eoe sub articles

EoE Sub Articles

1. Aggregate Generation
<ol>
	<li>Add 5 &#181;L of RA (retinoic acid) (1 mM stock dissolved in 99.8% ethanol, stored at -20 &#176;C) to the 10 mL of Differentiation Medium and mix well (final concentration of 0.5 &#181;M RA). 
	NOTE: RA is light-sensitive. A low concentration of ethanol does not affect cell differentiation.</li>
	<li>Add 10 mL of Differentiation Medium (with RA) to the 100 mm non-treated culture dish (dedicated to suspension culture).</li>
	<li>Seed the 1 x 106 cells in the 100 mm dish (Dish surface area 56.5 cm2).</li>
	<li>Put the flask with cells into the incubator at 37 &#176;C and 5% CO2 for 2 days to promote aggregate formation.</li>
	<li>After 2 days, exchange the Differentiation Medium. Aspirate medium containing aggregates using a 10 mL pipette and transfer to a 15 mL tube at RT.</li>
	<li>Allow the aggregates to settle by gravity for 1.5 min at RT.</li>
	<li>Discard the supernatant.</li>
	<li>Add a fresh 10 mL of Differentiation Medium with 0.5 &#181;M RA using a 10 mL serological pipette. 
	CAUTION: Do not pipette the cell aggregates up and down.</li>
	<li>Seed the aggregates into a new 100 mm non-treated culture dish (dedicated to suspension culture).</li>
	<li>Place the plate in the incubator (at 37 &#176;C and 5% CO2) for 2 days.</li>
</ol>
2. Aggregates Dissociation
<ol>
	<li>Aspirate the cell aggregates using a 10 mL pipette.</li>
	<li>Transfer the aggregates to a 15 mL tube. Allow the cell aggregates to settle by gravity for 1.5 min.</li>
	<li>Remove the supernatant.</li>
	<li>Wash the aggregates with DMEM alone (serum- and antibiotic-free).</li>
	<li>Allow the cell aggregates to settle by gravity sedimentation for 1.5 min at RT.</li>
	<li>Aspirate the supernatant and add 2 mL of trypsin-EDTA (0.25%).</li>
	<li>Place the cell aggregates into a water bath (37 &#176;C) for 10 min. Agitate the aggregates gently every 2 min by tapping with a hand.</li>
	<li>Stop the trypsinization process by adding 4 mL of Maintenance Medium.</li>
	<li>Pipette the aggregates up and down 20 times using a 1 mL pipette.</li>
	<li>Centrifuge cells for 5 min at 200 x g and RT.</li>
	<li>Remove the supernatant and resuspend the cell pellet in 5 mL of Maintenance Medium.</li>
	<li>Determine the cell number with a cell counter.</li>
</ol>
3. Plating Cells
<ol>
	<li>Add 3 mL per well of Maintenance Medium to a 6-well plate.</li>
	<li>Seed cells in the 6-well culture plate at a density of 0.5 x 106/well.</li>
	<li>Incubate at 37 &#176;C with 5% CO2 concentration.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM high glucose (4.5 g/L) with L-glutamine</td>
			<td>Lonza</td>
			<td>BE12-604Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 99.8%</td>
			<td>Chempur</td>
			<td>CHEM*613964202</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>EURx</td>
			<td>E5050-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin 10K/10K</td>
			<td>Lonza</td>
			<td>DE17-602E</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS), 1x concentrated without calcium and magnesium ions</td>
			<td>Lonza</td>
			<td>BE17- 517Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma- Aldrich</td>
			<td>R2625-50MG</td>
			<td>Dissolved in 99.8% ethanol; store in -20 &#176;C up to 6 months</td>
		</tr>
		<tr>
			<td>Trypsin 0.25% - EDTA in HBSS, without calcium and magnesium ions,with Phenol Red</td>
			<td>Biosera</td>
			<td>LM-T1720/500</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL Serological Pipettes</td>
			<td>Profilab</td>
			<td>515.01</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Serological Pipettes</td>
			<td>Profilab</td>
			<td>515.1</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm dish dedicated for suspension culture</td>
			<td>Corning</td>
			<td>C351029</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tubes</td>
			<td>Sigma- Aldrich</td>
			<td>CLS430791-500EA</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Serological Pipettes</td>
			<td>Profilab</td>
			<td>515.05</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Corning</td>
			<td>CLS3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture flasks, surface area 25 cm square</td>
			<td>Sigma- Aldrich</td>
			<td>CLS430639-200EA</td>
			<td></td>
		</tr>
	</tbody>
</table>

