Name: analysis of retinoic acid-induced neural differentiation of mouse embryonic stem cells in two and three-dimensional embryoid bodies
Uploaded: 2017-04-22T14:00:00
Description: Watch this Scientific Journal Video about Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies at JoVE.com

Questo studio è stato sostenuto da NIH concedere R01 HL119984 AH

1. Cultura di fibroblasti embrionali di topo (MEF) <ol><li> Preparare mezzo MEF, mezzo di Dulbecco modificato da Eagle (DMEM, high-glucosio), supplementato con siero fetale bovino 15% (FBS). </li><li> Cappotto 100 mm piatti di coltura di cellule con soluzione di gelatina allo 0,5% per 30 min a temperatura ambiente (RT). </li><li> Conte MEF usando un citometro. Rimuovere la soluzione di gelatina e versare immediatamente medio MEF pre-riscaldato a 37 ° C. Scongelare rapidamente fiale di mitomicina C MEF trattata in un ...

<ol>
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In questo protocollo vi presentiamo un metodo relativamente semplice e accessibile per studiare la differenziazione neuronale dei CES murini. In precedenti protocolli, RA è stato aggiunto al mezzo al giorno 2 o 4 giorni della EB hanging-goccia 8 o coltura in sospensione 7, rispettivamente, o immediatamente dopo l&#39;EB appeso aggregazione goccia 21. Nel protocollo abbiamo ideato, RA è stato aggiunto in precedenza. Nonostante l&#39;introduzione an...

