The authors would like to acknowledge Dr. Bence Daniel, Matt Peloquin, Dr. Endre Barta, Dr. Balint L Balint and members of the Nagy laboratory for discussions and comments on the manuscript. L.N is supported by grants from the Hungarian Scientific Research Fund (OTKA K100196 and K111941) and co-financed by the European Social Fund and the European Regional Development Fund and Hungarian Brain Research Program - Grant No. KTIA_13_NAP-A-I/9.

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The authors have nothing to disclose.

In recent years, advances in sequencing technology have allowed large-scale predictions of enhancers in many cell types and tissues 7-9. The workflow described above allows one to perform primary characterization of candidate enhancers chosen based on ChIP-seq data. The detailed steps and notes will help anyone to set up a routine enhancer validation in the lab.
The most critical step in the luciferase reporter assay is the transfection efficiency. It is recommended to include a GFP-construct in order to estimate the transfection efficiency in every experiment. Having fresh and good quality of plasmids can be critical and can significantly improve the transfection. The presented protocol can be adapted to any cellular model system. It is highly recommended to use the appropriate cell type for the characterization of tissue-specific enhancers. Thus, luciferase reporter assay requires that the cells chosen for the experiment should be efficiently transfectable. Alternatively, easily transfectable cells should be used where the transcription factor of interest is (over)expressed. This can be particularly important in case of some tissue-specific enhancers. 
In contrast, enhancer RNA can be easily used in any cell without such limitations to study enhancer activity in different conditions in vivo. Key parameters for efficient eRNA detection are listed above. Isolation of good quality, non-degraded RNA is essential. DNase treatment is very important in order to avoid the detection of genomic DNA contamination instead of the eRNA. As RNA is used for subsequent reverse transcription reaction to get cDNA, DNase should be appropriately inactivated. It is advisable to design primers for the detection of both sense and anti-sense eRNAs.
Carrying out the above described enhancer trap experiment (transfection of a construct containing an enhancer element and a luciferase-based reporter) can provide strong functional evidence for enhancer activity. However, the ultimate proof, which is rarely presented in research articles, would be genetic elimination of the designated enhancer and thus determination of its role in vivo or at least in a cell 28.
The above-described workflow does not give us information which gene is regulated by the validated enhancer. Further research could use 3C (Chromosome Conformation Capture) experiment that show whether the implicated enhancer is interacting with the promoter of the gene of interest.

Development of a multicellular organism requires precisely regulated expression of thousands of genes across developing tissues. Regulation of gene expression is accomplished in large part by enhancers. Enhancers are short non-coding DNA elements that can be bound with transcription factors (TFs) and act from a distance to activate transcription of a target gene1. Enhancers are generally cis-acting and most frequently found just upstream of the transcription start site (TSS), but recent studies also described examples where enhancers were found much further upstream, on the 3' of the gene or even within the introns and exons2. 
There are hundreds of thousands of potential enhancers in the vertebrate genomes1. Recent methods based on chromatin immunoprecipitation (ChIP) provide high-throughput data of the whole genome that can be used for enhancer analysis3-9. Though data obtained by ChIP-seq experiments greatly increases the likelihood to identify cell and tissue-specific enhancers, it is important to keep in mind that detected binding sites do not necessarily identify direct DNA binding and/or functional enhancers. Thus, further functional analysis of newly identified enhancers is indispensable. In this work, we present a basic three-step process of putative active enhancer identification and validation. This includes: 1) selection of putative transcription factor binding sites by bioinformatics analysis of ChIP-seq data, 2) cloning and validation of these regulatory sequences in reporter constructs, and 3) measurement of enhancer RNA (eRNA). 
Exposure of embryonic stem (ES) cells to retinoic acid (RA) is frequently used to promote neural differentiation of the pluripotent cells 10. RA exerts its effects by binding to RA receptors (RAR&#945;, &#946;, &#947;) and retinoid X receptors (RXR&#945;, &#946;, &#947;). RARs and RXRs in a form of heterodimer bind to DNA motifs called RA-response elements, that is typically arranged as direct repeats of AGGTCA sequence (called as half site) and regulate transcription. Ligand-treatment experiments allowed the identification of several retinoic acid regulated genes in ES cells 11,12. However, enhancer elements for many of these genes has not been described yet. To demonstrate how the here-described workflow can be used for enhancer identification and validation we show step-by-step the selection and characterization of two retinoic acid-dependent enhancers in embryonic stem cells.

