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

El estudio fue apoyado por la Fundación Universidad de Basilea Ciencia y la Fundación Nacional de Ciencia de Suiza con una donación de MHS y AG (SNF IZLIZ3_157230). Agradecemos a: Dr. Tania Rinaldi Burkat generosamente para proporcionar la infraestructura; todos los miembros del grupo Bettler para los debates y comentarios. Agradecemos a Gerhard Dorne (Leica Microsystems, Suiza) para la instalación profesional y competente de la cámara de vídeo de alta definición completa MC170 (Leica Microsystems, Suiza).

Todos los animales procedimientos siguieron la &#39;Guía para el Cuidado y Uso de Animales de Laboratorio &quot;(publicación NIH, 8ª edición, 2011) y fueron aprobados por el Comité de Protección de los Animales de Basilea (Suiza Directrices para el Cuidado y Uso de los Animales). Por estas directrices del protocolo animal se considera de &quot;grado de gravedad más bajo de los animales&quot;. 1. Preparación de medio de expansión Nota: El trabajo en condiciones asépticas como estándar ...

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En este estudio se describe un sistema estandarizado para el análisis de la EMT que utilizan células madre neurales (que se resumen en la Figura 3 complementario). La estandarización asegura la reproducibilidad (Tabla 1 y 2). Los NSCs se derivan de la corteza en desarrollo, un tejido que normalmente no experimenta EMT. Esto es ventajoso para el análisis de los primeros pasos en EMT. Los pasos iniciales en la EMT no pueden ser estudiados adecuadamente en las células tumorales que se...

Durante varias etapas del desarrollo embrionario, las células epiteliales pierden su fuerte adherencia entre sí (por ejemplo, las uniones estrechas) y adquieren un fenotipo migratorio en un proceso llamado transición epitelial a mesenquimal (EMT) 1. Se requiere EMT para la formación de tipos de células adicionales, tales como las células de la cresta neural mesenquimales, una población que segrega desde el neuroepitelio 2. EMT no sólo es esencial durante las fases embrionarias, sino también necesaria en etapas posteriores de la vida adulta para mantener los procesos fisiológicos en el organismo adulto, tales como la curación de heridas 3 y la regeneración del sistema nervioso central (SNC) en lesiones desmielinizantes 4. Los tumores epiteliales son conocidos para reactivar EMT como una etapa de iniciación para la migración, invasión y metástasis, en última instancia conduce a la progresión del cáncer 1,3. EMT es, en efecto vinculado centralmente a la fuerte migración de 1,3. Los pasos celulares de conditioning, iniciar, mantener y someterse a EMT no se entienden completamente y necesitan más investigaciones. Aquí, se presenta un sistema normalizado in vitro modelo EMT basado en NSCs, con factores de crecimiento definidos y medios (sin suero y sin el uso de la enzima). Este sistema modelo es de relevancia para los científicos que trabajan en la EMT. El caracol, Zeb y twist familias de proteínas han demostrado ser crucial para la EMT, tanto en el desarrollo y la enfermedad 1. El caracol, familias Zeb y la torcedura también están involucrados en el sistema presentado. El sistema se basa en una región específica del cerebro anterior que normalmente no experimenta EMT proporcionar una ventaja particular para el estudio de los acontecimientos iniciales durante la inducción de EMT. El sistema modelo potencialmente podría ser aplicado al estudio de EMT en el epitelio fuera del SNC, ya que los inductores de la EMT clave, como el caracol, Zeb y las proteínas de la torcedura, también se encuentran durante la EMT en sistemas de tejidos fuera del SNC. Este modelo sistema permite el aislamiento normalizado de células madre neurales de la corteza en desarrollo para estudiar las características de células madre en general y, en particular, la EMT. El uso de este sistema, se aislaron células madre neurales, EMT inducida y estudiamos la posterior migración bajo el efecto de FGF2 y BMP4. Hemos observado que interactúa FGF y BMP de señalización con TGF-señalización para promover la migración de células, validando así el sistema modelo.

