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

Denne undersøgelse blev støttet af NIH tilskud R01 HL119984 til AH

1. Dyrkning af muse embryonale fibroblaster (MEF) <ol><li> Forberede MEF medium, Dulbeccos modificerede Eagles medium (DMEM høj glucose) suppleret med 15% føtalt bovint serum (FBS). </li><li> Coat 100 mm celledyrkningsskåle med 0,5% gelatine-opløsning i 30 minutter ved stuetemperatur (RT). </li><li> Tæl MEF&#39;er ved hjælp af en cytometer. Fjern gelatineopløsningen og straks pour MEF medium forvarmet til 37 ° C. Hurtigt tø hætteglas med mitomycin C-behandlede MEF&#39;er i et 37 ° C vandbad i 2 min, derefte...

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I denne protokol præsenterer vi en relativt enkel og tilgængelig metode til at studere neural differentiering af murine ESC&#39;er. I tidligere protokoller blev RA tilsat til mediet på dag 2 eller dag 4 af EB hængende-dråbe 8 eller ved suspensionskultur 7 henholdsvis eller umiddelbart efter EB hængende dråbe aggregering 21. I den protokol vi udtænkt, blev RA tilføjet tidligere. Trods den tidligere introduktion af RA til EB&#39;er dannet ved...

Økonomiske og sociale råd stammer fra blastocyst indre cellemasse. Disse celler er pluripotente, dvs. at de har kapacitet til at differentiere til en hvilken som helst celletype i organismen for oprindelse. ESC in vitro differentiering har bred interesse som et eksperimentelt system til at undersøge udviklingsmæssige veje og mekanismer. Det giver en potent og fleksibel modelsystem til afprøvning af nye terapeutiske metoder til korrektion af celler og væv dysfunktion. EB rekapitulere mange aspekter af celledifferentiering under tidlig embryogenese. Især kan EB&#39;er anvendes, når embryoletalitet gør det vanskeligt at bestemme den cellulære grundlag af embryonale defekter 1, 2. EB&#39;er kan dannes enten af hængende dråbe eller flydende suspensionsteknikker 3. Fordelen ved førstnævnte er evnen til at generere EB&#39;er af ensartet størrelse og densitet, hvilket vil lette eksperimentelle reproducerbarhed. <p class = &quot;jove_content&quot;&gt; Interaktion med ekstracellulære matrix (ECM) adhæsionsproteiner kan påvirke motilitet og overlevelse af adhærente celler. I 2D-dyrkningssystem, der fibronectin ofte anvendes til at øge celle vedhæftning til underlaget. Fibronectin er et basalt lamina komponent anerkendt af 10 typer af celleoverflade-integrin heterodimerer 4. RA er en lille lipofil metabolit af vitamin A, som inducerer neural differentiering 5, 6. Høje koncentrationer af RA fremme neural genekspression og undertrykker mesodermal genekspression under EB formation 7, 8. RA fremstilles ved vitamin A oxidation til retinaldehyde ved enten alkohol eller retinol dehydrogenase, efterfulgt af retinaldehyde oxidation til slutproduktet ved retinaldehyde dehydrogenase 9. Neural differentiering kræver transport af RA fra cytoplasmaet<html> til kernen ved cellulær RA-bindende protein 2 (CRABP2). I kernen, RA binder til dens beslægtede receptor kompleks bestående af en RAR-RXR heterodimer 10. Dette resulterer i rekruttering af transkriptionelle co-aktivatorer, og initiering af transkription 9, 11. Endvidere RA fremmer nedbrydningen af phosphoryleret (aktiv) SMAD1, således antagonisere BMP og SMAD signalering 12. Ud over disse aktiviteter, RA øger Pax6-ekspression, en transkriptionsfaktor, der understøtter neural differentiering 13. RA signalering moduleres ved sirtuin-1 (SIRT1), et nukleart nicotinamidadenindinucleotid (NAD +) - afhængigt enzym, deacetylerer CRABP2, interferere med dets translokation til kernen, og dermed med RA binding til RAR-RXR heterodimer 14, 15, 16. e_content &quot;&gt; Vores mål i udformningen af RA-behandlede EB protokol beskrevet her er at optimere neural differentiering for at lette in vitro analyse af signalveje, der regulerer ESC differentiering til neuronale precursorceller. En af fordelene ved denne protokol er lettelse af analysen af ​​cellefunktion ved immunofluorescens. 3D EB&#39;er ikke godt gennemtrænges af antistoffer og er vanskelige at billedet. EB dissociation i en 2D monolag på bestemte tidspunkter i løbet af neural differentiering letter immunmærkning og afbildning af cellerne ved konfokal mikroskopi.