<table border="0" cellpadding="0" cellspacing="0" width="862">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">KOD DNA polymerase</td>
			<td style="width:121px;">Merck Millipore</td>
			<td style="width:119px;">71085-3</td>
			<td style="width:323px;">for PCR amplification of enhancer from gDNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNeasy Blood &#38; Tissue kit&#160;</td>
			<td>Qiagen</td>
			<td>69504</td>
			<td>for genomic DNA isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAquick PCR Purification kit</td>
			<td>Qiagen</td>
			<td>28106</td>
			<td>for PCR product purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gel extraction kit&#160;</td>
			<td>Qiagen</td>
			<td>28706</td>
			<td>for gel extraction if there are more PCR product</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HindIII</td>
			<td style="width:121px;">NEB</td>
			<td>R3104L</td>
			<td>restriction enzyme</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BamHI</td>
			<td style="width:121px;">NEB</td>
			<td>R3136L</td>
			<td>restriction enzyme</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FastAP</td>
			<td>Thermo Scientific</td>
			<td>EF0651</td>
			<td>release of 5&#39;- and 3&#39;-phosphate groups from DNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">T4 DNA ligase</td>
			<td style="width:121px;">NEB</td>
			<td>M0202</td>
			<td>for ligation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAprep Spin Miniprep kit</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td>for plasmid isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">DMEM</td>
			<td style="width:121px;">Gibco</td>
			<td style="width:119px;">31966-021</td>
			<td>ES media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">FBS</td>
			<td style="width:121px;">Hyclone</td>
			<td style="width:119px;">SH30070.03</td>
			<td>ES media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">MEM Non-Essential Amino Acid</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">M7145</td>
			<td>ES media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Penicillin-Streptomycin</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">P4333</td>
			<td>ES media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Beta Mercaptoethanol</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">M6250</td>
			<td>ES media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FuGENE HD&#160;</td>
			<td style="width:121px;">Promega</td>
			<td style="width:119px;">E2311</td>
			<td>transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Opti-MEM&#174; I Reduced Serum Medium</td>
			<td>Life Technologies</td>
			<td style="width:119px;">31985-062</td>
			<td>for transfection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">All-trans retinoic acid</td>
			<td style="width:121px;">Sigma</td>
			<td>R2625</td>
			<td>ligand, for activation of RAR/RXR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">96-well clear plate</td>
			<td style="width:121px;">Greiner</td>
			<td>655101</td>
			<td>for Beta galactosidase assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">96-well white plate</td>
			<td style="width:121px;">Greiner</td>
			<td>655075</td>
			<td>for Luciferase assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-luciferin, potassium salt</td>
			<td style="width:121px;">Goldbio.com</td>
			<td style="width:119px;">115144-35-9</td>
			<td>for Luciferase assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ATP salt</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">A7699-1G</td>
			<td>for Luciferase assay</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MgSO4x 7H2O</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">230391-25G</td>
			<td>for Luciferase assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">HEPES</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">H3375-25G</td>
			<td>for Luciferase assay</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Na2HPO4 x 7H2O</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">431478-50G</td>
			<td>for Beta galactosidase assay</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">NaH2PO4 x H2O</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">S9638-25G</td>
			<td>for Beta galactosidase assay</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MgSO4 x 7H2O</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">230391-25G</td>
			<td>for Beta galactosidase assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">KCl</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">P9541-500G</td>
			<td>for Beta galactosidase assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ONPG (o-nitrophenyl-&#946;-D-galactosidase)</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">N1127-1G</td>
			<td>for Beta galactosidase assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TRIzol&#174;</td>
			<td>Life Technologies</td>
			<td>15596-026</td>
			<td>RNA isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">High-Capacity cDNA Reverse Transcription Kit</td>
			<td>Life Technologies</td>
			<td>4368814</td>
			<td>reverse transcription of eRNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Rnase-free Dnase</td>
			<td style="width:121px;">Promega</td>
			<td>M6101</td>
			<td>Dnase treatment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">SsoFast Eva Green</td>
			<td style="width:121px;">BioRad</td>
			<td style="width:119px;">750000105</td>
			<td>RT-qPCR mastermix</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:300px;">CFX384 Touch&#8482; Real-Time PCR Detection System</td>
			<td style="width:121px;">BioRad</td>
			<td style="width:119px;"></td>
			<td>qPCR machine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">BioTek Synergy 4 microplate reader</td>
			<td style="width:121px;">BioTek</td>
			<td style="width:119px;"></td>
			<td>luminescent counter</td>
		</tr>
	</tbody>
</table>

prediction and validation of gene regulatory elements activated during retinoic acid induced embryonic stem cell differentiation