retinoic acid-induced neurogenesis using mouse embryonic carcinoma cells

Source: Leszczy&#324;ski, P., et al.,&#160; <a href="https://app.jove.com/v/58225">Neurogenesis Using P19 Embryonal Carcinoma Cells</a> J. Vis. Exp. (2019)
This video demonstrates a simple technique for retinoic acid-induced neurogenesis in P19 mouse embryonic carcinoma cells. Retinoic acid activates transcription factors and triggers gene expression for neuronal differentiation.

Retinoic Acid-Induced Neurogenesis using Mouse Embryonic Carcinoma Cells

1. Aggregate Generation

	Add 5 &#181;L of RA (retinoic acid) (1 mM stock dissolved in 99.8% ethanol, stored at -20 &#176;C) to the 10 mL of ...

Take a mouse embryonic carcinoma cell suspension capable of differentiating into various cell types.
Seed cells on a hydrophobic culture dish containing media and retinoic acid.
Incubate. Hydrophobic surfaces prevent cell adhesion, causing cells to form aggregates.
Retinoic acid enters cell aggregates, triggering signaling pathways and causing differentiation into neuronal progenitor cells.
Post-incubation, collect the cell aggregates.
Discard the media.
Add media containing retinoic acid and seed the aggregates onto a hydrophobic culture dish.
Incubate. Retinoic acid facilitates sustained cellular differentiation into neurons.
Collect the aggregates and remove the media.
Add a proteolytic enzyme to disrupt cell-cell adhesions within the aggregates.
Add media containing serum to stop the enzymatic activity.
Mechanically dissociate the aggregates, forming a neuronal suspension.
Centrifuge and discard the enzyme-rich supernatant.
Resuspend the neurons in media and seed them on an adherent multi-well plate containing media.
Incubate. Neurons adhere to the plate and mature, extending long projections.

retinoic-acid-induced-neurogenesis-using-mouse-embryonic-carcinoma

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

25 vues. Source : Leszczyński, P., et al., Neurogenèse à l’aide de cellules de carcinome embryonnaire P19 J. Vis. Exp. (2019) Cette vidéo présente une technique simple de neurogenèse induite par l’acide rétinoïque dans les cellules de carcinome embryonnaire de souris P19. L’acide rétinoïque active les facteurs de transcription et déclenche l’expression des gènes pour la différenciation neuronale. 

Vidéo: Neurogenèse induite par l’acide rétinoïque à l’aide de cellules de carcinome embryonnaire de souris - Concept

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

Trancher les cultures organotypiques d&#39;études de neurogenèse postnatale

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

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JoVE Journal

Biologie du développement

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

Différenciation efficace des cellules souches pluripotentes humaines en cellules hépatiques

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

The Mouse Hindbrain As a Model for Studying Embryonic Neurogenesis

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded forebrain neuroepithelium is not amenable to wholemount analysis to examine organ-wide neurogenesis patterns. Moreover, defining the mechanisms of forebrain neurogenesis is not necessarily predictive of neurogenesis in other parts of the brain; for example, due to the presence of forebrain-specific progenitor subtypes. The mouse hindbrain provides an alternative model for studying embryonic neurogenesis that is amenable to wholemount analysis, as well as tissue sections to observe the spatiotemporal distribution and behavior of neural progenitors. Moreover, it is easily dissected for other downstream applications, such as cell isolation or molecular biology analysis. As the mouse hindbrain can be readily analyzed in the vast number of cell lineage reporter and mutant mouse strains that have become available, it offers a powerful model for studying the cellular and molecular mechanisms of developmental neurogenesis in a mammalian organism. Here, we present a simple and quick method to use the mouse embryo hindbrain for analyzing mammalian neural progenitor cell (NPC) behavior in wholemount preparations and tissue sections.

This article demonstrates how the mouse embryonic hindbrain can be used as a model for studying developmental neurogenesis in both whole organ and tissue section preparations.

Retinoic Acid-Induced Neurogenesis using Mouse Embryonic Carcinoma Cells

Transcription

Retinoic Acid-Induced Neurogenesis using Mouse Embryonic Carcinoma Cells