Name: an enzyme- and serum-free neural stem cell culture model for emt investigation suited for drug discovery
Uploaded: 2016-08-23T13:00:00
Description: Watch this Scientific Journal Video about An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery at JoVE.com

Lo studio è stato sostenuto dall&#39;Università di Basilea Science Foundation e la National Science Foundation svizzero da una sovvenzione di MHS e AG (SNF IZLIZ3_157230). Ringraziamo: Dr. Tania Rinaldi Burkat per fornire generosamente infrastrutture; tutti i membri del gruppo Bettler per discussioni e commenti. Ringraziamo Gerhard Dorne (Leica Microsystems, Svizzera) per l&#39;installazione professionale e competente della telecamera video Full HD MC170 (Leica Microsystems, Svizzera).

Tutte le procedure sugli animali hanno seguito la &#39;Guida per la cura e l&#39;uso di animali da laboratorio &quot;(pubblicazione NIH, 8 ° edizione, 2011) e sono stati approvati dal Comitato per il benessere degli animali di Basilea (Linee direttive per la cura e l&#39;uso di animali). Con queste linee guida il protocollo animale è considerato di &quot;gravità grado più basso degli animali&quot;. 1. Preparazione del mezzo Expansion Nota: Il lavoro in condizioni asettiche come standard per ...

<ol>
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In questo studio un sistema standardizzato per EMT analisi NSC che utilizzano è descritta (riassunti nella figura supplementare 3). La standardizzazione assicura riproducibilità (Tabella 1 e 2). I NSCs sono derivati ​​dalla corteccia di sviluppo, un tessuto che normalmente non subisce EMT. Questo è un vantaggio per l&#39;analisi dei primi passi in EMT. Primi passi in EMT non possono essere adeguatamente studiati nelle cellule tumorali che si sono accumulati i cambiamenti genetici...