胚発生は、多くの遺伝子の活性化と抑制を含む多段階プロセスです。ゲノム中のエンハンサーエレメントは、組織及び細胞分化の間の遺伝子発現の細胞型特異的調節に寄与することが知られています。したがって、それらの同定およびさらなる調査は、細胞の運命を決定する方法を理解するために重要です。遺伝子発現データ（ 例えば、マイクロアレイまたはRNA-seqの）およびクロマチン免疫沈降（ChIPの）ベースのゲノムワイドな研究（チップ配列）の結果の統合は、これらの調節領域の大規模な同定を可能にします。しかしながら、細胞型特異的エンハンサーの機能的検証は、in vitroおよびin vivo実験手順でさらに必要とします。ここでは、アクティブなエンハンサーを同定し、実験的に検証できる方法について説明します。このプロトコルは、ステップバイステップのワークフローを提供します：1）のChIP-seqのデータ解析による調節領域の同定、2）クローニングおよびEXPERimentalレポーターアッセイで同定されたゲノム配列の推定調節の可能性の検証、およびエンハンサーRNA転写物のレベルを測定することにより、in vivoでのエンハンサー活性の3）決意。提示されたプロトコルは、ラボでは、このワークフローを設定するために誰を助けるために十分に詳述されています。重要なことに、このプロトコルは容易に適応し、任意の細胞モデル系において使用することができます。

