Name: an alternative culture method to maintain genomic hypomethylation of mouse embryonic stem cells using mek inhibitor pd0325901 and vitamin c
Uploaded: 2018-06-01T13:00:00
Description: Watch this Scientific Journal Video about An Alternative Culture Method to Maintain Genomic Hypomethylation of Mouse Embryonic Stem Cells Using MEK Inhibitor PD0325901 and Vitamin C at JoVE.com

这项工作得到了中国国家自然科学基金 (21435008 和 21327006) 和中国科学院战略优先研究项目 (XDB14030200) 的支持。

1. 筹备工作
<ol><li>
制备1.0 毫米 PD0325901 (酮抑制剂) 和3.0 毫米 CHIR99021 (GSK3 抑制剂) 的溶液。
	<ol>
<li>重2毫克的 PD0325901 和添加4.15 毫升亚砜在琥珀玻璃瓶。</li>
		<li>重2毫克的 CHIR99021 和添加1.43 毫升亚砜在琥珀玻璃瓶。</li>
		<li>重组后, 储存整除数 (50 µL/管在200µL PCR 管) 在-20 摄氏度和保护免受光。在使用前, 从冷藏库中取出含有冰冻库存的管子, 在室温下解冻。</li>
	</ol>
</li>
	<li>将 Vc ...

<ol>
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在这项工作中, 我们展示了一种新的方法, 结合 vc 和 PD0325901 维持小鼠 ES 细胞在未分化和 hypomethylated 的状态, 这是通过协同行动促进 dna 去甲基化的 Vc 和抑制从头 dna甲基化的 PD0325901。此外, 小鼠 ES 细胞在 Vc/PD0325901 培养体系下表现出很大的形态学。
为了更好地维持 Vc/PD0325901 培养系统中小鼠 ES 细胞的状态, 有一些关键步骤。首先, 需要预先筛选出血清批次, 以发现和储?...