<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

Muse embryonale stamceller (ESC&#39;er) isoleret fra den indre blastocyt (typisk ved dag E3.5), kan anvendes som in vitro modelsystem til undersøgelse tidlig fosterudvikling. I fravær af leukæmiinhiberende faktor (LIF), økonomiske og sociale råd differentiere som standard i neurale precursorceller. De kan samlet i en tredimensional (3D) sfæriske aggregat betegnes embryoid legeme (EB) på grund af dens lighed med den tidlige fase embryo. EB&#39;er kan podes på fibronectinovertrukne dækglas, hvor de ekspanderer ved dyrkning todimensionale (2D) udvidelser, eller indført i 3D collagenmatricer hvor de fortsat vokser som sfæroider, og differentierer til de tre kimlag: endodermal, mesodermal og ectodermal. 3D kollagen kultur efterligner in vivo miljø tættere end 2D EB&#39;er. 2D EB kultur letter analyse ved immunofluorescens og immunoblotting at spore differentiering. Vi har udviklet en to-trins neurale differentieringtion protokol. I det første trin, er EB&#39;er genereres af hængende-dråbe-teknikken, og samtidig induceres til at differentiere ved udsættelse for retinsyre (RA). I det andet trin, neurale differentierings forløber i en 2D- eller 3D-format i fravær af RA.

Oct4, Nanog, og SOX2 er de centrale transkriptionsfaktorer der bibringer ESC selvfornyelse og pluripotens. Vi anvendt den ovennævnte protokol til at sammenligne den neurale differentiering af økonomiske og sociale råd fra vild type og fra en stamme af genetisk modificerede mus, hvor Syx, et gen, der koder for RhoA-specifikke vekselkurs faktor Syx er afbrudt. Vi havde impliceret Syx i angiogenese 18. Vi har bemærket forskelle i adfærd af EB&#39;er agg...

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

Vi beskriver en teknik til anvendelse af murine embryonale stamceller til generering af to- eller tredimensionale embryoide legemer. Vi derefter forklare, hvordan at inducere neural differentiering af embryoide kropsceller ved retinsyre, og hvordan man analyserer deres tilstand af differentiering ved progenitorcelle markør immunofluorescens og immunoblotting.

Analyse af retinoidsyre-induceret Neural Differentiation af muse embryonale stamceller i to og tre-dimensionelle embryoide legemer

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

Følger retinale pigmentepitel (RPE) fra induceret Pluripotente Stem (IPS) Celler ved forskellige størrelser af embryoide legemer

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

Differentiering af en human neural stamcelle Line på tredimensionelle kulturer, Analyse af MicroRNA og formodede målgener

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

Neural Differentiering af Mouse Embryonale stamceller i Serumfrit monolagskultur

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

Epigenetisk forordning af Cardiac Differentiering af embryonale stamceller og væv

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

Forudsigelse og Validering af Gene Regulatory Elements Aktiveret Under retinoidsyre induceret embryonale stamceller Differentiering

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

Instrueret differentiering af Primitive og endelige hæmatopoietisk ophav fra humane pluripotente stamceller

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

Horisontal Hel Mount: En Novel Processing and Imaging Protocol for Tykke, Tredimensionale Tissue Tværsnit af Skin

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

Hurtig, instrueret differentiering af retinale Pigment epitel celler fra menneskelige embryonale eller induceret pluripotente stamceller

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

Generation af stillads-fri, tre-dimensionelle Insulin udtrykker Pancreatoids fra mus i bugspytkirtlen stamfaderen 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

Beregningsmæssige analyse af Caenorhabditis elegans kønscelleoverførsel at studere Distribution kerner, proteiner og cytoskelettet

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