood. To identify specific proteins involved in the process, we employed two-dimensional electrophoresis-based proteomics on glutamate untreated and treated retinal ex vivo sections. Rat retinal tissues were incubated with 1 mM glutamate for 1 h, followed by incubation in glutamate-free media for a total of 24 h. Consistent with prior reports, it was found that mitotic cells appeared in the outer nuclear layer without any histological damage. Immunohistological evaluations and immunoblotting confirmed the emergence of neuronal progenitor cells in the mature retina treated with glutamate. Proteomic analysis revealed the up-regulation of dihydropyrimidinase-related protein 3 (DRP-3), DRP-2 and stress-induced-phosphoprotein 1 (STIP1) during neural progenitor cell induction by glutamate. Moreover, mRNA expression of DRP-3, especially, its long isoform, robustly increased in the treated retina compared to that in the untreated retina. These results may indicate that glutamate induces neural progenitor cells in the mature rat retina by up-regulating the proteins which mediate cell mitosis and neurite growth.

  10. Lack of Diaph3 relaxes the spindle checkpoint causing the loss of neural progenitors.

    PubMed

    Damiani, Devid; Goffinet, André M; Alberts, Arthur; Tissir, Fadel

    2016-11-16

    The diaphanous homologue Diaph3 (aka mDia2) is a major regulator of actin cytoskeleton. Loss of Diaph3 has been constantly associated with cytokinesis failure ascribed to impaired accumulation of actin in the cleavage furrow. Here we report that Diaph3 is required before cell fission, to ensure the accurate segregation of chromosomes. Inactivation of the Diaph3 gene causes a massive loss of cortical progenitor cells, with subsequent depletion of intermediate progenitors and neurons, and results in microcephaly. In embryonic brain extracts, Diaph3 co-immunoprecipitates with BubR1, a key regulator of the spindle assembly checkpoint (SAC). Diaph3-deficient cortical progenitors have decreased levels of BubR1 and fail to properly activate the SAC. Hence, they bypass mitotic arrest and embark on anaphase in spite of incorrect chromosome segregation, generating aneuploidy. Our data identify Diaph3 as a major guard of cortical progenitors, unravel novel functions of Diaphanous formins and add insights into the pathobiology of microcephaly.

  11. Lack of Diaph3 relaxes the spindle checkpoint causing the loss of neural progenitors

    PubMed Central

    Damiani, Devid; Goffinet, André M.; Alberts, Arthur; Tissir, Fadel

    2016-01-01

    The diaphanous homologue Diaph3 (aka mDia2) is a major regulator of actin cytoskeleton. Loss of Diaph3 has been constantly associated with cytokinesis failure ascribed to impaired accumulation of actin in the cleavage furrow. Here we report that Diaph3 is required before cell fission, to ensure the accurate segregation of chromosomes. Inactivation of the Diaph3 gene causes a massive loss of cortical progenitor cells, with subsequent depletion of intermediate progenitors and neurons, and results in microcephaly. In embryonic brain extracts, Diaph3 co-immunoprecipitates with BubR1, a key regulator of the spindle assembly checkpoint (SAC). Diaph3-deficient cortical progenitors have decreased levels of BubR1 and fail to properly activate the SAC. Hence, they bypass mitotic arrest and embark on anaphase in spite of incorrect chromosome segregation, generating aneuploidy. Our data identify Diaph3 as a major guard of cortical progenitors, unravel novel functions of Diaphanous formins and add insights into the pathobiology of microcephaly. PMID:27848932

  12. Methylmercury Exposure during Early Xenopus laevis Development Affects Cell Proliferation and Death but not Neural Progenitor Specification

    PubMed Central

    Huyck, Ryan W.; Nagarkar, Maitreyi; Olsen, Nina; Clamons, Samuel E.; Saha, Margaret S.

    2015-01-01

    Methylmercury (MeHg) is a widespread environmental toxin that preferentially and adversely affects developing organisms. To investigate the impact of MeHg toxicity on the formation of the vertebrate nervous system at physiologically relevant concentrations, we designed a graded phenotype scale for evaluating Xenopus laevis embryos exposed to MeHg in solution. Embryos displayed a range of abnormalities in response to MeHg, particularly in brain development, which is influenced by both MeHg concentration and the number of embryos per ml of exposure solution. A TC50 of ~50 μg/l and LC50 of ~100 μg/l were found when maintaining embryos at a density of one per ml, and both increased with increasing embryo density. In situ hybridization and microarray analysis showed no significant change in expression of early neural patterning genes including sox2, en2, or delta; however a noticeable decrease was observed in the terminal neural differentiation genes GAD and xGAT, but not xVGlut. PCNA, a marker for proliferating cells, was negatively correlated with MeHg dose, with a significant reduction in cell number in the forebrain and spinal cord of exposed embryos by tadpole stages. Conversely, the number of apoptotic cells in neural regions detected by a TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay was significantly increased. These results provide evidence that disruption of embryonic neural development by MeHg may not be directly due to a loss of neural progenitor specification and gene transcription, but to a more general decrease in cell proliferation and increase in cell death throughout the developing nervous system. PMID:25496965

  13. Leukemia inhibitory factor (LIF) enhances MAP2 + and HUC/D + neurons and influences neurite extension during differentiation of neural progenitors derived from human embryonic stem cells.

    EPA Science Inventory

    Leukemia Inhibitory Factor (L1F), a member of the Interleukin 6 cytokine family, has a role in differentiation of Human Neural Progenitor (hNP) cells in vitro. hNP cells, derived from Human Embryonic Stem (hES) cells, have an unlimited capacity for self-renewal in monolayer cultu...

  14. Sonic hedgehog signaling coordinates the proliferation and differentiation of neural stem/progenitor cells by regulating cell cycle kinetics during development of the neocortex.

    PubMed

    Komada, Munekazu

    2012-06-01

    Sonic hedgehog (Shh) acts as a morphogen in normal development of various vertebrate tissues and organs. Shh signaling is essential for patterning and cell-fate specification, particularly in the central nervous system. Shh signaling plays different roles depending on its concentration, area, and timing of exposure. During the development of the neocortex, a low level of Shh is expressed in the neural stem/progenitor cells as well as in mature neurons in the dorsal telencephalon. Shh signaling in neocortex development has been shown to regulate cell cycle kinetics of radial glial cells and intermediate progenitor cells, thereby maintaining the proliferation, survival and differentiation of neurons in the neocortex. During the development of the telencephalon, endogenous Shh signaling is involved in the transition of slow-cycling neural stem cells to fast-cycling neural progenitor cells. It seems that high-level Shh signaling in the ventral telencephalon is essential for ventral specification, while low-level Shh signaling in the dorsal telencephalon plays important roles in the fine-tuning of cell cycle kinetics. The Shh levels and multiple functions of Shh signaling are important for proper corticogenesis in the developing brain. The present paper discusses the roles of Shh signaling in the proliferation and differentiation of neural stem/progenitor cells.

  15. In Vivo Labeling of Adult Neural Progenitors for MRI with Micron Sized Particles of Iron Oxide: Quantitation of Labeled Cell Phenotype

    PubMed Central

    Sumner, James P.; Shapiro, Erik M.; Maric, Dragan; Conroy, Richard; Koretsky, Alan P.

    2009-01-01

    The subventricular zone (SVZ) is a continual source of neural progenitors throughout adulthood. Many of the animal models designed to study the migration of these cells from the ventricle to places of interest like the olfactory bulb or an injury site require histology to localize precursor cells. Here, it is demonstrated that up to 30% of the neural progenitors that migrate along the rostral migratory stream (RMS) in an adult rodent can be labeled for MRI via intraventricular injection of micron sized particles of iron oxide (MPIOs). The precursors migrating from the SVZ along the RMS were found to populate the olfactory bulb with all three types of neural cells; neurons, oligodendrocytes, and astrocytes. In all cases 10-30% of these cells were labeled in the RMS en route to the olfactory bulb. Ara-C an anti-mitotic agent eliminated precursor cells at the SVZ, RMS, and olfactory bulb and also eliminated the MRI detection of the precursors. This indicates that MRI signal detected is due to progenitor cells that leave the SVZ and is not due to non-specific diffusion of MPIOs. Using MRI to visualize neural progenitor cell behavior in individual animals during plasticity or disease models should be a useful tool, especially in combination with other information that MRI can supply. PMID:18722534

  16. Leukemia inhibitory factor (LIF) enhances MAP2 + and HUC/D + neurons and influences neurite extension during differentiation of neural progenitors derived from human embryonic stem cells.

    EPA Science Inventory

    Leukemia Inhibitory Factor (L1F), a member of the Interleukin 6 cytokine family, has a role in differentiation of Human Neural Progenitor (hNP) cells in vitro. hNP cells, derived from Human Embryonic Stem (hES) cells, have an unlimited capacity for self-renewal in monolayer cultu...

  17. Fail-Safe Therapy by Gamma-Ray Irradiation Against Tumor Formation by Human-Induced Pluripotent Stem Cell-Derived Neural Progenitors.

    PubMed

    Katsukawa, Mitsuko; Nakajima, Yusuke; Fukumoto, Akiko; Doi, Daisuke; Takahashi, Jun

    2016-06-01

    Cell replacement therapy holds great promise for Parkinson's disease (PD), but residual undifferentiated cells and immature neural progenitors in the therapy may cause tumor formation. Although cell sorting could effectively exclude these proliferative cells, from the viewpoint of clinical application, there exists no adequate coping strategy in the case of their contamination. In this study, we analyzed a component of proliferative cells in the grafts of human-induced pluripotent stem cell-derived neural progenitors and investigated the effect of radiation therapy on tumor formation. In our differentiating protocol, analyses of neural progenitors (day 19) revealed that the proliferating cells expressed early neural markers (SOX1, PAX6) or a dopaminergic neuron progenitor marker (FOXA2). When grafted into the rat striatum, these immature neurons gradually became postmitotic in the brain, and the rosette structures disappeared at 14 weeks. However, at 4-8 weeks, the SOX1(+)PAX6(+) cells formed rosette structures in the grafts, suggesting their tumorigenic potential. Therefore, to develop a fail-safe therapy against tumor formation, we investigated the effect of radiation therapy. At 4 weeks posttransplantation, when KI67(+) cells comprised the highest ratio, radiation therapy with (137)Cs Gammacell Exactor for tumor-bearing immunodeficient rats showed a significant decrease in graft volume and percentage of SOX1(+)KI67(+) cells in the graft, thus demonstrating the preventive effect of gamma-ray irradiation against tumorigenicity. These results give us critical criteria for the safety of future cell replacement therapy for PD.

  18. Successful differentiation of neural stem/progenitor cells cultured on electrically adjustable indium tin oxide (ITO) surface.

    PubMed

    Lei, Kin Fong; Lee, I-Chi; Liu, Yung-Chiang; Wu, Yu-Chieh

    2014-12-02

    In order to control differentiation of neural cells and guide the developed neurites to targets, polyelectrolyte multilayer (PEM) films were used because of their capability of modulation of electrical charged characteristics, thickness, and stiffness. In this work, we suggested that indium tin oxide (ITO) is an alternative surface to achieve the above-mentioned objectives. A microfluidic system with four culture chambers was developed and each chamber consisted of parallel ITO surfaces for the application of adjustable electrical field. Neural stem/progenitor cells (NSPCs) were respectively cultured on the ITO surfaces with and without PEM film, constructed by alternate adsorption of poly(L-lysine) (PLL) and poly(L-glutamic acid) (PLGA). Analyses of cell morphology, cytotoxicity, process outgrowth, differentiated cell types, and neuron functionality were compared between both surfaces. In this study, NSPCs successfully differentiated on ITO surface with electrical stimulation. The optimal electrical potential was found to be 80 mV that could stimulate the longest process, i.e., >300 μm, after 3 days culture. Cell differentiation, process development, and functionality of differentiated neuron on ITO surface were shown to be strongly controlled by the electrical stimulation that can be simply adjusted by external equipment. The electrically adjustable cell differentiation reported here could potentially be applied to neurochip for the study of neural signal transmission in a well-constructed network.

  19. Poly-L-ornithine promotes preferred differentiation of neural stem/progenitor cells via ERK signalling pathway

    NASA Astrophysics Data System (ADS)

    Ge, Hongfei; Tan, Liang; Wu, Pengfei; Yin, Yi; Liu, Xin; Meng, Hui; Cui, Gaoyu; Wu, Nan; Lin, Jiangkai; Hu, Rong; Feng, Hua

    2015-10-01

    Neural stem/progenitor cells (NSPCs) replacement therapies are the most attractive strategies to restore an injured brain. Key challenges of such therapies are enriching NSPCs and directing them differentiation into specific neural cell types. Here, three biomaterial substrates Poly-L-ornithine (PO), Poly-L-lysine (PLL) and fibronectin (FN) were investigated for their effects on proliferation and differentiation of rat NSPCs, and the underlying mechanisms were also explored. The results showed PO significantly increased NSPCs proliferation and induced preferred differentiation, compared with PLL and FN. Checking protein markers of several neural cell subtypes, it is showed PO significantly induced NSPCs expressing Doublecortin (DCX) and Olig2, one for neuroblasts and young neurons and the other for young oligodendrocytes. It is suggested the ERK signaling pathway was involving in this process because an ERK antagonist U0126 could inhibit PO’s effects mentioned above, as well as an ERK pathway agonist Ceramide C6 could enhance them. Given that both neurons and oligodendrocytes are the most vulnerable cells in many neurological diseases, PO-induced preferred differentiation into neurons and oligodendrocytes is a potential paradigm for NSPCs-based therapies.

  20. Histone Methylation and microRNA-dependent Regulation of Epigenetic Activities in Neural Progenitor Self-Renewal and Differentiation.

    PubMed

    Cacci, Emanuele; Negri, Rodolfo; Biagioni, Stefano; Lupo, Giuseppe

    2017-01-01

    Neural stem/progenitor cell (NSPC) self-renewal and differentiation in the developing and the adult brain are controlled by extra-cellular signals and by the inherent competence of NSPCs to produce appropriate responses. Stage-dependent responsiveness of NSPCs to extrinsic cues is orchestrated at the epigenetic level. Epigenetic mechanisms such as DNA methylation, histone modifications and non-coding RNA-mediated regulation control crucial aspects of NSPC development and function, and are also implicated in pathological conditions. While their roles in the regulation of stem cell fate have been largely explored in pluripotent stem cell models, the epigenetic signature of NSPCs is also key to determine their multipotency as well as their progressive bias towards specific differentiation outcomes. Here we review recent developments in this field, focusing on the roles of histone methylation marks and the protein complexes controlling their deposition in NSPCs of the developing cerebral cortex and the adult subventricular zone. In this context, we describe how bivalent promoters, carrying antagonistic epigenetic modifications, feature during multiple steps of neural development, from neural lineage specification to neuronal differentiation. Furthermore, we discuss the emerging cross-talk between epigenetic regulators and microRNAs, and how the interplay between these different layers of regulation can finely tune the expression of genes controlling NSPC maintenance and differentiation. In particular, we highlight recent advances in the identification of astrocyte-enriched microRNAs and their function in cell fate choices of NSPCs differentiating towards glial lineages.

  1. Cell type-dependent Erk-Akt pathway crosstalk regulates the proliferation of fetal neural progenitor cells

    PubMed Central

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

    2016-01-01

    Neural progenitor (NP) cells are the multipotent cells that produce neurons and glia in the central nervous system. Compounds regulating their proliferation are key to both understanding brain development and unlocking their potential in regenerative repair. We discuss a chemical screen that unexpectedly identified inhibitors of Erk signaling potently promoting the self-renewing divisions of fetal NP cells. This occurred through crosstalk between Erk and Akt signaling cascades. The crosstalk mechanism is cell type-specific, and is not detected in adult NP cells as well as brain tumor cells. The mechanism was also shown to be independent from the GSK-3 signaling pathway, which has been reported to be a major regulator of NP cell homeostasis and inhibitors to which were also identified in the screen. In vitro Erk inhibition led to the prolonged rapid expansion of fetal NP cells while retaining their multipotency. In vivo inhibitor administration significantly inhibited the neuronal differentiation, and resulted in increased proliferative progenitor cells in the ventricular/subventricular zone (VZ/SVZ) of the embryonic cortex. Our results uncovered a novel regulating pathway for NP cell proliferation in the developing brain. The discovery provides a pharmacological basis for in vitro expansion and in vivo manipulation of NP cells. PMID:27211495

  2. Human skin neural crest progenitor cells are susceptible to BRAF(V600E)-induced transformation.

    PubMed

    Kumar, S M; Dai, J; Li, S; Yang, R; Yu, H; Nathanson, K L; Liu, S; Zhou, H; Guo, J; Xu, X

    2014-02-13

    Adult stem cells are multipotent and persist in small numbers in adult tissues throughout the lifespan of an organism. Unlike differentiated cells, adult stem cells are intrinsically resistant to senescence. It is unclear how adult stem cells in solid organs respond to oncogenic stimulation and whether these cells have a role in tumor initiation. We report here that expression of BRAF(V600E) in human neural crest progenitor cells (hNCPCs) did not induce growth arrest as seen in human melanocytes, but instead, increased their cell proliferation capacity. These cells (hNCPCs(V600E)) acquired anchorage-independent growth ability and were weakly tumorigenic in vivo. Unlike in human melanocytes, BRAF(V600E) expression in hNCPCs did not induce p16(INK4a) expression. BRAF(V600E) induced elevated expression of CDK2, CDK4, MITF and EST1/2 protein in hNCPCs, and also induced melanocytic differentiation of these cells. Furthermore, overexpression of MITF in hNCPCs(V600E) dramatically increased their tumorigenicity and resulted in fully transformed tumor cells. These findings indicate that hNCPCs are susceptible to BRAF(V600E)-induced transformation, and MITF potentiates the oncogenic effect of BRAF(V600E) in these progenitor cells. These results suggest that the hNCPCs are potential targets for BRAF(V600E)-induced melanocytic tumor formation.

  3. 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

  4. Numb regulates the balance between Notch recycling and late-endosome targeting in Drosophila neural progenitor cells

    PubMed Central

    Johnson, Seth A.; Zitserman, Diana; Roegiers, Fabrice

    2016-01-01

    The Notch signaling pathway plays essential roles in both animal development and human disease. Regulation of Notch receptor levels in membrane compartments has been shown to affect signaling in a variety of contexts. Here we used steady-state and pulse-labeling techniques to follow Notch receptors in sensory organ precursor cells in Drosophila. We find that the endosomal adaptor protein Numb regulates levels of Notch receptor trafficking to Rab7-labeled late endosomes but not early endosomes. Using an assay we developed that labels different pools of Notch receptors as they move through the endocytic system, we show that Numb specifically suppresses a recycled Notch receptor subpopulation and that excess Notch signaling in numb mutants requires the recycling endosome GTPase Rab11 activity. Our data therefore suggest that Numb controls the balance between Notch receptor recycling and receptor targeting to late endosomes to regulate signaling output after asymmetric cell division in Drosophila neural progenitors. PMID:27466320

  5. Suppression of Nestin reveals a critical role for p38-EGFR pathway in neural progenitor cell proliferation

    PubMed Central

    Hu, Wentao; Lu, Hong; Wang, Shang; Yin, Wenhan; Liu, Xujie; Dong, Lin; Chiu, Richard; Shen, Li; Lu, Wen-Jing; Lan, Feng

    2016-01-01

    The expression of intermediate filament Nestin is necessary for the neural progenitor cells (NPCs) to maintain stemness, but the underlying cellular and molecular mechanism remains unclear. In this study, we demonstrated that Nestin is required for the self-renew of NPCs through activating MAPK and EGFR pathways. Knockdown of Nestin by shRNA inhibited cell cycle progression and proliferation in mouse NPCs. Moreover, suppression of Nestin reduced expression of the epidermal growth factor receptor (EGFR) in NPCs and inhibited the mitogenic effects of EGF on these cells. Treatment of NPCs with p38-MAPK inhibitor PD169316 reversed cell cycle arrest caused by the knockdown of Nestin. Our findings indicate that Nestin promotes NPC proliferation via p38-MAPK and EGFR pathways, and reveals the necessity of these pathways in NPCs self-renewal. PMID:27894083

  6. CREB signalling in neural stem/progenitor cells: recent developments and the implications for brain tumour biology.

    PubMed

    Mantamadiotis, Theo; Papalexis, Nikos; Dworkin, Sebastian

    2012-04-01

    This paper discusses the evidence for the role of CREB in neural stem/progenitor cell (NSPC) function and oncogenesis and how these functions may be important for the development and growth of brain tumours. The cyclic-AMP response element binding (CREB) protein has many roles in neurons, ranging from neuronal survival to higher order brain functions such as memory and drug addiction behaviours. Recent studies have revealed that CREB also has a role in NSPC survival, differentiation and proliferation. Recent work has shown that over-expression of CREB in transgenic animals can impart oncogenic properties on cells in various tissues and that aberrant CREB expression is associated with tumours in patients. It is the central position of CREB, downstream of key developmental and growth signalling pathways, which give CREB the ability to influence a spectrum of cell activities, such as cell survival, growth and differentiation in both normal and cancer cells. Copyright © 2012 WILEY Periodicals, Inc.

  7. Increased abundance of translation machinery in stem cell-derived neural progenitor cells from four schizophrenia patients.

    PubMed

    Topol, A; English, J A; Flaherty, E; Rajarajan, P; Hartley, B J; Gupta, S; Desland, F; Zhu, S; Goff, T; Friedman, L; Rapoport, J; Felsenfeld, D; Cagney, G; Mackay-Sim, A; Savas, J N; Aronow, B; Fang, G; Zhang, B; Cotter, D; Brennand, K J

    2015-10-20

    The genetic and epigenetic factors contributing to risk for schizophrenia (SZ) remain unresolved. Here we demonstrate, for the first time, perturbed global protein translation in human-induced pluripotent stem cell (hiPSC)-derived forebrain neural progenitor cells (NPCs) from four SZ patients relative to six unaffected controls. We report increased total protein levels and protein synthesis, together with two independent sets of quantitative mass spectrometry evidence indicating markedly increased levels of ribosomal and translation initiation and elongation factor proteins, in SZ hiPSC NPCs. We posit that perturbed levels of global protein synthesis in SZ hiPSC NPCs represent a novel post-transcriptional mechanism that might contribute to disease progression.

