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

Diese Studie wurde von NIH Zuschusses R01 HL119984 zu AH unterstützt

1. Kultur von embryonalen Maus-Fibroblasten (MEF) <ol><li> Bereiten MEF Medium, nach Dulbecco modifiziertem Eagle-Medium (DMEM, high-glucose), ergänzt mit 15% fötalem Rinderserum (FBS). </li><li> Coat 100 mm Zellkulturschalen mit 0,5% Gelatine-Lösung für 30 min bei Raumtemperatur (RT). </li><li> Graf MEFs mit einem Durchflusszytometer. Entfernen der Gelatinelösung und sofort gießen MEF Medium vorgewärmt auf 37 ° C. Schnell auftauen Phiolen von Mitomycin C-behandeln MEFs in einem 37 ° C Wasserbad für 2 min, d...

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In diesem Protokoll stellen wir eine relativ einfache und zugängliche Methode neuronale Differenzierung von murinen WSR zu studieren. In vorangegangenen Protokollen wurde RA auf das Medium am Tag 2 oder Tag hinzugefügt 4 des EB hängenden Tropfen 8 oder durch Suspensionskultur bzw. 7, oder unmittelbar nach der Tropfenaggregation EB 21 hängt. In dem Protokoll wir erdacht wurde RA früher gegeben. Trotz der früheren Einführung von RA zu EBs durch...

WSR stammt aus der Blastozyste inneren Zellmasse. Diese Zellen sind pluripotent, dh sie haben die Fähigkeit zur Differenzierung zu jedem Zelltyp des Ursprungsorganismus haben. ESC in vitro Differenzierung von breitem Interesse als experimentelles System für die Entwicklungsweg und Mechanismen zu untersuchen. Es bietet ein starkes und flexibles Modellsystem neue therapeutische Ansätze zur Korrektur von Zell- und Gewebestörungen zu testen. EBs rekapitulieren viele Aspekte der Zelldifferenzierung während der frühen Embryonalentwicklung. Insbesondere kann EBs verwendet werden , wenn embryonale Letalität erschwert es , die zellulären Grundlagen der embryonalen Defekte 1, 2 zu bestimmen. EBs können 3 durch den hängenden Tropfen oder flüssigen Suspensionstechniken gebildet entweder werden. Der Vorteil des ersteren ist die Fähigkeit, EBs konsistenter Größe und Dichte zu erzeugen, so dass experimentelle Reproduzierbarkeit zu erleichtern. <p class = „jove_content“&gt; Wechselwirkungen mit extrazellulären Matrix (ECM) Adhäsionsproteine ​​kann die Beweglichkeit und das Überleben von adhärenten Zellen beeinflussen. In dem 2D-Kultursystem wird oft Fibronektin zu erhöhen Zelladhäsion auf das Substrat aufgebracht. Fibronectin ist eine Basallamina Komponente um 10 Arten von Zelloberflächen - Integrin - Heterodimeren erkannt 4. RA ist ein kleines lipophiles Metabolit von Vitamin A , das 5 neurale Differenzierung induziert, 6. Hohe Konzentrationen von RA Förderung neuronaler Genexpression reprimiert und mesodermalen Genexpression während der EB Bildung 7, 8. RA wird durch Vitamin - A - Oxidation Retinaldehyd entweder durch Alkohol oder Retinol - Dehydrogenase, gefolgt von Retinaldehyd Oxidation zum Endprodukt durch Retinaldehyd dehydrogenase 9 hergestellt. Neuronale Differenzierung erfordert Transport von RA aus dem Cytoplasma<html> an den Kern durch zelluläre RA-bindendes Protein 2 (CRABP2). Im Kern bindet RA an seinen zugehörigen Rezeptor - Komplex , bestehend aus einem RAR-RXR - Heterodimer 10. Dies führt bei der Rekrutierung von Transkriptions Co-Aktivator, und der Initiation der Transkription 9, 11. Darüber hinaus fördert die RA den Abbau von phosphoryliertem (aktiv) SMAD1, wodurch BMP und SMAD Antagonisierung 12 signalisiert. Zusätzlich zu diesen Aktivitäten erhöht RA Pax6 - Expression, einen Transkriptionsfaktor, 13 neuralen Differenzierung unterstützt. RA - Signalisierung moduliert durch sirtuin-1 (Sirt1), ein Kern Nicotinamidadenindinucleotid (NAD +) - abhängiges Enzym , die CRABP2 deacetyliert, mit seiner Translokation in den Zellkern zu stören, und damit mit RA der Bindung an den RAR-RXR - Heterodimer 14, 15, 16. e_content &quot;&gt; Unser Ziel der RA-behandelten EB - Protokoll bei der Gestaltung hier beschriebene neuronale Differenzierung zu optimieren , um in - vitro - Analyse der Signalwege zu erleichtern , die ESC Differenzierung in Zellen neuronalen Vorläufer regulieren. Einer der Vorteile dieses Protokolls ist die Erleichterung der die Analyse der Zellfunktion durch Immunofluoreszenz. 3D EBs ist gut nicht durch Antikörper durchdrungen und ist schwer zu Bild. EB Dissoziation in einen 2D-Monolayer zu bestimmten Zeitpunkt während der neuronalen Differenzierung und Immunomarkierung Bildgebung der Zellen durch konfokale Mikroskopie ermöglicht.

