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

Denne forskning blev støttet af Basic Science Research Program (2013R1A1A2060930) og Medical Research Center Program (2015R1A5A2009124) gennem Grundforskningsfonden Korea (NRF), finansieret af Ministeriet for Videnskab, IKT, og fremtidig planlægning. Denne forskning understøttes også af en start-up driftstilskud fra Hospital for Sick Children (HK Sung). Vi vil gerne takke Laura Barwell og Sarah JS Kim for deres fremragende hjælp i at redigere dette manuskript og Dr. Andras Nagy for at give G4 Mesc linje.

BEMÆRK: En skematisk af serum- og feeder-fri CM samling protokol er vist i figur 1. 1. Materialer (Udarbejdelse af MEF&#39;er, Medium, Plader, og løsninger) <ol><li> Forbered 500 ml medium til dyrkning af MEF. Supplement Dulbeccos modificerede Eagles medium (DMEM) med 10% FBS (ESC kvalitet), 50 enheder / ml penicillin og 50 mg / ml streptomycin. </li><li> Isoler MEF&#39;er fra embryoner efter en etableret rutine protokol 13 og vedligeholde dem i MEF medium. </li><li> Forbered 500 ...

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For en vellykket indsamling af serum- og feeder-fri Mesc-CM, bør tages hensyn til følgende forslag. Den mest kritiske faktor er at bruge tidlige passage mESCs for indsamling af Mesc-CM. Tidligere er det blevet vist, at tidlig passage Mesc-CM har bedre anti-aging effekter i forhold til sene passage mESCs. Passagen antal mESCs er blevet rapporteret til at påvirke deres udviklingsmæssige potentiale 16 og pluripotens 17. Mens yderligere forskning er nødvendig for at an...

Målet med denne protokol er at indsamle mus embryonale stamceller (Mesc) -konditioneret medium (Mesc-CM) fra serum- og feeder-fri dyrkningsbetingelser og at beskrive sine biologiske funktioner. Generelt embryonale stamceller (EKSF) har et stort potentiale for regenerativ medicin og celleterapi på grund af deres pluripotens og evne til selv-fornyelse 1-3. Men den direkte transplantation af stamceller har adskillige begrænsninger, såsom immun afstødning og tumordannelse 4,5. Derfor kan en celle-fri tilgang giver en alternativ terapeutisk strategi for regenerativ medicin og aldrende interventioner 6,7. Ældning ses som en cellulær modstykke til den aldrende væv og organer, kendetegnet ved en permanent tilstand af vækst anholdelse, ændret celle fysiologi og adfærd. Aldring er den vigtigste risikofaktor for udbredte sygdomme, herunder kræft, hjerte-karsygdomme, type 2-diabetes, og neurodegeneration 8. En af de åbenlyse karakteristika aldring er faldet i den regenerative potentiale af væv, som er forårsaget af stamceller aldring og udmattelse 9. Mange betydningsfulde undersøgelser har vist farmakologiske molekyler, såsom rapamycin 9, resveratrol 10, og metformin 11, og blodbårne systemiske faktorer, nemlig GDF11 12, der har evnen til konsekvent at forsinke ældning og forlænge levetiden. I den foreliggende undersøgelse har Mesc-CM blevet høstet uden serum (føtalt bovint serum, FBS) og føder (muse embryonale fibroblaster, MEF&#39;er) lag for at udelukke kontaminering af serum faktorer og sekretoriske faktorer fra MEF&#39;er. Disse betingelser er tilladt for en serum- og feeder-fri CM, der dermed muliggjort præcis identifikation af Mesc-specifikke sekretoriske faktorer. Denne foreslåede protokol er højeffektive, forholdsvis omkostningseffektive og letat operere. Denne teknik giver indblik i karakteriseringen af ​​Mesc-afledte opløselige faktorer, som kan mediere en anti-senescens virkning, som kan anvendes til udvikling af et sikkert og potentielt fordelagtig cellefri terapeutisk tilgang mod indgreb for aging-associerede sygdomme og andre regenerativ behandlinger.

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			<td height="43" style="height:43px;width:64px;">DMEM</td>
			<td style="width:64px;">Invitrogen</td>
			<td style="width:64px;">#11960-044</td>
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			<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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			<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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			<td height="22" style="height:22px;width:64px;">-mycin.</td>
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			<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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			<td style="width:64px;">Invitrogen</td>
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			<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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			<td height="85" style="height:85px;width:64px;">Leukemia inhibitory factor&#160;</td>
			<td style="width:64px;">Millipore</td>
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			<td style="width:64px;">100units/ml</td>
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			<td style="width:64px;">Invitrogen</td>
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			<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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			<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="64" style="height:64px;width:64px;">Dimethylformamide (DMF)</td>
			<td style="width:64px;">Sigma</td>
			<td style="width:64px;">#D4551</td>
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			<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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			<td height="85" style="height:85px;width:64px;">potassium ferrocyanide&#160;</td>
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			<td style="width:64px;">#455989</td>
			<td style="width:64px;">5mM</td>
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			<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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			<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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		<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>
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		<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>
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		<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>
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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>
		</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>
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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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		<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>
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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.

Oprindeligt er mESCs vedligeholdes på en MEF feeder i Mesc medium med FBS og andre kosttilskud (figur 1A og 2A). CM blev indsamlet fra mESCs i Reduceret Serum Media uden et fødelag, FBS, eller andre kosttilskud (figur 1B og 2B). Denne kultur tilstand tillader os at indsamle Mesc-specifikke medium uden potentiel forurening af faktorer fra feeder, FBS, eller andre kosttilskud. Kontrol medium blev indsamlet under de samme...

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

Denne protokol giver en metode til indsamling af musen embryonale stamceller (Mesc) -konditioneret medium (Mesc-CM) afledt af serum (føtalt bovint serum, FBS) - og feeder (mus embryonale fibroblaster, MEF&#39;er) -fri betingelser for en celle -fri tilgang. Det kan være anvendelig til behandling af aldring og aldring-associerede sygdomme.

Indsamling af serum- og Feeder-fri mus embryonale stamceller-conditioned Medium for en celle-fri tilgang

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 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 ...

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

Generering af induceret pluripotente stamceller fra humane perifere T-celler Brug af Sendai Virus i Feeder-fri Betingelser

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

Generering CRISPR / Cas9 Mediated monoallelisk Sletninger til Studieinformationen Enhancer funktion i mus embryonale 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-fri Udledning af melanocytter fra menneskelige pluripotente 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 mus embryonale fibroblaster med transkriptionsfaktorer at fremkalde 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 og funktion Studier i embryonale stamceller fra mus Brug rekombinase-medieret Cassette 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

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 ...

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

Definerede Xeno-fri og Feeder-fri kultur betingelser for den Generation af menneskelige iPSC-afledte Retinal Cell modeller

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 selvstyret metode til generering af hjerneorganoider fra menneskelige embryonale 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 af postganglioniske sympatiske neuroner ved hjælp af humane pluripotente stamceller under Feeder-fri og kemisk definerede kulturbetingelser

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