  8. Increased abundance of translation machinery in stem cell–derived neural progenitor cells from four schizophrenia patients

    PubMed Central

    Topol, A; English, J A; Flaherty, E; Rajarajan, P; Hartley, B J; Gupta, S; Desland, F; Zhu, S; Goff, T; Friedman, L; Rapoport, J; Felsenfeld, D; Cagney, G; Mackay-Sim, A; Savas, J N; Aronow, B; Fang, G; Zhang, B; Cotter, D; Brennand, K J

    2015-01-01

    The genetic and epigenetic factors contributing to risk for schizophrenia (SZ) remain unresolved. Here we demonstrate, for the first time, perturbed global protein translation in human-induced pluripotent stem cell (hiPSC)-derived forebrain neural progenitor cells (NPCs) from four SZ patients relative to six unaffected controls. We report increased total protein levels and protein synthesis, together with two independent sets of quantitative mass spectrometry evidence indicating markedly increased levels of ribosomal and translation initiation and elongation factor proteins, in SZ hiPSC NPCs. We posit that perturbed levels of global protein synthesis in SZ hiPSC NPCs represent a novel post-transcriptional mechanism that might contribute to disease progression. PMID:26485546

  9. 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

  10. Potential for cell therapy in Parkinson's disease using genetically programmed human embryonic stem cell-derived neural progenitor cells.

    PubMed

    Ambasudhan, Rajesh; Dolatabadi, Nima; Nutter, Anthony; Masliah, Eliezer; Mckercher, Scott R; Lipton, Stuart A

    2014-08-15

    Neural transplantation is a promising strategy for restoring dopaminergic dysfunction and modifying disease progression in Parkinson's disease (PD). Human embryonic stem cells (hESCs) are a potential resource in this regard because of their ability to provide a virtually limitless supply of homogenous dopaminergic progenitors and neurons of appropriate lineage. The recent advances in developing robust cell culture protocols for directed differentiation of hESCs to near pure populations of ventral mesencephalic (A9-type) dopaminergic neurons has heightened the prospects for PD cell therapy. Here, we focus our review on current state-of-the-art techniques for harnessing hESC-based strategies toward development of a stem cell therapeutic for PD. Importantly, we also briefly describe a novel genetic-programming approach that may address many of the key challenges that remain in the field and that may hasten clinical translation. © 2014 Wiley Periodicals, Inc.

  11. Polysaccharides from Ganoderma lucidum Promote Cognitive Function and Neural Progenitor Proliferation in Mouse Model of Alzheimer's Disease.

    PubMed

    Huang, Shichao; Mao, Jianxin; Ding, Kan; Zhou, Yue; Zeng, Xianglu; Yang, Wenjuan; Wang, Peipei; Zhao, Cun; Yao, Jian; Xia, Peng; Pei, Gang

    2017-01-10

    Promoting neurogenesis is a promising strategy for the treatment of cognition impairment associated with Alzheimer's disease (AD). Ganoderma lucidum is a revered medicinal mushroom for health-promoting benefits in the Orient. Here, we found that oral administration of the polysaccharides and water extract from G. lucidum promoted neural progenitor cell (NPC) proliferation to enhance neurogenesis and alleviated cognitive deficits in transgenic AD mice. G. lucidum polysaccharides (GLP) also promoted self-renewal of NPC in cell culture. Further mechanistic study revealed that GLP potentiated activation of fibroblast growth factor receptor 1 (FGFR1) and downstream extracellular signal-regulated kinase (ERK) and AKT cascades. Consistently, inhibition of FGFR1 effectively blocked the GLP-promoted NPC proliferation and activation of the downstream cascades. Our findings suggest that GLP could serve as a regenerative therapeutic agent for the treatment of cognitive decline associated with neurodegenerative diseases.

  12. A Method for 3D Immunostaining and Optical Imaging of the Mouse Brain Demonstrated in Neural Progenitor Cells

    PubMed Central

    Gleave, Jacqueline A.; Lerch, Jason P.; Henkelman, R. Mark; Nieman, Brian J.

    2013-01-01

    It is important to understand changes in cell distribution that occur as a part of disease progression. This is typically achieved using standard sectioning and immunostaining, however, many structures and cell distribution patterns are not readily appreciated in two-dimensions, including the distribution of neural stem and progenitor cells in the mouse forebrain. Three-dimensional immunostaining in the mouse brain has been hampered by poor penetration. For this reason, we have developed a method that allows for entire hemispheres of the mouse brain to be stained using commercially available antibodies. Brains stained for glial fibrillary acidic protein, doublecortin and nestin were imaged in three-dimensions using optical projection tomography and serial two-photon tomography. This staining method is simple, using a combination of heat, time and specimen preparation procedures readily available, so that it can be easily implemented without the need for specialized equipment, making it accessible to most laboratories. PMID:23936537

  13. The cerebrospinal fluid provides a proliferative niche for neural progenitor cells

    PubMed Central

    Lehtinen, Maria K.; Zappaterra, Mauro W.; Chen, Xi; Yang, Yawei J.; Hill, Anthony; Lun, Melody; Maynard, Thomas; Gonzalez, Dilenny; Kim, Seonhee; Ye, Ping; D’Ercole, A. Joseph; Wong, Eric T.; LaMantia, Anthony S.; Walsh, Christopher A.

    2011-01-01

    Cortical development depends on the active integration of cell autonomous and extrinsic cues, but the coordination of these processes is poorly understood. Here, we show that the apical complex protein Pals1 and Pten have opposing roles in localizing the Igf1R to the apical, ventricular domain of cerebral cortical progenitor cells. We found that the cerebrospinal fluid (CSF), which contacts this apical domain, has an age-dependent effect on proliferation, much of which is attributable to Igf2, but that CSF contains other signaling activities as well. CSF samples from patients with glioblastoma multiforme show elevated Igf2 and stimulate stem cell proliferation in an Igf2-dependent manner. Together, our findings demonstrate that the apical complex couples intrinsic and extrinsic signaling, enabling progenitors to sense and respond appropriately to diffusible CSF-borne signals distributed widely throughout the brain. The temporal control of CSF composition may have critical relevance to normal development and neuropathological conditions. PMID:21382550

  14. Embryonic exposure to ethanol disturbs regulation of mitotic spindle orientation via GABA(A) receptors in neural progenitors in ventricular zone of developing neocortex.

    PubMed

    Tochitani, Shiro; Sakata-Haga, Hiromi; Fukui, Yoshihiro

    2010-03-19

    Neural progenitors in the ventricular zone of the developing neocortex divide oriented either parallel or perpendicular to the ventricular surface based on their mitotic spindle orientation. It has been shown that the cleavage plane orientation is developmentally regulated and plays a crucial role in cell fate determination of neural progenitors or the maintenance of the proliferative ventricular zone during neocortical development. We tested if fetal exposure to ethanol, the most widely used psychoactive agent and a potent teratogen that may cause malformation in the central nervous system, alters mitotic cleavage orientation of the neural progenitors at the apical surface of the ventricular zone in the developing neocortex. Fetal exposure to ethanol on E10.5 and 11.5 increased the occurrence frequency of a horizontal cleavage plane that is parallel to the ventricular surface on E 12.5. Administration of picrotoxin, a GABA(A) receptor antagonist, prior to ethanol administration canceled the effect of ethanol with the frequency of horizontal division similar to the control level, although picrotoxin itself did not show any effect on cleavage plane orientation. Phenobarbital, a GABA(A) receptor agonist, induced horizontal cleavage to an extent similar to that induced by ethanol administration. (+)MK801, an antagonist of NMDA receptor that is another major target of ethanol in neural cells, did not affect the cleavage plane of dividing progenitors. These results suggest that fetal ethanol exposure induced alterations in the cleavage plane orientation of neural progenitors in the ventricular zone of the neocortex via the enhancement of the function of GABA(A) receptors.

  15. Cell-cycle-independent transitions in temporal identity of mammalian neural progenitor cells

    PubMed Central

    Okamoto, Mayumi; Miyata, Takaki; Konno, Daijiro; Ueda, Hiroki R.; Kasukawa, Takeya; Hashimoto, Mitsuhiro; Matsuzaki, Fumio; Kawaguchi, Ayano

    2016-01-01

    During cerebral development, many types of neurons are sequentially generated by self-renewing progenitor cells called apical progenitors (APs). Temporal changes in AP identity are thought to be responsible for neuronal diversity; however, the mechanisms underlying such changes remain largely unknown. Here we perform single-cell transcriptome analysis of individual progenitors at different developmental stages, and identify a subset of genes whose expression changes over time but is independent of differentiation status. Surprisingly, the pattern of changes in the expression of such temporal-axis genes in APs is unaffected by cell-cycle arrest. Consistent with this, transient cell-cycle arrest of APs in vivo does not prevent descendant neurons from acquiring their correct laminar fates. Analysis of cultured APs reveals that transitions in AP gene expression are driven by both cell-intrinsic and -extrinsic mechanisms. These results suggest that the timing mechanisms controlling AP temporal identity function independently of cell-cycle progression and Notch activation mode. PMID:27094546

  16. Human neural progenitor cells decrease photoreceptor degeneration, normalize opsin distribution and support synapse structure in cultured porcine retina.

    PubMed

    Mollick, Tanzina; Mohlin, Camilla; Johansson, Kjell

    2016-09-01

    Retinal neurodegenerative disorders like retinitis pigmentosa, age-related macular degeneration, diabetic retinopathy and retinal detachment decrease retinal functionality leading to visual impairment. The pathological events are characterized by photoreceptor degeneration, synaptic disassembly, remodeling of postsynaptic neurons and activation of glial cells. Despite intense research, no effective treatment has been found for these disorders. The current study explores the potential of human neural progenitor cell (hNPC) derived factors to slow the degenerative processes in adult porcine retinal explants. Retinas were cultured for 3 days with or without hNPCs as a feeder layer and investigated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), immunohistochemical, western blot and quantitative real time-polymerase chain reaction (qRT-PCR) techniques. TUNEL showed that hNPCs had the capacity to limit photoreceptor cell death. Among cone photoreceptors, hNPC coculture resulted in better maintenance of cone outer segments and reduced opsin mislocalization. Additionally, maintained synaptic structural integrity and preservation of second order calbindin positive horizontal cells was also observed. However, Müller cell gliosis only seemed to be alleviated in terms of reduced Müller cell density. Our observations indicate that at 3 days of coculture, hNPC derived factors had the capacity to protect photoreceptors, maintain synaptic integrity and support horizontal cell survival. Human neural progenitor cell applied treatment modalities may be an effective strategy to help maintain retinal functionality in neurodegenerative pathologies. Whether hNPCs can independently hinder Müller cell gliosis by utilizing higher concentrations or by combination with other pharmacological agents still needs to be determined. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Increased dentate neurogenesis after grafting of glial restricted progenitors or neural stem cells in the aging hippocampus.

    PubMed

    Hattiangady, Bharathi; Shuai, Bing; Cai, Jingli; Coksaygan, Turhan; Rao, Mahendra S; Shetty, Ashok K

    2007-08-01

    Neurogenesis in the dentate gyrus (DG) declines severely by middle age, potentially because of age-related changes in the DG microenvironment. We hypothesize that providing fresh glial restricted progenitors (GRPs) or neural stem cells (NSCs) to the aging hippocampus via grafting enriches the DG microenvironment and thereby stimulates the production of new granule cells from endogenous NSCs. The GRPs isolated from the spinal cords of embryonic day 13.5 transgenic F344 rats expressing human alkaline phosphatase gene and NSCs isolated from embryonic day 9 caudal neural tubes of Sox-2:EGFP transgenic mice were expanded in vitro and grafted into the hippocampi of middle-aged (12 months old) F344 rats. Both types of grafts survived well, and grafted NSCs in addition migrated to all layers of the hippocampus. Phenotypic characterization revealed that both GRPs and NSCs differentiated predominantly into astrocytes and oligodendrocytic progenitors. Neuronal differentiation of graft-derived cells was mostly absent except in the dentate subgranular zone (SGZ), where some of the migrated NSCs but not GRPs differentiated into neurons. Analyses of the numbers of newly born neurons in the DG using 5'-bromodeoxyuridine and/or doublecortin assays, however, demonstrated considerably increased dentate neurogenesis in animals receiving grafts of GRPs or NSCs in comparison with both naïve controls and animals receiving sham-grafting surgery. Thus, both GRPs and NSCs survive well, differentiate predominantly into glia, and stimulate the endogenous NSCs in the SGZ to produce more new dentate granule cells following grafting into the aging hippocampus. Grafting of GRPs or NSCs therefore provides an attractive approach for improving neurogenesis in the aging hippocampus. Disclosure of potential conflicts of interest is found at the end of this article.

  18. Neutralization of nerve growth factor impairs proliferation and differentiation of adult neural progenitors in the subventricular zone.

    PubMed

    Scardigli, Raffaella; Capelli, Paolo; Vignone, Domenico; Brandi, Rossella; Ceci, Marcello; La Regina, Federico; Piras, Eleonora; Cintoli, Simona; Berardi, Nicoletta; Capsoni, Simona; Cattaneo, Antonino

    2014-09-01

    Adult neurogenesis is a multistep process regulated by several extrinsic factors, including neurotrophins. Among them, little is known about the role of nerve growth factor (NGF) in the neurogenic niches of the mouse. Here we analyzed the biology of adult neural stem cells (NSCs) from the subventricular zone (SVZ) of AD11 anti-NGF transgenic mice, in which the expression of the recombinant antibody aD11 leads to a chronic postnatal neutralization of endogenous NGF. We showed that AD11-NSCs proliferate 10-fold less, with respect to their control counterparts, and display a significant impairment in their ability to differentiate into β-tubulin positive neurons. We found a considerable reduction in the number of SVZ progenitors and neuroblasts also in vivo, which correlates with a lower number of newborn neurons in the olfactory bulbs of AD11 mice and a severe deficit in the ability of these mice to discriminate between different odors. We also demonstrated that, in AD11 mice, the morphology of both SVZ-resident and neurosphere-derived astrocytes is significantly altered. We were able to reproduce the AD11 phenotype in vitro, by acutely treating wild type NSCs with the anti-NGF antibody, further demonstrating that both the proliferation and the differentiation defects are due to the NGF deprivation. Consistently, the proliferative impairment of AD11 progenitors, as well as the atrophic morphology of AD11 astrocytes, can be partly rescued in vitro and in vivo by exogenous NGF addition. Altogether, our results demonstrate a causal link between NGF signaling and proper proliferation and differentiation of neural stem cells from the SVZ.

  19. Overexpression of MCT8 enhances the differentiation of ES cells into neural progenitors.

    PubMed

    Sugiura, Mika; Nagaoka, Masato; Yabuuchi, Hikaru; Akaike, Toshihiro

    2007-09-07

    Embryonic stem (ES) cell differentiation is regulated by cytokines and growth factors, as well as small-compound chemicals incorporated into cells by transporter proteins. Little is known regarding the effect of transporters on ES cell differentiation. This study focused on the effect of transporters during the neural-lineage differentiation of ES cells. Among the 27 types of SLC family transporters, MCT8 expression was coincident with that of neural stem cell markers, and the overexpression of MCT8 accelerated the differentiation into neural cells. These results suggested that the transporters and their substrates also play a crucial role in the regulation of ES cell differentiation.

  20. Neuregulin 1 Type II-ErbB Signaling Promotes Cell Divisions Generating Neurons from Neural Progenitor Cells in the Developing Zebrafish Brain.

    PubMed

    Sato, Tomomi; Sato, Fuminori; Kamezaki, Aosa; Sakaguchi, Kazuya; Tanigome, Ryoma; Kawakami, Koichi; Sehara-Fujisawa, Atsuko

    2015-01-01

    Post-mitotic neurons are generated from neural progenitor cells (NPCs) at the expense of their proliferation. Molecular and cellular mechanisms that regulate neuron production temporally and spatially should impact on the size and shape of the brain. While transcription factors such as neurogenin1 (neurog1) and neurod govern progression of neurogenesis as cell-intrinsic mechanisms, recent studies show regulatory roles of several cell-extrinsic or intercellular signaling molecules including Notch, FGF and Wnt in production of neurons/neural progenitor cells from neural stem cells/radial glial cells (NSCs/RGCs) in the ventricular zone (VZ). However, it remains elusive how production of post-mitotic neurons from neural progenitor cells is regulated in the sub-ventricular zone (SVZ). Here we show that newborn neurons accumulate in the basal-to-apical direction in the optic tectum (OT) of zebrafish embryos. While neural progenitor cells are amplified by mitoses in the apical ventricular zone, neurons are exclusively produced through mitoses of neural progenitor cells in the sub-basal zone, later in the sub-ventricular zone, and accumulate apically onto older neurons. This neurogenesis depends on Neuregulin 1 type II (NRG1-II)-ErbB signaling. Treatment with an ErbB inhibitor, AG1478 impairs mitoses in the sub-ventricular zone of the optic tectum. Removal of AG1478 resumes sub-ventricular mitoses without precedent mitoses in the apical ventricular zone prior to basal-to-apical accumulation of neurons, suggesting critical roles of ErbB signaling in mitoses for post-mitotic neuron production. Knockdown of NRG1-II impairs both mitoses in the sub-basal/sub-ventricular zone and the ventricular zone. Injection of soluble human NRG1 into the developing brain ameliorates neurogenesis of NRG1-II-knockdown embryos, suggesting a conserved role of NRG1 as a cell-extrinsic signal. From these results, we propose that NRG1-ErbB signaling stimulates cell divisions generating neurons from

  1. Alternating Current Electric Fields of Varying Frequencies: Effects on Proliferation and Differentiation of Porcine Neural Progenitor Cells

    PubMed Central

    Lim, Ji-Hey; McCullen, Seth D.; Piedrahita, Jorge A.

    2013-01-01

    Abstract Application of sinusoidal electric fields (EFs) has been observed to affect cellular processes, including alignment, proliferation, and differentiation. In the present study, we applied low-frequency alternating current (AC) EFs to porcine neural progenitor cells (pNPCs) and investigated the effects on cell patterning, proliferation, and differentiation. pNPCs were grown directly on interdigitated electrodes (IDEs) localizing the EFs to a region accessible visually for fluorescence-based assays. Cultures of pNPCs were exposed to EFs (1 V/cm) of 1 Hz, 10 Hz, and 50 Hz for 3, 7, and 14 days and compared to control cultures. Immunocytochemistry was performed to evaluate the expression of neural markers. pNPCs grew uniformly with no evidence of alignment to the EFs and no change in cell numbers when compared with controls. Nestin expression was shown in all groups at 3 and 7 days, but not at 14 days. NG2 expression was low in all groups. Co-expression of glial fibrillary acidic protein (GFAP) and TUJ1 was significantly higher in the cultures exposed to 10- and 50-Hz EFs than the controls. In summary, sinusoidal AC EFs via IDEs did not alter the alignment and proliferation of pNPCs, but higher frequency stimulation appeared to delay differentiation into mature astrocytes. PMID:23961767

  2. Neuroprotective Effects of Transplanted Mesenchymal Stromal Cells-derived Human Umbilical Cord Blood Neural Progenitor Cells in EAE.

    PubMed

    Rafieemehr, Hassan; Kheyrandish, Maryam; Soleimani, Masoud

    2015-12-01

    Multiple Sclerosis (MS) is an autoimmune inflammatory demyelinating disease of the central nervous system. The aim of this study was to investigate the neuroprotective effects of transplanted human umbilical cord blood mesenchymal stromal cells (UCB-MSC) derived neural progenitor cell (MDNPC) in EAE, an experimental model of MS. To initiate neuronal differentiation of UCB-MSCs, the pre-induction medium was removed and replaced with induction media containing retinoic acid, b FGF, h EGF, NGF, IBMX and ascorbic acid for one week. The expression of neural genes was examined in comparison to control group by real-time PCR assay. Then, experimental autoimmune encephalitis (EAE) was induced using myelin oligodendrocyte glycoprotein (MOG, 35-55 peptides) in 24 C57BL/6 mice. After induction, the mice were divided in four groups (n=6) as follows: healthy, PBS, UCB-MSCs and MDNPC, respectively. At the end of the study, disease status in all the groups was analyzed using hematoxylin-eosin (H&E) staining of brain sections. We found that UCB-MSCs exhibit neuronal differentiation potential in vitro and transplanted MDNPC lowered clinical score and reduced CNS leukocyte infiltration compared to untreated mice. Our results showed that MDNPC from UCB may be a proper candidate for regenerative therapy in MS and other neurodegenerative diseases.

  3. Extremely low-frequency electromagnetic fields enhance the proliferation and differentiation of neural progenitor cells cultured from ischemic brains.

    PubMed

    Cheng, Yannan; Dai, Yiqin; Zhu, Ximin; Xu, Haochen; Cai, Ping; Xia, Ruohong; Mao, Lizhen; Zhao, Bing-Qiao; Fan, Wenying

    2015-10-21

    In the mammalian brain, neurogenesis persists throughout the embryonic period and adulthood in the subventricular zone of the lateral ventricle and the granular zone (dentate gyrus) of the hippocampus. Newborn neural progenitor cells (NPCs) in the two regions play a critical role in structural and functional plasticity and neural regeneration after brain injury. Previous studies have reported that extremely low-frequency electromagnetic fields (ELF-EMF) could promote osteogenesis, angiogenesis, and cardiac stem cells' differentiation, which indicates that ELF-EMF might be an effective tool for regenerative therapy. The present studies were carried out to examine the effects of ELF-EMF on hippocampal NPCs cultured from embryonic and adult ischemic brains. We found that exposure to ELF-EMF (50 Hz, 0.4 mT) significantly enhanced the proliferation capability both in embryonic NPCs and in ischemic NPCs. Neuronal differentiation was also enhanced after 7 days of cumulative ELF-EMF exposure, whereas glial differentiation was not influenced markedly. The expression of phosphorylated Akt increased during the proliferation process when ischemic NPCs were exposed to ELF-EMF. However, blockage of the Akt pathway abolished the ELF-EMF-induced proliferation of ischemic NPCs. These data show that ELF-EMF promotes neurogenesis of ischemic NPCs and suggest that this effect may occur through the Akt pathway.Video abstract, Supplemental Digital Content 1, http://links.lww.com/WNR/A347.

  4. Alternating current electric fields of varying frequencies: effects on proliferation and differentiation of porcine neural progenitor cells.

    PubMed

    Lim, Ji-Hey; McCullen, Seth D; Piedrahita, Jorge A; Loboa, Elizabeth G; Olby, Natasha J

    2013-10-01

    Application of sinusoidal electric fields (EFs) has been observed to affect cellular processes, including alignment, proliferation, and differentiation. In the present study, we applied low-frequency alternating current (AC) EFs to porcine neural progenitor cells (pNPCs) and investigated the effects on cell patterning, proliferation, and differentiation. pNPCs were grown directly on interdigitated electrodes (IDEs) localizing the EFs to a region accessible visually for fluorescence-based assays. Cultures of pNPCs were exposed to EFs (1 V/cm) of 1 Hz, 10 Hz, and 50 Hz for 3, 7, and 14 days and compared to control cultures. Immunocytochemistry was performed to evaluate the expression of neural markers. pNPCs grew uniformly with no evidence of alignment to the EFs and no change in cell numbers when compared with controls. Nestin expression was shown in all groups at 3 and 7 days, but not at 14 days. NG2 expression was low in all groups. Co-expression of glial fibrillary acidic protein (GFAP) and TUJ1 was significantly higher in the cultures exposed to 10- and 50-Hz EFs than the controls. In summary, sinusoidal AC EFs via IDEs did not alter the alignment and proliferation of pNPCs, but higher frequency stimulation appeared to delay differentiation into mature astrocytes.