<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

Embryonale Stammzellen der Maus (WSR) von der Innenmasse der Blastozyste isoliert ( in der Regel am Tag E3.5) können für die Untersuchung der frühe Embryonalentwicklung als in - vitro - Modellsystem verwendet werden. In Abwesenheit von Leukämie-Hemmfaktor (LIF), unterscheidet WSR standardmäßig in neuralen Vorläuferzellen. Sie können in einer dreidimensionalen (3D) angehäuft werden, um sphärische Aggregat bezeichnet Embryoidkörperbildung (EB) aufgrund seiner Ähnlichkeit mit dem frühen Stadium Embryo. EBs auf Fibronektin-beschichtete Deckgläser ausgesät werden, wo sie von wachsenden zweidimensionale (2D) Erweiterungen oder implantiert in 3D-Kollagen-Matrices erweitern, wo sie als Sphäroiden weiter wachsen und differenzieren sich in die drei Keimblätter: endodermisches, mesodermalen und ektodermalen. Die 3D - Kollagen Kultur ahmt die in - vivo - Umgebung näher als der 2D - EBs. Die 2D-EB Kultur erleichtert Analyse von Immunfluoreszenz und Immunoblotting Differenzierung zu verfolgen. Wir haben einen zweistufigen neuronalen Differenzierung entwickeltmunikationsprotokoll. Im ersten Schritt wird EBs durch die Hanging-Drop-Technik erzeugt werden, und, gleichzeitig, induziert durch Exposition gegenüber Retinsäure (RA) zu differenzieren. Im zweiten Schritt, neuronalen Differenzierung erfolgt in einem 2D- oder 3D-Format in Abwesenheit von RA.

Oct4, Nanog und SOX2 sind die Kerntranskriptionsfaktoren, die ESC Selbsterneuerung und Pluripotenz verleihen. Wir wandten das obige Protokoll , um die neuronale Differenzierung von WSR vom Wildtyp und aus einem Stamm von genetisch veränderten Mäusen , wo Syx, ein Gen , kodierend für das RhoA spezifische exchange factor Syx, gestört zu vergleichen. Wir hatten Syx in 18 Angiogenese beteiligt. Wir bemerkten Unterschiede im Verhalten von EBs von 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

Wir beschreiben eine Technik für die zur Erzeugung von zwei- oder dreidimensionalen Embryoidkörpern murinen embryonalen Stammzellen. Wir dann erklären, wie neuronale Differenzierung der Zellen Embryoidkörper von Retinsäure zu induzieren, und wie ihr Zustand der Differenzierung von Vorläuferzellmarker Immunofluoreszenz und Immunoblotting analysiert.