CES provengono dalla massa cellulare interna della blastocisti. Queste cellule sono pluripotenti, cioè hanno la capacità di differenziarsi in qualsiasi tipo di cellula dell&#39;organismo di origine. ESC differenziazione in vitro è di grande interesse, come un sistema sperimentale per studiare percorsi e meccanismi di sviluppo. Offre un potente e flessibile sistema modello per la sperimentazione di nuovi approcci terapeutici per la correzione della disfunzione delle cellule e dei tessuti. EBs ricapitolano molti aspetti della differenziazione cellulare durante l&#39;embriogenesi precoce. In particolare, EBs possono essere utilizzati quando letalità embrionale rende difficile determinare la base cellulare dei difetti embrionali 1, 2. EBs possono essere formate mediante la goccia sospesa o tecniche sospensione liquida 3. Il vantaggio del primo è la capacità di generare EBS di dimensioni e densità costante, facilitando così la riproducibilità sperimentale. <p class = &quot;jove_content&quot;&gt; interazione con proteine ​​di adesione extracellulari della matrice (ECM) può influenzare la motilità e la sopravvivenza delle cellule aderenti. Nel sistema di coltura 2D, fibronectina è spesso applicato per aumentare l&#39;adesione delle cellule al substrato. Fibronectina è un componente lamina basale riconosciuto da 10 tipi di superficie cellulare integrine eterodimeri 4. RA è una piccola metabolita della vitamina A lipofilo che induce neurale differenziazione 5, 6. Elevate concentrazioni di RA promuovere genica neurale e reprime l&#39;espressione genica mesoderma durante la formazione EB 7, 8. RA è prodotto da vitamina A ossidazione retinaldehyde da uno o alcol deidrogenasi retinolo, seguita da ossidazione retinaldehyde al prodotto finale retinaldehyde deidrogenasi 9. differenziazione neurale richiede il trasporto di RA dal citoplasma<html> al nucleo da cellule RA-binding protein 2 (CRABP2). Nel nucleo, RA si lega al suo recettore affine costituito da un eterodimero RAR-RXR 10. Ciò si traduce nel reclutamento di co-attivatori trascrizionali, e l&#39;inizio della trascrizione 9, 11. Inoltre, RA promuove la degradazione di fosforilato (attivo) SMAD1, contrapponendosi così BMP e SMAD segnalazione 12. Oltre a queste attività, RA aumenta l&#39;espressione di Pax6, un fattore di trascrizione che supporta neurale differenziazione 13. Segnalazione RA è modulata da sirtuin-1 (Sirt1), un dinucleotide adenina nicotinammide nucleare (NAD +) - enzima dipendente che deacetylates CRABP2, interferendo con la sua traslocazione al nucleo, e quindi con RA legame al eterodimero RAR-RXR 14, 15, 16. e_content &quot;&gt; Il nostro obiettivo nella progettazione del protocollo EB RA-trattati qui descritto è quello di ottimizzare la differenziazione neuronale al fine di facilitare l&#39;analisi in vitro delle vie di segnalazione che regolano la differenziazione ESC in cellule precursori neuronali. Uno dei vantaggi di questo protocollo è facilitazione l&#39;analisi della funzione delle cellule mediante immunofluorescenza. 3D EBs non sono ben penetrato da anticorpi e sono difficilmente immagine. EB dissociazione in un monostrato 2D in punti temporali specifici durante la differenziazione neuronale facilita immunomarcatura e l&#39;imaging delle cellule mediante microscopia confocale.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Materials</td>
		</tr>
		<tr>
			<td>MEFs</td>
			<td>EMD Millipore</td>
			<td>PMEF-CF</td>
			<td>ESC feeder layer</td>
		</tr>
		<tr>
			<td>ESC</td>
			<td>EMD Millipore</td>
			<td>CMTI-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture dish (60 mm)</td>
			<td>Eppendorf</td>
			<td>30701119</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Cell culture dish (100 mm)</td>
			<td>Falcon</td>
			<td>353003</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Petri dish (100 mm)</td>
			<td>Corning</td>
			<td>351029</td>
			<td>Hanging drops</td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Greiner Bio-One</td>
			<td>662160</td>
			<td>2D EBs</td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Eppendorf</td>
			<td>30720113</td>
			<td>Transfection</td>
		</tr>
		<tr>
			<td>Dark 1.5 ml centrifuge tube</td>
			<td>Celltreat Scientific Products</td>
			<td>229437</td>
			<td>RA stock solution</td>
		</tr>
		<tr>
			<td>Microscope cover-glass</td>
			<td>Fisherbrand</td>
			<td>12-545-80</td>
			<td>Circular, 12 mm diameter</td>
		</tr>
		<tr>
			<td>Superfrost-plus microscope slides</td>
			<td>Fisherbrand</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>3D collagen culture kit</td>
			<td>EMD Millipore</td>
			<td>ECM675</td>
			<td>3D culture</td>
		</tr>
		<tr>
			<td>Effectene Transfection Reagent</td>
			<td>Qiagen</td>
			<td>301427</td>
			<td>Stem cell transfection</td>
		</tr>
		<tr>
			<td>Microcon&#160;Centrifugal Filters (10 kDa)</td>
			<td>EMD Millipore</td>
			<td>MRCPRT010</td>
			<td>Protein concentration</td>
		</tr>
		<tr>
			<td>Name&#160;</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">Reagents</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Lonza</td>
			<td>12-709F</td>
			<td>MEFs culture</td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>Gibco</td>
			<td>12440-046</td>
			<td>ESCs culture</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>EMD Millipore</td>
			<td>ES-009-B</td>
			<td>ESCs culture</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>G2625</td>
			<td>Dish coating</td>
		</tr>
		<tr>
			<td>LIF</td>
			<td>R&#38;D Systems</td>
			<td>8878-LF-025</td>
			<td>To maintain ESC pluripotency</td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solutions</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gibco</td>
			<td>15750060</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>MycoZap Plus-PR</td>
			<td>Lonza</td>
			<td>VZA-2022</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-072</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>All-trans-retinoic acid</td>
			<td>Sigma-Aldrich</td>
			<td>R2625-50MG</td>
			<td>Induction of neural differentiation</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7030-50G</td>
			<td>Blocking and antibody dilution&#160;</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787-100ML</td>
			<td>Cell membrane permeabilization</td>
		</tr>
		<tr>
			<td>Cell strainer</td>
			<td>Corning</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolong Gold anti-fade reagent with DAPI</td>
			<td>Life Tech.</td>
			<td>P36931</td>
			<td>Mounting reagent</td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>Cell fixation</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>R&#38;D Systems</td>
			<td>1030-FN</td>
			<td>Dish coating</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010049</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type I</td>
			<td>Worthington Biochem. Corp</td>
			<td>LS004196</td>
			<td>EB dissociation</td>
		</tr>
		<tr>
			<td>Name&#160;</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">Primary Antibodies</td>
		</tr>
		<tr>
			<td>Nestin (Rat-401)</td>
			<td>Santa Cruz Biotech</td>
			<td>sc-33677</td>
			<td>Detection of neural differentiation</td>
		</tr>
		<tr>
			<td>Oct4</td>
			<td>Santa Cruz Biotech</td>
			<td>sc-5279</td>
			<td>Detection of neural differentiation</td>
		</tr>
		<tr>
			<td>Nanog</td>
			<td>Bethyl Laboratories</td>
			<td>A300-398A</td>
			<td>Detection of neural differentiation</td>
		</tr>
		<tr>
			<td>Sox2</td>
			<td>Cell Signaling</td>
			<td>3579</td>
			<td>Detection of neural differentiation</td>
		</tr>
		<tr>
			<td>Tubulin b3 (AA10)</td>
			<td>Santa Cruz Biotech</td>
			<td>sc-80016</td>
			<td>Detection of neural differentiation</td>
		</tr>
		<tr>
			<td>Name&#160;</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">Secondary Antibodies</td>
		</tr>
		<tr>
			<td>Donkey anti-Mouse-Alexa555</td>
			<td>Life Tech.</td>
			<td>A31570</td>
			<td>Immunofluorescence</td>
		</tr>
		<tr>
			<td>Donkey anti-mouse-Alexa488&#160;</td>
			<td>Life Tech.</td>
			<td>A21202</td>
			<td>Immunofluorescence</td>
		</tr>
		<tr>
			<td>Name&#160;</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td colspan="4">Instruments</td>
		</tr>
		<tr>
			<td>Wide-field microscope</td>
			<td>Nikon</td>
			<td>Eclipse TS100</td>
			<td>Cell culture imaging</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Nikon</td>
			<td>C2</td>
			<td>Immunofluorescence imaging</td>
		</tr>
	</tbody>
</table>