  5. The BAF complex interacts with Pax6 in adult neural progenitors to establish a neurogenic cross-regulatory transcriptional network.

    PubMed

    Ninkovic, Jovica; Steiner-Mezzadri, Andrea; Jawerka, Melanie; Akinci, Umut; Masserdotti, Giacomo; Petricca, Stefania; Fischer, Judith; von Holst, Alexander; Beckers, Johanes; Lie, Chichung D; Petrik, David; Miller, Erik; Tang, Jiong; Wu, Jiang; Lefebvre, Veronique; Demmers, Jeroen; Eisch, Amelia; Metzger, Daniel; Crabtree, Gerald; Irmler, Martin; Poot, Raymond; Götz, Magdalena

    2013-10-03

    Numerous transcriptional regulators of neurogenesis have been identified in the developing and adult brain, but how neurogenic fate is programmed at the epigenetic level remains poorly defined. Here, we report that the transcription factor Pax6 directly interacts with the Brg1-containing BAF complex in adult neural progenitors. Deletion of either Brg1 or Pax6 in the subependymal zone (SEZ) causes the progeny of adult neural stem cells to convert to the ependymal lineage within the SEZ while migrating neuroblasts convert to different glial lineages en route to or in the olfactory bulb (OB). Genome-wide analyses reveal that the majority of genes downregulated in the Brg1 null SEZ and OB contain Pax6 binding sites and are also downregulated in Pax6 null SEZ and OB. Downstream of the Pax6-BAF complex, we find that Sox11, Nfib, and Pou3f4 form a transcriptional cross-regulatory network that drives neurogenesis and can convert postnatal glia into neurons. Taken together, elements of our work identify a tripartite effector network activated by Pax6-BAF that programs neuronal fate.

  6. The role of the JAK-STAT pathway in neural stem cells, neural progenitor cells and reactive astrocytes after spinal cord injury.

    PubMed

    Wang, Tianyi; Yuan, Wenqi; Liu, Yong; Zhang, Yanjun; Wang, Zhijie; Zhou, Xianhu; Ning, Guangzhi; Zhang, Liang; Yao, Liwei; Feng, Shiqing; Kong, Xiaohong

    2015-03-01

    Patients with spinal cord injuries can develop severe neurological damage and dysfunction, which is not only induced by primary but also by secondary injuries. As an evolutionarily conserved pathway of eukaryotes, the JAK-STAT pathway is associated with cell growth, survival, development and differentiation; activation of the JAK-STAT pathway has been previously reported in central nervous system injury. The JAK-STAT pathway is directly associated with neurogenesis and glia scar formation in the injury region. Following injury of the axon, the overexpression and activation of STAT3 is exhibited specifically in protecting neurons. To investigate the role of the JAK-STAT pathway in neuroprotection, we summarized the effect of JAK-STAT pathway in the following three sections: Firstly, the modulation of JAK-STAT pathway in proliferation and differentiation of neural stem cells and neural progenitor cells is discussed; secondly, the time-dependent effect of JAK-STAT pathway in reactive astrocytes to reveal their capability of neuroprotection is revealed and lastly, we focus on how the astrocyte-secretory polypeptides (astrocyte-derived cytokines and trophic factors) accomplish neuroprotection via the JAK-STAT pathway.

  7. Tissue Engineering Special Feature: A macroporous hydrogel for the coculture of neural progenitor and endothelial cells to form functional vascular networks in vivo

    NASA Astrophysics Data System (ADS)

    Ford, Millicent C.; Bertram, James P.; Royce Hynes, Sara; Michaud, Michael; Li, Qi; Young, Michael; Segal, Steven S.; Madri, Joseph A.; Lavik, Erin B.

    2006-02-01

    A microvascular network is critical for the survival and function of most tissues. We have investigated the potential of neural progenitor cells to augment the formation and stabilization of microvascular networks in a previously uncharacterized three-dimensional macroporous hydrogel and the ability of this engineered system to develop a functional microcirculation in vivo. The hydrogel is synthesized by cross-linking polyethylene glycol with polylysine around a salt-leached polylactic-co-glycolic acid scaffold that is degraded in a sodium hydroxide solution. An open macroporous network is formed that supports the efficient formation of tubular structures by brain endothelial cells. After subcutaneous implantation of hydrogel cocultures in mice, blood flow in new microvessels was apparent at 2 weeks with perfused networks established on the surface of implants at 6 weeks. Compared to endothelial cells cultured alone, cocultures of endothelial cells and neural progenitor cells had a significantly greater density of tubular structures positive for platelet endothelial cell adhesion molecule-1 at the 6-week time point. In implant cross sections, the presence of red blood cells in vessel lumens confirmed a functional microcirculation. These findings indicate that neural progenitor cells promote the formation of endothelial cell tubes in coculture and the development of a functional microcirculation in vivo. We demonstrate a previously undescribed strategy for creating stable microvascular networks to support engineered tissues of desired parenchymal cell origin. microvasculature | neural stem cells | polymer | scaffold

  8. 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.

  9. Bone Morphogenetic Protein 4 Signalling in Neural Stem and Progenitor Cells during Development and after Injury

    PubMed Central

    Cole, Alistair E.; Murray, Simon S.; Xiao, Junhua

    2016-01-01

    Substantial progress has been made in identifying the extracellular signalling pathways that regulate neural stem and precursor cell biology in the central nervous system (CNS). The bone morphogenetic proteins (BMPs), in particular BMP4, are key players regulating neuronal and glial cell development from neural precursor cells in the embryonic, postnatal, and injured CNS. Here we review recent studies on BMP4 signalling in the generation of neurons, astrocytes, and oligodendroglial cells in the CNS. We also discuss putative mechanisms that BMP4 may utilise to influence glial cell development following CNS injury and highlight some questions for further research. PMID:27293450

  10. Human neural stem/progenitor cells derived from the olfactory epithelium express the TrkB receptor and migrate in response to BDNF.

    PubMed

    Ortiz-López, Leonardo; González-Olvera, Jorge Julio; Vega-Rivera, Nelly Maritza; García-Anaya, Maria; Carapia-Hernández, Ana Karen; Velázquez-Escobar, Julio César; Ramírez-Rodríguez, Gerardo Bernabé

    2017-07-04

    Neurogenesis constitutively occurs in the olfactory epithelium of mammals, including humans. The fact that new neurons in the adult olfactory epithelium derive from resident neural stem/progenitor cells suggests a potential use for these cells in studies of neural diseases, as well as in neuronal cell replacement therapies. In this regard, some studies have proposed that the human olfactory epithelium is a source of neural stem/progenitor cells for autologous transplantation. Although these potential applications are interesting, it is important to understand the cell biology and/or whether human neural stem/progenitor cells in the olfactory epithelium sense external signals, such as brain-derived neurotrophic factor (BDNF), that is also found in other pro-neurogenic microenvironments. BDNF plays a key role in several biological processes, including cell migration. Thus, we characterized human neural stem/progenitor cells derived from the olfactory epithelium (hNS/PCs-OE) and studied their in vitro migratory response to BDNF. In the present study, we determined that hNS/PCs-OE express the protein markers Nestin, Sox2, Ki67 and βIII-tubulin. Moreover, the doubling time of hNS/PCs-OE was approximately 38h. Additionally, we found that hNS/PCs-OE express the BDNF receptor TrkB, and pharmacological approaches showed that the BDNF-induced (40ng/ml) migration of differentiated hNS/PCs-OE was affected by the compound K252a, which prevents TrkB activation. This observation was accompanied by changes in the number of vinculin adhesion contacts. Our results suggest that hNS/PCs-OE exhibit a migratory response to BDNF, accompanied by the turnover of adhesion contacts. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

  11. IGF-1 enhances cell proliferation and survival during early differentiation of mesenchymal stem cells to neural progenitor-like cells.

    PubMed

    Huat, Tee Jong; Khan, Amir Ali; Pati, Soumya; Mustafa, Zulkifli; Abdullah, Jafri Malin; Jaafar, Hasnan

    2014-07-22

    There has been increasing interest recently in the plasticity of mesenchymal stem cells (MSCs) and their potential to differentiate into neural lineages. To unravel the roles and effects of different growth factors in the differentiation of MSCs into neural lineages, we have differentiated MSCs into neural lineages using different combinations of growth factors. Based on previous studies of the roles of insulin-like growth factor 1 (IGF-1) in neural stem cell isolation in the laboratory, we hypothesized that IGF-1 can enhance proliferation and reduce apoptosis in neural progenitor-like cells (NPCs) during differentiation of MSCs into NCPs.We induced MSCs differentiation under four different combinations of growth factors: (A) EGF + bFGF, (B) EGF + bFGF + IGF-1, (C) EGF + bFGF + LIF, (D) EGF + bFGF + BDNF, and (E) without growth factors, as a negative control. The neurospheres formed were characterized by immunofluorescence staining against nestin, and the expression was measured by flow cytometry. Cell proliferation and apoptosis were also studied by MTS and Annexin V assay, respectively, at three different time intervals (24 hr, 3 days, and 5 days). The neurospheres formed in the four groups were then terminally differentiated into neuron and glial cells. The four derived NPCs showed a significantly higher expression of nestin than was shown by the negative control. Among the groups treated with growth factors, NPCs treated with IGF-1 showed the highest expression of nestin. Furthermore, NPCs derived using IGF-1 exhibited the highest cell proliferation and cell survival among the treated groups. The NPCs derived from IGF-1 treatment also resulted in a better yield after the terminal differentiation into neurons and glial cells than that of the other treated groups. Our results suggested that IGF-1 has a crucial role in the differentiation of MSCs into neuronal lineage by enhancing the proliferation and reducing the apoptosis in the

  12. The Regenerative Response of Endogenous Neural Stem/Progenitor Cells to Traumatic Brain Injury

    DTIC Science & Technology

    2014-06-09

    for regeneration after injury. and promote the regenerative response from endogenous cells. Sonic hedgehog (Shh) has been shown to maintain neural...4 Sonic Hedgehog Signaling in the Mammalian Brain ..................................................... 8 CHAPTER 2...Traumatic Brain Injury Models Reveal Differential Effects in the Subventricular Zone and Divergent Sonic Hedgehog Signaling Pathways in Neuroblasts and

  13. Live imaging of mouse endogenous neural progenitors migrating in response to an induced tumor.

    PubMed

    Elvira, Gema; García, Isabel; Benito, Marina; Gallo, Juan; Desco, Manuel; Penadés, Soledad; Garcia-Sanz, Jose A; Silva, Augusto

    2012-01-01

    Adult neurogenesis is restricted to specific brain regions. Although involved in the continuous supply of interneurons for the olfactory function, the role of neural precursors in brain damage-repair remains an open question. Aiming to in vivo identify endogenous neural precursor cells migrating towards a brain damage site, the monoclonal antibody Nilo2 recognizing cell surface antigens on neuroblasts, was coupled to magnetic glyconanoparticles (mGNPs). The Nilo2-mGNP complexes allowed, by magnetic resonance imaging in living animals, the in vivo identification of endogenous neural precursors at their niche, as well as their migration to a lesion site (induced brain tumor), which was fast (within hours) and orderly. Interestingly, the rapid migration of neuroblasts towards a damage site is a characteristic that might be exploited to precisely localize early damage events in neurodegenerative diseases. In addition, it might facilitate the study of regenerative mechanisms through the activation of endogenous neural cell precursors. A similar approach, combining magnetic glyconanoparticles linked to appropriate antibodies could be applied to flag other small cell subpopulations within the organism, track their migration, localize stem cell niches, cancer stem cells or even track metastatic cells.

  14. Interactions of HIV and drugs of abuse: the importance of glia, neural progenitors, and host genetic factors.

    PubMed

    Hauser, Kurt F; Knapp, Pamela E

    2014-01-01

    Considerable insight has been gained into the comorbid, interactive effects of HIV and drug abuse in the brain using experimental models. This review, which considers opiates, methamphetamine, and cocaine, emphasizes the importance of host genetics and glial plasticity in driving the pathogenic neuron remodeling underlying neuro-acquired immunodeficiency syndrome and drug abuse comorbidity. Clinical findings are less concordant than experimental work, and the response of individuals to HIV and to drug abuse can vary tremendously. Host-genetic variability is important in determining viral tropism, neuropathogenesis, drug responses, and addictive behavior. However, genetic differences alone cannot account for individual variability in the brain "connectome." Environment and experience are critical determinants in the evolution of synaptic circuitry throughout life. Neurons and glia both exercise control over determinants of synaptic plasticity that are disrupted by HIV and drug abuse. Perivascular macrophages, microglia, and to a lesser extent astroglia can harbor the infection. Uninfected bystanders, especially astroglia, propagate and amplify inflammatory signals. Drug abuse by itself derails neuronal and glial function, and the outcome of chronic exposure is maladaptive plasticity. The negative consequences of coexposure to HIV and drug abuse are determined by numerous factors including genetics, sex, age, and multidrug exposure. Glia and some neurons are generated throughout life, and their progenitors appear to be targets of HIV and opiates/psychostimulants. The chronic nature of HIV and drug abuse appears to result in sustained alterations in the maturation and fate of neural progenitors, which may affect the balance of glial populations within multiple brain regions. © 2014 Elsevier Inc. All rights reserved.

  15. Interactions of HIV and drugs of abuse: the importance of glia, neural progenitors, and host genetic factors

    PubMed Central

    Hauser, Kurt F.; Knapp, Pamela E.

    2015-01-01

    Considerable insight has been gained into the comorbid, interactive effects of HIV and drug abuse in the brain using experimental models. This review, which considers opiates, methamphetamine, and cocaine, emphasizes the importance of host genetics and glial plasticity in driving the pathogenic neuron remodeling underlying neuro-acquired immunodeficiency syndrome (neuroAIDS) and drug abuse comorbidity. Clinical findings are less concordant than experimental work, and the response of individuals to HIV and to drug abuse can vary tremendously. Host-genetic variability is important in determining viral tropism, neuropathogenesis, drug responses, and addictive behavior. However, genetic differences alone cannot account for individual variability in the brain “connectome”. Environment and experience are critical determinants in the evolution of synaptic circuitry throughout life. Neurons and glia both exercise control over determinants of synaptic plasticity that are disrupted by HIV and drug abuse. Perivascular macrophages, microglia, and to a lesser extent astroglia can harbor the infection. Uninfected bystanders, especially astroglia, propagate and amplify inflammatory signals. Drug abuse by itself derails neuronal and glial function, and the outcome of chronic exposure is maladaptive plasticity. The negative consequences of coexposure to HIV and drug abuse are determined by numerous factors including genetics, sex, age, and multidrug exposure. Glia and some neurons are generated throughout life and their progenitors appear to be targets of HIV and opiates/psychostimulants. The chronic nature of HIV and drug abuse appears to result in sustained alterations in the maturation and fate of neural progenitors, which may affect the balance of glial populations within multiple brain regions. PMID:25175867

  16. Differential Subcellular Localization of the Glucocorticoid Receptor in Distinct Neural Stem and Progenitor Populations of the Mouse Telencephalon In Vivo

    PubMed Central

    Tsiarli, Maria A.; Monaghan, A. Paula; DeFranco, Donald B.

    2013-01-01

    Glucocorticoids are given to pregnant women at risk for premature delivery to promote lung maturation. Despite reports of detrimental effects of glucocorticoids on telencephalic neural stem/progenitor cells (NSPCs), the regional and cellular expression of the glucocorticoid receptor (GR) in various NSPC populations in the intact brain has not been thoroughly assessed. Therefore in this study we performed a detailed analysis of GR protein expression in the developing mouse ventral and dorsal telencephalon in vivo. At embryonic day 11.5 (E11.5), the majority of Pax6-positive radial glial cells (RGCs) and Tbr2-positive intermediate progenitor cells (IPCs) expressed nuclear GR, while a small number of RGCs on the apical ventricular zone (aVZ), expressed cytoplasmic GR. However, on E13.5, the latter population of RGCs increased in size, whereas abventricular NSPCs and especially neurons of the cortical plate, expressed nuclear GR. In IPCs, GR was always nuclear. A similar expression profile was observed throughout the ventral telencephalon, hippocampus and olfactory bulb, with NSPCs of the aVZ primarily expressing cytoplasmic GR, while abventricular NSPCs and mature cells primarily expressed nuclear GR. Close to birth, nuclear GR accumulated within specific cortical areas such as layer V, the subplate and CA1 area of the hippocampus. In summary, our data show that GR protein is present in early NSPCs of the dorsal and ventral telencephalon at E11.5 and primarily occupies the nucleus. Moreover, our study suggests that the subcellular localization of the receptor may be subjected to region and neurodevelopmental stage-specific regulation. PMID:23751362

  17. Transplantation of Human Neural Progenitor Cells Expressing IGF-1 Enhances Retinal Ganglion Cell Survival

    PubMed Central

    Guo, Caiwei; Sun, Yu; Liao, Tiffany; Beattie, Ursula; López, Francisco J.; Chen, Dong Feng; Lashkari, Kameran

    2015-01-01

    We have previously characterized human neuronal progenitor cells (hNP) that can adopt a retinal ganglion cell (RGC)-like morphology within the RGC and nerve fiber layers of the retina. In an effort to determine whether hNPs could be used a candidate cells for targeted delivery of neurotrophic factors (NTFs), we evaluated whether hNPs transfected with an vector that expresses IGF-1 in the form of a fusion protein with tdTomato (TD), would increase RGC survival in vitro and confer neuroprotective effects in a mouse model of glaucoma. RGCs co-cultured with hNPIGF-TD cells displayed enhanced survival, and increased neurite extension and branching as compared to hNPTD or untransfected hNP cells. Application of various IGF-1 signaling blockers or IGF-1 receptor antagonists abrogated these effects. In vivo, using a model of glaucoma we showed that IOP elevation led to reductions in retinal RGC count. In this model, evaluation of retinal flatmounts and optic nerve cross sections indicated that only hNPIGF-TD cells effectively reduced RGC death and showed a trend to improve optic nerve axonal loss. RT-PCR analysis of retina lysates over time showed that the neurotrophic effects of IGF-1 were also attributed to down-regulation of inflammatory and to some extent, angiogenic pathways. This study shows that neuronal progenitor cells that hone into the RGC and nerve fiber layers may be used as vehicles for local production and delivery of a desired NTF. Transplantation of hNPIGF-TD cells improves RGC survival in vitro and protects against RGC loss in a rodent model of glaucoma. Our findings have provided experimental evidence and form the basis for applying cell-based strategies for local delivery of NTFs into the retina. Application of cell-based delivery may be extended to other disease conditions beyond glaucoma. PMID:25923430

  18. 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

  19. Clinical and pathological effects of intrathecal injection of mesenchymal stem cell-derived neural progenitors in an experimental model of multiple sclerosis.

    PubMed

    Harris, Violaine K; Yan, Qi Jiang; Vyshkina, Tamara; Sahabi, Sadia; Liu, Xinhe; Sadiq, Saud A

    2012-02-15

    Multiple sclerosis (MS) is associated with irreversible disability in a significant proportion of patients. At present, there is no treatment to halt or reverse the progression of established disability. In an effort to develop cell therapy-based strategies for progressive MS, we investigated the pre-clinical efficacy of bone marrow mesenchymal stem cell-derived neural progenitors (MSC-NPs) as an autologous source of stem cells. MSC-NPs consist of a subpopulation of bone marrow MSCs with neural progenitor and immunoregulatory properties, and a reduced capacity for mesodermal differentiation, suggesting that this cell population may be appropriate for clinical application in the CNS. We investigated whether MSC-NPs could promote repair and recovery after intrathecal injection into mice with EAE. Multiple injections of MSC-NPs starting at the onset of the chronic phase of disease improved neurological function compared to controls, whereas a single injection had no effect on disease scores. Intrathecal injection of MSC-NPs correlated with reduced immune cell infiltration, reduced area of demyelination, and increased number of endogenous nestin-positive progenitor cells in EAE mice. These observations suggest that MSC-NPs may influence the rate of repair through effects on endogenous progenitors in the spinal cord. This study supports the use of autologous MSC-NPs in MS patients as a means of promoting CNS repair.

  20. Isolation of neural progenitor cells from the human adult subventricular zone based on expression of the cell surface marker CD271.

    PubMed

    van Strien, Miriam E; Sluijs, Jacqueline A; Reynolds, Brent A; Steindler, Dennis A; Aronica, Eleonora; Hol, Elly M

    2014-04-01

    Neural progenitor cells (NPCs) in the subventricular zone (SVZ) hold promise for future therapy for neurodegenerative disorders, because the stimulation of adult neurogenesis could potentially restore the function of degenerating neurons and glia. To obtain more knowledge on these NPCs, we developed a method to specifically isolate NPCs from postmortem adult human brains based on the expression of the specific human adult neural stem/progenitor cell marker glial fibrillary acidic protein δ (GFAPδ). An extensive immunophenotyping analysis for cell surface markers resulted in the observation that CD271 was limited to the SVZ-derived GFAPδ-positive cells. CD271(+) cells developed into neurospheres and could be differentiated into astrocytes, neurons, and oligodendrocytes. We are the first to show that a pure population of NPCs can be isolated from the adult human SVZ, which is highly instrumental for developing future therapies based on stimulating endogenous SVZ neurogenesis.