Analyse von Retinsäure-induzierte neuronale Differenzierung von embryonalen Stammzellen der Maus in zwei und drei-dimensionale Embryoidkörpern

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 ...

The overall goal of this procedure is to analyze the expression and production of genes and protein markers, respectively, of neural differentiation of mouse embryonic stem cells. This method can help determine the mechanism responsible for the inhibition of neural differentiation by the raw age of DPAs, and lead to better protocols to differentiate stem cells into neurons for therapeutic purposes. The main advantage of this technique is that it facilitates tracking the process of neural differentiation at the emerin and protein levels, including by optical microscopy.Demonstrating the procedure will be Dr.Junning Yang, a research associate in my laboratory. Begin embryoid body formation by harvesting embryonic stem cells with 0.25%Trypsin-EDTA for two to five minutes at 37 degrees celsius, 5%carbon dioxide. Add fresh Iscove's Modified Dulbecco's Medium, or IMDM, with 15%FBS to the detaching cells, and transfer the cell suspension to a 15 milliliter tube.Centrifuge at 160 x G for five minutes at room temperature. Then count the cells using a hemocytometer, and prepare a five x 10 to the fifth cells per milliliter suspension in IMDM with 0.5 micromolar retinoic acid. Next, use an eight-channel pipette and 200-microliter tips to plate 120-microliter drops per 100 millimeter petri dish.Invert the dish and fill the inverted lid with PBS to prevent hanging drops from drying. Culture at 37 degrees celsius, 5%carbon dioxide, for three to four days. Use 200-microliter pipette tips to harvest hanging drop embryoid bodies grown for three days.Touch an embryoid body with a pipette tip, and transfer it to a fibernectin-coated cover slip in a 24-well plate. Change the medium on the cultures every two to three days. When changing the medium, collect the used medium for protein secretion analysis as needed.To detect secreted proteins in the medium, transfer 500 microliters of embryoid body medium to 10 kilodalton cutoff centrifugal filters, and spin at 20, 000 x G at four degrees celsius. Examine the medium remaining inside the centrifugal filters every 15 to 20 minutes to prevent excessive enrichment. Stop centrifugation when the volume of the remaining medium has fallen to 25 microliters.Invert the filter and spin at 160 x G at four degrees celsius, then collect the remaining concentrated medium by following the manufacturer's instructions. For a 3D culture, harvest embryoid bodies grown for four days as hanging drops with 200-microliter pipette tips, as before. Transfer 30 embryoid bodies to a 1.5 milliliter centrifuge tube, then prepare collagen gel solution by diluting appropriate volumes of collagen solution and 5X DMEM, adding the neutralization solution, and immediately mixing well.Place the collagen gel on ice. Next, pipette an appropriate volume of chilled collagen solution into the tube containing the embryoid bodies, and then gently pipette the embryoid body suspension into the wells of a six-well plate without creating bubbles. Immediately transfer the plate to 37 degrees celsius, 5%carbon dioxide, for 60 minutes to initiate collagen polymerization.After the 60 minutes, overlay the plate containing the embryoid bodies with IMDM. For embryoid body dissociation, again collect the four-day hanging drop embryoid bodies with 200-microliter pipette tips. This time, transfer them to a non-adhesive bacteriological petri dish containing IMDM with 15%FBS and culture at 37 degrees celsius, 5%carbon dioxide, for four more days.Check the embryoid bodies twice every day to make sure they do not attach to the bottom. Gently shake the dish to prevent embryoid body attachment. After four days, transfer the embryoid bodies to a centrifuge tube and centrifuge at 185 x G for five minutes at room temperature.Following centrifugation, remove the supernatant. The palette volume should not exceed 100 microliters. Next, pipette one milliliter of 0.25%type I collagenase, supplemented with 20%FBS in PBS into the tube of embryoid bodies.Incubate the mixture for one hour at 37 degrees celsius and 5%carbon dioxide. Gently pipette the suspension using a one-milliliter pipette tip every 20 minutes. After the incubation, gently wash the cells three times with PBS.If cell aggregates are present, use a cell strainer with a 100-micron mesh to remove them. Plate the cells on gelatin-coated cover slips in IMDM, then incubate at 37 degrees celsius and 5%carbon dioxide. Wash the 2D or dissociated 3D embryoid bodies with PBS.Then fix with four percent paraformaldehyde and PBS for 30 minutes at room temperature. After washing the fixed cells three times with PBS to remove floating cell debris, add 1%triton X-100 in PBS to permeabilize the cells for 20 minutes at room temperature. After washing as before, remove the last wash and block with 5%BSA in PBS for 30 minutes.Towards the end of the incubation, prepare a primary antibody dillution in 1%BSA in PBS supplemented with 0.03%triton X-100. When the 30 minutes has elapsed, replace the blocking solution with the primary antibody solution. Incubate for three hours at room temperature, or overnight, at four degrees celsius.After washing, apply a secondary antibody in PBS and incubate for one hour at room temperature. Finally, mount cover slips on glass slides with an anti-fade medium for optical microscopy. This image shows sprout emergence from an embryoid body generated from ESCs, positive for two copies of the Syx gene, which codes for a guanine exchange factor that activates the GTPase RhoA.The image was taken after six days in 3D culture. This embryoid body formed from Syx KnockOut ESCs demonstrates increased sprouting after the same time in culture. Here, phase images of 2D embryoid body edges show that cells expanded faster from Syx KnockOuts than from Syx wild-type.These immunofluorescence images illustrate that the neural differentiation marker, Nestin, shown in red, was more abundant in cells expanding from six-day 2D6 KnockOut embryoid bodies than from their Syx wild-type counterparts. Here, immunofluorescence images show that the neural differentiation markers Nestin and beta III Tubulin were more abundant in cells dissociated from 13-day 3D6 KnockOut embryoid bodies than from their Syx wild-type counterparts. This is a time-consuming procedure because of the duration of embryoid body growth and differentiation.It not only requires at least eight days, but in some cases we follow differentiation up to 24 days. While attempting this procedure, it is important to remember to limit the viability of embryoid body diameter to ensure similar differentiation rates along the embryoid bodies.