ES 细胞来源于胚泡1的 ICM。细胞处于干细胞状态, 可以形成所有体细胞血统和生殖细胞2。ES 细胞的建立为研究体外的发育过程提供了机会, 并可根据其干细胞3生成再生医学的医学相关性细胞。
两组 seminally 在1981年建立了小鼠 ES 细胞系, 当从早期小鼠胚胎中提取的细胞以小鼠胚胎成纤维细胞 (MEFs) 的胎牛血清 (含血清) 培养, 作为进纸层1,4.MEFs 被灭活 mitotically, 在培养 ES 细胞前先在菜肴中生长。MEFs 为小鼠 ES 细胞附着提供支持, 并产生生长因子, 促进传播和抑制分化5, 而血清为细胞增殖提供必需的营养因子和荷尔蒙。随后的研究表明, 饲养细胞产生的 lif 是小鼠 ES 细胞干细胞自我更新和维持的关键细胞因子, 在培养基中加入 lif 可以替代馈线细胞6。目前, 喂食器中小鼠 ES 细胞的维持仍然是许多研究者采用的标准方法。然而, 这一古典文化方法也出现了一些问题。首先, 饲养细胞分泌过量和不受控制的因素, 可能导致致病性污染7。为避免这种干扰, 用明胶对菜肴表面进行涂敷, 并在含血清培养基中添加 LIF, 是维持不带进料层细胞的小鼠 ES 细胞的替代方法。此外, 在血清/LIF 条件下生长的小鼠 ES 细胞在细胞群中呈现形态学异质性, 甚至在干细胞相关因子的表达水平上表现为8。最近的研究表明, 在血清/生命条件下, 与干细胞相关的核心转录因子 (包括 SOX2, 北美和 OCT4) 可以维持干细胞通过 LIF 和 WNT 信号;然而, 值得注意的是, 它们还激活了成纤维细胞生长因子信号, 触发分化8。由于核心转录因子的矛盾双重作用, 在血清中培养的小鼠 ES 细胞呈现出不均一性, 由两个可互换的种群组成, 一个类似于 ICM, 另一个相似于引啮鼠动物状态8。此外, 血清中的小鼠 ES 细胞通常表现为全球甲基9, 而 ICM 和 PGCs 处于全局 hypomethylated 状态, 与干细胞9、10紧密相连。
开发新的培养小鼠 ES 细胞的方法有相当大的需求。自 2003年11以来已建立了若干改进的协议, 但仍然存在一些限制和缺点7。自2008年以来, 两个小分子激酶抑制剂 PD0325901 (丝裂原活化蛋白激酶 (MAPK)/胞外信号调节激酶 (ERK)) 和 CHIR99021 (糖原合成酶抑制剂) 的联合利用激酶 3 (GSK3)), 在 N2B27培养基与 LIF 和没有血清已经打开新的观点培养小鼠 ES 细胞12。这种新定义的培养基的特点是使用两种抑制剂 (2i)。在2干细胞培养基中培养的小鼠 ES 细胞在细胞群中比较均匀, 表达的因子也较多。此外, 2 的 i/低甲基化培养的小鼠 ES 细胞在全球范围内呈现 DNA, 它接近于 ICM 样细胞 9, 13.即便如此, 2i 文化也有其弊端。PD0325901 和 CHIR99021 不溶于水, 一般被溶于二甲基亚砜 (双砜) 的库存溶液中, 添加到培养基中。研究表明, 长期和低剂量的细胞暴露于亚砜可能导致细胞毒性14。
在这里, 我们利用了两个小分子化合物, 并开发了一种新的培养方法的小鼠 ES 细胞。这种新的培养方法结合 Vc 和甲酮抑制剂 PD0325901, 以促进 DNA 低甲基化迅速和有效地向可比水平的 PGC, 命名为 Vc/PD0325901 培养协议。Vc/PD0325901-added 血清中的小鼠 ES 细胞在形态学上表现为同质性, 并维持在基态。与2i 培养相比, Vc/PD0325901 条件下培养的小鼠 ES 细胞具有更快的 DNA 去甲基化动力学, 可达到与 PGC 相比的低甲基化水平。另外, 使用单一抑制剂 (PD0325901) 将亚砜的输入量减少到培养基中, 与 2i (PD0325901/CHIR99021) 中使用的相比, 减少了对细胞的损伤。

