an-optogenetic-approach-for-assessing-formation-neuronal-connections

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

DUKE-NUS Graduate Medical School

Using flow cytometry measurements combined with quantitative analysis of cell cycle kinetics, we show that rhesus monkey embryonic stem cells (ESCs) are characterized by an extremely rapid transit through the G1 phase, which accounts for 15% of the total cell cycle duration. Monkey ESCs exhibit a non-phasic expression of cyclin E, which is detected during all phases of the cell cycle, and do not growth-arrest in G1 after gamma-irradiation, reflecting the absence of a G1 checkpoint. Serum deprivation or pharmacological inhibition of mitogen-activated protein kinase kinase (MEK) did not result in any alteration in the cell cycle distribution, indicating that ESC growth does not rely on mitogenic signals transduced by the Ras/Raf/MEK pathway. Taken together, these data indicate that rhesus monkey ESCs, like their murine counterparts, exhibit unusual cell cycle features in which cell cycle control mechanisms operating during the G1 phase are reduced or absent.

Cell cycle features of primate embryonic stem cells.

Embryonic stem cells (ESC) have the ability of indefinite self-renewal and multilineage differentiation, and they carry great potential in cell-based therapies. The rhesus macaque is the most relevant preclinical model for assessing the benefit, safety, and efficacy of ESC-based transplantations in the treatment of neurodegenerative diseases. In the case of neural cell grafting, tracing both the neurons and their axonal projections in vivo is essential for studying the integration of the grafted cells in the host brain. Tau-Green fluorescent protein (tau-GFP) is a powerful viable lineage tracer, allowing visualization of cell bodies, dendrites, and axons in exquisite detail. Here, we report the first rhesus monkey ESC line that ubiquitously and stably expresses tau-GFP. First, we derived a new line of rhesus monkey ESC (LYON-ES1) that show marker expression and cell cycle characteristics typical of primate ESCs. LYON-ES1 cells are pluripotent, giving rise to derivatives of the three germ layers in vitro and in vivo through teratoma formation. They retain all their undifferentiated characteristics and a normal karyotype after prolonged culture. Using lentiviral infection, we then generated a monkey ESC line stably expressing tau-GFP that retains all the characteristics of the parental wild-type line and is clonogenic. We show that neural precursors derived from the tau-GFP ESC line are multipotent and that their fate can be precisely mapped in vivo after grafting in the adult rat brain. Disclosure of potential conflicts of interest is found at the end of this article.

Derivation and cloning of a novel rhesus embryonic stem cell line stably expressing tau-green fluorescent protein.

The link between cortical precursors G1 duration (TG1) and their mode of division remains a major unresolved issue of potential importance for regulating corticogenesis. Here, we induced a 25% reduction in TG1 in mouse cortical precursors via forced expression of cyclin D1 and cyclin E1. We found that in utero electroporation-mediated gene transfer transfects a cohort of synchronously cycling precursors, necessitating alternative methods of measuring cell-cycle phases to those classical used. TG1 reduction promotes cell-cycle reentry at the expense of differentiation and increases the self-renewal capacities of Pax6 precursors as well as of Tbr2 basal precursors (BPs). A population level analysis reveals sequential and lineage-specific effects, showing that TG1 reduction: (i) promotes Pax6 self-renewing proliferative divisions before promoting divisions wherein Pax6 precursors generate Tbr2 BPs and (ii) promotes self-renewing proliferative divisions of Tbr2 precursors at the expense of neurogenesis, thus leading to an amplification of the BPs pool in the subventricular zone and the dispersed mitotic compartment of the intermediate zone. These results point to the G1 mode of division relationship as an essential control mechanism of corticogenesis. This is further supported by long-term studies showing that TG1 reduction results in cytoarchitectural modifications including supernumerary supragranular neuron production. Modeling confirms that the TG1-induced changes in neuron production and laminar fate are mediated via the changes in the mode of division. These findings also have implications for understanding the mechanisms that have contributed to brain enlargement and complexity during evolution.