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

Ableiten Retinapigmentepithel (RPE) von induzierten pluripotenten Stammzellen (iPS-Zellen) von verschiedenen Größen von Embryoidkörpern

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

Forschung

Entwicklungsbiologie

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

Differenzierung einer Menschen Neural Stem Cell Line auf Dreidimensional Kulturen, Analyse der MicroRNA und mutmaßlichen Zielgene

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

Neuronale Differenzierung von embryonalen Stammzellen der Maus in Serum-freiem Monolayer-Kultur

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

Epigenetische Regulation der Herz Differenzierung von embryonalen Stammzellen und Gewebe

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

Vorhersage und Validierung von Gen-regulatorischen Elementen Activated Während Retinsäure Induced Embryonic Stem Zelldifferenzierung

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to ...

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

Horizontale Ganze Mount: Ein neuartiges Verarbeitungs- und Imaging-Protokoll für dickes, dreidimensionales Gewebequerschnitt der Haut

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

Schnelle, gezielte Differenzierung der retinalen Pigment epithelialen Zellen aus menschlichen embryonalen oder induzierte pluripotente Stammzellen

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 von Gerüst-frei, dreidimensionale Insulin mit dem Ausdruck Pancreatoids vom Maus Pankreas Stammväter 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

Computergestützte Analyse der Keimbahn Caenorhabditis Elegans , die Verteilung der Kerne, Proteine und das Zytoskelett zu studieren

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

Generation of Human Primordial Germ Cell-like Cells at the Surface of Embryoid Bodies from Primed-pluripotency Induced Pluripotent Stem Cells

Generation von menschlichen Ur Keimzelle-wie Zellen an der Oberfläche der Embryoid Körper von grundiert Pluripotenz induzierte pluripotente Stammzellen

Primordial germ cells (PGCs) are common precursors of all germline cells. In mouse embryos, a founding population of ~40 PGCs are induced from pluripotent ...