<table border="0" cellpadding="0" cellspacing="0" width="790">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">fetal bovine serum Australia source</td>
			<td style="width:205px;">Corning</td>
			<td style="width:119px;">35-076-CV</td>
			<td style="width:251px;">Component of mouse ES cell medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/high glucose</td>
			<td style="width:205px;">Hyclone</td>
			<td style="width:119px;">SH30022</td>
			<td>Component of mouse ES cell medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LIF</td>
			<td>Millipore</td>
			<td style="width:119px;">ESG1107</td>
			<td>Component of mouse ES cell medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">non-essential amino acids (NEAA)</td>
			<td style="width:205px;">Gibco</td>
			<td style="width:119px;">11140050</td>
			<td>Component of mouse ES cell medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium pyruvate</td>
			<td style="width:205px;">Gibco</td>
			<td style="width:119px;">11360070</td>
			<td>Component of mouse ES cell medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine</td>
			<td style="width:205px;">Gibco</td>
			<td style="width:119px;">25030081</td>
			<td>Component of mouse ES cell medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">penicillin streptomycin solution</td>
			<td style="width:205px;">Gibco</td>
			<td style="width:119px;">15140122</td>
			<td>Component of mouse ES cell medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:119px;">P5493</td>
			<td>Cells rinse</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">trypsin</td>
			<td style="width:205px;">Hyclone</td>
			<td style="width:119px;">SH30042</td>
			<td>Cell dissociation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">gelatin</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:119px;">48722</td>
			<td>Dishes coating</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PD0325901</td>
			<td>Stemolecule</td>
			<td>04-0006-10</td>
			<td>small-molecule chemical for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CHIR99021</td>
			<td>Stemolecule</td>
			<td>04-0004</td>
			<td>small-molecule chemical for cell culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">vitamin C</td>
			<td style="width:205px;">Sinopharm Chemical Reagent Co.,Ltd&#160;</td>
			<td style="width:119px;">XW00508171</td>
			<td>small-molecule chemical for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra Bioscience water purification systemr</td>
			<td style="width:205px;">Purelab</td>
			<td style="width:119px;">Ultra</td>
			<td>Ultra water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:119px;">D8418</td>
			<td>Cell freezing medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">centrifuger</td>
			<td style="width:205px;">Eppendorf</td>
			<td style="width:119px;">5427R</td>
			<td>DNA and protein extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">protease inhibitor cocktail</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:119px;">P8340</td>
			<td>Component of RIPA buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">PMSF Protease Inhibitor</td>
			<td style="width:205px;">ThermoFisher Scientific</td>
			<td style="width:119px;">36978</td>
			<td>Component of RIPA buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DTT</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:119px;">43815</td>
			<td>Component of RIPA buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGTA</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:119px;">E3889</td>
			<td>Component of RIPA buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Genomic DNA Purification Kit&#160;</td>
			<td style="width:205px;">Promega</td>
			<td style="width:119px;">A1125</td>
			<td>Genomic DNA extraction</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">NanoDrop 2000</td>
			<td style="width:205px;">ThermoFisher Scientific</td>
			<td style="width:119px;">NanoDrop 2000</td>
			<td>Determination of DNA concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">calf intestinal phosphatase</td>
			<td>New England Biolabs</td>
			<td style="width:119px;">M0290</td>
			<td>DNA digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase I</td>
			<td>New England Biolabs</td>
			<td style="width:119px;">M0303</td>
			<td>DNA digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">snake venom phosphodiesterase I</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:119px;">P4506</td>
			<td>DNA digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nanosep 3K Omega</td>
			<td>Pall</td>
			<td style="width:119px;">OD003C35</td>
			<td>filtration of digested DNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1290 UHPLC system</td>
			<td style="width:205px;">Agilent&#160;</td>
			<td style="width:119px;">1290</td>
			<td>UHPLC separation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">G6410B triple quadrupole mass spectrometer</td>
			<td style="width:205px;">Agilent&#160;</td>
			<td style="width:119px;">G6410B</td>
			<td>MS/MS analysis</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Zorbax Eclipse Plus C18 column</td>
			<td style="width:205px;">Agilent&#160;</td>
			<td style="width:119px;">Zorbax Eclipse Plus</td>
			<td>colume for UHPLC separation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">5-methylcytosine</td>
			<td>Solarbio Life Sciences</td>
			<td style="width:119px;">SM8900&#160;</td>
			<td>standard 5mC</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">5-hydroxymethylcytosine (standard)</td>
			<td>Toronto Research Chemicals</td>
			<td style="width:119px;">M295900</td>
			<td>standard 5hmC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prdm14 antibody</td>
			<td>bioworld</td>
			<td>BS7634</td>
			<td>Western blot analysis-primary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dnmt3a antibody</td>
			<td>bioworld</td>
			<td>BS6587</td>
			<td>Western blot analysis-primary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dnmt3b antibody</td>
			<td>abcam</td>
			<td>ab13604</td>
			<td>Western blot analysis-primary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dnmt3l antibody</td>
			<td>abcam</td>
			<td>ab3493</td>
			<td>Western blot analysis-primary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dnmt1 antibody</td>
			<td>abcam</td>
			<td>ab13537</td>
			<td>Western blot analysis-primary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#946;-tubulin antibody</td>
			<td>bioworld</td>
			<td>BS1482MH</td>
			<td>Western blot analysis-primary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat anti-rabbit IgG</td>
			<td>abcam</td>
			<td style="width:119px;">ab6721</td>
			<td>Western blot analysis-secondary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat anti-mouse IgG</td>
			<td>abcam</td>
			<td style="width:119px;">ab6789</td>
			<td>Western blot analysis-secondary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trizol</td>
			<td style="width:205px;">Invitrogen</td>
			<td>15596-026</td>
			<td>RNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">reverse transcription system</td>
			<td style="width:205px;">Promega</td>
			<td style="width:119px;">A3500</td>
			<td>RNA reverse transcription</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GoTaq qPCR Master Mix</td>
			<td style="width:205px;">Promega</td>
			<td style="width:119px;">A6001</td>
			<td>RT-PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">StemTAG alkaline phosphatase staining and activity assay kit</td>
			<td style="width:205px;">Cells Biolabs, Inc.</td>
			<td>CBA-302</td>
			<td>&#160;alkaline phosphatase staining analysis</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">mouse ES cells: WT, 129 SvEv</td>
			<td style="width:205px;">Provided by Professor Guoliang Xu (Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China)</td>
			<td style="width:119px;"></td>
			<td>cell strain of mouse ES cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">microscope</td>
			<td style="width:205px;">Zeiss</td>
			<td style="width:119px;">LSM510</td>
			<td>cells observation</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:216px;">GraphPad Prism 5.0</td>
			<td style="width:205px;">GraphPad Prism Software Inc.</td>
			<td style="width:119px;">5.0</td>
			<td>statistical analysis</td>
		</tr>
	</tbody>
</table>

