Name: collection of serum- and feeder-free mouse embryonic stem cell-conditioned medium for a cell-free approach
Uploaded: 2017-01-08T15:00:00
Description: Watch this Scientific Journal Video about Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach at JoVE.com

Cette recherche a été soutenue par le programme de base de recherche en sciences (2013R1A1A2060930) et le Programme Medical Research Center (2015R1A5A2009124) par la National Research Foundation de Corée (NRF), financé par le ministère de la Science, les TIC et la planification future. Cette recherche est également soutenu par un démarrage Subvention de fonctionnement de The Hospital for Sick Children (HK Sung). Nous tenons à remercier Laura Barwell et Sarah JS Kim pour leur excellente aide à la rédaction de ce manuscrit et le Dr Andras Nagy pour fournir la ligne G4 MESC.

REMARQUE: Un schéma du sérum et le protocole de collecte de CM sans chargeur-est représenté sur la figure 1. 1. Matériaux (Préparation du MEF, Medium, Plaques et Solutions) <ol><li> Préparer 500 ml de milieu de culture les MEF. Supplément milieu de Eagle modifié par Dulbecco (DMEM) avec 10% de FBS (qualité ESC), 50 unités / ml de pénicilline et 50 mg / ml de streptomycine. </li><li> Isoler MEFs à partir d&#39; embryons suivant un protocole de routine établie 13 et les ...

<ol>
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Pour le succès de la collecte de sérum et de MESC-CM sans alimentation, les suggestions suivantes devraient être prises en considération. Le facteur le plus important est l&#39;utilisation précoce mESCs de passage pour la collecte des MESC-CM. Auparavant, il a été montré que le passage précoce MESC-CM a de meilleurs effets anti-âge par rapport à mESCs de passage tardif. Le nombre de passage mESCs a été rapporté à affecter leur potentiel de développement 16 et 17 pluripotence. <p...

L&#39;objectif de ce protocole est de recueillir des souris embryonnaires cellules souches (MESC) moyenne Climatisé (MESC-CM) à partir des conditions de culture sans sérum et sans alimentation et de caractériser ses fonctions biologiques. En général, les cellules souches embryonnaires (CSE) ont un grand potentiel pour la médecine régénérative et la thérapie cellulaire en raison de leur pluripotence et la capacité d&#39;auto-renouvellement 1-3. Cependant, la transplantation directe de cellules souches a plusieurs limitations, telles que le rejet immunitaire et la formation de tumeurs 4,5. Par conséquent, une approche sans cellule peut fournir une stratégie thérapeutique alternative pour la médecine régénérative et les interventions de vieillissement 6,7. La sénescence est considérée comme une contrepartie cellulaire au vieillissement des tissus et des organes, caractérisé par un état permanent d&#39;arrêt de la croissance, la physiologie cellulaire altérée, et les comportements. Le vieillissement est le principal facteur de risque pour les maladies courantes, y compris le cancer, les maladies cardiovasculaires, type 2 diabète, et la neurodégénérescence 8. Une des caractéristiques évidentes du vieillissement est la baisse du potentiel de régénération des tissus, qui est causée par le vieillissement de cellules souches et de l&#39; épuisement 9. De nombreuses études ont montré d&#39; importantes molécules pharmacologiques, tels que la rapamycine 9, le resvératrol 10, et de la metformine 11, et les facteurs systémiques sang, à savoir GDF11 12, qui ont la capacité de retarder constamment le vieillissement et de prolonger la durée de vie. Dans la présente étude, MESC-CM a été récolté sans sérum (sérum fœtal bovin, FBS) et alimentation (fibroblastes de souris embryonnaires, MEF) couches d&#39;exclure la contamination des facteurs sériques et les facteurs sécrétoires de MEF. Ces conditions ont permis une CM et sans sérum d&#39;alimentation qui a permis par conséquent l&#39;identification précise des facteurs sécrétoires MESC spécifiques. Ce protocole proposé est très efficace, relativement rentable et facileopérer. Cette technique donne un aperçu de la caractérisation des facteurs solubles MESC dérivés qui peuvent arbitrer un effet anti-sénescence, qui peut être utilisé pour le développement d&#39;une approche thérapeutique acellulaire sûre et potentiellement avantageuse vers les interventions pour les maladies liées au vieillissement associées et autres régénérative traitements.