  1. Isolation of Neural Progenitor Cells From the Human Adult Subventricular Zone Based on Expression of the Cell Surface Marker CD271

    PubMed Central

    Sluijs, Jacqueline A.; Reynolds, Brent A.; Steindler, Dennis A.; Aronica, Eleonora

    2014-01-01

    Neural progenitor cells (NPCs) in the subventricular zone (SVZ) hold promise for future therapy for neurodegenerative disorders, because the stimulation of adult neurogenesis could potentially restore the function of degenerating neurons and glia. To obtain more knowledge on these NPCs, we developed a method to specifically isolate NPCs from postmortem adult human brains based on the expression of the specific human adult neural stem/progenitor cell marker glial fibrillary acidic protein δ (GFAPδ). An extensive immunophenotyping analysis for cell surface markers resulted in the observation that CD271 was limited to the SVZ-derived GFAPδ-positive cells. CD271+ cells developed into neurospheres and could be differentiated into astrocytes, neurons, and oligodendrocytes. We are the first to show that a pure population of NPCs can be isolated from the adult human SVZ, which is highly instrumental for developing future therapies based on stimulating endogenous SVZ neurogenesis. PMID:24604282

  2. Transcriptional Regulation of Notch1 Expression by Nkx6.1 in Neural Stem/Progenitor Cells during Ventral Spinal Cord Development

    PubMed Central

    Li, Ying; Tzatzalos, Evangeline; Kwan, Kelvin Y.; Grumet, Martin; Cai, Li

    2016-01-01

    Notch1 signaling plays a critical role in maintaining and determining neural stem/progenitor cell (NSPC) fate, yet the transcriptional mechanism controlling Notch1 specific expression in NSPCs remains incomplete. Here, we show transcription factor Nkx6.1 interacts with a cis-element (CR2, an evolutionarily conserved non-coding fragment in the second intron of Notch1 locus) and regulates the expression of Notch1 in ventral NSPCs of the developing spinal cord. We show that the Notch1 expression is modulated by the interaction of Nkx6.1 with a 139 bp enhancer sequence within CR2. Knockdown or overexpression of Nkx6.1 leads to down- or up-regulated Notch1 expression, respectively. In CR2-GFP transgenic mouse, GFP expression was found prominent in the ventricular zone and neural progenitor cells from embryonic day 9.5 to postnatal day 7. GFP+ cells were mainly neural progenitors for interneurons and not for motoneurons or glial cells. Moreover, GFP expression persisted in a subset of ependymal cells in the adult spinal cord, suggesting that CR2 is active in both embryonic and adult NSPCs. Together our data reveal a novel mechanism of Notch1 transcriptional regulation in the ventral spinal cord by Nkx6.1 via its binding with Notch1 enhancer CR2 during embryonic development. PMID:27924849

  3. NKCC1 knockdown decreases neuron production through GABA(A)-regulated neural progenitor proliferation and delays dendrite development.

    PubMed

    Young, Stephanie Z; Taylor, M Morgan; Wu, Sharon; Ikeda-Matsuo, Yuri; Kubera, Cathryn; Bordey, Angélique

    2012-09-26

    Signaling through GABA(A) receptors controls neural progenitor cell (NPC) development in vitro and is altered in schizophrenic and autistic individuals. However, the in vivo function of GABA(A) signaling on neural stem cell proliferation, and ultimately neurogenesis, remains unknown. To examine GABA(A) function in vivo, we electroporated plasmids encoding short-hairpin (sh) RNA against the Na-K-2Cl cotransporter NKCC1 (shNKCC1) in NPCs of the neonatal subventricular zone in mice to reduce GABA(A)-induced depolarization. Reduced GABA(A) depolarization identified by a loss of GABA(A)-induced calcium responses in most electroporated NPCs led to a 70% decrease in the number of proliferative Ki67(+) NPCs and a 60% reduction in newborn neuron density. Premature loss of GABA(A) depolarization in newborn neurons resulted in truncated dendritic arborization at the time of synaptic integration. However, by 6 weeks the dendritic tree had partially recovered and displayed a small, albeit significant, decrease in dendritic complexity but not total dendritic length. To further examine GABA(A) function on NPCs, we treated animals with a GABA(A) allosteric agonist, pentobarbital. Enhancement of GABA(A) activity in NPCs increased the number of proliferative NPCs by 60%. Combining shNKCC1 and pentobarbital prevented the shNKCC1 and the pentobarbital effects on NPC proliferation, suggesting that these manipulations affected NPCs through GABA(A) receptors. Thus, dysregulation in GABA(A) depolarizing activity delayed dendritic development and reduced NPC proliferation resulting in decreased neuronal density.

  4. CXCR7 Participates in CXCL12-mediated Cell Cycle and Proliferation Regulation in Mouse Neural Progenitor Cells

    PubMed Central

    Wang, Y.; Xu, P.; Qiu, L.; Zhang, M.; Huang, Y.; Zheng, J.C.

    2016-01-01

    Background: Cell cycle regulation of neural progenitor cells (NPCs) is an essential process for neurogenesis, neural development, and repair after brain trauma. Stromal cell-derived factor-1 (SDF-1, CXCL12) and its receptors CXCR4 and CXCR7 are well known in regulating the migration and survival of NPCs. The effects of CXCL12 on NPCs proliferation, cell cycle regulation, and their associated signaling pathways remain unclear. Cyclin D1 is a protein required for progression through the G1 phase of the cell cycle and a known downstream target of β-catenin. Therefore, cyclin D1 plays critical roles of cell cycle regulation, proliferation, and survival in NPCs. Methods: Primary mouse NPCs (mNPCs) were derived from brain tissues of wild-type, Cxcr4 knockout, or Cxcr7 knockout mice at mouse embryonic day 13.5 (E13.5). Flow cytometry was used to perform cell cycle analysis by quantitation of DNA content. Real-time PCR and Western blot were used to evaluate mRNA and protein expressions, respectively. Ki67 immunostaining and TUNEL assay were used to assess the proliferation and survival of mNPCs, respectively. Results: CXCL12 pretreatment led to the shortening of G0/G1 phase and lengthening of S phase, suggesting that CXCL12 regulates cell cycle progression in mNPCs. Consistently, CXCL12 treatment increased the expression of CyclinD1 and β-catenin, and promoted proliferation and survival of mNPCs. Cxcr7 knockout of mNPCs blocked CXCL12-mediated mNPCs proliferation, whereas Cxcr4 knockout mNPC did not significantly effect CXCL12- mediated mNPCs proliferation. Conclusion: CXCR7 plays an important role in CXCL12-mediated mNPC cell cycle regulation and proliferation. PMID:27573194

  5. Brain self-protection: the role of endogenous neural progenitor cells in adult brain after cerebral cortical ischemia.

    PubMed

    Li, Bin; Piao, Chun-Shu; Liu, Xiao-Yun; Guo, Wen-Ping; Xue, Yue-Qiang; Duan, Wei-Ming; Gonzalez-Toledo, Maria E; Zhao, Li-Ru

    2010-04-23

    Convincing evidence has shown that brain ischemia causes the proliferation of neural stem cells/neural progenitor cells (NSCs/NPCs) in both the subventricular zone (SVZ) and the subgranular zone (SGZ) of adult brain. The role of brain ischemia-induced NSC/NPC proliferation, however, has remained unclear. Here we have determined whether brain ischemia-induced amplification of the NSCs/NPCs in adult brain is required for brain self-protection. The approach of intracerebroventricular (ICV) infusion of cytosine arabinoside (Ara-C), an inhibitor for cell proliferation, for the first 7days after brain ischemia was used to block ischemia-induced NSC/NPC proliferation. We observed that ICV infusion of Ara-C caused a complete blockade of NSC/NPC proliferation in the SVZ and a dramatic reduction of NSC/NPC proliferation in the SGZ. Additionally, as a result of the inhibition of ischemia-induced NSC/NPC pool amplification, the number of neurons in the hippocampal CA1 and CA3 was significantly reduced, the infarction size was significantly enlarged, and neurological deficits were significantly worsened after focal brain ischemia. We also found that an NSC/NPC-conditioned medium showed neuroprotective effects in vitro and that adult NSC/NPC-released brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) are required for NSC/NPC-conditioned medium-induced neuroprotection. These data suggest that NSC/NPC-generated trophic factors are neuroprotective and that brain ischemia-triggered NSC/NPC proliferation is crucial for brain protection. This study provides insights into the contribution of endogenous NSCs/NPCs to brain self-protection in adult brain after ischemia injury.

  6. Chemotactic recruitment of adult neural progenitor cells into multifunctional hydrogels providing sustained SDF-1α release and compatible structural support.

    PubMed

    Lim, Teck Chuan; Rokkappanavar, Swetha; Toh, Wei Seong; Wang, Li-Shan; Kurisawa, Motoichi; Spector, Myron

    2013-03-01

    Without chemotactic cues and structural support, cavitary brain lesions typically fail to recruit endogenous neural progenitor cells (NPCs). Toward resolving this, we engineered multifunctional biomaterials comprising injectable gelatin-hydroxyphenylpropionic acid (Gtn-HPA) hydrogels and dextran sulfate/chitosan polyelectrolyte complex nanoparticles (PCNs) that delivered stromal cell-derived factor-1α (SDF-1α). Over 7 d of interface with in vitro tissue simulant containing adult rat hippocampal NPCs (aNPCs) and their neuronal progeny, Gtn-HPA/SDF-1α-PCN hydrogels promoted chemotactic recruitment to enhance infiltration of aNPCs by 3- to 45-fold relative to hydrogels that lacked SDF-1α or vehicles to sustain SDF-1α release. When cross-linked with 0.85-0.95 mM HO, Gtn-HPA/SDF-1α-PCN hydrogels provided optimally permissive structural support for migration of aNPCs. Specific matrix metalloproteinase (MMP) inhibitors revealed that 42, 30, and 55% of cell migration into Gtn-HPA/SDF-1α-PCN hydrogels involved MMP-2, 3, and 9, respectively, demonstrating the hydrogels to be compatible toward homing endogenous NPCs, given their expression of similar MMPs. Interestingly, PCNs utilized FGF-2 found in situ to induce chemokinesis, potentiate SDF-1α chemotactic recruitment, and increase proliferation of recruited cells, which collectively orchestrated a higher number of migrated aNPCs. Overall, Gtn-HPA/SDF-1α-PCN hydrogels prove to be promising biomaterials for injection into cavitary brain lesions to recruit endogenous NPCs and enhance neural tissue repair/regeneration.

  7. CXCR7 Participates in CXCL12-mediated Cell Cycle and Proliferation Regulation in Mouse Neural Progenitor Cells.

    PubMed

    Wang, Y; Xu, P; Qiu, L; Zhang, M; Huang, Y; Zheng, J C

    2016-01-01

    Cell cycle regulation of neural progenitor cells (NPCs) is an essential process for neurogenesis, neural development, and repair after brain trauma. Stromal cell-derived factor-1 (SDF-1, CXCL12) and its receptors CXCR4 and CXCR7 are well known in regulating the migration and survival of NPCs. The effects of CXCL12 on NPCs proliferation, cell cycle regulation, and their associated signaling pathways remain unclear. Cyclin D1 is a protein required for progression through the G1 phase of the cell cycle and a known downstream target of β -catenin. Therefore, cyclin D1 plays critical roles of cell cycle regulation, proliferation, and survival in NPCs. Primary mouse NPCs (mNPCs) were derived from brain tissues of wild-type, Cxcr4 knockout, or Cxcr7 knockout mice at mouse embryonic day 13.5 (E13.5). Flow cytometry was used to perform cell cycle analysis by quantitation of DNA content. Real-time PCR and Western blot were used to evaluate mRNA and protein expressions, respectively. Ki67 immunostaining and TUNEL assay were used to assess the proliferation and survival of mNPCs, respectively. CXCL12 pretreatment led to the shortening of G0/G1 phase and lengthening of S phase, suggesting that CXCL12 regulates cell cycle progression in mNPCs. Consistently, CXCL12 treatment increased the expression of CyclinD1 and β -catenin, and promoted proliferation and survival of mNPCs. Cxcr7 knockout of mNPCs blocked CXCL12-mediated mNPCs proliferation, whereas Cxcr4 knockout mNPC did not significantly effect CXCL12- mediated mNPCs proliferation. CXCR7 plays an important role in CXCL12-mediated mNPC cell cycle regulation and proliferation.

  8. Spatiotemporal recapitulation of central nervous system development by murine embryonic stem cell-derived neural stem/progenitor cells.

    PubMed

    Okada, Yohei; Matsumoto, Arifumi; Shimazaki, Takuya; Enoki, Ryosuke; Koizumi, Amane; Ishii, Seiji; Itoyama, Yasuto; Sobue, Gen; Okano, Hideyuki

    2008-12-01

    Neural stem/progenitor cells (NS/PCs) can generate a wide variety of neural cells. However, their fates are generally restricted, depending on the time and location of NS/PC origin. Here we demonstrate that we can recapitulate the spatiotemporal regulation of central nervous system (CNS) development in vitro by using a neurosphere-based culture system of embryonic stem (ES) cell-derived NS/PCs. This ES cell-derived neurosphere system enables the efficient derivation of highly neurogenic fibroblast growth factor-responsive NS/PCs with early temporal identities and high cell-fate plasticity. Over repeated passages, these NS/PCs exhibit temporal progression, becoming epidermal growth factor-responsive gliogenic NS/PCs with late temporal identities; this change is accompanied by an alteration in the epigenetic status of the glial fibrillary acidic protein promoter, similar to that observed in the developing brain. Moreover, the rostrocaudal and dorsoventral spatial identities of the NS/PCs can be successfully regulated by sequential administration of several morphogens. These NS/PCs can differentiate into early-born projection neurons, including cholinergic, catecholaminergic, serotonergic, and motor neurons, that exhibit action potentials in vitro. Finally, these NS/PCs differentiate into neurons that form synaptic contacts with host neurons after their transplantation into wild-type and disease model animals. Thus, this culture system can be used to obtain specific neurons from ES cells, is a simple and powerful tool for investigating the underlying mechanisms of CNS development, and is applicable to regenerative treatment for neurological disorders.

  9. Human Neural Progenitor Transplantation Rescues Behavior and Reduces α-Synuclein in a Transgenic Model of Dementia with Lewy Bodies.

    PubMed

    Goldberg, Natalie R S; Marsh, Samuel E; Ochaba, Joseph; Shelley, Brandon C; Davtyan, Hayk; Thompson, Leslie M; Steffan, Joan S; Svendsen, Clive N; Blurton-Jones, Mathew

    2017-02-22

    Synucleinopathies are a group of neurodegenerative disorders sharing the common feature of misfolding and accumulation of the presynaptic protein α-synuclein (α-syn) into insoluble aggregates. Within this diverse group, Dementia with Lewy Bodies (DLB) is characterized by the aberrant accumulation of α-syn in cortical, hippocampal, and brainstem neurons, resulting in multiple cellular stressors that particularly impair dopamine and glutamate neurotransmission and related motor and cognitive function. Recent studies show that murine neural stem cell (NSC) transplantation can improve cognitive or motor function in transgenic models of Alzheimer's and Huntington's disease, and DLB. However, examination of clinically relevant human NSCs in these models is hindered by the challenges of xenotransplantation and the confounding effects of immunosuppressant drugs on pathology and behavior. To address this challenge, we developed an immune-deficient transgenic model of DLB that lacks T-, B-, and NK-cells, yet exhibits progressive accumulation of human α-syn (h-α-syn)-laden inclusions and cognitive and motor impairments. We demonstrate that clinically relevant human neural progenitor cells (line CNS10-hNPCs) survive, migrate extensively and begin to differentiate preferentially into astrocytes following striatal transplantation into this DLB model. Critically, grafted CNS10-hNPCs rescue both cognitive and motor deficits after 1 and 3 months and, furthermore, restore striatal dopamine and glutamate systems. These behavioral and neurochemical benefits are likely achieved by reducing α-syn oligomers. Collectively, these results using a new model of DLB demonstrate that hNPC transplantation can impact a broad array of disease mechanisms and phenotypes and suggest a cellular therapeutic strategy that should be pursued. © Stem Cells Translational Medicine 2017.

  10. Recombinant neural progenitor transplants in the spinal dorsal horn alleviate chronic central neuropathic pain.

    PubMed

    Jergova, Stanislava; Gajavelli, Shyam; Pathak, Nirmal; Sagen, Jacqueline

    2016-04-01

    Neuropathic pain induced by spinal cord injury (SCI) is clinically challenging with inadequate long-term treatment options. Partial pain relief offered by pharmacologic treatment is often counterbalanced by adverse effects after prolonged use in chronic pain patients. Cell-based therapy for neuropathic pain using GABAergic neuronal progenitor cells (NPCs) has the potential to overcome untoward effects of systemic pharmacotherapy while enhancing analgesic potency due to local activation of GABAergic signaling in the spinal cord. However, multifactorial anomalies underlying chronic pain will likely require simultaneous targeting of multiple mechanisms. Here, we explore the analgesic potential of genetically modified rat embryonic GABAergic NPCs releasing a peptidergic NMDA receptor antagonist, Serine-histogranin (SHG), thus targeting both spinal hyperexcitability and reduced inhibitory processes. Recombinant NPCs were designed using either lentiviral or adeno-associated viral vectors (AAV2/8) encoding single and multimeric (6 copies of SHG) cDNA. Intraspinal injection of recombinant cells elicited enhanced analgesic effects compared with nonrecombinant NPCs in SCI-induced pain in rats. Moreover, potent and sustained antinociception was achieved, even after a 5-week postinjury delay, using recombinant multimeric NPCs. Intrathecal injection of SHG antibody attenuated analgesic effects of the recombinant grafts suggesting active participation of SHG in these antinociceptive effects. Immunoblots and immunocytochemical assays indicated ongoing recombinant peptide production and secretion in the grafted host spinal cords. These results support the potential for engineered NPCs grafted into the spinal dorsal horn to alleviate chronic neuropathic pain.

  11. Rationale and Methodology of Reprogramming for Generation of Induced Pluripotent Stem Cells and Induced Neural Progenitor Cells

    PubMed Central

    Tian, Zuojun; Guo, Fuzheng; Biswas, Sangita; Deng, Wenbin

    2016-01-01

    Great progress has been made regarding the capabilities to modify somatic cell fate ever since the technology for generation of induced pluripotent stem cells (iPSCs) was discovered in 2006. Later, induced neural progenitor cells (iNPCs) were generated from mouse and human cells, bypassing some of the concerns and risks of using iPSCs in neuroscience applications. To overcome the limitation of viral vector induced reprogramming, bioactive small molecules (SM) have been explored to enhance the efficiency of reprogramming or even replace transcription factors (TFs), making the reprogrammed cells more amenable to clinical application. The chemical induced reprogramming process is a simple process from a technical perspective, but the choice of SM at each step is vital during the procedure. The mechanisms underlying cell transdifferentiation are still poorly understood, although, several experimental data and insights have indicated the rationale of cell reprogramming. The process begins with the forced expression of specific TFs or activation/inhibition of cell signaling pathways by bioactive chemicals in defined culture condition, which initiates the further reactivation of endogenous gene program and an optimal stoichiometric expression of the endogenous pluri- or multi-potency genes, and finally leads to the birth of reprogrammed cells such as iPSCs and iNPCs. In this review, we first outline the rationale and discuss the methodology of iPSCs and iNPCs in a stepwise manner; and then we also discuss the chemical-based reprogramming of iPSCs and iNPCs. PMID:27104529

  12. Comparison of Neurite Outgrowth Induced by Erythropoietin (EPO) and Carbamylated Erythropoietin (CEPO) in Hippocampal Neural Progenitor Cells.

    PubMed

    Oh, Dong Hoon; Lee, In Young; Choi, Miyeon; Kim, Seok Hyeon; Son, Hyeon

    2012-08-01

    A previous animal study has shown the effects of erythropoietin (EPO) and its non-erythropoietic carbamylated derivative (CEPO) on neurogenesis in the dentate gyrus. In the present study, we sought to investigate the effect of EPO on adult hippocampal neurogenesis, and to compare the ability of EPO and CEPO promoting dendrite elongation in cultured hippocampal neural progenitor cells. Two-month-old male BALB/c mice were given daily injections of EPO (5 U/g) for seven days and were sacrificed 12 hours after the final injection. Proliferation assays demonstrated that EPO treatment increased the density of bromodeoxyuridine (BrdU)-labeled cells in the subgranular zone (SGZ) compared to that in vehicle-treated controls. Functional differentiation studies using dissociated hippocampal cultures revealed that EPO treatment also increased the number of double-labeled BrdU/microtubule-associated protein 2 (MAP2) neurons compared to those in vehicle-treated controls. Both EPO and CEPO treatment significantly increased the length of neurites and spine density in MAP2(+) cells. In summary, these results provide evidences that EPO and CEPO promote adult hippocampal neurogenesis and neuronal differentiation. These suggest that EPO and CEPO could be a good candidate for treating neuropsychiatric disorders such as depression and anxiety associated with neuronal atrophy and reduced hippocampal neurogenesis.

  13. HDAC3 controls gap 2/mitosis progression in adult neural stem/progenitor cells by regulating CDK1 levels.

    PubMed

    Jiang, Yindi; Hsieh, Jenny

    2014-09-16

    The maintenance of the resident adult neural stem/progenitor cell (NSPC) pool depends on the precise balance of proliferation, differentiation, and maintenance of the undifferentiated state. Identifying the mechanisms that regulate this balance in adult hippocampal NSPCs can provide insight into basic stem cell self-renewal principles important for tissue homeostasis and preventing tumor formation. Pharmacological inhibition of histone deacetylases (HDACs), a class of histone-modifying enzymes, have promising effects in cancer cells, yet the specific roles of individual HDACs in stem cell proliferation is unclear. Here using conditional KO (cKO) mice and in vitro cell culture, we show that histone deacetylase 3 (HDAC3) is required for the proliferation of adult NSPCs. Detailed cell cycle analysis of NSPCs from Hdac3 cKO mice reveals a defect in cell cycle progression through the gap 2/mitosis (G2/M) but not the S phase. Moreover, HDAC3 controls G2/M phase progression mainly through posttranslational stabilization of the G2/M cyclin-dependent kinase 1 (CDK1). These results demonstrate that HDAC3 plays a critical role in NSPC proliferation and suggest that strategies aimed at pharmacological modulation of HDAC3 may be beneficial for tissue regeneration and controlling tumor cell growth.

  14. Delayed epidural transplantation of human induced pluripotent stem cell-derived neural progenitors enhances functional recovery after stroke.

    PubMed

    Lee, I-Hui; Huang, Shiang-Suo; Chuang, Ching-Yu; Liao, Ko-Hsun; Chang, Li-Hsin; Chuang, Chia-Chi; Su, Yu-Shih; Lin, Hung-Jui; Hsieh, Jui-Yu; Su, Shu-Han; Lee, Oscar Kuang-Sheng; Kuo, Hung-Chih

    2017-05-16

    Induced pluripotent stem cell-derived neural progenitor cells (iPSC-NPCs) are a promising source of tailor-made cell therapy for neurological diseases. However, major obstacles to clinical use still exist. To circumvent complications related to intracerebral administration, we implanted human iPSC-NPCs epidurally over the peri-infarct cortex 7 days after permanent middle cerebral artery occlusion in adult rats. Compared to controls, cell-treated rats showed significant improvements in paretic forelimb usage and grip strength from 10 days post-transplantation (dpt) onwards, as well as reductions in lesion volumes, inflammatory infiltration and astrogliosis at 21 dpt. Few iPSC-NPCs migrated into rat peri-infarct cortices and exhibited poor survival in tissue. To examine the paracrine therapeutic mechanisms of epidural iPSC-NPC grafts, we used transmembrane co-cultures of human iPSC-NPCs with rat cortical cells subjected to oxygen-glucose deprivation. Compared to other human stem cells, iPSC-NPCs were superior at promoting neuronal survival and outgrowth, and mitigating astrogliosis. Using comparative whole-genome microarrays and cytokine neutralization, we identified a neurorestorative secretome from iPSC-NPCs, and neutralizing enriched cytokines abolished neuroprotective effects in co-cultures. This proof-of-concept study demonstrates a relatively safe, yet effective epidural route for delivering human iPSC-NPCs, which acts predominately through discrete paracrine effects to promote functional recovery after stroke.