an alternative culture method to maintain genomic hypomethylation of mouse embryonic stem cells using mek inhibitor pd0325901 and vitamin c

胚胎干细胞有可能分化为三种细菌层 (内胚层、胚层或外胚层) 中的任何一种, 并能为再生医学产生许多血统。ES 细胞培养体外长期以来一直是人们普遍关注的问题。经典的, 小鼠 ES 细胞维持在血清和白血病抑制因子 (LIF) 的含培养基。然而, 在血清/生长条件下, 细胞表现出异质性和干细胞相关基因的表达谱, 大多处于亚稳态状态。此外, 培养的 es 细胞呈现全球甲基, 但内细胞质量 (ICM) 和原始生殖细胞 (PGCs) 的天真 ES 细胞处于全球低甲基化状态。ICM 和 PGCs 的 hypomethylated 状态与他们的干细胞紧密相关。为了改善小鼠 ES 细胞培养方法, 我们最近开发了一种新的方法, 在选择性地联合利用两个小分子化合物, 以维持 DNA hypomethylated 和多能状态。在这里, 我们提出的合作治疗维生素 C (Vc) 和 PD0325901 可以清除约 90% 5 甲基胞嘧啶 (5mC) 5 天的小鼠 ES 细胞。生成的5mC 内容与 PGCs 中的相同。机械调查显示, 通过减少 PD0325901 5mC 合成, Prdm14 表达式抑制 Dnmt3b (DNA 甲基) 和 Dnmt3l (余子). Vc 促进5mC 到 5-hydroxymethylcytosine (5hmC) 的转换主要由 Tet1 和 Tet2, 表明参与的被动和主动 DNA demethylations。此外, 在 Vc/PD0325901 条件下, 小鼠 ES 细胞呈均匀形态学和多能态。我们共同提出了一种新的化学协同培养方法来实现小鼠 ES 细胞 DNA 低甲基化和干细胞的维持。小分子化学依赖性方法克服了血清培养的主要缺陷, 并有望生成均质 ES 细胞, 进一步临床应用和研究。

Vc/PD0325901 协同诱导的小鼠 ES 细胞的全局擦除。血清中的小鼠 ES 细胞表现出 dna 甲基, 而多能 ICM 细胞和 PGCs 显示全球对 dna 甲基化的擦除, hypomethylated 状态与干细胞9、10密切相关。
以前, 我们和其他人发现 Vc 可以增强5mC 去甲基化15, 17.同时,...

