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

Deze studie werd ondersteund door NIH-subsidie ​​R01 HL119984 AH

1. Cultuur van de muis embryonale fibroblasten (MEF) <ol><li> Bereiden MEF medium, Dulbecco&#39;s gemodificeerd Eagle&#39;s medium (DMEM hoge glucose) aangevuld met 15% foetaal runderserum (FBS). </li><li> Coat 100 mm celcultuurschalen met 0,5% gelatine gedurende 30 minuten bij kamertemperatuur (RT). </li><li> Count MEF met behulp van een cytometer. Verwijder de gelatineoplossing en giet onmiddellijk MEF medium voorverwarmd tot 37 ° C. Snel ontdooien flesjes mitomycine-C behandelde MEFs in een 37 ° C waterbad gedure...

<ol>
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In dit protocol presenteren we een betrekkelijk eenvoudige en toegankelijke wijze neurale differentiatie van muis SER bestuderen. In eerdere protocollen werd RA toegevoegd aan het medium op dag 2 en dag 4 van de EB hangende-druppel 8 of suspensiecultuur 7, respectievelijk, of onmiddellijk na de EB opknoping druppel 21 aggregatie. In het protocol we bedacht, werd RA eerder toegevoegd. Ondanks de eerdere invoering van RA EBS gevormd door suspensiekwee...

SER afkomstig van de blastocyst binnenste celmassa. Deze cellen zijn pluripotent, dat wil zeggen zij hebben het vermogen om te differentiëren in elk celtype van het organisme van herkomst. ESC in vitro differentiatie is van groot belang als een experimenteel systeem voor het onderzoeken van ontwikkelingstrajecten en mechanismen. Het biedt een krachtige en flexibele modelsysteem om nieuwe therapeutische benaderingen voor de correctie van cellen en weefsels dysfunctie te testen. EBS recapituleren vele aspecten van celdifferentiatie tijdens de vroege embryogenese. In het bijzonder kan EBS worden gebruikt wanneer embryonische letaliteit bemoeilijkt de cellulaire basis van de embryonale gebreken 1, 2 bepalen. EBS kan hetzij worden gevormd door de hangende druppel of vloeibare suspensietechnieken 3. Het voordeel van de eerste is de mogelijkheid om EB consistente grootte en dichtheid te genereren, waardoor experimentele reproduceerbaarheid vergemakkelijkt. <p class = &quot;jove_content&quot;&gt; interactie met extracellulaire matrix (ECM) adhesie eiwitten kunnen de motiliteit en overleving van hechtende cellen beïnvloeden. In de 2D-cultuur systeem, wordt fibronectine vaak toegepast op cel adhesie te verhogen tot het substraat. Fibronectine is een basale lamina component herkend door 10 soorten celoppervlak integrine heterodimeren 4. RA is een kleine lipofiele metaboliet van vitamine A die neurale differentiatie 5, 6 induceert. Hoge concentraties van RA bevorderen neurale genexpressie en onderdrukt mesodermale genexpressie tijdens EB vorming 7, 8. RA wordt door vitamine A oxidatie tot retinaldehyde door een alcohol of retinol dehydrogenase, gevolgd door retinaldehyde oxidatie tot het uiteindelijke product door retinaldehyde dehydrogenase 9. Neurale differentiatie vereist transport van RA uit het cytoplasma<html> de kern van cellulaire RA-bindend eiwit 2 (CRABP2). In de kern, RA bindt aan de verwante receptor complex van een RAR-RXR heterodimeer 10. Dit leidt tot rekrutering van transcriptionele co-activatoren, en initiatie van transcriptie 9, 11. Bovendien RA bevordert de afbraak van gefosforyleerd (actieve) Smad1, dus tegenwerken van BMP en Smad signalering 12. Naast deze activiteiten RA verhoogt Pax6 expressie, een transcriptiefactor die neuronale differentiatie 13 ondersteunt. RA signalering wordt gemoduleerd door sirtuine-1 (Sirt1), een nucleair nicotinamide adenine dinucleotide (NAD +) - afhankelijk enzym dat CRABP2 deacetylates interfereren met zijn translocatie naar de kern, en dus met RA binding aan de RAR-RXR heterodimeer 14, 15, 16. e_content &quot;&gt; Ons doel bij het ontwerpen van de RA-behandelde EB protocol hier beschreven neurale differentiatie optimaliseren om in vitro analyse van de signaleringspaden die ESC differentiatie reguleren in neuronale voorlopercellen te vergemakkelijken. Een voordeel van dit protocol is vergemakkelijking van de analyse van celfunctie door immunofluorescentie. 3D EB&#39;s niet goed gepenetreerd door antilichamen en moeilijk beeld. EB dissociatie in een 2D monolaag op specifieke tijdstippen tijdens neurale differentiatie bevordert immunokleuring en beeldvorming van de cellen met confocale microscopie.

