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

Çalışma MHS ve AG (SNF IZLIZ3_157230) bir hibe ile Basel Bilim Vakfı Üniversitesi ve İsviçre Ulusal Bilim Vakfı tarafından desteklenmiştir. Biz teşekkür ederiz: Dr. Tania Rinaldi Burkat cömertçe altyapının sağlanması için; tartışmalar ve yorumlar için Bettler grubun tüm üyeleri. Biz Gerhard Dorne (Leica Microsystems, İsviçre) Full HD MC170 video kamera profesyonel ve yetkin montaj için (Leica Microsystems, İsviçre) teşekkür ederim.

Tüm hayvan prosedürleri &#39;Laboratuvar Hayvanları Bakım ve Kullanım Kılavuzu&#39; (NIH yayın, 8 th edition, 2011) izledi ve Basel Hayvan Refahı Komitesi (Hayvanlarının Bakımı ve Kullanımı için İsviçre Kuralları) tarafından onaylanmıştır. bu kurallara hayvan protokolü &quot;düşük hayvan şiddeti notu&quot; olarak kabul edilir. Genişletme maddesi hazırlanması 1. Not: Doku kültürü için standart olarak aseptik koşullarda çalışmak. <ol><li> İki adet 15 ml t...

<ol>
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EMT analizi kullanan NSC&#39;lerde için standart bir sistem tarif edilmektedir Bu çalışmada, (Ek Şekil 3&#39;te özetlenmiştir). Standardizasyon tekrarlanabilirliği (Tablo 1 ve 2) sağlamaktadır. NSC&#39;lerde gelişmekte korteksinden normal EMT uğramayan bir doku elde edilir. Bu EMT erken aşamada analizi için avantajlıdır. EMT içinde ilk adımlar yeterli genetik değişiklikler birikmiş ve zaten EMT özelliklerini benimsemiş olabilir tümör hücrelerinde okudu edilemez...

Embriyonik gelişme birkaç aşamalarında, epitel hücreleri birbirine (örneğin, sıkı kavşaklar) için güçlü bağlılığı kaybeder ve mezenkimal geçiş (EMT) 1 bir süreç olarak adlandırılan epitel bir göçmen fenotip kazanır. EMT gibi mezenkimal nöral tepe hücreleri gibi ek hücre tiplerinin oluşumu neuroepithelium 2 ayıran bir popülasyon için gereklidir. EMT evrelerinde sadece gerekli değil, aynı zamanda lezyonlar 4 demiyelinizan böyle yara iyileşmesi 3 ve merkezi sinir sistemi (MSS) rejenerasyon olarak yetişkin organizmada fizyolojik süreçleri, korumak için yetişkin hayatının ileriki aşamalarında gerekli değildir. Epitelyal tümörler sonuçta kanser oluşumunu 1,3 giden, göç, istila ve metastaz için bir başlangıç ​​adımı olarak EMT yeniden bilinmektedir. EMT gerçekten merkezi güçlü göç 1,3 bağlantılıdır. c hücresel adımlarıonditioning, başlatılması geçiren ve EMT sürdürmek tam olarak anlaşılamamıştır ve daha fazla araştırma ihtiyaç değildir. Burada tanımlanan büyüme faktörleri ve ortam (hiç serum ve enzim) kullanılmasına NSC&#39;lerde göre standart bir in vitro EMT model sistem sunulmuştur. Bu model sistem EMT üzerinde çalışan bilim adamları için öneme sahiptir. Salyangoz, Zeb ve büküm protein aileleri Geliştirme ve hastalık 1 EMT için kritik olduğu gösterilmiştir. Salyangoz, Zeb ve Büküm aileler de sunulan sistemde yer almaktadır. Sistem normal EMT EMT indüksiyon sırasında ilk olayların incelenmesi için özel bir avantaj sağlayarak uğramayan önbeyin belirli bir bölgeye dayanmaktadır. Bu Salyangoz, Zeb ve büküm proteinleri gibi önemli EMT indükleyiciler, aynı zamanda merkezi sinir sistemi dışındaki doku sistemleri EMT sırasında bulunan yana model sistem, potansiyel olarak, merkezi sinir sistemi dışındaki epitelde EMT çalışma uygulanabilir. Bu model sistem, özellikle, kök hücre, genel olarak özellikleri ve EMT çalışma gelişmekte korteksten NSC&#39;lerde standart izolasyon sağlar. Bu sistemi kullanarak, biz, NSC&#39;lerde izole kaynaklı EMT ve FGF2&#39;deki ve BMP4 etkisindeki sonraki göç okudu. Biz FGF- ve böylece model sistem doğrulayarak, hücre göçü teşvik etmek TGFβ-sinyal ile etkileşime BMP-sinyalizasyon görülmektedir.