Durante diversi stadi di sviluppo embrionale, le cellule epiteliali perdono la loro forte aderenza tra loro (ad esempio, giunzioni strette) e acquisiscono un fenotipo migratorio in un processo chiamato la transizione epitelio mesenchimale (EMT) 1. EMT è necessaria per la formazione di tipi cellulari aggiuntivi, come le cellule della cresta neurale mesenchimali, una popolazione che segrega dal neuroepitelio 2. EMT non è essenziale solo durante le fasi embrionali, ma richiesto anche nelle fasi successive della vita adulta a mantenere processi fisiologici nell&#39;organismo adulto, come ad esempio la guarigione della ferita 3 e del sistema nervoso centrale di rigenerazione (SNC) in lesioni demielinizzanti 4. Tumori epiteliali sono noti per riattivare EMT come un passo di iniziazione per la migrazione, invasione e metastasi, in ultima analisi, che porta alla progressione del cancro 1,3. EMT è infatti legata in posizione centrale ad una forte migrazione 1,3. I passi cellulari di conditioning, avviando, in fase e il mantenimento di EMT non sono pienamente compresi e hanno bisogno di ulteriori indagini. Qui, viene presentato un standardizzato in vitro sistema modello EMT sulla base di NSC, con fattori di crescita definiti e media (senza siero e senza l&#39;utilizzo di enzimi). Questo sistema modello è rilevante per gli scienziati che lavorano su EMT. Le famiglie di proteine ​​Lumaca, Zeb e Twist hanno dimostrato di essere fondamentale per EMT, sia in fase di sviluppo e di malattia 1. La lumaca, Zeb e Twist famiglie sono anche coinvolti nel sistema presentato. Il sistema è basato su una specifica regione del proencefalo che normalmente non subisce EMT fornire un vantaggio particolare per lo studio di eventi iniziali durante EMT induzione. Il sistema modello potrebbe potenzialmente essere applicato a studiare EMT in epiteli all&#39;esterno del SNC, poiché induttori EMT chiave, come la chiocciola, Zeb e proteine ​​torsione, si riscontrano anche durante EMT in sistemi tessuto fuori del SNC. Questo modello sistema permette l&#39;isolamento standardizzato di NSC dalla corteccia in via di sviluppo per studiare le caratteristiche di cellule staminali in generale e, in particolare, EMT. Utilizzando questo sistema, abbiamo isolato le cellule staminali neurali, EMT indotta e studiato la successiva migrazione sotto l&#39;effetto di FGF2 e BMP4. Abbiamo osservato che interagisce FGF- e BMP di segnalazione con TGFβ di segnalazione per promuovere la migrazione delle cellule, convalidando così il modello di sistema.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BMP4, rhBMP4</td>
			<td>RnD Systems&#160;</td>
			<td>314-BP-01M</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine pancreas insulin</td>
			<td>Sigma&#160;</td>
			<td>I1882</td>
			<td></td>
		</tr>
		<tr>
			<td>Boyden chamber, CytoSelect cell invasion assay</td>
			<td>Cell Biolabs</td>
			<td>CBA-110</td>
			<td>24 well plate system</td>
		</tr>
		<tr>
			<td>Cell culture dish with grid</td>
			<td>Ibidi 500 mm dish, 35 mm</td>
			<td>80156</td>
			<td></td>
		</tr>
		<tr>
			<td>CellMask Orange</td>
			<td>Life Technologies</td>
			<td>C10045</td>
			<td>Plasma membrane dye, use at 1:1000 .</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>LifeTechnologies</td>
			<td>D1306</td>
			<td>Stock at 5mg/ml. Use at 1:10000. Cancerogenic. Appropriate protection (gloves, coat, goggles) required.</td>
		</tr>
		<tr>
			<td>DMEM/F12 1:1 medium bottle</td>
			<td>Gibco Invitrogen</td>
			<td>21331-020</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF2, rhFGF2</td>
			<td>RnD Systems</td>
			<td>233-FB-01M</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectine, bovine</td>
			<td>Sigma&#160;</td>
			<td>F4759</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax supplement&#160;</td>
			<td>Gibco Invitrogen&#160;</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphics software with pixel measurement feature</td>
			<td>Fiji</td>
			<td>fiji.sc/Fiji</td>
			<td>version 2.0.0-rc-30/1.49s</td>
		</tr>
		<tr>
			<td>HBSS media</td>
			<td>Sigma&#160;</td>
			<td>H9394</td>
			<td></td>
		</tr>
		<tr>
			<td>Human apo-Transferrin</td>
			<td>Sigma</td>
			<td>T1147</td>
			<td>Possible lung irritant. Avoid inhalation. Use appropriate protection.</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco Invitrogen&#160;</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Nestin, Mouse anti Nestin antibody</td>
			<td>Genetex</td>
			<td>GTX26142</td>
			<td>Use at 1:100, 4% PFA fixation, Triton X100 at 0.1%</td>
		</tr>
		<tr>
			<td>Olig2, Rabbit anti Olig2 antibody</td>
			<td>Provided by Hirohide Takebayash</td>
			<td>Personal stock</td>
			<td>Use at 1:2000, 4% PFA fixation, Triton X100 at 0.1%</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin/Fungizone</td>
			<td>Gibco Invitrogen&#160;</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Podoplanin, Mouse anti Podoplanin antibody</td>
			<td>Acris</td>
			<td>DM3614P</td>
			<td>Use at 1:250, 4% PFA fixation, avoid Triton X100</td>
		</tr>
		<tr>
			<td>Poly-L-ornithine</td>
			<td>Sigma&#160;</td>
			<td>P3655</td>
			<td></td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>Sigma&#160;</td>
			<td>P5780</td>
			<td>Skin and eye irritant. Appropriate protection required.</td>
		</tr>
		<tr>
			<td>Sodium selenite</td>
			<td>Sigma&#160;</td>
			<td>S5261</td>
			<td></td>
		</tr>
		<tr>
			<td>Sox10, Rabbit anti Sox10 antibody</td>
			<td>Millipore Chemicon</td>
			<td>AB5774</td>
			<td>Use at 1:200, 4% PFA fixation, Triton X100 at 0.1%</td>
		</tr>
		<tr>
			<td>TGFb1, rhTGFb1</td>
			<td>RnD Systems</td>
			<td>240-B-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Uncoated Petri dishes</td>
			<td>Falcon Corning</td>
			<td>351029</td>
			<td></td>
		</tr>
	</tbody>
</table>

an enzyme- and serum-free neural stem cell culture model for emt investigation suited for drug discovery