<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

Muis embryonale stamcellen (SER) geïsoleerd uit de binnenste massa van de blastocyst (gewoonlijk op dag E3.5), kan worden gebruikt als in vitro modelsysteem voor het bestuderen vroege embryonale ontwikkeling. Bij afwezigheid van leukemie remmende factor (LIF), SER differentiëren standaard tot neurale voorlopercellen. Ze kunnen worden vergaard in een driedimensionale (3D) sferisch aggregaat aangeduid embryoiden lichaam (EB) vanwege zijn gelijkenis met het vroege stadium embryo. EBS kunnen worden gezaaid op fibronectine beklede dekglaasjes, waar ze uitbreiden door kweken tweedimensionale (2D) extensies of geïmplanteerd in 3D collageenmatrices waar ze blijven groeien als sferoïden en differentiëren tot drie kiembladen: endodermale, mesodermale en ectodermale. De 3D collageen cultuur bootst de in vivo omgeving beter dan de 2D-EBS. De 2D EB cultuur vergemakkelijkt de analyse van immunofluorescentie en immunoblotting om differentiatie te volgen. We hebben een twee-staps neurale differentia ontwikkeldtie protocol. In de eerste stap worden EB opgewekt door de hangende druppel techniek en tegelijkertijd worden geïnduceerd om te differentiëren door blootstelling aan retinoïnezuur (RA). In de tweede stap neurale differentiatie verloopt in een 2D- of 3D-formaat bij afwezigheid van RA.

Oct4, Nanog, en SOX2 de kern transcriptiefactoren die ESC zelfvernieuwing en pluripotentie verlenen. We pasten bovenstaande protocol bij de neurale differentiatie van SER vergelijken van wild type en van een stam van genetisch gemodificeerde muizen waarbij Syx, een gen dat codeert voor het RhoA specifieke exchange factor Syx wordt verstoord. We hadden Syx betrokken bij angiogenese 18. Wij merkten verschillen in het gedrag van EBS geaggregeerde van Syx...

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

We beschrijven een werkwijze voor het gebruiken van muriene embryonale stamcellen voor het genereren van twee- of driedimensionaal embryoïde lichamen. Vervolgens hebben we uitgelegd hoe neurale differentiatie van de embryoid lichaamscellen door retinolzuur, en hoe ertoe te bewegen hun staat van differentiatie te analyseren door voorlopercellen cel marker immunofluorescentie en immunoblotting.

Analyse van retinoïnezuur geïnduceerde neurale differentiatie van muis embryonale stamcellen in twee en drie-dimensionale embryoiden organen

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 ...

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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

Afleiden van retinale pigment epitheel (RPE) van induced pluripotent stem (iPS) cellen door verschillende maten van embryoiden Bodies

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

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

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

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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

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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

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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

Regie van differentiatie van primitieve en definitieve hematopoietische progenitorcellen van menselijke pluripotente stamcellen

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

Horizontale gehele berging: Een nieuw verwerkings- en beeldprotocol voor dikke, driedimensionale weefsel dwarsdoorsneden van de huid

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

Snelle, gerichte differentiatie van de epitheliale cellen van de retinale Pigment van menselijke embryonale of geïnduceerde pluripotente stamcellen

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

Steiger-vrije, driedimensionale insuline uitdrukken Pancreatoids van muis alvleesklier voorlopercellen In Vitro generatie

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

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