Forced G1-phase reduction alters mode of division, neuron number, and laminar phenotype in the cerebral cortex.

Directing differentiation of neural stem/progenitor cells (NPCs) to produce functional neurons is a promising remedy for neural pathological conditions. The major challenge, however, lies in the effective and efficient generation of a sizable population of neurons. A potential strategy is to incorporate RNA interference (RNAi) during directed stem cell differentiation to recapitulate the complex cell-signaling cascades that often occurs during the process. In this study, in vitro silencing of RE1-silencing transcription factor (REST) was carried out using small-interfering RNAs (siRNAs) to evaluate the efficacy of combining REST knockdown with conventional differentiation approaches to enhance neurogenesis. While earlier studies have demonstrated enhanced neuronal lineage commitment from embryonic stem cells and mesenchymal stem cells upon REST knockdown, the effects of REST silencing during other stages of neural development have not been extensively evaluated. We hypothesize that REST knockdown would enhance NPC development to mature neurons and that induced REST silencing can serve as a potential biochemical approach to direct cell fate. Under nonspecific induction conditions, REST knockdown induced eightfold higher Tuj1 mRNA expression at day 14 compared with untransfected cells and cells subjected to scrambled-siRNA treatment (controls). Immunostaining also revealed greater percentage of Tuj1 positive cells with REST knockdown. Combined with neuronal induction, REST silencing enhanced the kinetics of neuronal differentiation and the rate of maturation of committed neuronal cells. Specifically, upregulation of MAP2 occurred as early as 3 days after induction with REST silencing and the expression was comparable to the controls at day 14. Likewise, downregulation of REST generated more than twice the percentage of Tuj1 and MAP2 positive cells compared with controls at day 5 (p<0.05). Morphologically, REST-silencing enhanced the number and length of neurite extensions from Tuj1 positive cells (p<0.05), which was not evaluated in previous differentiation studies with REST knockdown. Taken together, these results demonstrate the efficacy of combining REST silencing during directed NPC differentiation to enhance the rate of differentiation and subsequent maturation of NPCs. This study also highlights the potential of RNAi as a biomedical strategy for guided stem cell differentiation.

Directing neuronal differentiation of primary neural progenitor cells by gene knockdown approach.

Gliazelle-Nanofasern Interaktionen werden durch die Z&uuml;chtung der prim&auml;ren Ratte kortikale Astrozyten auf Poly [Caprolacton - co-(Ethyl-Ethylen-Phosphat)] Electrospun Nanofasern und L&ouml;sungsmittel-Darsteller Filme (zweidimensionale Steuerung) studiert. Die Ergebnisse zeigen, dass Nanofaser Topographie erheblich Gliazelle Verbreitung unterdr&uuml;ckt und verbessert die Apoptose, ohne Ver&auml;nderung der zellul&auml;re Aktivierung im Vergleich zu Filmen. Dar&uuml;ber hinaus erh&ouml;ht Nanofaser Topographie gen-silencing Effizienz in Astrozyten. Die Ergebnisse legen nahe, dass Nanofasern als potentielle Substrate f&uuml;r Nervenregeneration durch die Unterdr&uuml;ckung von Gliazelle Wachstum dienen und weiter die Verwendung erleichtern k&ouml;nnen von Gen-silencing zur Verbesserung der CNS-Regenerierung.

Die Auswirkungen von Nanofaser Topographie auf Gliazelle Verhalten und Gene Silencing Effizienz.

Astrocita-nanofibra interazioni sono studiate da coltura astrociti corticale primario ratto su poli [caprolattone - co-(fosfato di etilene etilico)] elettrofilate nanofibre e solvente-cast film (controllo bidimensionale). I risultati indicano che nanofibra topografia significativamente sopprime la proliferazione di astrociti e migliora il apoptosis, senza alterare l&#39;attivazione cellulare rispetto al film. Inoltre, nanofibra topografia migliora l&#39;efficienza di silenziamento genico negli astrociti. I risultati suggeriscono che le nanofibre possono servire come potenziali substrati per la rigenerazione del nervo di sopprimere la crescita di astrociti e possono agevolare ulteriormente l&#39;utilizzo di silenziamento genico per migliorare la rigenerazione del CNS.