<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

Transición epitelial a mesenquimal (EMT) describe el proceso de epitelio mesénquima en transdifferentiating. EMT es un proceso fundamental durante el desarrollo embrionario que también ocurre comúnmente en glioblastoma, el tumor cerebral maligno más frecuente. EMT también se ha observado en múltiples carcinomas fuera del cerebro incluyendo cáncer de mama, cáncer de pulmón, cáncer de colon, cáncer gástrico. EMT está vinculado centralmente a la malignidad mediante la promoción de la migración, la invasión y la formación de metástasis. Los mecanismos de inducción EMT no se entienden completamente. Aquí se describe un sistema in vitro para el aislamiento normalizado de las células corticales neuronales madre (NSC) y posterior EMT-inducción. Este sistema ofrece la flexibilidad de utilizar cualquiera de las células individuales o cultivo de explantes. En este sistema, de rata o de ratón NSCs del cerebro anterior de embriones se cultivan en un medio definido, desprovisto de suero y enzimas. Las células madre neurales expresan Olig2 y Sox10, dos factores de transcripción se observa en oligodendroccélulas precursoras yte (OPC). Utilizando este sistema, las interacciones entre FGF, BMP y TGF-señalización que implica ZEB1, Zeb2, y Twist2 fueron observada en la que TGF-activación mejora de forma significativa la migración de células, lo que sugiere un efecto sinérgico BMP / TGF-interacción. Los resultados apuntan a una red de FGF, BMP y TGF-señalización de estar involucrados en la inducción y mantenimiento de EMT. Este sistema modelo es relevante para investigar EMT in vitro. Es rentable y muestra una alta reproducibilidad. También permite la comparación de los diferentes compuestos con respecto a sus respuestas de migración (medición de la distancia cuantitativo), y cribado de alto rendimiento de los compuestos para inhibir o potenciar EMT (medición cualitativa). El modelo es, por tanto, muy adecuado para poner a prueba las bibliotecas de drogas para las sustancias que afectan a la EMT.

Este sistema modelo de EMT se basa en el aislamiento normalizado de NSCs tanto como células individuales o como explantes de una región específica del tubo neural en desarrollo, la corteza central (Figuras 1 y 2). Para la evaluación cuantitativa, los explantes se sembraron a la derecha en el centro de una placa de cultivo de rejilla 500 micras (Figura 3). Explantes de la corteza por el medio fueron expuestos primero en FGF2 durante d...

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 modelo enzima y Cultura Neural Stem Cell libre de suero para la EMT Investigación Adecuado para el descubrimiento de fármacos

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

Diferenciación neural de células madre embrionarias de ratón en el suero libre de monocapa Cultura

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

Investigación

Biología del Desarrollo

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

El cultivo humanas pluripotentes y células madre neurales en un sistema de cultivo celular cerrado para la investigación básica y preclínica

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

Predicción y validación de los elementos reguladores de genes activados durante ácido retinoico inducida por la diferenciación de células madre embrionarias

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

La producción a gran escala de los cardiomiocitos de células madre pluripotentes utilizando un pequeño protocolo de diferenciación basada en la molécula altamente reproducible

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

Infección por el Virus Zika de células madre neurales de cerebro Fetal humano cultivadas para análisis de 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

Usando humanos inducida por células de hepatocito-como derivado de células madre pluripotentes de descubrimiento de fármacos

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

Imágenes de células vivas de la segregación de cromosomas durante la Mitosis

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

Citometría de flujo en tiempo real de células vivas para investigar la afluencia del calcio, formación de poro y fagocitosis por los receptores P2X7 en células progenitoras neurales adultas

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

Modelo de impacto cortical controlado de lesión cerebral de ratón con trasplante terapéutico de células neuronales derivadas de células madre pluripotentes inducidas por el hombre

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

Generación de células madre neuronales inducidas a partir de células mononucleares periféricas y diferenciación hacia precursores de neuronas dopaminérgicas para estudios de trasplante