我々はゲノムワイドRA調節遺伝子は、その近接して受容体濃縮を持っている識別するために、汎特異的RXR抗体を使用します。レチノイン酸で処理されたES細胞から得られたRXRチップ、配列データのバイオインフォマティクス解析は、RXR部位を占領下で核内受容体半部位（AGGTCA）（ 図1）の濃縮が明らかになりました。バイオインフォマティクスのアルゴリズムを用いて、我々はRXRのChIP-seqのデータ（ 図2）に半分サイトのモチーフ検索結果をバックマッピングされました。この分析は、正確に標準的な核内受容体結合部位と重複したものをChIPのピークを識別するために私たちを助けてくれました。 IGVにおけるこれらの部 ​​位の視覚化はHOXA1、以前に特徴付けRAR / RXRターゲット（ 図2及び図3）に近接して、これらの転写因子の濃縮を示しました。我々はまた、新規RA標的遺伝子、すなわちPRMT8 27を同定しました 。このラtterの領域は、2つのハーフサイト（AGGTCAAGGTCA）との間にはスペーサーヌクレオチド（ 図3）との直接の繰り返しが含まれています。機能的にHOXA1のために識別された要素が実際にRAR / RXRに結合することができることを検証し、RAによって調節するために、我々は、識別された要素を含むゲノム領域300bpの〜を含まTK-ルシフェラーゼレポーターベクターを、構築しました。また、応答エレメント（ 図5）せずにベクトルを構築しました。 ES細胞は、トランスフェクトし、ルシフェラーゼ活性を、レチノイン酸（ 図5）の非存在下および存在下で測定しました。 RAREなしの構築物が誘導されなかったが、レチノイン酸応答エレメント（RARE）を含む構築物は、ショーは、RAの治療の際に、ルシフェラーゼシグナル強度を増加しました。 また、HOXA1エンハンサーのレチノイン酸依存性活性を検討しました。 HOXA1 RAR / RXRに結合したエンハンサーcのセンスおよびアンチセンスエルナ式かどうかを確認するには遺伝子のmRNA発現とorrelateは、我々はまた、HOXA1のmRNA（ 図6）のレベルを測定しました。これらの結果は、エンハンサー活性は、従ってエンハンサーはおそらくRA依存性エンハンサーの調節に関与していることを確認し、短期のRA処理（3時間）により誘導されることを確認しました。 <img alt="図1" src="/files/ftp_upload/53978/53978fig1.jpg" /> 図1. ホーマーD Eノボ Motifの結果。例RXRのChIP-seqのためのホーマーによって生成された出力HTML（homerResults.html）が示されています。シーケンスロゴはRXRに結合した遺伝子座でのde novoモチーフ発見によって識別された濃縮された転写因子のモチーフをトップに対応します。 7463 RXRは、ゲノム領域は、モチーフ検索のために使用された占有しました。これらの領域の77.66パーセントは、（＃1としてランク付け）AGGTCA様モチーフが含まれています。詳細な説明をご覧ください：（http://homer.salk.edu/ho<html>マー/モチーフ/） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53978/53978fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図2" src="/files/ftp_upload/53978/53978fig2.jpg" /> 図 関心のモチーフを含む 2. 検索のゲノム領域。整列RXRのChIP-seqが統合ゲノミクスビューア（IGV）によって（mm_ES_RXR_24h_ATRA.bamとAGGTCAモチーフの発生（mm_ES_RXR_24h_ATRA_homerpeaks_motif1_mm10s_200_remaped_mbed.bed）読み込みの可視化。アラインメントは、リードストランドによって着色されている（読み取り、赤、 -鎖：+鎖上。青）ゲノムは、位置合わせのために使用：MM10。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53978/53978fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> JPG &quot;/&gt; HOXA1とPRMT8エンハンサーの 図3. ゲノム遺伝子座27 が示されている未処理およびRA処理した（24時間、1μM）胚性幹細胞から得られたRXRのChIP-のSeq信号のIGVのスナップショット。結合配列が赤色に着色されている識別された。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53978/53978fig3large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図4" src="/files/ftp_upload/53978/53978fig4.jpg" /> 。最も近い遺伝子の転写開始部位（TSS）に離れて相対2キロバイト- テストエルナコード配列図 4. 実験デザインエンハンサーRNA（エルナ）測定のために選択されたエンハンサーは、少なくとも1.5を配置する必要があります。関心とモチーフ解析の転写因子（TF）チップ、配列データは、ゲノム全体の直接の結合部位を同定するために使用され得ます。 transcripの結合として、的なコアクチベーター（ 例えば、P300）は、多くの場合、遠位エンハンサーをマークし、TFとP300の両方に富む領域が良好なエンハンサー候補です。 eRNAsの発現は、積極的に活性化されたエンハンサーヒストンマークH3K27acの濃縮と相関されます。 200の地域- 。離れてセンスまたはアンチセンス方向の転写因子結合の中心から千塩基対がエルナプライマーの設計のために推奨され<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53978/53978fig4large.jpg" target="_blank">、この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図5" src="/files/ftp_upload/53978/53978fig5.jpg" /> 示されたレポーターの 図5. ルシフェラーゼ活性は、ES細胞で構築します。 HOXA1エンハンサー示すゲノム領域は、TK-Lucベクターにクローン化し、胚性幹細胞にトランスフェクトしました。 1と2は、レチノイン酸応答エレメント（RARE、国連を含ま構築derlinedシーケンス）。細胞は、RA（24時間、1μM）で処理しました。 βGalは、正規化のための内部対照として使用しました。バーは4生物学的複製からの平均正規化した値を表します。 ±SD *** P &lt;0.005 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53978/53978fig5large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図6" src="/files/ftp_upload/53978/53978fig6.jpg" /> RT-qPCRにより測定されるように、図6レチノイン酸誘導性エンハンサーRNA転写。HOXA1のmRNAとエルナレベル。全RNAを、レチノイン酸（RA）またはビヒクルとしてDMSO（VEH）で3時間処理したES細胞から単離しました。センスおよびアンチセンスエルナ検出とPCRアンプリコンのためのフォワードプライマーを示しています。 RXR結合部位の中心からエルナRT-qPCRにより検出された領域の距離が示されています。値は、36B4のMR平均値に正規化しましたNA。データは平均値±s *** P &lt;0.005を意味表す<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53978/53978fig6large.jpg" target="_blank">。この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation at JoVE.com

Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation

In this work we provide an experimental workflow of how active enhancers can be identified and experimentally validated.

レチノイン酸誘導された胚性幹細胞分化の間に活性化された予測と遺伝子調節要素の検証

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to ...

prediction-validation-gene-regulatory-elements-activated-during

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

8.1K ビュー.  Sanford-Burnham-Prebys Medical Discovery Institute at Lake Nona. In this work we provide an experimental workflow of how active enhancers can be identified and experimentally validated.

ビデオ: Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation

Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes available for basic research and translational applications. To differentiate hiPSCs into cardiomyocytes, various protocols including embryoid body (EB)-based differentiation and growth factor induction have been developed. However, these protocols are inefficient and highly variable in their ability to generate purified cardiomyocytes. Recently, a small molecule-based protocol utilizing modulation of Wnt/&#946;-Catenin signaling was shown to promote cardiac differentiation with high efficiency. With this protocol, greater than 50%-60% of differentiated cells were cardiac troponin-positive cardiomyocytes were consistently observed. To further increase cardiomyocyte purity, the differentiated cells were subjected to glucose starvation to specifically eliminate non-cardiomyocytes based on the metabolic differences between cardiomyocytes and non-cardiomyocytes. Using this selection strategy, we consistently obtained a greater than 30% increase in the ratio of cardiomyocytes to non-cardiomyocytes in a population of differentiated cells. These highly purified cardiomyocytes should enhance the reliability of results from human iPSC-based in vitro disease modeling studies and drug screening assays.