Gli effetti della topografia nanofibra sul comportamento di astrociti e l&#39;efficienza di silenziamento genico.

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Astrocyte-nanofiber interactions are studied by culturing primary rat cortical astrocytes on poly[caprolactone-co-(ethyl ethylene phosphate)] electrospun nanofibers and solvent-cast films (two-dimensional control). The results indicate that nanofiber topography significantly suppresses astrocyte proliferation and enhances apoptosis, without altering cellular activation as compared to films. Moreover, nanofiber topography enhances gene-silencing efficiency in astrocytes. The results suggest that nanofibers may serve as potential substrates for nerve regeneration by suppressing astrocyte growth and may further facilitate the use of gene-silencing to enhance CNS regeneration.

The Effects of Nanofiber Topography on Astrocyte Behavior and Gene Silencing Efficiency.

Astrocyte-nanofibres interactions sont &eacute;tudi&eacute;es en cultivant les astrocytes corticaux primaires de rat sur nanofibres &eacute;lectrofil&eacute;es poly [caprolactone - co-(phosphate d&#39;&eacute;thyl&egrave;ne &eacute;thylique)] et des films de solvant-cast (contr&ocirc;le &agrave; deux dimensions). Les r&eacute;sultats indiquent que topographie de nanofibres significativement supprime la prolif&eacute;ration astrocytaire et am&eacute;liore l&#39;apoptose, sans alt&eacute;rer l&#39;activation cellulaire par rapport aux films. En outre, topographie de nanofibres am&eacute;liore l&#39;efficacit&eacute; de silen&ccedil;age g&eacute;nique dans les astrocytes. Les r&eacute;sultats sugg&egrave;rent que les nanofibres peuvent servir de substrats potentiels pour la r&eacute;g&eacute;n&eacute;ration des nerfs en supprimant la croissance astrocyte et peut faciliter l&#39;utilisation du g&egrave;ne-baffle pour am&eacute;liorer la r&eacute;g&eacute;n&eacute;ration de la CNS.

Les effets de la topographie de nanofibres sur le comportement de l&#39;Astrocyte et efficacit&eacute; de silen&ccedil;age g&eacute;nique.

