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

Dit onderzoek werd gesteund door de Basic Science Research Program (2013R1A1A2060930) en de Medical Research Center Program (2015R1A5A2009124) door de National Research Foundation Korea (NRF), gefinancierd door het ministerie van Wetenschap, ICT, en Toekomst Planning. Dit onderzoek wordt ook ondersteund door een Start-up exploitatiesubsidie ​​van de Hospital for Sick Children (HK Sung). We willen graag Laura Barwell en Sarah JS Kim bedanken voor hun uitstekende hulp bij het bewerken van dit manuscript en Dr. Andras Nagy voor het verstrekken van de G4 mES lijn.

OPMERKING: Een schema van de serum- en voedingsvrije CM collectie protocol wordt weergegeven in figuur 1. 1. Materialen (Voorbereiding van de MEF, Medium, platen, en oplossingen) <ol><li> Bereid 500 ml van medium tot de cultuur van de MEF. Aangevuld Dulbecco&#39;s gemodificeerd Eagle&#39;s medium (DMEM) met 10% FBS (ESC kwaliteit), 50 eenheden / ml penicilline en 50 mg / ml streptomycine. </li><li> Isoleer MEF uit embryo&#39;s na een vaste routine protocol 13 en te onderhouden in ME...

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Voor de succesvolle collectie van serum en feeder-free mES-CM, moeten de volgende suggesties in overweging worden genomen. De meest kritische factor is het gebruik van vroege passage mESCs voor het verzamelen van mES-CM. Eerder is aangetoond dat vroege passage mES-CM heeft betere anti-aging effect vergeleken met late passage mESCs. De passage aantal mESCs gemeld hun ontwikkelingspotentieel 16 en pluripotentie 17 beïnvloeden. Terwijl aanvulling behoeft de bijzondere ken...

Het doel van dit protocol is om de muis te verzamelen embryonale stamcellen (mES) -geconditioneerd medium (mES-CM) van serum en feeder-vrije kweekomstandigheden en zijn biologische functies te karakteriseren. In het algemeen zijn embryonale stamcellen (SER) hebben een groot potentieel voor regeneratieve geneeskunde en celtherapie als gevolg van hun pluripotentie en capaciteit voor zelf-vernieuwing 1-3. De directe transplantatie van stamcellen heeft verschillende beperkingen, zoals afstoting en tumorvorming 4,5. Daarom kan een cel-vrije benadering van een alternatieve therapeutische strategie voor de regeneratieve geneeskunde en veroudering interventies 6,7 bieden. Senescence wordt gezien als een cellulaire tegenhanger van de veroudering van weefsels en organen, gekenmerkt door een permanente staat van groeistop, veranderde cel fysiologie en gedrag. Veroudering is de belangrijkste risicofactor voor voorkomende ziekten zoals kanker, hart- en vaatziekten, tYpe 2 diabetes, en neurodegeneratie 8. Een voor de hand liggende kenmerken van veroudering is de daling van het regeneratieve vermogen van weefsels, die wordt veroorzaakt door stamcellen veroudering en uitputting 9. Veel belangrijke studies farmacologische moleculen, zoals rapamycine 9, resveratrol 10 en metformine 11, bloed overgedragen systemische factoren, namelijk GDF11 12, dat het vermogen om consequent veroudering te vertragen en verlengt de levensduur te laten geven. In de onderhavige studie werd mES-CM geoogst zonder serum (foetaal bovine serum, FBS) en feeder (muis embryonale fibroblasten MEF) lagen op de verontreiniging van serumfactoren en secretoire factoren uit MEFs sluiten. Deze voorwaarden zijn toegestaan ​​voor een serum en feeder-vrij CM dat bijgevolg kon de nauwkeurige identificatie van mES specifieke secretoire factoren. Deze voorgestelde protocol is zeer efficiënt, relatief kosteneffectief en eenvoudigopereren. Deze techniek geeft inzicht in de karakterisering van mES-afgeleide oplosbare factoren die een anti-veroudering effect kan bemiddelen, die kunnen worden gebruikt voor de ontwikkeling van een veilig en potentieel voordelige celvrije therapeutische benadering van interventies voor veroudering geassocieerde ziekten en andere regeneratieve behandelingen.

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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 height="22" style="height:22px;width:64px;">X-gal&#160;</td>
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			<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="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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		<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>
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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.

Oorspronkelijk worden mESCs bijgehouden op een MEF feeder in mES medium met FBS en andere supplementen (figuren 1A en 2A). CM werd verzameld mESCs verminderd serum medium zonder voedsterlaag, FBS of andere supplementen (figuren 1B en 2B). Deze cultuur voorwaarde stelt ons in staat om te verzamelen mES-specifieke geconditioneerd medium zonder mogelijke besmetting door de factoren uit de feeder, FBS of andere supplementen....

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

Dit protocol verschaft een werkwijze voor het verzamelen van muis embryonale stamcellen (mES) -geconditioneerd medium (mES-CM) verkregen uit serum (foetaal bovine serum, FBS) - en feeder (muis embryonale fibroblasten MEF) vrij voorwaarden voor een cel -gratis aanpak. Het kan dienstig voor de behandeling van veroudering en veroudering-geassocieerde ziekten.

Het verzamelen van serum en Feeder-vrij muis embryonale stamcellen geconditioneerd medium voor een 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 ...

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JoVE Journal

Developmental Biology

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

Neurale differentiatie van muis embryonale stamcellen in serum-vrije monolaagkweek

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

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

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Snelle, gerichte differentiatie van de epitheliale cellen van de retinale Pigment van menselijke embryonale of geïnduceerde pluripotente stamcellen

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

Gedefinieerde Xeno-vrij en kweekomstandigheden Feeder-gratis voor de generatie van menselijke iPSC afkomstige Retinal cel modellen

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

Een statische zelfsturende methode voor het genereren van hersenorganoïden uit menselijke embryonale stamcellen

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

Efficiënte differentiatie van postganglionic Sympathische neuronen met behulp van menselijke pluripotente stamcellen onder Feeder-vrije en chemisch gedefinieerde cultuurvoorwaarden

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