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

Denna forskning stöddes av Basic Science Research Program (2013R1A1A2060930) och Medical Research Center Program (2015R1A5A2009124) genom National Research Foundation of Korea (NRF), som finansieras av ministeriet för vetenskap, IT och framtida planering. Denna forskning stöds också av en start-up driftsbidrag från sjukhuset för sjuka barn (HK Sung). Vi vill tacka Laura Barwell och Sarah JS Kim för deras utmärkta hjälp att redigera detta manuskript och Dr Andras Nagy för att tillhandahålla G4 Mesc linje.

ANMÄRKNING: En schematisk bild av serum- och feeder-fria CM samling protokoll visas i figur 1. 1. Material (Beredning av MEF, Medium, tallrikar, och lösningar) <ol><li> Bered 500 ml medium att odla MEF. Komplettera Dulbeccos modifierade Eagles medium (DMEM) med 10% FBS (ESC kvalitet), 50 enheter / ml penicillin och 50 mg / ml streptomycin. </li><li> Isolera MEF från embryon efter en etablerad rutin protokoll 13 och hålla dem i MEF medium. </li><li> Bered 500 ml medium att odla m...

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För en framgångsrik insamling av serum-och feeder-fri Mesc-CM, bör följande förslag beaktas. Den mest kritiska faktorn är att använda tidig passage mESCs för insamling av Mesc-CM. Tidigare har det visat sig att en tidig passage Mesc-CM har bättre anti-aging effekter jämfört med slutet av passage mESCs. Passagen antalet mESCs har rapporterats påverka deras utvecklingspotential 16 och pluripotens 17. Medan ytterligare forskning behövs för att analysera de sp...

Målet med detta protokoll är att samla mus embryonala stamceller (Mesc) -konditionerat medium (Mesc-CM) från serum-och matarfria odlingsbetingelser och för att karakterisera dess biologiska funktioner. I allmänhet, embryonala stamceller (ESC) har stor potential för regenerativ medicin och cellterapi på grund av deras pluripotens och förmåga till självförnyelse 1-3. Emellertid den direkta transplantation av stamceller har flera begränsningar, såsom immunavstötning och tumörbildning 4,5. Därför kan ett cellfritt förhållningssätt ger en alternativ terapeutisk strategi för regenerativ medicin och åldrande insatser 6,7. Åldrande ses som en cellulär motsvarighet till den åldrande vävnader och organ, som kännetecknas av ett permanent tillstånd av tillväxthämning, förändrad cellfysiologi, och beteenden. Åldrande är den viktigaste riskfaktorn för sjukdomarna inklusive cancer, hjärt- och kärlsjukdomar, tYP 2-diabetes, och neurodegeneration 8. En av de uppenbara egenskaperna hos åldrande är nedgången i regenerativ potential vävnader, som orsakas av stamcells åldrande och utmattning 9. Många betydande studier har visat farmakologiska molekyler, såsom rapamycin 9, resveratrol 10, och metformin 11, och blodburna systemiska faktorer, nämligen GDF11 12, som har förmågan att konsekvent fördröja åldrande och förlänga livslängden. I den aktuella studien har Mesc-CM skördats utan serum (fetalt bovinserum, FBS) och matare (mus embryonala fibroblaster, MEF) skikt för att utesluta kontaminering av serumfaktorer och sekretoriska faktorer från MEF. Dessa villkor är tillåtna för en serum-och matarfria CM som därmed möjliggjorde korrekt identifiering av Mesc specifika sekretoriska faktorer. Denna föreslagna protokoll är mycket effektiv, relativt kostnadseffektiv och lättatt driva. Denna teknik ger insikter i karakterisering av Mesc härledda lösliga faktorer som kan förmedla en anti-åldrande effekt, som kan användas för att utveckla en säker och potentiellt fördelaktiga cellfritt terapeutiskt tillvägagångssätt mot insatser för äldre relaterade sjukdomar och andra regenerativ behandlingar.

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			<td height="43" style="height:43px;width:64px;">DMEM</td>
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			<td style="width:64px;">50units/ml penicillin and 50mg/ml strepto</td>
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			<td style="width:64px;">100uM</td>
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			<td height="22" style="height:22px;width:64px;">X-gal&#160;</td>
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			<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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			<td height="22" style="height:22px;width:64px;">NaCl&#160;</td>
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			<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>
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			<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>
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			<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>
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			<td height="43" style="height:43px;width:64px;">TRIzol</td>
			<td style="width:64px;">Ambion</td>
			<td style="width:64px;">#15596018</td>
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		<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>
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		<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>
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		<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>
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			<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>
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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.

Ursprungligen är mESCs hölls på en MEF matare i Mesc medium med FBS och andra kosttillskott (figurerna 1A och 2A). CM samlades in från mESCs i minskade serum Media utan ett matarskikt, FBS, eller andra tillskott (figurerna 1B och 2B). Denna kultur tillstånd tillåter oss att samla Mesc specifika konditionerat medium utan potentiell förorening av faktorerna från mataren, FBS, eller andra kosttillskott. Kontrollmedi...

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

Detta protokoll ger en metod för insamling av mus embryonala stamceller (Mesc) -konditionerat medium (Mesc-CM) härstammar från serum (fetalt bovint serum, FBS) - och matar (mus embryonala fibroblaster, MEF) -fria förutsättningar för en cell -fri tillvägagångssätt. Det kan vara tillämplig för behandling av åldrande och åldersassocierade sjukdomar.

Insamling av Serum och Feeder fria Mouse embryonala stamceller konditionerade medium för ett cellfritt 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 ...

Research

JoVE Journal

Developmental Biology

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

Neural differentiering av Mouse embryonala stamceller i Serumfri enskiktskultur

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

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

Generering av inducerade pluripotenta stamceller från humana perifera T-celler med Sendai virus i Feeder fria förhållanden

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

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Generera crispr / Cas9 medierad monoallel Strykningar att studera Enhancer Funktion i mus embryonala stamceller

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Feeder-fria Derivering av Melanocyter från Human pluripotenta stamceller

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

Omprogrammering Mouse Embryonala fibroblasts med transkriptionsfaktorer att inducera en Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Struktur-funktionsstudier i mus embryonala stamceller Använda Recombinase-medierad kassett Exchange

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

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

Snabb, regisserad differentiering av Retinal Pigment epitelial celler från mänskliga embryonala eller inducerade pluripotenta stamceller

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

Definierade Xeno-fri och Feeder-fri odlingsbetingelser för den Generation av mänskliga iPSC-derived Retinal cellmodeller

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

En statisk självstyrd metod för att generera hjärnorganoider från mänskliga embryonala stamceller

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

Effektiv differentiering av postganglitoniska sympatiska nervceller med hjälp av mänskliga pluripotenta stamceller under Feeder-fria och kemiskt definierade kulturförhållanden

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