&#x30A2;&#x30B9;&#x30C8;&#x30ED; &#x30B5;&#x30A4;&#x30C8;&#x521D;&#x4EE3;&#x30E9;&#x30C3;&#x30C8;&#x76AE;&#x8CEA;&#x4E0A;&#x30DD;&#x30EA; [&#x30AB;&#x30D7;&#x30ED;&#x30E9;&#x30AF;&#x30C8;&#x30F3; - co - &#xFF08;&#x30A8;&#x30C1;&#x30EB; &#x30A8;&#x30C1;&#x30EC;&#x30F3; &#x30EA;&#x30F3;&#x9178;&#xFF09;] &#x30A8;&#x30EC;&#x30AF;&#x30C8;&#x30ED;&#x30B9;&#x30D4;&#x30CB;&#x30F3;&#x30B0; &#x30CA;&#x30CE;&#x30D5;&#x30A1;&#x30A4;&#x30D0;&#x30FC;&#x3092;&#x57F9;&#x990A;&#x30A2;&#x30B9;&#x30C8;&#x30ED; &#x30CA;&#x30CE;&#x30D5;&#x30A1;&#x30A4;&#x30D0;&#x30FC;&#x306E;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x3092;&#x7814;&#x7A76;&#x3057;&#x3001;&#x6EB6;&#x5A92;&#x30AD;&#x30E3;&#x30B9;&#x30C8;&#x819C; &#xFF08;&#x4E8C;&#x6B21;&#x5143;&#x5236;&#x5FA1;)&#x3002;&#x30CA;&#x30CE;&#x30D5;&#x30A1;&#x30A4;&#x30D0;&#x30FC;&#x306E;&#x5730;&#x5F62;&#x304C;&#x5927;&#x5E45;&#x306B;&#x30A2;&#x30B9;&#x30C8;&#x30ED;&#x306E;&#x5897;&#x6B96;&#x3092;&#x6291;&#x5236;&#x3057;&#x3001;&#x30A2;&#x30DD;&#x30C8;&#x30FC;&#x30B7;&#x30B9;&#x3001;&#x7D30;&#x80DE;&#x6D3B;&#x6027;&#x5316;&#x819C;&#x3068;&#x6BD4;&#x8F03;&#x3057;&#x3066;&#x5909;&#x66F4;&#x3059;&#x308B;&#x3053;&#x3068;&#x306A;&#x304F;&#x304C;&#x5F37;&#x5316;&#x3092;&#x793A;&#x5506;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x307E;&#x305F;&#x3001;&#x30CA;&#x30CE;&#x30D5;&#x30A1;&#x30A4;&#x30D0;&#x30FC;&#x5730;&#x5F62;&#x30A2;&#x30B9;&#x30C8;&#x30ED; &#x30B5;&#x30A4;&#x30C8;&#x306B;&#x304A;&#x3051;&#x308B;&#x907A;&#x4F1D;&#x5B50;&#x30B5;&#x30A4;&#x30EC;&#x30F3;&#x30B7;&#x30F3;&#x30B0;&#x306E;&#x52B9;&#x7387;&#x304C;&#x5411;&#x4E0A;&#x3057;&#x307E;&#x3059;&#x3002;&#x30CA;&#x30CE;&#x30D5;&#x30A1;&#x30A4;&#x30D0;&#x30FC; &#x30A2;&#x30B9;&#x30C8;&#x30ED; &#x30B5;&#x30A4;&#x30C8;&#x306E;&#x6210;&#x9577;&#x3092;&#x6291;&#x5236;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;&#x795E;&#x7D4C;&#x518D;&#x751F;&#x306E;&#x305F;&#x3081;&#x306E;&#x6F5C;&#x5728;&#x7684;&#x306A;&#x57FA;&#x677F;&#x3068;&#x3057;&#x3066;&#x5F79;&#x7ACB;&#x3064;&#x304B;&#x3082;&#x3057;&#x308C;&#x306A;&#x3044;&#x3001;&#x3055;&#x3089;&#x306B;&#x4E2D;&#x67A2;&#x795E;&#x7D4C;&#x7CFB;&#x306E;&#x518D;&#x751F;&#x3092;&#x5F37;&#x5316;&#x3059;&#x308B;&#x907A;&#x4F1D;&#x5B50;&#x30B5;&#x30A4;&#x30EC;&#x30F3;&#x30B7;&#x30F3;&#x30B0;&#x306E;&#x4F7F;&#x7528;&#x3092;&#x4FC3;&#x9032;&#x3059;&#x308B;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x5506;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

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&#xC0AC;&#xC774;&#xD1A0; nanofiber &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC740; &#xD3F4; &#xB9AC; [caprolactone-&#xCF5C;&#xB85C;&#xB77C;&#xB3C4;-(&#xC5D0;&#xD2F8; &#xC5D0;&#xD2F8;&#xB80C; &#xC778;&#xC0B0; &#xC5FC;)] electrospun nanofibers &#xBC0F; &#xC6A9; &#xB9E4;-&#xCE90;&#xC2A4;&#xD2B8; &#xD544;&#xB984; (2 &#xCC28;&#xC6D0; &#xCEE8;&#xD2B8;&#xB864;)&#xC5D0; &#xC8FC; &#xC950; &#xB300;&#xB1CC; &#xD53C; &#xC9C8;&#xC758; &#xC774;&#xB2E4; &#xACBD;&#xC791; &#xD558; &#xC5EC; &#xACF5;&#xBD80; &#xD588;&#xB2E4;. &#xACB0;&#xACFC; nanofiber &#xC9C0;&#xD615;&#xC744; &#xD06C;&#xAC8C; &#xC0AC;&#xC774;&#xD1A0; &#xD655;&#xC0B0;&#xC744; &#xC5B5;&#xC81C; &#xD558; &#xACE0; &#xC601;&#xD654;&#xC5D0; &#xBE44;&#xD574; &#xC138;&#xD3EC; &#xD65C;&#xC131;&#xD654;&#xB97C; &#xBCC0;&#xACBD; &#xD558;&#xC9C0; &#xC54A;&#xACE0; apoptosis, &#xD5A5;&#xC0C1;&#xC744; &#xB098;&#xD0C0;&#xB0C5;&#xB2C8;&#xB2E4;. &#xB610;&#xD55C;, nanofiber &#xC9C0;&#xD615; &#xC774;&#xB2E4;&#xC5D0;&#xC11C; &#xC720;&#xC804;&#xC790; &#xCE68;&#xBB35; &#xD6A8;&#xC728;&#xC131;&#xC744; &#xD5A5;&#xC0C1; &#xC2DC;&#xD0B5;&#xB2C8;&#xB2E4;. &#xACB0;&#xACFC; &#xC81C;&#xC548; nanofibers &#xC0AC;&#xC774;&#xD1A0; &#xC131;&#xC7A5;&#xC744; &#xC5B5;&#xC81C; &#xD558; &#xC5EC; &#xC2E0;&#xACBD; &#xC7AC;&#xC0DD;&#xC5D0; &#xB300; &#xD55C; &#xC7A0;&#xC7AC;&#xC801;&#xC778; &#xAE30;&#xD310;&#xC73C;&#xB85C; &#xC81C;&#xACF5;&#xD560; &#xC218; &#xC788;&#xB2E4; CNS &#xC7AC;&#xC0DD;&#xC744; &#xD5A5;&#xC0C1; &#xC2DC;&#xD0A4;&#xAE30; &#xC704;&#xD574; &#xC720;&#xC804;&#xC790; &#xCE68;&#xBB35; &#xC2DC;&#xD0A4;&#xAE30;&#xC758; &#xC0AC;&#xC6A9;&#xC744; &#xCD09;&#xC9C4; &#xCD94;&#xAC00; &#xB420; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;.

&#xC0AC;&#xC774;&#xD1A0;&#xC758; &#xD589;&#xB3D9;&#xC5D0; Nanofiber &#xC9C0;&#xD615;&#xACFC; &#xC720;&#xC804;&#xC790; &#xCE68;&#xBB35; &#xD6A8;&#xC728;&#xC758; &#xD6A8;&#xACFC;.

Astrocito-nanofibras interacciones son estudiadas por cultivo astrocitos cortical primaria rata en poly [caprolactone - co-(fosfato de etileno de etilo)] electrospun nanofibras y solvente-reparto de pel&iacute;culas (control de dos dimensiones). Los resultados indican que nanofibras topograf&iacute;a significativamente suprime la proliferaci&oacute;n de astrositos y aumenta la apoptosis, sin alterar la activaci&oacute;n celular en comparaci&oacute;n con las pel&iacute;culas. Por otra parte, topograf&iacute;a de nanofibras mejora la eficacia de silenciamiento del gen en los astrocitos. Los resultados sugieren que nanofibras sirvan como sustratos potenciales para la regeneraci&oacute;n del nervio al suprimir el crecimiento de astrositos y pueden facilitar a&uacute;n m&aacute;s el uso de silenciamiento del gen para realzar la regeneraci&oacute;n de la CNS.

Los efectos de la topograf&iacute;a de nanofibras en Astrocito comportamiento y eficiencia de silenciamiento g&eacute;nico.

