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 forskningen ble støttet av Basic Science Research Program (2013R1A1A2060930) og Medical Research Center Program (2015R1A5A2009124) gjennom National Research Foundation of Korea (NRF), finansiert av departementet for vitenskap, IKT, og fremtidig planlegging. Denne forskningen er også støttet av en start-up Drifts Grant fra The Hospital for Sick Children (HK Sung). Vi vil gjerne takke Laura Barwell og Sarah JS Kim for deres gode hjelp i å endre denne manuskript og Dr. Andras Nagy for å gi G4 Mesc linje.

MERK: En skjematisk av serum-og mater fritt CM samling protokollen er vist i figur 1. 1. Materialer (Utarbeidelse av MEFs, Medium, plater og løsninger) <ol><li> Forbered 500 ml medium til kultur de MEFs. Supplement Dulbeccos modifiserte Eagles medium (DMEM) med 10% FBS (ESC kvalitet), 50 enheter / ml penicillin og 50 mg / ml streptomycin. </li><li> Isolere MEFs fra embryoer etter en etablert rutine protokoll 13 og holde dem i MEF medium. </li><li> Forbered 500 ml medium til kultur ...

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For en vellykket samling av serum-og mater fritt Mesc-CM, bør følgende forslag tas i betraktning. Den mest kritiske faktoren er å bruke tidlige passasje mESCs for innsamling av Mesc-CM. Tidligere har det vist seg at tidlig passasje Mesc-CM har bedre anti-aging effekt sammenlignet med senere passasje mESCs. Passasjen antall mESCs har blitt rapportert å påvirke deres utviklingspotensial 16 og pluripotency 17. Selv om ytterligere forskning er nødvendig for å analyse...

Målet med denne protokollen er å samle mus embryonale stamceller (Mesc) -conditioned medium (Mesc-CM) fra serum-og mater frie dyrkningsforhold og for å karakterisere sine biologiske funksjoner. Generelt, embryonale stamceller (ESCS) har et stort potensiale for regenerativ medisin og celleterapi på grunn av deres pluripotency og evne til selvfornyelse 1-3. Imidlertid er direkte transplantasjon av stamceller har flere begrensninger, for eksempel immunavstøtning og tumordannelse 4,5. Derfor kan en celle-fri tilnærming gir en alternativ behandlingsstrategi for regenerativ medisin og aldring intervensjoner 6,7. Senescence blir sett på som en mobil motstykke til aldring av vev og organer, preget av en permanent tilstand av vekst arrest, endret cellefysiologi og atferd. Aldring er den viktigste risikofaktor for utbredte sykdommer, inkludert kreft, hjerte- og karsykdommer, type 2 diabetes, og neurodegeneration 8. En av de åpenbare karakteristikkene av aldring er nedgangen i den regenerative potensialet i vev, som er forårsaket av stamcelle aldring og utmattelse 9. Mange betydelige studier har vist farmakologiske molekyler, slik som rapamycin 9, resveratrol 10, og metformin 11, og blodbårne systemiske faktorer, nemlig GDF11 12, som har evnen til å gjennomgående forsinke aldring og forlenge levetiden. I denne studien har Mesc-CM blitt høstet uten serum (føtalt bovint serum, FBS) og mater (mus embryonale fibroblaster, MEFs) lag for å utelukke forurensning av serumfaktorer og sekretoriske faktorer fra MEFs. Disse forholdene er tillatt for en serum-og mater fritt CM som dermed muliggjorde nøyaktig identifikasjon av Mesc-spesifikke sekretoriske faktorer. Denne foreslåtte protokoll er svært effektiv, forholdsvis kostnadseffektiv og lettå operere. Denne teknikken gir innsikt i den karakteriseringen av Mesc-avledet oppløselige faktorer som kan mediere en anti-senescens virkning, som kan anvendes for utvikling av en sikker og potensielt fordelaktige cellefritt terapeutisk tilnærming mot intervensjoner for aldring-assosierte sykdommer og andre regenerative 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;">20%, ES cell quality</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;">2mM</td>
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			<td style="width:64px;">100uM</td>
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			<td height="85" style="height:85px;width:64px;">Leukemia inhibitory factor&#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="85" style="height:85px;width:64px;">potassium ferrocyanide&#160;</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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			<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.

Opprinnelig er mESCs opprettholdes på en MEF mater i Mesc medium med FBS og andre kosttilskudd (figur 1A og 2A). CM ble samlet inn fra mESCs i Redusert Serum Media uten en mater lag, FBS, eller andre kosttilskudd (Tall 1B og 2B). Denne kulturen tilstanden tillater oss å samle Mesc spesifikke kondisjonert medium uten potensiell forurensning av faktorene fra materen, FBS, eller andre kosttilskudd. Kontrollmediet ble saml...

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 protokollen gir en fremgangsmåte for innsamling av muse-embryonale stamceller (Mesc) -conditioned medium (Mesc-CM) avledet fra serum (føtalt bovint serum, FBS) - og mater (mus embryonale fibroblaster, MEFs) -fri forhold for en celle -gratis tilnærming. Det kan være aktuelt for behandling av aldring og aldring-assosierte sykdommer.

Innsamling av serum-og mater-fri Mouse Embryonic Stem Cell kondisjonerte Medium for en Cell-fri tilnærming

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 Differensiering av mus embryonale stamceller i Serum-free enlagskultur

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 Induced Pluripotent stamceller fra menneske perifere T-celler Bruke Sendai Virus i Feeder-free 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 mediert Monoallelic Slett å studere Enhancer funksjon 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 Utledning av Melanocytter fra Menneskelig Pluripotent 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 transkripsjonsfaktorer for å indusere 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-funksjon studier i mus embryonale stamceller Bruke recombinase-mediert Cassette Veksling

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

Rask, rettet differensiering av netthinnens Pigment epitelceller fra menneskelige embryonale eller indusert Pluripotent 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

Definerte Xeno-fri og mater-fri kultur betingelser for generering av menneskelig iPSC-avledet Retinal celle 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 selvrettet metode for å generere hjerneorganoider fra humane 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 differensiering av postganglioniske sympatiske nevroner ved hjelp av humane pluripotente stamceller under materfrie og kjemisk definerte kulturforhold

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