Epiteliale di transizione mesenchimale (EMT) descrive il processo dell&#39;epitelio transdifferentiating in mesenchima. EMT è un processo fondamentale durante lo sviluppo embrionale, che comunemente si verifica nel glioblastoma, la più frequente tumore maligno al cervello. EMT è stato osservato anche in più carcinomi di fuori del cervello, tra cui il cancro al seno, il cancro del polmone, cancro del colon, cancro gastrico. EMT è legata in posizione centrale per malignità, promuovendo la migrazione, l&#39;invasione e la formazione di metastasi. I meccanismi di EMT induzione non sono pienamente compresi. Qui si descrive un sistema in vitro per l&#39;isolamento standardizzato di cellule corticali neurali staminali (NSC) e la successiva EMT-induzione. Questo sistema fornisce la flessibilità necessaria per utilizzare sia singole cellule o espianto. In questo sistema, ratto o topo embrionali NSC proencefalo sono coltivate in un mezzo definito, privo di siero e enzimi. Le NSC espresso Olig2 e Sox10, due fattori di trascrizione osservata in oligodendroccellule precursori yte (OPC). Utilizzando questo sistema, le interazioni tra FGF-, BMP- e TGFβ di segnalazione che coinvolge Zeb1, Zeb2, e Twist2 sono stati osservati in cui TGFβ-attivazione significativamente migliorata la migrazione delle cellule, suggerendo un sinergico / TGFβ-interazione BMP-. I risultati indicano una rete di FGF-, BMP- e TGFβ-segnalazione di essere coinvolti in induzione e mantenimento EMT. Questo sistema modello è rilevante per indagare EMT in vitro. E &#39;conveniente e mostra elevata riproducibilità. Permette anche per il confronto delle mescole diverse rispetto alle loro risposte migrazione (misurazione quantitativa di distanza), e high-throughput screening di composti di inibire o migliorare EMT (misurazione qualitativa). Il modello è quindi adatto per testare le librerie di droga per le sostanze che interessano EMT.

Questo sistema modello EMT è basato sull&#39;isolamento standardizzata di NSC sia come singole cellule o espianti come da una regione specifica del tubo neurale sviluppo, la corteccia centrale (figure 1 e 2). Per la valutazione quantitativa, espianti sono stati seminati proprio al centro di 500 micron cultura griglia piatto (Figura 3). Espianti da corteccia centrale sono stati esposti a FGF2 per due giorni, seguiti da altri due giorni i...

Watch this Scientific Journal Video about An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery at JoVE.com

An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery

Epithelial to mesenchymal transition (EMT) allows cancers to become invasive. To investigate EMT, a neural stem cell (NSC)-based in vitro model devoid of serum and enzymes is described. This standardized system allows quantitative and qualitative assessment of cell migration, gene and protein expression. The model is suited for drug discovery.

Un modello enzima e Neural Stem Cell Culture siero-libero per EMT Investigation Adatto per Drug Discovery

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during ...