During development and tissue repair, progenitor cells are guided by both biochemical and biophysical cues of their microenvironment, including topographical signals. The topographical cues have been shown to play an important role in controlling the fate of cells. Systematic investigation of topographical structures with different geometries and sizes under the identical experimental conditions on the same chip will enhance the understanding of the role of shape and size in cell-topography interactions. A simple customizable multi-architecture chip (MARC) array is therefore developed to incorporate, on a single chip, distinct topographies of various architectural complexities, including both isotropic and anisotropic features, in nano- to micrometer dimensions, with different aspect ratios and hierarchical structures. Polydimethylsiloxane (PDMS) replicas of MARC are used to investigate the influence of different geometries and sizes in neural differentiation of primary murine neural progenitor cells (mNPCs). Anisotropic gratings (2 μm gratings, 250 nm gratings) and isotropic 1 μm pillars significantly promote differentiation of mNPCs into neurons, as indicated by expression of β-III-tubulin (59%, 58%, and 58%, respectively, compared to 30% on the control). In contrast, glial differentiation is enhanced on isotropic 2 μm holes and 1 μm pillars. These results illustrate that anisotropic topographies enhance neuronal differentiation while isotropic topographies enhance glial differentiation on the same chip under the same conditions. MARC enables simultaneous cost-effective investigation of multiple topographies, allowing efficient optimization of topographical and biochemical cues to modulate cell differentiation.

Microarray with micro- and nano-topographies enables identification of the optimal topography for directing the differentiation of primary murine neural progenitor cells.

Taurine is a sulfur-containing amino acid present in high concentrations in mammalian tissues. It has been implicated in several processes involving brain development and neurotransmission. However, the role of taurine in hippocampal neurogenesis during brain development is still unknown. Here we show that taurine regulates neural progenitor cell (NPC) proliferation in the dentate gyrus of the developing brain as well as in cultured early postnatal (P5) hippocampal progenitor cells and hippocampal slices derived from P5 mice brains. Taurine increased cell proliferation without having a significant effect on neural differentiation both in cultured P5 NPCs as well as cultured hippocampal slices and in vivo. Expression level analysis of synaptic proteins revealed that taurine increases the expression of Synapsin 1 and PSD 95. We also found that taurine stimulates the phosphorylation of ERK1/2 indicating a possible role of the ERK pathway in mediating the changes that we observed, especially in proliferation. Taken together, our results demonstrate a role for taurine in neural stem/progenitor cell proliferation in developing brain and suggest the involvement of the ERK1/2 pathways in mediating these actions. Our study also shows that taurine influences the levels of proteins associated with synapse development. This is the first evidence showing the effect of taurine on early postnatal neuronal development using a combination of in vitro, ex-vivo and in vivo systems.

Taurine induces proliferation of neural stem cells and synapse development in the developing mouse brain.

Neural stem/progenitor cells (NSC) have the potential for treatment of a wide range of neurological diseases such as Parkinson Disease and multiple sclerosis. Currently, NSC have been isolated only from hippocampus and subventricular zone (SVZ) of the adult brain. It is not known whether NSC can be found in all parts of the developing mid-trimester central nervous system (CNS) when the brain undergoes massive transformation and growth. Multipotent NSC from the mid-trimester cerebra, thalamus, SVZ, hippocampus, thalamus, cerebellum, brain stem and spinal cord can be derived and propagated as clonal neurospheres with increasing frequencies with increasing gestations. These NSC can undergo multi-lineage differentiation both in vitro and in vivo, and engraft in a developmental murine model. Regionally-derived NSC are phenotypically distinct, with hippocampal NSC having a significantly higher neurogenic potential (53.6%) over other sources (range of 0%-27.5%, p<0.004). Whole genome expression analysis showed differential gene expression between these regionally-derived NSC, which involved the Notch, epidermal growth factor as well as interleukin pathways. We have shown the presence of phenotypically-distinct regionally-derived NSC from the mid-trimester CNS, which may reflect the ontological differences occurring within the CNS. Aside from informing on the role of such cells during fetal growth, they may be useful for different cellular therapy applications.

Guillaume Marcy

Neuroscience & Behavioral Disorders

DUKE-NUS Graduate Medical School

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Guillaume Marcy

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Neuroscience & Behavioral Disorders

DUKE-NUS Graduate Medical School

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Neuroscience & Behavioral Disorders