  15. Background migration of USPIO/MLs is a major drawback for in situ labeling of endogenous neural progenitor cells.

    PubMed

    Vreys, Ruth; Soenen, Stefaan J H; De Cuyper, Marcel; Van der Linden, Annemie

    2011-01-01

    MR-labeling of endogenous neural progenitor cells (NPCs) to follow up cellular migration with in vivo magnetic resonance imaging (MRI) is a very promising tool in the rapidly growing field of cellular imaging. To date, most of the in situ labeling work has been performed using micron-sized iron oxide particles. In this work magnetoliposomes (MLs), i.e. ultrasmall superparamagnetic iron oxide cores (USPIOs), each individually coated by a phospholipid bilayer, were used as the MR contrast agent. One of the main advantages of MLs is that the phospholipid bilayer allows easy modification of the surface, which creates the opportunity to construct a wide range of MLs optimized for specific biomedical applications. We have investigated the ability of MLs to label endogenous NPCs after direct injection into the adult mouse brain. Whereas MRI revealed contrast relocation towards the olfactory bulb, our data strongly imply that this relocation is independent of the migration of endogenous NPCs but represents background migration of MLs along a white matter tract. Our findings suggest that the small size of USPIOs/MLs intrinsically limits their potential for in situ labeling of NPCs. Copyright © 2010 John Wiley & Sons, Ltd.

  16. A Hydrogel Bridge Incorporating Immobilized Growth Factors and Neural Stem/Progenitor Cells to Treat Spinal Cord Injury.

    PubMed

    Li, Hang; Ham, Trevor R; Neill, Nicholas; Farrag, Mahmoud; Mohrman, Ashley E; Koenig, Andrew M; Leipzig, Nic D

    2016-04-06

    Spinal cord injury (SCI) causes permanent, often complete disruption of central nervous system (CNS) function below the damaged region, leaving patients without the ability to regenerate lost tissue. To engineer new CNS tissue, a unique spinal cord bridge is created to deliver stem cells and guide their organization and development with site-specifically immobilized growth factors. In this study, this bridge is tested, consisting of adult neural stem/progenitor cells contained within a methacrylamide chitosan (MAC) hydrogel and protected by a chitosan conduit. Interferon-γ (IFN-γ) and platelet-derived growth factor-AA (PDGF-AA) are recombinantly produced and tagged with an N-terminal biotin. They are immobilized to streptavidin-functionalized MAC to induce either neuronal or oligodendrocytic lineages, respectively. These bridges are tested in a rat hemisection model of SCI between T8 and T9. After eight weeks treatments including chitosan conduits result in a significant reduction in lesion area and macrophage infiltration around the lesion site (p < 0.0001). Importantly, neither immobilized IFN-γ nor PDGF-AA increased macrophage infiltration. Retrograde tracing demonstrates improved neuronal regeneration through the use of immobilized growth factors. Immunohistochemistry staining demonstrates that immobilized growth factors are effective in differentiating encapsulated cells into their anticipated lineages within the hydrogel, while qualitatively reducing glial fibrillary acid protein expression.

  17. Functional Recovery from Neural Stem/Progenitor Cell Transplantation Combined with Treadmill Training in Mice with Chronic Spinal Cord Injury

    PubMed Central

    Tashiro, Syoichi; Nishimura, Soraya; Iwai, Hiroki; Sugai, Keiko; Zhang, Liang; Shinozaki, Munehisa; Iwanami, Akio; Toyama, Yoshiaki; Liu, Meigen; Okano, Hideyuki; Nakamura, Masaya

    2016-01-01

    Most studies targeting chronic spinal cord injury (SCI) have concluded that neural stem/progenitor cell (NS/PC) transplantation exerts only a subclinical recovery; this in contrast to its remarkable effect on acute and subacute SCI. To determine whether the addition of rehabilitative intervention enhances the effect of NS/PC transplantation for chronic SCI, we used thoracic SCI mouse models to compare manifestations secondary to both transplantation and treadmill training, and the two therapies combined, with a control group. Significant locomotor recovery in comparison with the control group was only achieved in the combined therapy group. Further investigation revealed that NS/PC transplantation improved spinal conductivity and central pattern generator activity, and that treadmill training promoted the appropriate inhibitory motor control. The combined therapy enhanced these independent effects of each single therapy, and facilitated neuronal differentiation of transplanted cells and maturation of central pattern generator activity synergistically. Our data suggest that rehabilitative treatment represents a therapeutic option for locomotor recovery after NS/PC transplantation, even in chronic SCI. PMID:27485458

  18. Rationale and Methodology of Reprogramming for Generation of Induced Pluripotent Stem Cells and Induced Neural Progenitor Cells.

    PubMed

    Tian, Zuojun; Guo, Fuzheng; Biswas, Sangita; Deng, Wenbin

    2016-04-20

    Great progress has been made regarding the capabilities to modify somatic cell fate ever since the technology for generation of induced pluripotent stem cells (iPSCs) was discovered in 2006. Later, induced neural progenitor cells (iNPCs) were generated from mouse and human cells, bypassing some of the concerns and risks of using iPSCs in neuroscience applications. To overcome the limitation of viral vector induced reprogramming, bioactive small molecules (SM) have been explored to enhance the efficiency of reprogramming or even replace transcription factors (TFs), making the reprogrammed cells more amenable to clinical application. The chemical induced reprogramming process is a simple process from a technical perspective, but the choice of SM at each step is vital during the procedure. The mechanisms underlying cell transdifferentiation are still poorly understood, although, several experimental data and insights have indicated the rationale of cell reprogramming. The process begins with the forced expression of specific TFs or activation/inhibition of cell signaling pathways by bioactive chemicals in defined culture condition, which initiates the further reactivation of endogenous gene program and an optimal stoichiometric expression of the endogenous pluri- or multi-potency genes, and finally leads to the birth of reprogrammed cells such as iPSCs and iNPCs. In this review, we first outline the rationale and discuss the methodology of iPSCs and iNPCs in a stepwise manner; and then we also discuss the chemical-based reprogramming of iPSCs and iNPCs.

  19. Three-Dimensional Normal Human Neural Progenitor Tissue-Like Assemblies: A Model of Persistent Varicella-Zoster Virus Infection

    PubMed Central

    Goodwin, Thomas J.; McCarthy, Maureen; Osterrieder, Nikolaus; Cohrs, Randall J.; Kaufer, Benedikt B.

    2013-01-01

    Varicella-zoster virus (VZV) is a neurotropic human alphaherpesvirus that causes varicella upon primary infection, establishes latency in multiple ganglionic neurons, and can reactivate to cause zoster. Live attenuated VZV vaccines are available; however, they can also establish latent infections and reactivate. Studies of VZV latency have been limited to the analyses of human ganglia removed at autopsy, as the virus is strictly a human pathogen. Recently, terminally differentiated human neurons have received much attention as a means to study the interaction between VZV and human neurons; however, the short life-span of these cells in culture has limited their application. Herein, we describe the construction of a model of normal human neural progenitor cells (NHNP) in tissue-like assemblies (TLAs), which can be successfully maintained for at least 180 days in three-dimensional (3D) culture, and exhibit an expression profile similar to that of human trigeminal ganglia. Infection of NHNP TLAs with cell-free VZV resulted in a persistent infection that was maintained for three months, during which the virus genome remained stable. Immediate-early, early and late VZV genes were transcribed, and low-levels of infectious VZV were recurrently detected in the culture supernatant. Our data suggest that NHNP TLAs are an effective system to investigate long-term interactions of VZV with complex assemblies of human neuronal cells. PMID:23935496

  20. Three-dimensional normal human neural progenitor tissue-like assemblies: a model of persistent varicella-zoster virus infection.

    PubMed

    Goodwin, Thomas J; McCarthy, Maureen; Osterrieder, Nikolaus; Cohrs, Randall J; Kaufer, Benedikt B

    2013-01-01

    Varicella-zoster virus (VZV) is a neurotropic human alphaherpesvirus that causes varicella upon primary infection, establishes latency in multiple ganglionic neurons, and can reactivate to cause zoster. Live attenuated VZV vaccines are available; however, they can also establish latent infections and reactivate. Studies of VZV latency have been limited to the analyses of human ganglia removed at autopsy, as the virus is strictly a human pathogen. Recently, terminally differentiated human neurons have received much attention as a means to study the interaction between VZV and human neurons; however, the short life-span of these cells in culture has limited their application. Herein, we describe the construction of a model of normal human neural progenitor cells (NHNP) in tissue-like assemblies (TLAs), which can be successfully maintained for at least 180 days in three-dimensional (3D) culture, and exhibit an expression profile similar to that of human trigeminal ganglia. Infection of NHNP TLAs with cell-free VZV resulted in a persistent infection that was maintained for three months, during which the virus genome remained stable. Immediate-early, early and late VZV genes were transcribed, and low-levels of infectious VZV were recurrently detected in the culture supernatant. Our data suggest that NHNP TLAs are an effective system to investigate long-term interactions of VZV with complex assemblies of human neuronal cells.

  1. Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

    PubMed Central

    Ross, Heather H.; Sandhu, Milap S.; Sharififar, Sharareh; Fuller, David D.

    2015-01-01

    Extended culture of neural stem/progenitor cells facilitates in vitro analyses to understand their biology while enabling expansion of cell populations to adequate numbers prior to transplantation. Identifying approaches to refine this process, to augment the production of all CNS cell types (i.e., neurons), and to possibly contribute to therapeutic cell therapy protocols is a high research priority. This report describes an easily applied in vivo “pre-conditioning” stimulus which can be delivered to awake, non-anesthetized animals. Thus, it is a non-invasive and non-stressful procedure. Specifically described are the procedures for exposing mouse or rat pups (aged postnatal day 1-8) to a brief (40-80 min) period of intermittent hypoxia (AIH). The procedures included in this video protocol include calibration of the whole-body plethysmography chamber in which pups are placed during AIH and the technical details of AIH exposure. The efficacy of this approach to elicit tissue-level changes in the awake animal is demonstrated through the enhancement of subsequent in vitro expansion and neuronal differentiation in cells harvested from the subventricular zone (SVZ). These results support the notion that tissue level changes across multiple systems could be observed following AIH, and support the continued optimization and establishment of AIH as a priming or conditioning modality for therapeutic cell populations. PMID:26556530

  2. Lithium increases proliferation of hippocampal neural stem/progenitor cells and rescues irradiation-induced cell cycle arrest in vitro.

    PubMed

    Zanni, Giulia; Di Martino, Elena; Omelyanenko, Anna; Andäng, Michael; Delle, Ulla; Elmroth, Kecke; Blomgren, Klas

    2015-11-10

    Radiotherapy in children causes debilitating cognitive decline, partly linked to impaired neurogenesis. Irradiation targets primarily cancer cells but also endogenous neural stem/progenitor cells (NSPCs) leading to cell death or cell cycle arrest. Here we evaluated the effects of lithium on proliferation, cell cycle and DNA damage after irradiation of young NSPCs in vitro.NSPCs were treated with 1 or 3 mM LiCl and we investigated proliferation capacity (neurosphere volume and bromodeoxyuridine (BrdU) incorporation). Using flow cytometry, we analysed apoptosis (annexin V), cell cycle (propidium iodide) and DNA damage (γH2AX) after irradiation (3.5 Gy) of lithium-treated NSPCs.Lithium increased BrdU incorporation and, dose-dependently, the number of cells in replicative phase as well as neurosphere growth. Irradiation induced cell cycle arrest in G1 and G2/M phases. Treatment with 3 mM LiCl was sufficient to increase NSPCs in S phase, boost neurosphere growth and reduce DNA damage. Lithium did not affect the levels of apoptosis, suggesting that it does not rescue NSPCs committed to apoptosis due to accumulated DNA damage.Lithium is a very promising candidate for protection of the juvenile brain from radiotherapy and for its potential to thereby improve the quality of life for those children who survive their cancer.

  3. 532 nm Low-Power Laser Irradiation Facilitates the Migration of GABAergic Neural Stem/Progenitor Cells in Mouse Neocortex

    PubMed Central

    Fukuzaki, Yumi; Shin, Hyeryun; Kawai, Hideki D.; Yamanoha, Banri; Kogure, Shinichi

    2015-01-01

    Background and Objective Accumulating evidence has shown that low-power laser irradiation (LLI) affects cell proliferation and survival, but little is known about LLI effects on neural stem/progenitor cells (NSPCs). Here we investigate whether transcranial 532 nm LLI affects NSPCs in adult murine neocortex and in neurospheres from embryonic mice. Study Design/Materials and Methods We applied 532 nm LLI (Nd:YVO4, CW, 60 mW) on neocortical surface via cranium in adult mice and on cultured cells from embryonic mouse brains in vitro to investigate the proliferation and migration of NSPCs and Akt expression using immunohistochemical assays and Western blotting techniques. Results In vivo experiments demonstrated that 532 nm LLI significantly facilitated the migration of GABAergic NSPCs that were induced to proliferate in layer 1 by mild ischemia. In vitro experiments using GABAergic NSPCs derived from embryonic day 14 ganglionic eminence demonstrated that 532 nm LLI for 60 min promoted the migration of GAD67-immunopositive NSPCs with a significant increase of Akt expression. Meanwhile, the LLI induced proliferation, but not migration, of NSPCs that give rise to excitatory neurons. Conclusion It is concluded that 532 nm LLI promoted the migration of GABAergic NSPCs into deeper layers of the neocortex in vivo by elevating Akt expression. PMID:25919297

  4. Lithium increases proliferation of hippocampal neural stem/progenitor cells and rescues irradiation-induced cell cycle arrest in vitro

    PubMed Central

    Omelyanenko, Anna; Andäng, Michael; Delle, Ulla; Elmroth, Kecke; Blomgren, Klas

    2015-01-01

    Radiotherapy in children causes debilitating cognitive decline, partly linked to impaired neurogenesis. Irradiation targets primarily cancer cells but also endogenous neural stem/progenitor cells (NSPCs) leading to cell death or cell cycle arrest. Here we evaluated the effects of lithium on proliferation, cell cycle and DNA damage after irradiation of young NSPCs in vitro. NSPCs were treated with 1 or 3 mM LiCl and we investigated proliferation capacity (neurosphere volume and bromodeoxyuridine (BrdU) incorporation). Using flow cytometry, we analysed apoptosis (annexin V), cell cycle (propidium iodide) and DNA damage (γH2AX) after irradiation (3.5 Gy) of lithium-treated NSPCs. Lithium increased BrdU incorporation and, dose-dependently, the number of cells in replicative phase as well as neurosphere growth. Irradiation induced cell cycle arrest in G1 and G2/M phases. Treatment with 3 mM LiCl was sufficient to increase NSPCs in S phase, boost neurosphere growth and reduce DNA damage. Lithium did not affect the levels of apoptosis, suggesting that it does not rescue NSPCs committed to apoptosis due to accumulated DNA damage. Lithium is a very promising candidate for protection of the juvenile brain from radiotherapy and for its potential to thereby improve the quality of life for those children who survive their cancer. PMID:26397227

  5. Definition of genetic events directing the development of distinct types of brain tumors from postnatal neural stem/progenitor cells.

    PubMed

    Hertwig, Falk; Meyer, Katharina; Braun, Sebastian; Ek, Sara; Spang, Rainer; Pfenninger, Cosima V; Artner, Isabella; Prost, Gaëlle; Chen, Xinbin; Biegel, Jaclyn A; Judkins, Alexander R; Englund, Elisabet; Nuber, Ulrike A

    2012-07-01

    Although brain tumors are classified and treated based upon their histology, the molecular factors involved in the development of various tumor types remain unknown. In this study, we show that the type and order of genetic events directs the development of gliomas, central nervous system primitive neuroectodermal tumors, and atypical teratoid/rhabdoid-like tumors from postnatal mouse neural stem/progenitor cells (NSC/NPC). We found that the overexpression of specific genes led to the development of these three different brain tumors from NSC/NPCs, and manipulation of the order of genetic events was able to convert one established tumor type into another. In addition, loss of the nuclear chromatin-remodeling factor SMARCB1 in rhabdoid tumors led to increased phosphorylation of eIF2α, a central cytoplasmic unfolded protein response (UPR) component, suggesting a role for the UPR in these tumors. Consistent with this, application of the proteasome inhibitor bortezomib led to an increase in apoptosis of human cells with reduced SMARCB1 levels. Taken together, our findings indicate that the order of genetic events determines the phenotypes of brain tumors derived from a common precursor cell pool, and suggest that the UPR may represent a therapeutic target in atypical teratoid/rhabdoid tumors.

  6. Differentiated human midbrain-derived neural progenitor cells express excitatory strychnine-sensitive glycine receptors containing α2β subunits.

    PubMed

    Wegner, Florian; Kraft, Robert; Busse, Kathy; Härtig, Wolfgang; Ahrens, Jörg; Leffler, Andreas; Dengler, Reinhard; Schwarz, Johannes

    2012-01-01

    Human fetal midbrain-derived neural progenitor cells (NPCs) may deliver a tissue source for drug screening and regenerative cell therapy to treat Parkinson's disease. While glutamate and GABA(A) receptors play an important role in neurogenesis, the involvement of glycine receptors during human neurogenesis and dopaminergic differentiation as well as their molecular and functional characteristics in NPCs are largely unknown. Here we investigated NPCs in respect to their glycine receptor function and subunit expression using electrophysiology, calcium imaging, immunocytochemistry, and quantitative real-time PCR. Whole-cell recordings demonstrate the ability of NPCs to express functional strychnine-sensitive glycine receptors after differentiation for 3 weeks in vitro. Pharmacological and molecular analyses indicate a predominance of glycine receptor heteromers containing α2β subunits. Intracellular calcium measurements of differentiated NPCs suggest that glycine evokes depolarisations mediated by strychnine-sensitive glycine receptors and not by D-serine-sensitive excitatory glycine receptors. Culturing NPCs with additional glycine, the glycine-receptor antagonist strychnine, or the Na(+)-K(+)-Cl(-) co-transporter 1 (NKCC1)-inhibitor bumetanide did not significantly influence cell proliferation and differentiation in vitro. These data indicate that NPCs derived from human fetal midbrain tissue acquire essential glycine receptor properties during neuronal maturation. However, glycine receptors seem to have a limited functional impact on neurogenesis and dopaminergic differentiation of NPCs in vitro.

  7. CXCL12 N-terminal end is sufficient to induce chemotaxis and proliferation of neural stem/progenitor cells.

    PubMed

    Filippo, Thais R M; Galindo, Layla T; Barnabe, Gabriela F; Ariza, Carolina B; Mello, Luiz E; Juliano, Maria A; Juliano, Luiz; Porcionatto, Marimélia A

    2013-09-01

    Neural stem/progenitor cells (NSC) respond to injury after brain injuries secreting IL-1, IL-6, TNF-α, IL-4 and IL-10, as well as chemokine members of the CC and CXC ligand families. CXCL12 is one of the chemokines secreted at an injury site and is known to attract NSC-derived neuroblasts, cells that express CXCL12 receptor, CXCR4. Activation of CXCR4 by CXCL12 depends on two domains located at the N-terminal of the chemokine. In the present work we aimed to investigate if the N-terminal end of CXCL12, where CXCR4 binding and activation domains are located, was sufficient to induce NSC-derived neuroblast chemotaxis. Our data show that a synthetic peptide analogous to the first 21 amino acids of the N-terminal end of CXCL12, named PepC-C (KPVSLSYRCPCRFFESHIARA), is able to promote chemotaxis of neuroblasts in vivo, and stimulate chemotaxis and proliferation of CXCR4+ cells in vitro, without affecting NSC fate. We also show that PepC-C upregulates CXCL12 expression in vivo and in vitro. We suggest the N-terminal end of CXCL12 is responsible for a positive feedback loop to maintain a gradient of CXCL12 that attracts neuroblasts from the subventricular zone into an injury site.

  8. Cell-permeable p38 MAP kinase promotes migration of adult neural stem/progenitor cells

    PubMed Central

    Hamanoue, Makoto; Morioka, Kazuhito; Ohsawa, Ikuroh; Ohsawa, Keiko; Kobayashi, Masaaki; Tsuburaya, Kayo; Akasaka, Yoshikiyo; Mikami, Tetsuo; Ogata, Toru; Takamatsu, Ken

    2016-01-01

    Endogenous neural stem/progenitor cells (NPCs) can migrate toward sites of injury, but the migration activity of NPCs is insufficient to regenerate damaged brain tissue. In this study, we showed that p38 MAP kinase (p38) is expressed in doublecortin-positive adult NPCs. Experiments using the p38 inhibitor SB203580 revealed that endogenous p38 participates in NPC migration. To enhance NPC migration, we generated a cell-permeable wild-type p38 protein (PTD-p38WT) in which the HIV protein transduction domain (PTD) was fused to the N-terminus of p38. Treatment with PTD-p38WT significantly promoted the random migration of adult NPCs without affecting cell survival or differentiation; this effect depended on the cell permeability and kinase activity of the fusion protein. These findings indicate that PTD-p38WT is a novel and useful tool for unraveling the roles of p38, and that this protein provides a reasonable approach for regenerating the injured brain by enhancing NPC migration. PMID:27067799

  9. NR2B-containing NMDA receptors promote neural progenitor cell proliferation through CaMKIV/CREB pathway

    SciTech Connect

    Li, Mei; Zhang, Dong-Qing; Wang, Xiang-Zhen; Xu, Tie-Jun

    2011-08-12

    Highlights: {yields} The NR2B component of the NMDARs is important for the NSPC proliferation. {yields} pCaMKIV and pCREB exist in NSPCs. {yields} The CaMKIV/CREB pathway mediates NSPC proliferation. -- Abstract: Accumulating evidence indicates the involvement of N-methyl-D-aspartate receptors (NMDARs) in regulating neural stem/progenitor cell (NSPC) proliferation. Functional properties of NMDARs can be markedly influenced by incorporating the regulatory subunit NR2B. Here, we aim to analyze the effect of NR2B-containing NMDARs on the proliferation of hippocampal NSPCs and to explore the mechanism responsible for this effect. NSPCs were shown to express NMDAR subunits NR1 and NR2B. The NR2B selective antagonist, Ro 25-6981, prevented the NMDA-induced increase in cell proliferation. Moreover, we demonstrated that the phosphorylation levels of calcium/calmodulin-dependent protein kinase IV (CaMKIV) and cAMP response element binding protein (CREB) were increased by NMDA treatment, whereas Ro 25-6981 decreased them. The role that NR2B-containing NMDARs plays in NSPC proliferation was abolished when CREB phosphorylation was attenuated by CaMKIV silencing. These results suggest that NR2B-containing NMDARs have a positive role in regulating NSPC proliferation, which may be mediated through CaMKIV phosphorylation and subsequent induction of CREB activation.