The overall goal of this procedure is to induce migration in typically non-migratory cells, to identify specific compounds that activate or block migration. This method can help to answer key questions in the field of cell migration and invasion, and may be of particular interest to scientists working with neural stem cells. The main advantage of this technique is that non-migratory and non-invasive cells can be analyzed during the process of becoming migratory and invasive.The implications of this technique extend toward the identification of novel compounds that block epithelial to mesenchymal transition, or EMT, which occurs in human high-grade gliomas believed to derive from neural stem cells. Though this method can provide insight into EMT and neural stem cells, it can also be used to study other epithelial cancers, such as breast, lung, or colon cancer. On embryonic day 14.5, confirm the correct age of the rat embryos by the presence of beginning digit formation in a rat forelimb.Next, transfer the embryos into individual, uncoated petri dishes filled with ice-cold PBS, which are placed under a stereo microscope. Use a pair of fine-tipped forceps to remove the embryonic hulls. Keep embryos in expansion medium on ice.Using two autoclave-sterilized fine forceps, and starting at the intersection of the developing telencephalon and diencephalon, pull simultaneously anteriorly and posteriorly, to remove the head skin and skull. Then, identify the midbrain-hindbrain boundary separating the mesencephalon from the rhombencephalon, and cut the rhombencephalon just at or below the midbrain-hindbrain boundary. Holding the block of the mesencephalon with a pair of forceps, sever the connection between the telencephalon and diencephalon at the skull base with the facial skeleton, and immerse the telencephalon, diencephalon, mesencephalon block in an uncoated petri dish of expansion medium on ice.When all of the neural blocks have been harvested in the same manner, transfer one telencephalon, diencephalon, mesencephalon block into fresh, ice-cold PBS under the microscope. To separate the mesencephalon from the diencephalon, first sever the tissue between the diencephalon and the telencephalon. Next, split the two telencephalic hemispheres, and identify the medial and lateral ganglionic eminences, which are visible through the future foramen interventriculare.Dissect along a straight line at the intersection between the medial and lateral ganglionic eminences and the anterior cortex, followed by a straight line through the cortex, hippocampus, and choroid plexus. Cut a second straight line through the cortex, hippocampus, and choroid plexus at the intersection of the caudal ganglionic eminence and the posterior cortex. Then, flatten out the telencephalon, identify the cortical hem containing the hippocampus and the choroid plexus.Separate the cortical hem from the cortex, leaving the size of the cortex identical to the size of the ganglionic eminences. Flip the cortex-ganglionic eminence block so that the ventricular side is touching the dish surface, and the meninges are facing up. Pinning the ganglionic eminences to the dish surface with one hand, use forceps in the other hand to peel off the meninges.Then, cut each cortex along the lateral ganglionic eminence, about half of the distance of the diameter of the lateral ganglionic eminence. To prepare the explant cultures, cut the cortices into explants of less than 400 microns in diameter, using a 400 micron grid below the dissection dish as a reference if necessary. Then, transfer all of the explants into a fresh petri dish with ice cold expansion medium.Next, remove the excess fibronectin from a previously-coated 35 millimeter tissue culture dish and allow the dish to dry. Then, place the fibronectin-coated dish over a 10 by 10 millimeter grid and add one milliliter of cold expansion medium to the center of the dish, allowing the medium to form a spherical drop. Place up to eight explants into the center of the drop with at least three millimeters of space between each piece of tissue.Then, carefully place the dish in a cell culture incubator without shaking to allow the explants to attach to the fibronectin-coated surface. After one hour, fill the dish to a final volume of two milliliters of expansion medium plus the growth factors or test substances of interest, and return the plate to the incubator. In this experiment, explants were seeded at the center of 500 micron-grid culture dishes and cultured in different growth factors.In the control and TGF Beta 1 alone treated explants, a strong proliferations, coupled with almost no migration was observed. BMP4 however, induced an intermediate cell migration and proliferation response. With the combination of BMP4 and TGF Beta 1 inducing the strongest migration and proliferation of all the groups.Once mastered, the neural stem cell can be isolated from 10 to 15 embryos in approximately two hours. While attempting this procedure, it is important to maintain all of the embryos and cell explants in ice-cold medium. This cost-efficient and highly-reproducible system provides the flexibility of using either single cells or explant cultures to study cell migration and invasion in vitro.Culturing the cells in large multi-well plates allows analysis of the effects of a variety of compounds on migration and invasion in parallel. Further, the system can be easily adapted for high throughput screening of novel compounds. After watching this video, you should have a good understanding of how to isolate neural stem cells with the potential to undergo EMT from the appropriate region of the developing cortex.

Research

JoVE Journal

Developmental Biology

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

Differenziamento neurale delle cellule staminali embrionali di topo in siero privo monostrato Cultura

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Ricerca

Biologia dello sviluppo

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

Coltura umane pluripotenti e neurali cellule staminali in un sistema di coltura cellulare chiuso per base e preclinica di ricerca

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

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

Previsione e Validazione del gene regolatore elementi attivato durante retinoico Acid Induced embrionali cellule staminali Differenziazione

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Produzione su larga scala dei cardiomiociti da cellule staminali umane pluripotenti usando un altamente riproducibile piccole molecole-Based Differenziazione protocollo

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis

Infezione del Virus di Zika delle cellule staminali neurali del cervello fetale umano colto per analisi Immunocytochemical

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

Utilizzando umani indotti dell'epatocita-come di derivati da cellule staminali pluripotenti per scoperta della droga

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Live Cell Imaging della segregazione cromosomica durante la mitosi

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

In tempo reale Live-cella flusso Cytometry per indagare l'afflusso del calcio, la formazione del poro e fagocitosi dai recettori P2X7 in cellule progenitrici neurali adulto

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Modello di impatto corticale controllato di lesioni cerebrali del topo con trapianto terapeutico di cellule neurali derivate da cellule staminali indotte dall'uomo

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Generazione di cellule staminali neurali indotte da cellule mononucleari periferiche e differenziazione verso i precursori dei neuroni dopaminergici per gli studi di trapianto