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		<tr height="43">
			<td height="43" style="height:43px;width:64px;">DMEM</td>
			<td style="width:64px;">Invitrogen</td>
			<td style="width:64px;">#11960-044</td>
			<td style="width:64px;"></td>
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			<td height="64" style="height:64px;width:64px;">FBS</td>
			<td style="width:64px;">Invitrogen</td>
			<td style="width:64px;">#30044333</td>
			<td style="width:64px;">20%, ES cell quality</td>
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		<tr height="126">
			<td height="148" rowspan="2" style="height:148px;width:64px;">Penicillin and streptomycin&#160;</td>
			<td rowspan="2" style="width:64px;">Invitrogen</td>
			<td rowspan="2" style="width:64px;">#15140</td>
			<td style="width:64px;">50units/ml penicillin and 50mg/ml strepto</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:64px;">-mycin.</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:64px;">L-glutamine&#160;</td>
			<td style="width:64px;">Invitrogen</td>
			<td style="width:64px;">#25030</td>
			<td style="width:64px;">2mM</td>
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		<tr height="106">
			<td height="106" style="height:106px;width:64px;">Nonessential amino acids (NEAA)&#160;</td>
			<td style="width:64px;">Invitrogen</td>
			<td style="width:64px;">#11140</td>
			<td style="width:64px;">100uM</td>
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			<td height="64" style="height:64px;width:64px;">&#946;-mercaptoethanol&#160;</td>
			<td style="width:64px;">Sigma</td>
			<td style="width:64px;">#M3148</td>
			<td style="width:64px;">100uM</td>
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		<tr height="85">
			<td height="85" style="height:85px;width:64px;">Leukemia inhibitory factor&#160;</td>
			<td style="width:64px;">Millipore</td>
			<td style="width:64px;">#ESG1107</td>
			<td style="width:64px;">100units/ml</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:64px;">OPTI-MEM</td>
			<td style="width:64px;">Invitrogen</td>
			<td style="width:64px;">#22600</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:64px;">X-gal&#160;</td>
			<td style="width:64px;">Sigma</td>
			<td style="width:64px;">#B4252</td>
			<td style="width:64px;">1mg/ml</td>
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		<tr height="64">
			<td height="64" style="height:64px;width:64px;">Paraformaldehyde (PFA)</td>
			<td style="width:64px;">Sigma</td>
			<td style="width:64px;">P6148</td>
			<td style="width:64px;">3.70%</td>
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		<tr height="64">
			<td height="64" style="height:64px;width:64px;">Dimethylformamide (DMF)</td>
			<td style="width:64px;">Sigma</td>
			<td style="width:64px;">#D4551</td>
			<td style="width:64px;"></td>
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		<tr height="85">
			<td height="85" style="height:85px;width:64px;">Potassium ferricyanide&#160;</td>
			<td style="width:64px;">Aldrich</td>
			<td style="width:64px;">#455946</td>
			<td style="width:64px;">5mM</td>
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		<tr height="85">
			<td height="85" style="height:85px;width:64px;">potassium ferrocyanide&#160;</td>
			<td style="width:64px;">Aldrich</td>
			<td style="width:64px;">#455989</td>
			<td style="width:64px;">5mM</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:64px;">NaCl&#160;</td>
			<td style="width:64px;">Sigma</td>
			<td style="width:64px;">#S7653</td>
			<td style="width:64px;">150mM</td>
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		<tr height="26">
			<td height="26" style="height:26px;width:64px;">MgCl2&#160;</td>
			<td style="width:64px;">Sigma</td>
			<td style="width:64px;">#M2393</td>
			<td style="width:64px;">2mM</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:64px;">Mytomycin C&#160;</td>
			<td style="width:64px;">Sigma</td>
			<td style="width:64px;">#M4287</td>
			<td style="width:64px;">10ug/ml</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:64px;">Propidium iodide&#160;</td>
			<td style="width:64px;">Sigma</td>
			<td style="width:64px;">#P4170</td>
			<td style="width:64px;">50ug/ml</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:64px;">TRIzol</td>
			<td style="width:64px;">Ambion</td>
			<td style="width:64px;">#15596018</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:64px;">M-MLV reverse transcript-tase</td>
			<td style="width:64px;">Promega</td>
			<td style="width:64px;">#M170B</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:64px;">Power SYBR Green PCR master mix&#160;</td>
			<td style="width:64px;">Applied Biosystems</td>
			<td style="width:64px;">#4367659</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:64px;">HDFs, NHDF-Ad-Der-Fibroblast&#160;</td>
			<td style="width:64px;">LONZA</td>
			<td style="width:64px;">#CC-2511</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:64px;">Bottle top filter,&#160;</td>
			<td style="width:64px;">Corning</td>
			<td style="width:64px;">#430513</td>
			<td style="width:64px;">0.2&#956;m</td>
		</tr>
	</tbody>
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collection of serum- and feeder-free mouse embryonic stem cell-conditioned medium for a cell-free approach

The capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate various cell types has opened new avenues in the field of regenerative medicine. However, despite their benefits, the tumorigenic potential of ESCs and iPSCs has long been a barrier for clinical applications. Interestingly, it has been shown that ESCs produce several soluble factors that can promote tissue regeneration and delay cellular aging, suggesting that ESCs and iPSCs can also be utilized as a cell-free intervention method. Therefore, the method for harvesting mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) with minimal contamination of serum components (fetal bovine serum, FBS) and feeder cells (mouse embryonic fibroblasts, MEFs) has been highly demanded. Here, the present study demonstrates an optimized method for the collection of mESC-CM under serum- and feeder-free conditions and for the characterization of mESC-CM using senescence-associated multiple readouts. This protocol will provide a method to collect pure mESC-specific secretory factors without serum and feeder contamination.

A l&#39; origine, mESCs sont maintenus sur un dispositif d&#39; alimentation en milieu MEF MESC avec du FBS et d&#39; autres suppléments (Figures 1A et 2A). CM ont été recueillies auprès de mESCs réduit Sérum médias sans couche d&#39;alimentation, FBS, ou d&#39; autres suppléments (figures 1B et 2B). Cette condition de culture nous permet de recueillir MESC spécifique milieu conditionné sans contamination poten...

Watch this Scientific Journal Video about Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach at JoVE.com

Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach

Ce protocole fournit une méthode pour la collecte de cellules souches embryonnaires de souris (MESC) de moyenne Climatisé (MESC-CM) dérivé de sérum (sérum fœtal bovin, FBS) - et d&#39;alimentation (fibroblastes embryonnaires de souris, MEF) conditions sans gluten pour une cellule approche -free. Il peut être applicable pour le traitement du vieillissement et de maladies associées au vieillissement.

Collection de Serum et libre Feeder-Mouse Embryonic Stem Cell conditionné moyenne pour une approche sans cellule

The capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate various cell types has opened new avenues in the field ...

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Developmental Biology

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

Différenciation neurale des souris de cellules souches embryonnaires dans le sérum sans culture monocouche

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

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Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Génération de cellules souches pluripotentes induites à partir de cellules T périphériques humains à l&#39;aide de virus Sendai dans des conditions libres Feeder-

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

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

Rapide, réalisée la différenciation des cellules épithéliales pigmentaire rétinien de cellules souches pluripotentes humaines embryonnaires ou induit

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

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

Xeno-libre défini et exempte d’engraissement des Conditions de Culture pour les modèles de cellule rétinienne Generation of Human dérivés iPSC

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of ...

A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells

Une méthode statique auto-dirigée pour générer des organoïdes cérébraux à partir de cellules souches embryonnaires humaines

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a ...

Efficient Differentiation of Postganglionic Sympathetic Neurons using Human Pluripotent Stem Cells under Feeder-free and Chemically Defined Culture Conditions

Différenciation efficace des neurones sympathiques postganglioniques utilisant des cellules souches pluripotentes humaines dans des conditions de culture sans alimentation et définies chimiquement

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously ...