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

Bu araştırma Temel Bilimler Araştırma Programı (2013R1A1A2060930) ve Tıbbi Araştırma Merkezi Programı (2015R1A5A2009124) Kore Ulusal Araştırma Vakfı aracılığıyla (UÇK), Bilim, BİT Bakanlığı tarafından finanse edilen ve Gelecek Planlama tarafından desteklenmiştir. Bu araştırma aynı zamanda Hastanesi Çocuk Hastalıkları (HK Sung) bir Başlangıç ​​İşletim Grant tarafından desteklenmektedir. Biz G4 Mesc hattını sağlamak için bu el yazması ve Dr. Andras Nagy düzenleme onların mükemmel yardım için Laura Barwell ve Sarah JS Kim teşekkür etmek istiyorum.

Not: serum- ve besleyici-CM toplama protokolün bir şeması, Şekil 1 &#39;de gösterilmiştir. 1. Malzemeler (MEFS Hazırlanması, Orta, Tabaklar, ve Çözümler) <ol><li> Kültür MEF&#39;ler orta 500 ml hazırlayın. % 10 FBS (ESC kalitesi), 50 birim / ml penisilin ve 50 mg / ml streptomisin içeren Dulbecco Modifiye Kartal Ortamı (DMEM) Supplement. </li><li> Kurulmuş bir rutin protokolü 13 aşağıdaki embriyolar MEFS ayırmak ve MEF ortamda onları korumak. </li><li> kült?...

<ol>
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serum- ve besleyici içermeyen Mesc-CM başarıyla toplanması için aşağıdaki öneriler dikkate alınmalıdır. En önemli faktör Mesc-CM toplanması için erken geçiş mESCs kullanıyor. Daha önce, bu erken geçiş Mesc-CM geç geçişli mESCs göre daha iyi bir anti-yaşlanma etkileri olduğu gösterilmiştir. MESCs geçiş sayısı gelişim potansiyeli 16 ve pluripotensini 17 etkilediği bildirilmiştir. Ek araştırmalar, anti-yaşlılık etkilere neden Mesc ...

Bu protokolün amacı, serum- ve besleyici içermeyen kültür koşullarından fareye embriyonik kök hücre (Mesc) -conditioned ortamı (Mesc-CM) toplamak ve biyolojik fonksiyonları karakterize etmektir. Genel olarak, embriyonik kök hücreler (EKH) nedeniyle kendini yenileme 1-3 onların pluripotensin ve kapasite rejeneratif tıp ve hücre tedavisinde büyük bir potansiyele sahiptir. Ancak, kök hücrelerin doğrudan nakli gibi bağışıklık reddi ve tümör oluşumu 4,5 gibi çeşitli sınırlamalar vardır. Bu nedenle, bir hücre barındırmayan bir yaklaşım rejeneratif tıpta ve yaşlanma müdahaleler 6,7 için alternatif bir tedavi stratejisini sağlayabilir. Yaşlılık büyüme durması, değişmiş hücre fizyolojisi ve davranışları sürekli devlet tarafından karakterize doku ve organların yaşlanması, bir hücresel meslektaşı olarak görülüyor. kanser dahil yaygın hastalıklar, kardiyovasküler hastalık, t ana risk faktörüdür Yaşlanma2 diyabet ype ve nörodejenerasyon 8. Yaşlanmanın bariz özelliklerinden biri kök hücre yaşlanması ve bitkinlik 9 kaynaklanan dokuların rejeneratif potansiyelinin, düşüş olduğunu. Birçok önemli çalışmalar, sürekli yaşlanmayı geciktirmek ve ömrünü uzatmak için yeteneği var rapamisin 9, resveratrol 10, ve metformin 11 ve kan yoluyla bulaşan sistemik faktörler, yani GDF11 12 gibi farmakolojik molekülleri göstermiştir. Bu çalışmada, Mesc-CM, serum faktörleri ve MEF&#39;lerden salgısal faktör kontaminasyonu ortadan kaldırır serumsuz (fetal inek serumu, FBS) ve besleyici (fare embriyonik fibroblastlar, MEF&#39;ler) katmanları hasat edilmiştir. dolayısıyla Mesc özgü salgı faktörlerin doğru tespit etkin bir serum- ve besleyici içermeyen CM izin Bu koşullar. Bu önerilen protokol nispeten yüksek verimli, etkin maliyet ve kolayçalıştırmak için. Bu teknik, yaşlanma ile ilişkili hastalıklar ve diğer yenileme için girişimler karşı güvenli ve potansiyel olarak avantajlı bir hücresiz terapötik bir yaklaşım geliştirilmesi için kullanılabilecek bir anti-yaşlılık etkisi aracılık edebilen Mesc türetilen çözünür faktörlerin tanımlanması, kavranmasını sağlar tedaviler.

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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>
			<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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			<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 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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			<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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			<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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			<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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			<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>
			<td style="width:64px;">Aldrich</td>
			<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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		<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>
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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>
		</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>
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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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		<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.

Başlangıçta, mESCs FBS ve diğer ek (Şekil 1 A ve 2A) ile Mesc ortam içinde bir MEF besleyici tutulur. CM besleyici tabaka FBS ya da diğer ek (Şekil 1B ve 2B) olmadan düşük serum ortamında mESCs toplanmıştır. Bu kültür durum bize besleyici, FBS veya diğer takviyeleri faktörler tarafından potansiyel kirlenme olmadan klimalı ortam Mesc-özgü toplamak için izin verir. Kontrol ortamı mESCs olmadan ayn?...

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

Bu protokol, serumdan türetilmiş fare embriyonik kök hücrelerin içinde (Mesc) -conditioned ortamı (Mesc-CM) için bir yöntem sağlar (fetal inek serumu, FBS) - ve besleyici (fare embriyonik fibroblastlar, MEF&#39;ler) bir hücrenin içermeyen koşullar -ücretsiz yaklaşım. Bu yaşlanma ve yaşlanma ile ilişkili hastalıkların tedavisi için uygun olabilir.

Serum- toplanması ve Besleyici serbest Fare Embriyonik Hücre içermeyen Yaklaşım Hücre klimalı Orta Kök

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

Serum serbest Tek tabakalı Kültüründe Fare Embriyonik Kök Hücre Sinir Farklılaşma

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

Besleyici içermeyen şartlar Sendai virüs kullanılarak insan periferik T hücrelerinden uyarılmış pluripotent kök hücrelerin üretilmesi

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

Yaratma CRISPR / Cas9 aracılığı monoallelik Delesyonlar Fare Embriyonik Kök Hücreleri Artırıcı Fonksiyonu Eğitim için

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

Insan pluripotent kök hücreleri gelen melanosit besleyici içermeyen türetilmesi

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

Transkripsiyon Faktörleri ile fare embriyonik fibroblastlar yeniden programlama bir Hemogenic Programı tetikleyebilecek

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

Recombinase aracılı Kaset Değişimi Kullanarak Fare Embriyonik Kök Hücre yapı-fonksiyon Çalışmaları

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

Hızlı, yönlendirilmiş farklılaşma insan embriyonik veya İndüklenmiş Pluripotent kök hücrelerinden retina Pigment epitel hücrelerinin

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

Tanımlanmış Xeno-alerjik ve besleyici-Alerjik kültür koşulları üretimi insan IPSC kaynaklı Retina hücre modelleri için

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

İnsan Embriyonik Kök Hücrelerinden Beyin Organoidleri Üretmek için Statik Kendi Kendini Yöneten Bir Yöntem

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

Besleyicisiz ve Kimyasal Olarak Tanımlanmış Kültür Koşulları Altında İnsan Pluripotent Kök Hücreleri Kullanan Postgangliyonik Sempatik Nöronların Verimli Farklılaşması

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