  10. Transcription-associated processes cause DNA double-strand breaks and translocations in neural stem/progenitor cells.

    PubMed

    Schwer, Bjoern; Wei, Pei-Chi; Chang, Amelia N; Kao, Jennifer; Du, Zhou; Meyers, Robin M; Alt, Frederick W

    2016-02-23

    High-throughput, genome-wide translocation sequencing (HTGTS) studies of activated B cells have revealed that DNA double-strand breaks (DSBs) capable of translocating to defined bait DSBs are enriched around the transcription start sites (TSSs) of active genes. We used the HTGTS approach to investigate whether a similar phenomenon occurs in primary neural stem/progenitor cells (NSPCs). We report that breakpoint junctions indeed are enriched around TSSs that were determined to be active by global run-on sequencing analyses of NSPCs. Comparative analyses of transcription profiles in NSPCs and B cells revealed that the great majority of TSS-proximal junctions occurred in genes commonly expressed in both cell types, possibly because this common set has higher transcription levels on average than genes transcribed in only one or the other cell type. In the latter context, among all actively transcribed genes containing translocation junctions in NSPCs, those with junctions located within 2 kb of the TSS show a significantly higher transcription rate on average than genes with junctions in the gene body located at distances greater than 2 kb from the TSS. Finally, analysis of repair junction signatures of TSS-associated translocations in wild-type versus classical nonhomologous end-joining (C-NHEJ)-deficient NSPCs reveals that both C-NHEJ and alternative end-joining pathways can generate translocations by joining TSS-proximal DSBs to DSBs on other chromosomes. Our studies show that the generation of transcription-associated DSBs is conserved across divergent cell types.

  11. Applications of stripe assay in the study of CXCL12-mediated neural progenitor cell migration and polarization.

    PubMed

    Zhang, Min; Song, Aihong; Lai, Siqiang; Qiu, Lisha; Huang, Yunlong; Chen, Qiang; Zhu, Bing; Xu, Dongsheng; Zheng, Jialin C

    2015-12-01

    The polarization and migration of neural progenitor cells (NPCs) are critical for embryonic brain development and neurogenesis after brain injury. Although stromal-derived factor-1α (SDF-1α, CXCL12) and its receptor CXCR4 are well-known to mediate the migration of NPCs in the developing brain, the dynamic cellular processes and structure-related molecular events remain elusive. Transwell and microfluidic-based assays are classical assays to effectively study cellular migration. However, both of them have limitations in the analysis of a single cell. In this study, we modified the stripe assay and extended its applications in the study of NPC polarization and intracellular molecular events associated with CXCL12-mediated migration. In response to localized CXCL12, NPCs formed lamellipodia in the stripe assay. Furthermore, CXCR4 and Rac1 quickly re-distributed to the area of lamellipodia, indicating their roles in NPC polarization upon CXCL12 stimulation. Although the chemokine stripes in the assay provided concentration gradients that can be best used to study cellular polarization and migration through immunocytochemistry, they can also generate live imaging data with comparable quality. In conclusion, stripe assay is a visual, dynamic and economical tool to study cellular mobility and its related molecule mechanisms. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. 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

  13. Transcription Mapping of Embryonic Rat Brain Reveals EGR-1 Induction in SOX2 Neural Progenitor Cells.

    PubMed

    Wells, Timothy; Rough, Kirsty; Carter, David A

    2011-01-01

    Neuronal expression of the early growth response-1 (EGR-1; NGFI-A/Zif268) transcription factor has been extensively studied in the adult mammalian brain and linked to aspects of mature physiological/behavioral function. In contrast, this factor has not been studied in detail in the embryonic brain. Here, we used a fluorescent protein-encoding Egr-1 transgene to map the cellular distribution of Egr-1 transcription in embryonic rat brain. We identified a novel, widely distributed population of GFP(+) cells, characterized as a precursor/stem cell phenotype by co-localization with SOX2/nestin/vimentin/S-100β and exclusion from other known cellular markers including DCX/BLBP/TBR2/NURR1. At both E18 and E20, these cells were located across the developing brain but concentrated in the subplate and intermediate zones. The transgene was also highly expressed in developing (NeuN(+)) striatal neurons. The authentic expression pattern that we observed for the rEgr-1 transgene sequence indicates that restriction to neuronal/precursor cells is largely driven by proximal 5(') sequence. Deletion of conserved Egr-1 silencer (neuron restrictive silencer factor) elements did not markedly alter transcriptional activity in transfected cells; this is consistent with a dominant role for positive factors in the control of cell-specific Egr-1 expression. Induction of Egr-1 in a population of SOX2(+) cells indicates a co-incidence of extrinsic (EGR-1) and cell-intrinsic (SOX2) cellular signals that may form a novel level of progenitor cell regulation. The wide distribution of EGR-1 signaling in SOX2(+) cells suggests an organizational role during late embryonic brain development.

  14. Birth of neural progenitors during the embryonic period of sexual differentiation in the Japanese quail brain.

    PubMed

    Bardet, Sylvia M; Mouriec, Karen; Balthazart, Jacques

    2012-12-15

    Several brain areas in the diencephalon are involved in the activation and expression of sexual behavior, including in quail the medial preoptic nucleus (POM). However, the ontogeny of these diencephalic brain nuclei has not to this date been examined in detail. We investigated the ontogeny of POM and other steroid-sensitive brain regions by injecting quail eggs with 5-bromo-2-deoxyuridine (BrdU) at various stages between embryonic day (E)3 and E16 and killing animals at postnatal (PN) days 3 or 56. In the POM, large numbers of BrdU-positive cells were observed in subjects injected from E3-E10, the numbers of these cells was intermediate in birds injected on E12, and most cells were postmitotic in both sexes on E14-E16. Injections on E3-E4 labeled large numbers of Hu-positive cells in POM. In contrast, injections performed at a later stage labeled cells that do not express aromatase nor neuronal markers such as Hu or NeuN in the POM and other steroid-sensitive nuclei and thus do not have a neuronal phenotype in these locations, contrary to what is observed in the telencephalon and cerebellum. No evidence could also be collected to demonstrate that these cells have a glial nature. Converging data, including the facts that these cells divide in the brain mantle and express proliferating cell nuclear antigen (PCNA), a cell cycling marker, indicate that cells labeled by BrdU during the second half of embryonic life are slow-cycling progenitors born and residing in the brain mantle. Future research should now identify their functional significance. Copyright © 2012 Wiley Periodicals, Inc.

  15. Transcription Mapping of Embryonic Rat Brain Reveals EGR-1 Induction in SOX2+ Neural Progenitor Cells

    PubMed Central

    Wells, Timothy; Rough, Kirsty; Carter, David A.

    2011-01-01

    Neuronal expression of the early growth response-1 (EGR-1; NGFI-A/Zif268) transcription factor has been extensively studied in the adult mammalian brain and linked to aspects of mature physiological/behavioral function. In contrast, this factor has not been studied in detail in the embryonic brain. Here, we used a fluorescent protein-encoding Egr-1 transgene to map the cellular distribution of Egr-1 transcription in embryonic rat brain. We identified a novel, widely distributed population of GFP+ cells, characterized as a precursor/stem cell phenotype by co-localization with SOX2/nestin/vimentin/S-100β and exclusion from other known cellular markers including DCX/BLBP/TBR2/NURR1. At both E18 and E20, these cells were located across the developing brain but concentrated in the subplate and intermediate zones. The transgene was also highly expressed in developing (NeuN+) striatal neurons. The authentic expression pattern that we observed for the rEgr-1 transgene sequence indicates that restriction to neuronal/precursor cells is largely driven by proximal 5′ sequence. Deletion of conserved Egr-1 silencer (neuron restrictive silencer factor) elements did not markedly alter transcriptional activity in transfected cells; this is consistent with a dominant role for positive factors in the control of cell-specific Egr-1 expression. Induction of Egr-1 in a population of SOX2+ cells indicates a co-incidence of extrinsic (EGR-1) and cell-intrinsic (SOX2) cellular signals that may form a novel level of progenitor cell regulation. The wide distribution of EGR-1 signaling in SOX2+ cells suggests an organizational role during late embryonic brain development. PMID:21629823

  16. Birth of neural progenitors during the embryonic period of sexual differentiation in the Japanese quail brain

    PubMed Central

    Bardet, Sylvia M.; Mouriec, Karen; Balthazart, Jacques

    2012-01-01

    Several brain areas in the diencephalon are involved in the activation and expression of sexual behavior, including in quail, the medial preoptic nucleus (POM). However, the ontogeny of these diencephalic brain nuclei has not to this date been examined in detail. We investigated the ontogeny of POM and other steroid-sensitive brain regions by injecting quail eggs with 5-bromo-2-deoxyuridine (BrdU) at various stages between E3 and E16 and killing animals at postnatal (PN) days 3 or 56. In the POM, large numbers of BrdU-positive cells were observed in subjects injected from E3 to E10, the numbers of these cells was intermediate in birds injected on E12, and most cells were post-mitotic in both sexes on E14-E16. Injections on E3-E4 labeled large numbers of Hu positive cells in POM. In contrast, injections performed at a later stage labeled cells that do not express aromatase nor neuronal markers such as Hu or NeuN in the POM and other steroid-sensitive nuclei and thus do not have a neuronal phenotype in these locations contrary to what is observed in the telencephalon and cerebellum. No evidence could also be collected to demonstrate that these cells have a glial nature. Converging data, including the facts that these cells divide in the brain mantle and express PCNA, a cell cycling marker, indicate that cells labeled by BrdU during the second half of embryonic life are slow cycling progenitors born and residing in the brain mantle. Future research should now identify their functional significance. PMID:22628012

  17. The CSF-1 receptor ligands IL-34 and CSF-1 exhibit distinct developmental brain expression patterns and regulate neural progenitor cell maintenance and maturation

    PubMed Central

    Nandi, Sayan; Gokhan, Solen; Dai, Xu-Ming; Wei, Suwen; Enikolopov, Grigori; Lin, Haishan; Mehler, Mark F.; Stanley, E. Richard

    2012-01-01

    The CSF-1 receptor (CSF-1R) regulates CNS microglial development. However, the localization and developmental roles of this receptor and its ligands, IL-34 and CSF-1, in the brain are poorly understood. Here we show that compared to wild type mice, CSF-1R-deficient (Csf1r−/−) mice have smaller brains of greater mass. They further exhibit an expansion of lateral ventricle size, an atrophy of the olfactory bulb and a failure of midline crossing of callosal axons. In brain, IL-34 exhibited a broader regional expression than CSF-1, mostly without overlap. Expression of IL-34, CSF-1 and the CSF-1R were maximal during early postnatal development. However, in contrast to the expression of its ligands, CSF-1R expression was very low in adult brain. Postnatal neocortical expression showed that CSF-1 was expressed in layer VI, whereas IL-34 was expressed in the meninges and layers II–V. The broader expression of IL-34 is consistent with its previously implicated role in microglial development. The differential expression of CSF-1R ligands, with respect to CSF-1R expression, could reflect their CSF-1R-independent signaling. Csf1r−/− mice displayed increased proliferation and apoptosis of neocortical progenitors and reduced differentiation of specific excitatory neuronal subtypes. Indeed, addition of CSF-1 or IL-34 to microglia-free, CSF-1R-expressing dorsal forebrain clonal cultures, suppressed progenitor self-renewal and enhanced neuronal differentiation. Consistent with a neural developmental role for the CSF-1R, ablation of the Csf1r gene in Nestin-positive neural progenitors led to a smaller brain size, an expanded neural progenitor pool and elevated cellular apoptosis in cortical forebrain. Thus our results also indicate novel roles for the CSF-1R in the regulation of corticogenesis. PMID:22542597

  18. Electric signals regulate directional migration of ventral midbrain derived dopaminergic neural progenitor cells via Wnt/GSK3β signaling.

    PubMed

    Liu, Jia; Zhu, Bangfu; Zhang, Gaofeng; Wang, Jian; Tian, Weiming; Ju, Gong; Wei, Xiaoqing; Song, Bing

    2015-01-01

    Neural progenitor cell (NPC) replacement therapy is a promising treatment for neurodegenerative disorders including Parkinson's disease (PD). It requires a controlled directional migration and integration of NPCs, for example dopaminergic (DA) progenitor cells, into the damaged host brain tissue. There is, however, only limited understanding of how to regulate the directed migration of NPCs to the diseased or damaged brain tissues for repair and regeneration. The aims of this study are to explore the possibility of using a physiological level of electrical stimulation to regulate the directed migration of ventral midbrain NPCs (NPCs(vm)), and to investigate their potential regulation via GSK3β and associated downstream effectors. We tested the effects of direct-current (DC) electric fields (EFs) on the migration behavior of the NPCs(vm). A DC EF induced directional cell migration toward the cathode, namely electrotaxis. Reversal of the EF polarity triggered a sharp reversal of the NPC(vm) electrotaxis. The electrotactic response was both time and EF voltage dependent. Pharmacologically inhibiting the canonical Wnt/GSK3β pathway significantly reduced the electrotactic response of NPCs(vm), which is associated with the down-regulation of GSK3β phosphorylation, β-catenin activation and CLASP2 expression. This was further proved by RNA interference of GSK3β, which also showed a significantly reduced electrotactic response in association with reduced β-catenin activation and CLASP2 expression. In comparison, RNA interference of β-catenin slightly reduced electrotactic response and CLASP2 expression. Both pharmacological inhibition of Wnt/GSK3β and RNA interference of GSK3β/β-catenin clearly reduced the asymmetric redistribution of CLASP2 and its co-localization with α-tubulin. These results suggest that Wnt/GSK3β signaling contributes to the electrotactic response of NPCs(vm) through the coordination of GSK3β phosphorylation, β-catenin activation, CLASP2

  19. Characterization of the Filum terminale as a neural progenitor cell niche in both rats and humans.

    PubMed

    Chrenek, Ryan; Magnotti, Laura M; Herrera, Gabriella R; Jha, Ruchira M; Cardozo, David L

    2017-02-15

    Neural stem cells (NSCs) reside in a unique microenvironment within the central nervous system (CNS) called the NSC niche. Although they are relatively rare, niches have been previously characterized in both the brain and spinal cord of adult animals. Recently, another potential NSC niche has been identified in the filum terminale (FT), which is a thin band of tissue at the caudal end of the spinal cord. While previous studies have demonstrated that NSCs can be isolated from the FT, the in vivo architecture of this tissue and its relation to other NSC niches in the CNS has not yet been established. In this article we report a histological analysis of the FT NSC niche in postnatal rats and humans. Immunohistochemical characterization reveals that the FT is mitotically active and its cells express similar markers to those in other CNS niches. In addition, the organization of the FT most closely resembles that of the adult spinal cord niche. J. Comp. Neurol. 525:661-675, 2017. © 2016 Wiley Periodicals, Inc.

  20. Characterization of the Filum terminale as a neural progenitor cell niche in both rats and humans

    PubMed Central

    Chrenek, Ryan; Herrera, Gabriella R.; Jha, Ruchira M.; Cardozo, David L.

    2016-01-01

    Abstract Neural stem cells (NSCs) reside in a unique microenvironment within the central nervous system (CNS) called the NSC niche. Although they are relatively rare, niches have been previously characterized in both the brain and spinal cord of adult animals. Recently, another potential NSC niche has been identified in the filum terminale (FT), which is a thin band of tissue at the caudal end of the spinal cord. While previous studies have demonstrated that NSCs can be isolated from the FT, the in vivo architecture of this tissue and its relation to other NSC niches in the CNS has not yet been established. In this article we report a histological analysis of the FT NSC niche in postnatal rats and humans. Immunohistochemical characterization reveals that the FT is mitotically active and its cells express similar markers to those in other CNS niches. In addition, the organization of the FT most closely resembles that of the adult spinal cord niche. J. Comp. Neurol. 525:661–675, 2017. © 2016 Wiley Periodicals, Inc. PMID:27511739

  1. MUTATIONS IN KATNB1 CAUSE COMPLEX CEREBRAL MALFORMATIONS BY DISRUPTING ASYMMETRICALLY DIVIDING NEURAL PROGENITORS

    PubMed Central

    Mishra-Gorur, Ketu; Çağlayan, Ahmet Okay; Schaffer, Ashleigh E.; Chabu, Chiswili; Henegariu, Octavian; Vonhoff, Fernando; Akgümüş, Gözde Tuğce; Nishimura, Sayoko; Han, Wenqi; Tu, Shu; Baran, Burcin; Gumus, Hakan; Dilber, Cengiz; Zaki, Maha S.; Hossni, Heba AA; Rivière, Jean-Baptiste; Kayserili, Hülya; Spencer, Emily G.; Rosti, Rasim O.; Schroth, Jana; Per, Hüseyin; Cağlar, Caner; Cağlar, Cagri; Dölen, Duygu; Baranoski, Jacob F.; Kumandaş, Sefer; Minja, Frank J.; Erson-Omay, E. Zeynep; Mane, Shrikant M.; Lifton, Richard P.; Xu, Tian; Keshishian, Haig; Dobyns, William B; Chi, Neil C.; Šestan, Nenad; Louvi, Angeliki; Bilgüvar, Kaya; Yasuno, Katsuhito; Gleeson, Joseph G.; Günel, Murat

    2016-01-01

    SUMMARY Exome sequencing analysis of over 2,000 children with complex malformations of cortical development identified 5 independent homozygous deleterious mutations in KATNB1, encoding the regulatory subunit of the microtubule severing enzyme katanin. Mitotic spindle formation is defective in patient-derived fibroblasts, a consequence of disrupted interactions of mutant KATNB1 with KATNA1, the catalytic subunit of katanin, and other microtubule associated proteins. Loss of KATNB1 orthologs in zebrafish (katnb1) and flies (kat80) results in microcephaly, recapitulating the human phenotype. In the developing Drosophila optic lobe, kat80 loss specifically affects the asymmetrically dividing neuroblasts, which display supernumerary centrosomes and spindle abnormalities during mitosis, leading to cell cycle progression delays and reduced cell numbers. Furthermore, kat80 depletion results in dendritic arborization defects in sensory and motor neurons, affecting neural architecture. Taken together, we provide insight into the mechanisms by which KATNB1 mutations cause human cerebral cortical malformations, demonstrating its fundamental role during brain development. PMID:25521378

  2. Notch signaling induces retinal stem-like properties in perinatal neural retina progenitors and promotes symmetric divisions in adult retinal stem cells.

    PubMed

    Balenci, Laurent; van der Kooy, Derek

    2014-02-01

    Understanding the mechanisms regulating retinal stem cell (RSC) activity is fundamental for future stem cell-based therapeutic purposes. By combining gain and loss of function approaches, we addressed whether Notch signaling may play a selective role in retinal stem versus retinal progenitor cells in both developing and adult eyes. Inhibition of either Notch or fibroblast growth factor signaling reduced proliferation of retinal stem and retinal progenitor cells, and inhibited RSC self-renewal. Conversely, exogenous Delta-like 3 and direct intrinsic Notch activation stimulated expansionary symmetric divisions in adult RSCs with the concomitant upregulation of Hes5. Knocking down Hes5 expression specifically decreased the numbers, but not the diameters, of adult RSC primary spheres, indicating that HES5 is the downstream effector of Notch receptor in controlling adult RSC proliferation. In addition, constitutive Notch activation induced retinal stem-like asymmetric self-renewal properties, with no expansion (no symmetrical division) in perinatal neural retina progenitor cells. These findings highlight central roles of Notch signaling activity in regulating the modes of division of retinal stem and retinal progenitor cells.

  3. FGF8 signaling sustains progenitor status and multipotency of cranial neural crest-derived mesenchymal cells in vivo and in vitro

    PubMed Central

    Shao, Meiying; Liu, Chao; Song, Yingnan; Ye, Wenduo; He, Wei; Yuan, Guohua; Gu, Shuping; Lin, Congxin; Ma, Liang; Zhang, Yanding; Tian, Weidong; Hu, Tao; Chen, YiPing

    2015-01-01

    The cranial neural crest (CNC) cells play a vital role in craniofacial development and regeneration. They are multi-potent progenitors, being able to differentiate into various types of tissues. Both pre-migratory and post-migratory CNC cells are plastic, taking on diverse fates by responding to different inductive signals. However, what sustains the multipotency of CNC cells and derivatives remains largely unknown. In this study, we present evidence that FGF8 signaling is able to sustain progenitor status and multipotency of CNC-derived mesenchymal cells both in vivo and in vitro. We show that augmented FGF8 signaling in pre-migratory CNC cells prevents cell differentiation and organogenesis in the craniofacial region by maintaining their progenitor status. CNC-derived mesenchymal cells with Fgf8 overexpression or control cells in the presence of exogenous FGF8 exhibit prolonged survival, proliferation, and multi-potent differentiation capability in cell cultures. Remarkably, exogenous FGF8 also sustains the capability of CNC-derived mesenchymal cells to participate in organogenesis such as odontogenesis. Furthermore, FGF8-mediated signaling strongly promotes adipogenesis but inhibits osteogenesis of CNC-derived mesenchymal cells in vitro. Our results reveal a specific role for FGF8 in the maintenance of progenitor status and in fate determination of CNC cells, implicating a potential application in expansion and fate manipulation of CNC-derived cells in stem cell-based craniofacial regeneration. PMID:26243590

  4. Gene Expression Profiling Supports the Neural Crest Origin of Adult Rodent Carotid Body Stem Cells and Identifies CD10 as a Marker for Mesectoderm-Committed Progenitors.

    PubMed

    Navarro-Guerrero, Elena; Platero-Luengo, Aida; Linares-Clemente, Pedro; Cases, Ildefonso; López-Barneo, José; Pardal, Ricardo

    2016-06-01

    Neural stem cells (NSCs) are promising tools for understanding nervous system plasticity and repair, but their use is hampered by the lack of markers suitable for their prospective isolation and characterization. The carotid body (CB) contains a population of peripheral NSCs, which support organ growth during acclimatization to hypoxia. We have set up CB neurosphere (NS) cultures enriched in differentiated neuronal (glomus) cells versus undifferentiated progenitors to investigate molecular hallmarks of cell classes within the CB stem cell (CBSC) niche. Microarray gene expression analysis in NS is compatible with CBSCs being neural crest derived-multipotent progenitor cells able to sustain CB growth upon exposure to hypoxia. Moreover, we have identified CD10 as a marker suitable for isolation of a population of CB mesectoderm-committed progenitor cells. CD10 + cells are resting in normoxia, and during hypoxia they are activated to proliferate and to eventually complete maturation into mesectodermal cells, thus participating in the angiogenesis necessary for CB growth. Our results shed light into the molecular and cellular mechanisms involved in CBSC fate choice, favoring a potential use of these cells for cell therapy. Stem Cells 2016;34:1637-1650. © 2016 AlphaMed Press.