Watch this Scientific Journal Video about An Alternative Culture Method to Maintain Genomic Hypomethylation of Mouse Embryonic Stem Cells Using MEK Inhibitor PD0325901 and Vitamin C at JoVE.com

An Alternative Culture Method to Maintain Genomic Hypomethylation of Mouse Embryonic Stem Cells Using MEK Inhibitor PD0325901 and Vitamin C

用酮抑制剂 PD0325901 和维生素 C 维持小鼠胚胎干细胞基因组低甲基化的一种替代培养方法

我们详细描述了两种基于化学的小鼠胚胎干细胞培养协议。这种新方法采用了促进甲基胞嘧啶介导氧化 (维生素 C) 的协同机制, 并抑制了 5-PD0325901 的合成, 以维持小鼠胚胎干细胞的 DNA 低甲基化和干细胞.

Embryonic stem (ES) cells have the potential to differentiate into any of the three germ layers (endoderm, mesoderm, or ectoderm), and can generate many ...

Research

JoVE Journal

Biochemistry

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

在利什曼原虫、人体细胞和小鼠睾丸中的 Polysome 分析

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For ...

科研

生物化学

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

用石英谐振器研究磷脂结合蛋白附件 a2 与固体支持的脂质层结合动力学的耗散微重法

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

DNA Electrophoresis Using Thiazole Orange Instead of Ethidium Bromide or Alternative Dyes

用噻唑橙代替溴代或替代染料进行 DNA 电泳

DNA gel electrophoresis using agarose is a common tool in molecular biology laboratories, allowing separation of DNA fragments by size. After separation, ...

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

DNA序列识别利用高通量原酶分析的DNAPrimase

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. ...

Using an Extracellular Flux Analyzer to Measure Changes in Glycolysis and Oxidative Phosphorylation during Mouse Sperm Capacitation

使用细胞外通量分析仪测量小鼠精子发作期间糖解和氧化磷酸化的变化

Mammalian sperm acquire fertilization capacity in the female reproductive tract in a process known as capacitation. Capacitation-associated processes ...

Improved UPLC-UV Method for the Quantification of Vitamin C in Lettuce Varieties (Lactuca sativa L.) and Crop Wild Relatives (Lactuca spp.)

Improved UPLC-UV Method for the Quantification of Vitamin C in Lettuce Varieties Lactuca sativa L. and Crop Wild Relatives Lactuca spp.

改进的 UPLC-UV 方法，用于在莱图斯品种（Lactuca sativa L.）和作物野生亲亲（Lactuca spp.） 中对维生素 C 进行定量化

Vitamins, especially vitamin C, are important micronutrients found in fruits and vegetables. Vitamin C is also a major contributor to their antioxidant ...

Synthesis of Masarimycin, a Small Molecule Inhibitor of Gram-Positive Bacterial Growth

马萨霉素的合成，一种革兰氏阳性细菌生长的小分子抑制剂

Peptidoglycan (PG) in the cell wall of bacteria is a unique macromolecular structure that confers shape, and protection from the surrounding environment. ...

Deciphering Molecular Mechanism of Histone Assembly by DNA Curtain Technique

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in ...

Isolation of Mitochondrial Contact Sites

An Improved Method to Isolate Mitochondrial Contact Sites

Unveiling Mitochondrial Contact Sites and Architectural Insights (Video) | JoVE

一种分离线粒体接触位点的改进方法

Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, the ...

A Fibrinolytic Enzyme-Based Method to Study Cerebral Thrombus Cell Components

A Comprehensive Approach to Analyze the Cell Components of Cerebral Blood Clots

A Novel Method for Comprehensive Cell Component Analysis of Cerebral Blood Clots (Video) | JoVE

一种分析脑血凝块细胞成分的综合方法

Cerebral thrombosis, a blood clot in a cerebral artery or vein, is the most common type of cerebral infarction. The study of the cell components of ...