<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

mezenkimal geçiş (EMT) için epitel epitel mezenşimin içine transdifferentiating işlemi anlatılmaktadır. EMT da yaygın, glioblastoma en sık malign beyin tümörü meydana embriyonik gelişim sırasında temel bir süreçtir. EMT, aynı zamanda, göğüs kanseri, akciğer kanseri, kolon kanseri, mide kanseri de dahil olmak üzere beyin dışında çok karsinom gözlenmiştir. EMT merkezi göç, işgali ve metastaz oluşumunu teşvik ederek malignite bağlantılıdır. EMT indüksiyon mekanizmaları tam olarak anlaşılmış değildir. Burada kortikal nöral kök hücre (NSC&#39;lerde) ve daha sonra EMT indüksiyonu standart izolasyonu için in vitro bir sistem tarif eder. Bu sistem tek hücreler veya eksplant kültürü ya da kullanmak için esneklik sağlar. Bu sistemde, sıçan veya fare embriyonik ön beyin NSC&#39;lerde, tanımlanmış bir ortam içinde kültürlenmiş ve serum enzimlerin yoksundur. NSC&#39;lerde oligodendrositlerin gözlenen olig2 ve Sox10 iki transkripsiyon faktörleri olarak ifadeYTE ön-madde hücreleri (OPC). Bu sistemi kullanarak, FGF-, BMP- arasındaki etkileşimler Zeb1, Zeb2 içeren TGFβ-sinyal ve Twist2 TGFβ etkinleştirme önemli ölçüde sinerjistik BMP- / TGFβ-etkileşim olduğunu, hücre göçü geliştirilmiş burada gözlenmiştir. Sonuçlar EMT indüksiyonu ve bakım dahil olmak FGF-, BMP- ve TGFβ-sinyal ağı işaret eder. Bu model sistemi, in vitro EMT araştırmak için uygundur. Bu düşük maliyetli ve yüksek tekrarlanabilirlik göstermektedir. Ayrıca, göç yanıtlara göre (kantitatif mesafe ölçümü) ve inhibe etmek veya EMT (kalitatif ölçümü) geliştirmek için bileşikler yüksek verimli tarama ile farklı bileşiklerin karşılaştırılmasına imkan verir. modeli bu nedenle de EMT etkileyen maddeler için ilaç kütüphaneleri test etmek için uygundur.

Bu EMT model sistem olarak tek hücre ya da gelişmekte olan sinir tüpün belirli bir bölgeden eksplant olarak iki NSC&#39;lerde standart izolasyonuna dayanmaktadır, merkezi korteks (Şekiller 1 ve 2). Nicel değerlendirmesi için, eksplantlar 500 mikron ızgara kültürü çanak (Şekil 3) merkezinde hakkı ekildi. Merkezi korteksten eksplantlar ilk büyüme faktörleri (Tablo 1) farklı kombinasyonları ilave iki g?...

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.

Ilaç keşfi için uygundur EMT araştırma için enzim ve serum içermeyen nöral kök hücre kültürü modeli

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

Serum serbest Tek tabakalı Kültüründe Fare Embriyonik Kök Hücre Sinir Farklılaşma

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

Gelişim Biyolojisi

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

Temel ve Klinik öncesi Araştırma bir Kapalı Hücre Kültürü Sistemi İnsan Pluripotent ve Sinir Kök Hücre Kültürleme

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

Tahmin ve Gen Düzenleme Elementlerin Doğrulama Retinoik Asit Kaynaklı Embriyonik sırasında Aktif Hücre Farklılaşma Kök

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

Oldukça Tekrarlanabilir Küçük Molekül Tabanlı Farklılaşma Protokolü kullanarak insan pluripotent kök hücreleri gelen kardiyomiyositlerde Büyük Ölçekli Üretim

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

Kültürlü insan Fetal beyin sinir kök hücreleri Zika virüs enfeksiyonu Immunocytochemical analiz için

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

İnsan kullanarak Pluripotent kök hücre kaynaklı hepatosit benzeri hücreler indüklenen ilaç bulma için

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

Kromozom segregasyon mitoz sırasında canlı hücre görüntüleme

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

Kalsiyum akını, gözenek oluşumu ve P2X7 reseptörleri yetişkin nöral progenitör hücreler tarafından fagositoz araştırmak için gerçek zamanlı canlı hücre Akış Sitometresi

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

Insan kaynaklı pluripotent kök hücre türetilen nöral hücrelerin terapötik transplantasyonu ile fare beyin yaralanması kontrollü Kortiik darbe modeli

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

Periferik mononükleer hücrelerden Indüklenen nöral kök hücrelerin üretimi ve transplantasyon çalışmaları için Dopaminergic neuron precursors doğru farklılaşma