analysis of retinoic acid-induced neural differentiation of mouse embryonic stem cells in two and three-dimensional embryoid bodies

Le cellule staminali embrionali di topo (CES) isolati dalla massa interna della blastocisti (tipicamente a giorno E3.5), può essere utilizzato come sistema modello in vitro per lo studio sviluppo embrionale precoce. In assenza di fattore inibitorio della leucemia (LIF), CES differenziano per default in cellule precursori neurali. Essi possono essere ammassati in una tridimensionale (3D) aggregato sferico definito corpo embrioide (EB) a causa della sua somiglianza con l&#39;embrione in fase iniziale. EBs possono essere seminati su vetrini fibronectina rivestite, dove si espandono coltivando due estensioni (2D) dimensionali, o impiantati in matrici di collagene 3D dove continuano a crescere come sferoidi, e si differenziano in tre strati germinali: endodermica, mesoderma e ectodermica. La cultura collagene 3D simula l&#39;ambiente in vivo più da vicino di EBS 2D. La cultura 2D EB facilita l&#39;analisi per immunofluorescenza e immunoblotting per monitorare differenziazione. Abbiamo sviluppato una differenziazione neuronale in due fasiprotocollo zione. Nella prima fase, EBs sono generati dalla sospensione tecnica-drop, e, contemporaneamente, vengono indotte a differenziarsi da esposizione all&#39;acido retinoico (RA). Nella seconda fase, neurali procede differenziazione in formato 2D o 3D in assenza di RA.

Oct4, Nanog, e SOX2 sono i fattori di trascrizione fondamentali che conferiscono ESC auto-rinnovamento e pluripotenza. Abbiamo applicato il protocollo di cui sopra per confrontare la differenziazione neuronale dei CES da wild type e da un ceppo di topi geneticamente modificati in cui Syx, un gene che codifica per il fattore di scambio RhoA-specifica Syx, è interrotta. Avevamo implicati Syx nell&#39;angiogenesi 18. Abbiamo notato differenze nei comportame...

Watch this Scientific Journal Video about Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies at JoVE.com

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Descriviamo una tecnica per utilizzare le cellule staminali embrionali murine per generare due o tre corpi embrionali tridimensionali. Abbiamo poi spiegato come indurre la differenziazione neuronale delle cellule del corpo embrionali di acido retinoico, e come analizzare il loro stato di differenziazione dal marcatore di cellule progenitrici immunofluorescenza e immunoblotting.

Analisi di acido retinoico-indotta neurale differenziazione di cellule staminali embrionali di topo a organi embrionali due e tridimensionali

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

Research

JoVE Journal

Developmental Biology

Deriving Retinal Pigment Epithelium (RPE) from Induced Pluripotent Stem (iPS) Cells by Different Sizes of Embryoid Bodies

Deriving Retinal Pigment Epithelium RPE from Induced Pluripotent Stem iPS Cells by Different Sizes of Embryoid Bodies

Derivazione retina epitelio pigmentato (RPE) da pluripotenti indotte (IPS) Stem Cells dalle diverse dimensioni delle corpi embrionali

Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of ...

Ricerca

Biologia dello sviluppo

Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes

Differenziazione di una linea di cellule staminali neurali umane in Tre Culture dimensionali, Geni Analisi di MicroRNA e putativo di destinazione

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local ...

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 ...

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Regolazione epigenetica del Cardiac differenziazione delle cellule staminali embrionali e Tessuti

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

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 ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

Regia di differenziazione dei progenitori ematopoietici primitivi e definitivo da cellule staminali pluripotenti umane

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Supporto orizzontale intero: un protocollo di elaborazione e imaging novello per le sezioni trasversali tessute spesse e tridimensionali della pelle

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

Rapida, regia di differenziazione delle cellule epiteliali del pigmento retinico da cellule staminali pluripotenti umane embrionali o indotto

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Generazione di insulina senza impalcatura, tridimensionale esprimendo Pancreatoids dal Mouse progenitori pancreatico In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Analisi computazionale della linea germinale Caenorhabditis elegans di studiare la distribuzione dei Nuclei, le proteine ed il citoscheletro

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...