  5. Inhibition of Sirt1 promotes neural progenitors toward motoneuron differentiation from human embryonic stem cells

    SciTech Connect

    Zhang, Yun; Wang, Jing; Chen, Guian; Fan, Dongsheng; Deng, Min

    2011-01-14

    Research highlights: {yields} Nicotinamide inhibit Sirt1. {yields} MASH1 and Ngn2 activation. {yields} Increase the expression of HB9. {yields} Motoneurons formation increases significantly. -- Abstract: Several protocols direct human embryonic stem cells (hESCs) toward differentiation into functional motoneurons, but the efficiency of motoneuron generation varies based on the human ESC line used. We aimed to develop a novel protocol to increase the formation of motoneurons from human ESCs. In this study, we tested a nuclear histone deacetylase protein, Sirt1, to promote neural precursor cell (NPC) development during differentiation of human ESCs into motoneurons. A specific inhibitor of Sirt1, nicotinamide, dramatically increased motoneuron formation. We found that about 60% of the cells from the total NPCs expressed HB9 and {beta}III-tubulin, commonly used motoneuronal markers found in neurons derived from ESCs following nicotinamide treatment. Motoneurons derived from ESC expressed choline acetyltransferase (ChAT), a positive marker of mature motoneuron. Moreover, we also examined the transcript levels of Mash1, Ngn2, and HB9 mRNA in the differentiated NPCs treated with the Sirt1 activator resveratrol (50 {mu}M) or inhibitor nicotinamide (100 {mu}M). The levels of Mash1, Ngn2, and HB9 mRNA were significantly increased after nicotinamide treatment compared with control groups, which used the traditional protocol. These results suggested that increasing Mash1 and Ngn2 levels by inhibiting Sirt1 could elevate HB9 expression, which promotes motoneuron differentiation. This study provides an alternative method for the production of transplantable motoneurons, a key requirement in the development of hESC-based cell therapy in motoneuron disease.

  6. Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

    PubMed Central

    Donegà, Matteo; Giusto, Elena; Cossetti, Chiara; Schaeffer, Julia; Pluchino, Stefano

    2014-01-01

    Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases. These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson's disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS). This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v.) or intracerebroventricular (i.c.v.) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo. Here we describe the methods that we have developed for the i.v. and i.c.v. delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage

  7. Neural Progenitor Cell Transplantation Promotes Neuroprotection, Enhances Hippocampal Neurogenesis, and Improves Cognitive Outcomes after Traumatic Brain Injury

    PubMed Central

    Blaya, Meghan O.; Tsoulfas, Pantelis; Bramlett, Helen M.; Dietrich, W. Dalton

    2014-01-01

    Transplantation of neural progenitor cells (NPCs) may be a potential treatment strategy for traumatic brain injury (TBI) due to their intrinsic advantages, including the secretion of neurotrophins. Neurotrophins are critical for neuronal survival and repair, but their clinical use is limited. In this study, we hypothesized that pericontusional transplantation of NPCs genetically modified to secrete a synthetic, human multineurotrophin (MNTS1) would overcome some of the limitations of traditional neurotrophin therapy. MNTS1 is a multifunctional neurotrophin that binds all three tropomyosin-related kinase (Trk) receptors, recapitulating the prosurvival activity of 3 endogenous mature neurotrophins. NPCs obtained from rat fetuses at E15 were transduced with lentiviral vectors containing MNTS1 and GFP constructs (MNTS1-NPCs) or fluorescent constructs alone (control GFP-NPCs). Adult rats received fluid percussion-induced TBI or sham surgery. Animals were transplanted 1 week later with control GFP-NPCs, MNTS1-NPCs, or injected with saline (vehicle). At five weeks, animals were evaluated for hippocampal-dependent spatial memory. Six weeks post surgery, we observed significant survival and neuronal differentiation of MNTS1-NPCs and injury-activated tropism towards contused regions. NPCs displayed processes that extended into several remote structures, including the hippocampus and contralateral cortex. Both GFP- and MNTS1-NPCs conferred significant preservation of pericontusional host tissues and enhanced hippocampal neurogenesis. NPC transplantation improved spatial memory capacity on the Morris water maze (MWM) task. Transplant recipients exhibited escape latencies approximately half that of injured vehicle controls. While we observed greater transplant survival and neuronal differentiation of MNTS1-NPCs, our collective findings suggest that MNTS1 may be superfluous in terms of preserving the cytoarchitecture and rescuing behavioral deficits given the lack of significant

  8. Neural progenitor cell transplantation promotes neuroprotection, enhances hippocampal neurogenesis, and improves cognitive outcomes after traumatic brain injury.

    PubMed

    Blaya, Meghan O; Tsoulfas, Pantelis; Bramlett, Helen M; Dietrich, W Dalton

    2015-02-01

    Transplantation of neural progenitor cells (NPCs) may be a potential treatment strategy for traumatic brain injury (TBI) due to their intrinsic advantages, including the secretion of neurotrophins. Neurotrophins are critical for neuronal survival and repair, but their clinical use is limited. In this study, we hypothesized that pericontusional transplantation of NPCs genetically modified to secrete a synthetic, human multineurotrophin (MNTS1) would overcome some of the limitations of traditional neurotrophin therapy. MNTS1 is a multifunctional neurotrophin that binds all three tropomyosin-related kinase (Trk) receptors, recapitulating the prosurvival activity of 3 endogenous mature neurotrophins. NPCs obtained from rat fetuses at E15 were transduced with lentiviral vectors containing MNTS1 and GFP constructs (MNTS1-NPCs) or fluorescent constructs alone (control GFP-NPCs). Adult rats received fluid percussion-induced TBI or sham surgery. Animals were transplanted 1week later with control GFP-NPCs, MNTS1-NPCs, or injected with saline (vehicle). At five weeks, animals were evaluated for hippocampal-dependent spatial memory. Six weeks post-surgery, we observed significant survival and neuronal differentiation of MNTS1-NPCs and injury-activated tropism toward contused regions. NPCs displayed processes that extended into several remote structures, including the hippocampus and contralateral cortex. Both GFP- and MNTS1-NPCs conferred significant preservation of pericontusional host tissues and enhanced hippocampal neurogenesis. NPC transplantation improved spatial memory capacity on the Morris water maze (MWM) task. Transplant recipients exhibited escape latencies approximately half that of injured vehicle controls. While we observed greater transplant survival and neuronal differentiation of MNTS1-NPCs, our collective findings suggest that MNTS1 may be superfluous in terms of preserving the cytoarchitecture and rescuing behavioral deficits given the lack of significant

  9. Neural progenitor cells isolated from the subventricular zone present hemichannel activity and form functional gap junctions with glial cells.

    PubMed

    Talaverón, Rocío; Fernández, Paola; Escamilla, Rosalba; Pastor, Angel M; Matarredona, Esperanza R; Sáez, Juan C

    2015-01-01

    The postnatal subventricular zone (SVZ) lining the walls of the lateral ventricles contains neural progenitor cells (NPCs) that generate new olfactory bulb interneurons. Communication via gap junctions between cells in the SVZ is involved in NPC proliferation and in neuroblast migration towards the olfactory bulb. SVZ NPCs can be expanded in vitro in the form of neurospheres that can be used for transplantation purposes after brain injury. We have previously reported that neurosphere-derived NPCs form heterocellular gap junctions with host glial cells when they are implanted after mechanical injury. To analyze functionality of NPC-glial cell gap junctions we performed dye coupling experiments in co-cultures of SVZ NPCs with astrocytes or microglia. Neurosphere-derived cells expressed mRNA for at least the hemichannel/gap junction channel proteins connexin 26 (Cx26), Cx43, Cx45 and pannexin 1 (Panx1). Dye coupling experiments revealed that gap junctional communication occurred among neurosphere cells (incidence of coupling: 100%). Moreover, hemichannel activity was also detected in neurosphere cells as evaluated in time-lapse measurements of ethidium bromide uptake. Heterocellular coupling between NPCs and glial cells was evidenced in co-cultures of neurospheres with astrocytes (incidence of coupling: 91.0 ± 4.7%) or with microglia (incidence of coupling: 71.9 ± 6.7%). Dye coupling in neurospheres and in co-cultures was inhibited by octanol, a gap junction blocker. Altogether, these results suggest the existence of functional hemichannels and gap junction channels in postnatal SVZ neurospheres. In addition, they demonstrate that SVZ-derived NPCs can establish functional gap junctions with astrocytes or microglia. Therefore, cell-cell communication via gap junctions and hemichannels with host glial cells might subserve a role in the functional integration of NPCs after implantation in the damaged brain.

  10. Progesterone Increases Circulating Endothelial Progenitor Cells and Induces Neural Regeneration after Traumatic Brain Injury in Aged Rats

    PubMed Central

    Li, Zhanying; Wang, Bin; Kan, Zhisheng; Zhang, Baoliang; Yang, Zhuo; Chen, Jieli; Wang, Dong; Wei, Huijie

    2012-01-01

    Abstract Vascular remodeling plays a key role in neural regeneration in the injured brain. Circulating endothelial progenitor cells (EPCs) are a mediator of the vascular remodeling process. Previous studies have found that progesterone treatment of traumatic brain injury (TBI) decreases cerebral edema and cellular apoptosis and inhibits inflammation, which in concert promote neuroprotective effects in young adult rats. However, whether progesterone treatment regulates circulating EPC level and fosters vascular remodeling after TBI have not been investigated. In this study, we hypothesize that progesterone treatment following TBI increases circulating EPC levels and promotes vascular remodeling in the injured brain in aged rats. Male Wistar 20-month-old rats were subjected to a moderate unilateral parietal cortical contusion injury and were treated with or without progesterone (n=54/group). Progesterone was administered intraperitoneally at a dose of 16mg/kg at 1 h post-TBI and was subsequently injected subcutaneously daily for 14 days. Neurological functional tests and immnunostaining were performed. Circulating EPCs were measured by flow cytometry. Progesterone treatment significantly improved neurological outcome after TBI measured by the modified neurological severity score, Morris Water Maze and the long term potentiation in the hippocampus as well as increased the circulating EPC levels compared to TBI controls (p<0.05). Progesterone treatment also significantly increased CD34 and CD31 positive cell number and vessel density in the injured brain compared to TBI controls (p<0.05). These data indicate that progesterone treatment of TBI improves multiple neurological functional outcomes, increases the circulating EPC level, and facilitates vascular remodeling in the injured brain after TBI in aged rats. PMID:21534727

  11. Assessing cell cycle progression of neural stem and progenitor cells in the mouse developing brain after genotoxic stress.

    PubMed

    Etienne, Olivier; Bery, Amandine; Roque, Telma; Desmaze, Chantal; Boussin, François D

    2014-05-07

    Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a pseudostratified epithelium lining the lateral ventricles of the embryonic brain. Genotoxic stresses, such as ionizing radiation, have highly deleterious effects on the developing brain related to the high sensitivity of NSPC. Elucidation of the cellular and molecular mechanisms involved depends on the characterization of the DNA damage response of these particular types of cells, which requires an accurate method to determine NSPC progression through the cell cycle in the damaged tissue. Here is shown a method based on successive intraperitoneal injections of EdU and BrdU in pregnant mice and further detection of these two thymidine analogues in coronal sections of the embryonic brain. EdU and BrdU are both incorporated in DNA of replicating cells during S phase and are detected by two different techniques (azide or a specific antibody, respectively), which facilitate their simultaneous detection. EdU and BrdU staining are then determined for each NSPC nucleus in function of its distance from the ventricular margin in a standard region of the dorsal telencephalon. Thus this dual labeling technique allows distinguishing cells that progressed through the cell cycle from those that have activated a cell cycle checkpoint leading to cell cycle arrest in response to DNA damage. An example of experiment is presented, in which EdU was injected before irradiation and BrdU immediately after and analyzes performed within the 4 hr following irradiation. This protocol provides an accurate analysis of the acute DNA damage response of NSPC in function of the phase of the cell cycle at which they have been irradiated. This method is easily transposable to many other systems in order to determine the impact of a particular treatment on cell cycle progression in living tissues.

  12. Neural progenitor cell implants modulate vascular endothelial growth factor and brain-derived neurotrophic factor expression in rat axotomized neurons.

    PubMed

    Talaverón, Rocío; Matarredona, Esperanza R; de la Cruz, Rosa R; Pastor, Angel M

    2013-01-01

    Axotomy of central neurons leads to functional and structural alterations which largely revert when neural progenitor cells (NPCs) are implanted in the lesion site. The new microenvironment created by NPCs in the host tissue might modulate in the damaged neurons the expression of a high variety of molecules with relevant roles in the repair mechanisms, including neurotrophic factors. In the present work, we aimed to analyze changes in neurotrophic factor expression in axotomized neurons induced by NPC implants. For this purpose, we performed immunofluorescence followed by confocal microscopy analysis for the detection of vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and nerve growth factor (NGF) on brainstem sections from rats with axotomy of abducens internuclear neurons that received NPC implants (implanted group) or vehicle injections (axotomized group) in the lesion site. Control abducens internuclear neurons were strongly immunoreactive to VEGF and BDNF but showed a weak staining for NT-3 and NGF. Comparisons between groups revealed that lesioned neurons from animals that received NPC implants showed a significant increase in VEGF content with respect to animals receiving vehicle injections. However, the immunoreactivity for BDNF, which was increased in the axotomized group as compared to control, was not modified in the implanted group. The modifications induced by NPC implants on VEGF and BDNF content were specific for the population of axotomized abducens internuclear neurons since the neighboring abducens motoneurons were not affected. Similar levels of NT-3 and NGF immunolabeling were obtained in injured neurons from axotomized and implanted animals. Among all the analyzed neurotrophic factors, only VEGF was expressed by the implanted cells in the lesion site. Our results point to a role of NPC implants in the modulation of neurotrophic factor expression by lesioned central neurons, which might

  13. Neural Progenitor Cell Implants Modulate Vascular Endothelial Growth Factor and Brain-Derived Neurotrophic Factor Expression in Rat Axotomized Neurons

    PubMed Central

    Talaverón, Rocío; Matarredona, Esperanza R.; de la Cruz, Rosa R.; Pastor, Angel M.

    2013-01-01

    Axotomy of central neurons leads to functional and structural alterations which largely revert when neural progenitor cells (NPCs) are implanted in the lesion site. The new microenvironment created by NPCs in the host tissue might modulate in the damaged neurons the expression of a high variety of molecules with relevant roles in the repair mechanisms, including neurotrophic factors. In the present work, we aimed to analyze changes in neurotrophic factor expression in axotomized neurons induced by NPC implants. For this purpose, we performed immunofluorescence followed by confocal microscopy analysis for the detection of vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and nerve growth factor (NGF) on brainstem sections from rats with axotomy of abducens internuclear neurons that received NPC implants (implanted group) or vehicle injections (axotomized group) in the lesion site. Control abducens internuclear neurons were strongly immunoreactive to VEGF and BDNF but showed a weak staining for NT-3 and NGF. Comparisons between groups revealed that lesioned neurons from animals that received NPC implants showed a significant increase in VEGF content with respect to animals receiving vehicle injections. However, the immunoreactivity for BDNF, which was increased in the axotomized group as compared to control, was not modified in the implanted group. The modifications induced by NPC implants on VEGF and BDNF content were specific for the population of axotomized abducens internuclear neurons since the neighboring abducens motoneurons were not affected. Similar levels of NT-3 and NGF immunolabeling were obtained in injured neurons from axotomized and implanted animals. Among all the analyzed neurotrophic factors, only VEGF was expressed by the implanted cells in the lesion site. Our results point to a role of NPC implants in the modulation of neurotrophic factor expression by lesioned central neurons, which might

  14. Pronounced hypoxia in the subventricular zone following traumatic brain injury and the neural stem/progenitor cell response.

    PubMed

    Baumann, Gisela; Travieso, Lissette; Liebl, Daniel J; Theus, Michelle H

    2013-07-01

    Traumatic brain injury (TBI) elicits identifiable changes within the adult subventricular zone (SVZ). Previously, we demonstrated that EphB3/ephrinB3 interaction inhibits neural stem/progenitor cell (NSPC) proliferation and downregulating this pathway following TBI plays a pivotal role in the expansion of the SVZ neurogenic compartment. It remains unclear, however, what early initiating factors may precede these changes. Using hypoxyprobe-1 (HPb) to identify regions of low oxygen tension or hypoxia (<1%), we found HPb uptake throughout the cortex (CTX), corpus callosum (CC) and SVZ within the first 24 h following controlled cortical impact (CCI) injury. At this early time point, HPb co-localized with EphB3 in the SVZ. NSPC specific markers also co-localized with HPb staining throughout the lateral wall of the ventricle. To determine the cell autonomous effects of hypoxia on EphB3/ephrinB3 signaling in NSPCs, we used an in vitro model of hypoxia to mimic 1% oxygen in the presence and absence of soluble aggregated ephrinB3 (eB3). As expected, hypoxia stimulated the uptake of 5-bromo-2'-deoxyuridine (BrdU) and reduced cell death. Coincident with these proliferative changes, both Hif1-α and phospho (p)-AKT were increased while EphB3 expression was decreased. Stimulation of EphB3 attenuated hypoxia-induced proliferation and prevented phosphorylation of AKT. Hif1-α accumulation, on the other hand, was not affected by EphB3/ephrinB3 signaling. These findings indicate that this pathway limits the NSPC response to hypoxic stimuli. These studies also suggest that early transient changes in oxygen tension following localized cortical injury may initiate a growth-promoting response in the SVZ.

  15. Role of low voltage activated calcium channels in neuritogenesis and active migration of embryonic neural progenitor cells.

    PubMed

    Louhivuori, Lauri M; Louhivuori, Verna; Wigren, Henna-Kaisa; Hakala, Elina; Jansson, Linda C; Nordström, Tommy; Castrén, Maija L; Akerman, Karl E

    2013-04-15

    The central role of calcium influx and electrical activity in embryonic development raises important questions about the role and regulation of voltage-dependent calcium influx. Using cultured neural progenitor cell (NPC) preparations, we recorded barium currents through voltage-activated channels using the whole-cell configuration of the patch-clamp technique and monitored intracellular free calcium concentrations with Fura-2 digital imaging. We found that NPCs as well as expressing high-voltage-activated (HVA) calcium channels express functional low-threshold voltage-dependent calcium channels in the very early stages of differentiation (5 h to 1 day). The size of the currents recorded at -50 versus -20 mV after 1 day in differentiation was dependent on the nature of the charge carrier. Peak currents measured at -20 mV in the presence 10 mM Ca2+ instead of 10 mM Ba2+ had a tendency to be smaller, whereas the nature of the divalent species did not influence the amplitude measured at -50 mV. The T-type channel blockers mibefradil and NNC 55-0396 significantly reduced the calcium responses elicited by depolarizing with extracellular potassium, while the overall effect of the HVA calcium channel blockers was small at differentiation day 1. At differentiation day 20, the calcium responses were effectively blocked by nifedipine. Time-lapse imaging of differentiating neurospheres cultured in the presence of low-voltage-activated (LVA) blockers showed a significant decrease in the number of active migrating neuron-like cells and neurite extensions. Together, these data provide evidence that LVA calcium channels are involved in the physiology of differentiating and migrating NPCs.

  16. Neural progenitor cells isolated from the subventricular zone present hemichannel activity and form functional gap junctions with glial cells

    PubMed Central

    Talaverón, Rocío; Fernández, Paola; Escamilla, Rosalba; Pastor, Angel M.; Matarredona, Esperanza R.; Sáez, Juan C.

    2015-01-01

    The postnatal subventricular zone (SVZ) lining the walls of the lateral ventricles contains neural progenitor cells (NPCs) that generate new olfactory bulb interneurons. Communication via gap junctions between cells in the SVZ is involved in NPC proliferation and in neuroblast migration towards the olfactory bulb. SVZ NPCs can be expanded in vitro in the form of neurospheres that can be used for transplantation purposes after brain injury. We have previously reported that neurosphere-derived NPCs form heterocellular gap junctions with host glial cells when they are implanted after mechanical injury. To analyze functionality of NPC-glial cell gap junctions we performed dye coupling experiments in co-cultures of SVZ NPCs with astrocytes or microglia. Neurosphere-derived cells expressed mRNA for at least the hemichannel/gap junction channel proteins connexin 26 (Cx26), Cx43, Cx45 and pannexin 1 (Panx1). Dye coupling experiments revealed that gap junctional communication occurred among neurosphere cells (incidence of coupling: 100%). Moreover, hemichannel activity was also detected in neurosphere cells as evaluated in time-lapse measurements of ethidium bromide uptake. Heterocellular coupling between NPCs and glial cells was evidenced in co-cultures of neurospheres with astrocytes (incidence of coupling: 91.0 ± 4.7%) or with microglia (incidence of coupling: 71.9 ± 6.7%). Dye coupling in neurospheres and in co-cultures was inhibited by octanol, a gap junction blocker. Altogether, these results suggest the existence of functional hemichannels and gap junction channels in postnatal SVZ neurospheres. In addition, they demonstrate that SVZ-derived NPCs can establish functional gap junctions with astrocytes or microglia. Therefore, cell-cell communication via gap junctions and hemichannels with host glial cells might subserve a role in the functional integration of NPCs after implantation in the damaged brain. PMID:26528139

  17. Senescence Mediated by p16INK4a Impedes Reprogramming of Human Corneal Endothelial Cells into Neural Crest Progenitors

    PubMed Central

    Lu, Wen-Juan; Tseng, Scheffer C. G.; Chen, Shuangling; Tighe, Sean; Zhang, Yuan; Liu, Xin; Chen, Szu-Yu; Su, Chen-Wei; Zhu, Ying-Ting

    2016-01-01

    Human corneal endothelial cells (HCECs) have limited proliferative capacity due to “contact-inhibition” at G1 phase. Such contact-inhibition can be delayed from Day 21 to Day 42 by switching EGF-containing SHEM to LIF/bFGF-containing MESCM through transient activation of LIF-JAK1-STAT3 signaling that delays eventual nuclear translocation of p16INK4a. Using the latter system, we have reported a novel tissue engineering technique by implementing 5 weekly knockdowns with p120 catenin (p120) and Kaiso siRNAs since Day 7 to achieve effective expansion of HCEC monolayers to a transplantable size with a normal HCEC density, through reprogramming of HCECs into neural crest progenitors by activating p120-Kaiso-RhoA-ROCK-canonical BMP signaling. Herein, we noted that a single knockdown with p120-Kaiso siRNAs at Day 42 failed to achieve such reprogramming when contact inhibition transitioned to senescence with nuclear translocation of p16INK4a. In contrast, 5 weekly knockdowns with p120-Kaiso siRNAs since Day 7 precluded senescence mediated by p16INK4a by inducing nuclear translocation of Bmi1 because of sustained activation of JAK2-STAT3 signaling downstream of p120-Kaiso-RhoA-ROCK signaling. STAT3 or Bmi1 siRNA impeded nuclear exclusion of p16INK4a and suppressed the reprogramming induced by p120-Kaiso siRNAs, suggesting that another important engineering strategy of HCEC lies in prevention of senescence mediated by nuclear translocation of p16INK4a. PMID:27739458

  18. 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.