Here, we describe a robust protocol for human cardiomyocyte derivation that combines small molecule-modulated cardiac differentiation and glucose deprivation-mediated cardiomyocyte purification, enabling production of purified cardiomyocytes for the purposes of cardiovascular disease modeling and drug screening.

小分子変調分化とその後のグルコース飢餓を用いたヒト人工多能性幹細胞からの高度に精製された心筋細胞の導出

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes ...

発生生物学

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

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

無血清単層培養中のマウス胚性幹細胞の神経分化

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

胚性幹細胞や組織の心臓分化のエピジェネティック制御

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

2および3次元の胚様体中のマウス胚性幹細胞のレチノイン酸誘発神経分化の分析

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early embryonic development, kinase activity is highly dynamic and widespread across the embryo. Pharmacological and genetic approaches are commonly used to probe kinase activities. Unfortunately, it is challenging to achieve superior spatial and temporal resolution using these strategies. Furthermore, it is not feasible to control the kinase activity in a reversible fashion in live cells and multicellular organisms. Such a limitation remains a bottleneck for achieving a quantitative understanding of kinase activity during development and differentiation. This work presents an optogenetic strategy that takes advantage of a bicistronic system containing photoactivatable proteins Arabidopsis thaliana cryptochrome 2 (CRY2) and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). Reversible activation of the mitogen-activated protein kinase (MAPK) signaling pathway is achieved through light-mediated protein translocation in live cells. This approach can be applied to mammalian cell cultures and live vertebrate embryos. This bicistronic system can be generalized to control the activity of other kinases with similar activation mechanisms and can be applied to other model systems.

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

This protocol describes an optogenetic strategy to modulate mitogen-activated protein kinase (MAPK) activity during cell differentiation and Xenopus embryonic development. This method allows for the reversible activation of the MAPK signaling pathway in mammalian cell culture and in multicellular live organisms, like Xenopus embryos, with high spatial and temporal resolution.

細胞分化中のマイトジェン活性化プロテインキナーゼ経路の光媒介可逆的調節<em&gt;アフリカツメガエル</em&gt;胚発生

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

人間の胚または誘導多能性幹細胞からの網膜色素上皮細胞の急速な監督の分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

細胞分裂時の染色体分配の生きているセルイメージ投射

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

ヒト多能性幹細胞の肝細胞への効率的な分化

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during heart development using ventricular specific fluorescent reporter knock-in mice (MLC-2v-tdTomato mice). Heart development involves a linear heart tube formation, the heart tube looping, and four chamber septation. These complex processes are highly conserved in all vertebrates. The mouse embryonic heart has been widely used for heart developmental studies. However, due to their extremely small size, dissecting mouse embryonic hearts is technically challenging. In addition, visualization of cardiac chamber formation often needs in situ hybridization, beta-galactosidase staining using LacZ reporter mice, or immunostaining of sectioned embryonic hearts. Here, we describe how to dissect mouse embryonic hearts and directly visualize ventricular chamber formation of MLC-2v-tdTomato mice using whole mount epifluorescent microscopy. With this method, it is possible to directly examine heart tube formation and looping, and four chamber formation without further experimental manipulation of mouse embryos. Although the MLC-2v-tdTomato reporter knock-in mouse line is used in this protocol as an example, this protocol can be applied to other heart-specific fluorescent reporter transgenic mouse lines.

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

落射蛍光マウント全体を用いたマウス胚発生中に心臓商工会議所発展の分析

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM). Cell replacement therapy holds great promise for treatment of PD. Recently, induced neural stem cells (iNSCs) have emerged as a potential candidate for cell replacement therapy due to the reduced risk of tumor formation and the plasticity to give rise to region-specific neurons and glia. iNSCs can be reprogrammed from autologous somatic cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells. Compared with other types of somatic cells, PBMNCs are an appealing starter cell type because of the ease to access and expand in culture. Sendai virus (SeV), an RNA non-integrative virus, encoding reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming. In this study, we describe a protocol in which iNSCs are obtained by reprogramming PBMNCs, and differentiated into specialized VM DA neurons by a two-stage method. Then DA precursors are transplanted into unilaterally 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models to evaluate the safety and efficacy for treatment of PD. This method provides a platform to investigate the functions and therapeutic effects of patient-specific DA neural cells in vitro and in vivo.

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson&#39;s disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.