  19. Controlled microfluidics to examine growth-factor induced migration of neural progenitors in the Drosophila visual system.

    PubMed

    Beck, Cade; Singh, Tanya; Farooqi, Angela; Venkatesh, Tadmiri; Vazquez, Maribel

    2016-03-15

    The developing visual system in Drosophila melanogaster provides an excellent model with which to examine the effects of changing microenvironments on neural cell migration via microfluidics, because the combined experimental system enables direct genetic manipulation, in vivo observation, and in vitro imaging of cells, post-embryo. Exogenous signaling from ligands such as fibroblast growth factor (FGF) is well-known to control glia differentiation, cell migration, and axonal wrapping central to vision. The current study employs a microfluidic device to examine how controlled concentration gradient fields of FGF are able to regulate the migration of vision-critical glia cells with and without cellular contact with neuronal progenitors. Our findings quantitatively illustrate a concentration-gradient dependent chemotaxis toward FGF, and further demonstrate that glia require collective and coordinated neuronal locomotion to achieve directionality, sustain motility, and propagate long cell distances in the visual system. Conventional assays are unable to examine concentration- and gradient-dependent migration. Our data illustrate quantitative correlations between ligand concentration/gradient and glial cell distance traveled, independent or in contact with neurons. Microfluidic systems in combination with a genetically-amenable experimental system empowers researchers to dissect the signaling pathways that underlie cellular migration during nervous system development. Our findings illustrate the need for coordinated neuron-glia migration in the Drosophila visual system, as only glia within heterogeneous populations exhibited increasing motility along distances that increased with increasing FGF concentration. Such coordinated migration and chemotactic dependence can be manipulated for potential therapeutic avenues for NS repair and/or disease treatment. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Direct Stimulation of Adult Neural Stem/Progenitor Cells In Vitro and Neurogenesis In Vivo by Salvianolic Acid B

    PubMed Central

    Zhuang, Pengwei; Zhang, Yanjun; Cui, Guangzhi; Bian, Yuhong; Zhang, Mixia; Zhang, Jinbao; Liu, Yang; Yang, Xinpeng; Isaiah, Adejobi Oluwaniyi; Lin, Yingxue; Jiang, Yongbo

    2012-01-01

    Background Small molecules have been shown to modulate the neurogenesis processes. In search for new therapeutic drugs, the herbs used in traditional medicines for neurogenesis are promising candidates. Methodology and Principal Findings We selected a total of 45 natural compounds from Traditional Chinese herbal medicines which are extensively used in China to treat stroke clinically, and tested their proliferation-inducing activities on neural stem/progenitor cells (NSPCs). The screening results showed that salvianolic acid B (Sal B) displayed marked effects on the induction of proliferation of NSPCs. We further demonstrated that Sal B promoted NSPCs proliferation in dose- and time-dependent manners. To explore the molecular mechanism, PI3K/Akt, MEK/ERK and Notch signaling pathways were investigated. Cell proliferation assay demonstrated that Ly294002 (PI3K/Akt inhibitor), but neither U0126 (ERK inhibitor) nor DAPT (Notch inhibitor) inhibited the Sal B-induced proliferation of cells. Western Blotting results showed that stimulation of NSPCs with Sal B enhanced the phosphorylation of Akt, and Ly294002 abolished this effect, confirming the role of Akt in Sal B mediated proliferation of NSPCs. Rats exposed to transient cerebral ischemia were treated for 4 weeks with Sal B from the 7th day after stroke. BrdU incorporation assay results showed that exposure Sal B could maintain the proliferation of NSPCs after cerebral ischemia. Morris water maze test showed that delayed post-ischemic treatment with Sal B improved cognitive impairment after stroke in rats. Significance Sal B could maintain the NSPCs self-renew and promote proliferation, which was mediated by PI3K/Akt signal pathway. And delayed post-ischemic treatment with Sal B improved cognitive impairment after stroke in rats. These findings suggested that Sal B may act as a potential drug in treatment of brain injury or neurodegenerative diseases. PMID:22545124

  1. Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress

    PubMed Central

    Etienne, Olivier; Bery, Amandine; Roque, Telma; Desmaze, Chantal; Boussin, François D.

    2014-01-01

    Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a pseudostratified epithelium lining the lateral ventricles of the embryonic brain. Genotoxic stresses, such as ionizing radiation, have highly deleterious effects on the developing brain related to the high sensitivity of NSPC. Elucidation of the cellular and molecular mechanisms involved depends on the characterization of the DNA damage response of these particular types of cells, which requires an accurate method to determine NSPC progression through the cell cycle in the damaged tissue. Here is shown a method based on successive intraperitoneal injections of EdU and BrdU in pregnant mice and further detection of these two thymidine analogues in coronal sections of the embryonic brain. EdU and BrdU are both incorporated in DNA of replicating cells during S phase and are detected by two different techniques (azide or a specific antibody, respectively), which facilitate their simultaneous detection. EdU and BrdU staining are then determined for each NSPC nucleus in function of its distance from the ventricular margin in a standard region of the dorsal telencephalon. Thus this dual labeling technique allows distinguishing cells that progressed through the cell cycle from those that have activated a cell cycle checkpoint leading to cell cycle arrest in response to DNA damage. An example of experiment is presented, in which EdU was injected before irradiation and BrdU immediately after and analyzes performed within the 4 hr following irradiation. This protocol provides an accurate analysis of the acute DNA damage response of NSPC in function of the phase of the cell cycle at which they have been irradiated. This method is easily transposable to many other systems in order to determine the impact of a particular treatment on cell cycle progression in living tissues. PMID:24837791

  2. TNF-α Affects Human Cortical Neural Progenitor Cell Differentiation through the Autocrine Secretion of Leukemia Inhibitory Factor

    PubMed Central

    Lan, Xiqian; Chen, Qiang; Wang, Yongxiang; Jia, Beibei; Sun, Lijun; Zheng, Jialin; Peng, Hui

    2012-01-01

    Proinflammatory cytokine tumor necrosis factor-alpha (TNF-α) is a crucial effector of immune responses in the brain that participates in the pathogenesis of several acute and chronic neurodegenerative disorders. Accumulating evidence has suggested that TNF-α negatively regulates embryonic and adult neurogenesis. However, the effect of TNF-α on cell fate decision in human neural progenitor cells (NPCs) has rarely been studied. Our previous studies have shown that recombinant TNF-α enhances astrogliogenesis and inhibits neurogenesis of human NPCs through the STAT3 (signal transducer and activator of transcription 3) pathway. In the current study, we further elucidated the specific mechanism involved in TNF-α-induced astrogliogenesis. We found that TNF-α activated STAT3 at delayed time points (6 h and 24 h), whereas conditioned medium collected from TNF-α-treated NPCs induced an immediate STAT3 activation. These data suggest TNF-α plays an indirect role on STAT3 activation and the subsequent NPC differentiation. Further, we showed that TNF-α induced abundant amounts of the IL-6 family cytokines, including Leukemia inhibitory factor (LIF) and Interleukin 6 (IL-6), in human NPCs. TNF-α-induced STAT3 phosphorylation and astrogliogenesis were abrogated by the addition of neutralizing antibody for LIF, but not for IL-6, revealing a critical role of autocrine secretion of LIF in TNF-α-induced STAT3 activation and astrogliogenesis. This study generates important data elucidating the role of TNF-α in neurogenesis and may provide insight into new therapeutic strategies for brain inflammation. PMID:23236394

  3. Short Term Morphine Exposure In Vitro Alters Proliferation and Differentiation of Neural Progenitor Cells and Promotes Apoptosis via Mu Receptors

    PubMed Central

    Willner, Dafna; Cohen-Yeshurun, Ayelet; Avidan, Alexander; Ozersky, Vladislav; Shohami, Esther; Leker, Ronen R.

    2014-01-01

    Background Chronic morphine treatment inhibits neural progenitor cell (NPC) progression and negatively effects hippocampal neurogenesis. However, the effect of acute opioid treatment on cell development and its influence on NPC differentiation and proliferation in vitro is unknown. We aim to investigate the effect of a single, short term exposure of morphine on the proliferation, differentiation and apoptosis of NPCs and the mechanism involved. Methods Cell cultures from 14-day mouse embryos were exposed to different concentrations of morphine and its antagonist naloxone for 24 hours and proliferation, differentiation and apoptosis were studied. Proliferating cells were labeled with bromodeoxyuridine (BrdU) and cell fate was studied with immunocytochemistry. Results Cells treated with morphine demonstrated decreased BrdU expression with increased morphine concentrations. Analysis of double-labeled cells showed a decrease in cells co-stained for BrdU with nestin and an increase in cells co-stained with BrdU and neuron-specific class III β-tubuline (TUJ1) in a dose dependent manner. Furthermore, a significant increase in caspase-3 activity was observed in the nestin- positive cells. Addition of naloxone to morphine-treated NPCs reversed the anti-proliferative and pro-apoptotic effects of morphine. Conclusions Short term morphine exposure induced inhibition of NPC proliferation and increased active caspase-3 expression in a dose dependent manner. Morphine induces neuronal and glial differentiation and decreases the expression of nestin- positive cells. These effects were reversed with the addition of the opioid antagonist naloxone. Our results demonstrate the effects of short term morphine administration on the proliferation and differentiation of NPCs and imply a mu-receptor mechanism in the regulation of NPC survival. PMID:25072277

  4. Electric field-controlled directed migration of neural progenitor cells in 2D and 3D environments.

    PubMed

    Meng, Xiaoting; Li, Wenfei; Young, Fraser; Gao, Runchi; Chalmers, Laura; Zhao, Min; Song, Bing

    2012-02-16

    Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system(1,2). These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans(3,4). In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types(5,6), including neural progenitor cells (NPCs)(7,8). Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously(5,11). Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures(9,10). Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

  5. Transcription-associated processes cause DNA double-strand breaks and translocations in neural stem/progenitor cells

    PubMed Central

    Schwer, Bjoern; Wei, Pei-Chi; Chang, Amelia N.; Kao, Jennifer; Du, Zhou; Meyers, Robin M.; Alt, Frederick W.

    2016-01-01

    High-throughput, genome-wide translocation sequencing (HTGTS) studies of activated B cells have revealed that DNA double-strand breaks (DSBs) capable of translocating to defined bait DSBs are enriched around the transcription start sites (TSSs) of active genes. We used the HTGTS approach to investigate whether a similar phenomenon occurs in primary neural stem/progenitor cells (NSPCs). We report that breakpoint junctions indeed are enriched around TSSs that were determined to be active by global run-on sequencing analyses of NSPCs. Comparative analyses of transcription profiles in NSPCs and B cells revealed that the great majority of TSS-proximal junctions occurred in genes commonly expressed in both cell types, possibly because this common set has higher transcription levels on average than genes transcribed in only one or the other cell type. In the latter context, among all actively transcribed genes containing translocation junctions in NSPCs, those with junctions located within 2 kb of the TSS show a significantly higher transcription rate on average than genes with junctions in the gene body located at distances greater than 2 kb from the TSS. Finally, analysis of repair junction signatures of TSS-associated translocations in wild-type versus classical nonhomologous end-joining (C-NHEJ)–deficient NSPCs reveals that both C-NHEJ and alternative end-joining pathways can generate translocations by joining TSS-proximal DSBs to DSBs on other chromosomes. Our studies show that the generation of transcription-associated DSBs is conserved across divergent cell types. PMID:26873106

  6. 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.

  7. Early appearance of stem/progenitor cells with neural-like characteristics in human cord blood mononuclear fraction cultured in vitro.

    PubMed

    Habich, Aleksandra; Jurga, Marcin; Markiewicz, Inga; Lukomska, Barbara; Bany-Laszewicz, Urszula; Domanska-Janik, Krystyna

    2006-07-01

    The exposure of human umbilical cord blood mononuclear cells devoid of hematopoietic stem cells (HUCB-MNCsCD34-) to defined culture condition promotes their conversion into neural lineage. We have asked the question if observed fate change of HUCB-MNCsCD34- results from direct conversion of hematopoietic precursors into neural-like phenotypes due to expression of overlapping genetic program or, alternatively, these neural phenotypes arise from sequential differentiation of more primitive progenitors (embryonic-like cells) preexisting in HUCB-MNCsCD34- fraction. HUCB-MNCs negatively selected for CD34 antigens were cultured in vitro up to 14 days. Changes in stem/neural cell genes and proteins were successively evaluated during this period and after evoked neuronal differentiation of cells in the presence of RA or BDNF or cocultured with neonatal rat brain astrocytes. Freshly isolated HUCB-MNCsCD34- expressed pluripotent cell markers: Oct3/4, Sox2, and Rex1 genes. During 24 hours of culture the frequency of Oct3/4 immunopositive cells increased markedly with parallel enlargement of "side population" and CD133+ cell appearance. Concomitantly, cultured cells start to form aggregates and express pro-neural genes, i.e., enhanced Sox2, OTX1, Nestin, GFAP, and NF-200. During the next days of culture immunoreactions for beta-tubulin III, MAP2, GFAP, S100beta, Doublecortin, and GalC were induced with reciprocal lowering of stem cell gene and protein markers. At this stage cells successively adhered to the bottom, dispersed, and decreased proliferation rate (Ki67 expression). Additional treatments with neuromorphogenes or coculturing with rat brain primary culture induced further differentiation of these neural precursors toward more advanced neuronal phenotypes. HUCB-MNCs(CD34-) fraction contains embryonic-like stem/progenitor cells which increase rapidly but transiently in culture, then differentiate spontaneously after cell aggregate adhesion toward neural lineage

  8. Function of Armcx3 and Armc10/SVH Genes in the Regulation of Progenitor Proliferation and Neural Differentiation in the Chicken Spinal Cord

    PubMed Central

    Mirra, Serena; Ulloa, Fausto; Gutierrez-Vallejo, Irene; Martì, Elisa; Soriano, Eduardo

    2016-01-01

    The eutherian X-chromosome specific family of Armcx genes has been described as originating by retrotransposition from Armc10/SVH, a single Arm-containing somatic gene. Armcx3 and Armc10/SVH are characterized by high expression in the central nervous system and they play an important role in the regulation of mitochondrial distribution and transport in neurons. In addition, Armcx/Arm10 genes have several Armadillo repeats in their sequence. In this study we address the potential role of this gene family in neural development by using the chick neural tube as a model. We show that Armc10/SVH is expressed in the chicken spinal cord, and knocking-down Armc10/SVH by sh-RNAi electroporation in spinal cord reduces proliferation of neural precursor cells (NPCs). Moreover, we analyzed the effects of murine Armcx3 and Armc10 overexpression, showing that both proteins regulate progenitor proliferation, while Armcx3 overexpression also specifically controls neural maturation. We show that the phenotypes found following Armcx3 overexpression require its mitochondrial localization, suggesting a novel link between mitochondrial dynamics and regulation of neural development. Furthermore, we found that both Armcx3 and Armc10 may act as inhibitors of Wnt-β-catenin signaling. Our results highlight both common and differential functions of Armcx/Armc10 genes in neural development in the spinal cord. PMID:26973462

  9. N-cadherin promotes recruitment and migration of neural progenitor cells from the SVZ neural stem cell niche into demyelinated lesions.

    PubMed

    Klingener, Michael; Chavali, Manideep; Singh, Jagdeep; McMillan, Nadia; Coomes, Alexandra; Dempsey, Peter J; Chen, Emily I; Aguirre, Adan

    2014-07-16

    Discrete cellular microenvironments regulate stem cell pools and their development, as well as function in maintaining tissue homeostasis. Although the signaling elements modulating neural progenitor cells (NPCs) of the adult subventricular zone (SVZ) niche are fairly well understood, the pathways activated following injury and the resulting outcomes, are less clear. In the present study, we used mouse models of demyelination and proteomics analysis to identify molecular cues present in the adult SVZ niche during injury, and analyzed their role on NPCs in the context of promoting myelin repair. Proteomic analysis of SVZ tissue from mice with experimental demyelination identified several proteins that are known to play roles in NPC proliferation, adhesion, and migration. Among the proteins found to be upregulated were members of the N-cadherin signaling pathway. During the onset of demyelination in the subcortical white matter (SCWM), activation of epidermal growth factor receptor (EGFR) signaling in SVZ NPCs stimulates the interaction between N-cadherin and ADAM10. Upon cleavage and activation of N-cadherin signaling by ADAM10, NPCs undergo cytoskeletal rearrangement and polarization, leading to enhanced migration out of the SVZ into demyelinated lesions of the SCWM. Genetically disrupting either EGFR signaling or ADAM10 inhibits this pathway, preventing N-cadherin regulated NPC polarization and migration. Additionally, in vivo experiments using N-cadherin gain- and loss-of-function approaches demonstrated that N-cadherin enhances the recruitment of SVZ NPCs into demyelinated lesions. Our data revealed that EGFR-dependent N-cadherin signaling physically initiated by ADAM10 cleavage is the response of the SVZ niche to promote repair of the injured brain.

  10. Upregulation of Slc38a1 Gene Along with Promotion of Neurosphere Growth and Subsequent Neuronal Specification in Undifferentiated Neural Progenitor Cells Exposed to Theanine.

    PubMed

    Takarada, Takeshi; Ogura, Masato; Nakamichi, Noritaka; Kakuda, Takami; Nakazato, Ryota; Kokubo, Hiroshi; Ikeno, Shinsuke; Nakamura, Saki; Kutsukake, Takaya; Hinoi, Eiichi; Yoneda, Yukio

    2016-02-01

    We have shown marked promotion of both cluster growth and neuronal specification in pluripotent P19 cells with overexpression of solute carrier 38a1 (Slc38a1), which is responsible for membrane transport of glutamine. In this study, we evaluated pharmacological profiles of the green tea amino acid ingredient theanine, which is a good substrate for glutamine transporters, on proliferation and neuronal specification in neural progenitor cells from embryonic rat neocortex. Sustained exposure to theanine, but not glutamine, accelerated the growth of neurospheres composed of proliferating cells and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reducing activity at concentrations of 1-100 μM in undifferentiated progenitor cells. Such prior exposure to theanine promoted spontaneous and induced commitment to a neuronal lineage with concomitant deteriorated astroglial specification. Selective upregulation was seen in the expression of Slc38a1 in progenitor cells cultured with theanine. Similarly significant increases in cluster growth and MTT reducing activity were found in P19 cells cultured with theanine for 4 days. Luciferase activity was doubled in a manner sensitive to the deletion of promoter regions in P19 cells with a luciferase reporter plasmid of the Slc38a1 promoter after sustained exposure to theanine for 4 days. Overexpression of X-box binding protein-1 led to a marked increase in luciferase activity in P19 cells transfected with the Slc38a1 reporter plasmid. These results suggest that theanine accelerates cellular proliferation and subsequent neuronal specification through a mechanism relevant to upregulation of Slc38a1 gene in undifferentiated neural progenitor cells.

  11. A novel 2q37 microdeletion containing human neural progenitors genes including STK25 results in severe developmental delay, epilepsy, and microcephaly.

    PubMed

    Imitola, Jaime; Khurana, Divya S; Teplyuk, Nadiya M; Zucker, Mark; Jethva, Reena; Legido, Agustin; Krichevsky, Ana M; Frangieh, Michael; Walsh, Christopher A; Carvalho, Karen S

    2015-11-01

    2q37 microdeletion syndrome is a rare syndrome characterized by neurodevelopmental delay, bone, cardiovascular, and neurological alterations. This syndrome is typically associated with loss of genetic material of approximately 100 genes in the 2q37 band. However, the genes associated with neurodevelopmental phenotype in this syndrome are still unknown. We identified a deleted region of 496 kb by whole genome array CGH in a patient who fulfilled criteria for 2q37 microdeletion syndrome with developmental delay, microcephaly, hypoplasia of the corpus callosum, hand wringing, toe walking, and seizures. The deleted segment contains genes that are highly expressed in the developing human cortical plate and the subventricular zone (SVZ) in vivo and human neural progenitors in vitro, including SEPT2, THAP4, ATG4B, PPP1R7, and STK25. Network analysis revealed that STK25 was the most interacting gene associated with neural development in this deletion. Our report narrows the likely causative genomic region for microcephaly and neurodevelopmental delay in 2q37 microdeletion syndrome to a small genomic region enriched with neural progenitor genes that may represent an important locus for the development of the human cortex and corpus callosum. © 2015 Wiley Periodicals, Inc.

  12. Cystein-serine-rich nuclear protein 1, Axud1/Csrnp1, is essential for cephalic neural progenitor proliferation and survival in zebrafish.

    PubMed

    Feijóo, Carmen G; Sarrazin, Andres F; Allende, Miguel L; Glavic, Alvaro

    2009-08-01

    The CSRNP (cystein-serine-rich nuclear protein) family has been conserved from Drosophila to human. Although knockout mice for each of the mammalian proteins have been generated, their function during vertebrate development has remained elusive. As an alternative to obtain insights on CSRNP's role in development, we have analysed the expression pattern and function of one member of this family, axud1, during zebrafish development. Our expression analysis indicates that axud1 is expressed from cleavage to larval stages in a dynamic pattern, becoming restricted after gastrulation to anterior regions of the developing neuraxis and later on concentrated predominantly in proliferating domains of the brain. Knockdown analysis using antisense morpholinos shows that reducing Axud1 levels impairs neural progenitor cell proliferation and survival, revealing an essential function of this gene for the growth of cephalic derivatives. The brain growth phenotypes elicited by decreasing Axud1 expression are specific and independent of anterior-posterior patterning events, initial establishment of neural progenitors, or neural differentiation occurring in this tissue. However, Axud1 is necessary for six3.1 expression and is positively regulated by sonic hedgehog. Phylogenetic examination shows that axud1 is likely to be the ortholog of the only member of this family present in Drosophila, as well as to the previously described mouse CSRNP1 and to human AXUD1 (Axin upregulated-1). Thus, we provide evidence as to the role of axud1 in brain growth in vertebrates.

  13. Self-renewal and differentiation of reactive astrocyte-derived neural stem/progenitor cells isolated from the cortical peri-infarct area after stroke.

    PubMed

    Shimada, Issei S; LeComte, Matthew