We thank Dr. Leonid Gnatovskiy for his carefully and critical reading of the paper. This work was supported in part by grants from the National Institutes of Health (HL109054) and the Samuel and Jean Frankel Cardiovascular Center, University of Michigan (Inaugural Fund) to WZ and from the Leon H Charney Division of Cardiology, New York University School of Medicine to BL.

マウスのESCからマウス胚性CPCの1導出<ol><li>マウス胚性線維芽細胞（MEF）フィーダー層を準備します。 <ol><li>暖かいMEF培地を37℃まで（DMEM中10％FBS）。 </li><li>ゼラチンでコーティングしたプレートを準備します。 <ol><li> 10cmディッシュに6ウェルプレートのウェルまたは5中への水の0.1％ゼラチンを1ml加える。 </li><li>少なくとも30分間37°Cまたは室温でプレートや食器のままにしておきます。使用前にゼラチンを熱望。 </li></ol></li><li>融解は、穏やかに振盪しながら、37℃の水浴中で迅速にMEFを照射。 </li><li> 15ミリリットルまたは50ミリリットルコニカルチューブに静かに細胞を移す。予め温めMEF培地5mlを追加します。ゆっくり細胞を旋回し、5分間200×gで遠心する。 </li><li> MEF培地中の細胞を再懸濁し、生存細胞を数える。 6ウェルプレートからウェルあたりの細胞のための2ミリリットルと10cmディッシュから細胞について10ミリリットル：MEF培地の以下のボリュームを使用してください。 </li><li> 1-2で6ウェルプレートまたは10cmの皿にプレート細胞プレート/皿当たり×10 6。 </li><li> 37℃でのMEFプレート/皿をインキュベートし、5％CO 2の少なくとも24時間前に使用する。 注：週より古いMEFプレート/皿を捨てる。 </li></ol></li><li>マウスのESCを準備<ol><li>文化ISL1-CRE。 Rosa26YFP / MEF2C-CRE。 90％のコンフルエントまでMEF上Rosa26YFPマウスのESC。使用ES細胞培地は、410ミリリットルDMEM、75ミリリットルのFBS、0.1mMのMEM NEAA、2mMのL-グルタミン、0.1mMのピルビン酸、100 Uペニシリン/ストレプトマイシン、および0.1mMのβメルカプトエタノールからなる。 </li><li> 1.1.2で説明したように、ゼラチンコーティングした6ウェルプレートを準備します。 </li><li>プレートから培地を吸引し、DPBSで細胞をすすいでください。 <ol><li>吸引しDPBSと6ウェルプレートの1ウェルに0.4ミリリットル0.25％トリプシンを追加します。 5分間37℃、5％CO 2で細胞をインキュベートする。トリプシン反応を停止するために1ミリリットル予め温めたES培地を追加します。 </li></ol></li><li> 200 XGと吸引媒体での収穫細胞と遠心細胞。 1ミリリットルのES細胞培地中で細胞を再懸濁し、細胞をカウントします。&lt;/ LI&gt; <li> 3×10 4 / cm 2の密度でゼラチンコートプレート上プレートのESC。 ESCは約90％コンフルエントに達したときに、約2日間、37℃、5％CO 2でインキュベートする。メディア毎日を変更します。 </li><li> ESCは、90％コンフルエントである場合には、完全にMEFフィーダーを削除する手順1.2.2-1.2.5を繰り返します。 </li></ol></li><li> EB形成によってCPCの分化を誘導する<ol><li>続くように分化培地を準備します36ミリリットルIMDM、12ミリリットルハムF12、2mMのL-グルタミン、0.34ミリリットルBSA（7.5％）、0.25ミリリットルN2、0.5ミリリットルのB27、50μg/ mlのAA、および0.45 mMの1-チオグリセロールを。 </li><li>吸引し細胞からの培地およびDPBSですすぐ。吸引しDPBSと6ウェルプレートの1ウェルに0.4ミリリットル0.25％トリプシンを追加します。 5分間37℃でインキュベートする。 </li><li>単一細胞に細胞クラスターを分散させるために上下に反応し、ピペットを中止する1ミリリットルの分化培地を追加します。 </li><li> 200×gで遠心したESC 5分間、中を捨てる。 <ol><li>再懸 ​​濁し、1×10 6 &lt;/商標&gt; 1ミリリットル分化培地中のESC。第EBの凝集のための細胞、培養37℃で分化培地中で1×10 5細胞/ mlの濃度の低い剥離ペトリ皿で細胞を数える。 </li></ol></li><li>二日EB形成した後、50ミリリットルチューブに皿からのEBを転送する。 1分間200×gでスピン。培地を除去およびCa 2+およびMg 2+なしで10ミリリットルのDPBSでのEBをすすぐ。 </li><li>吸引しDPBSと1ミリリットル0.25％トリプシンを追加します。 5分間37℃でインキュベートする。反応を停止する4.5ミリリットル分化培地を加える。上下にピペットで単一細胞にしたEBを解離する。 </li><li> 5分間200×gで細胞を遠心します。 1ミリリットルの分化培地で培地を吸引し、細胞を再懸濁。 </li><li>細胞をカウントし、分化培地中で1×10 5細胞/ mlに2×10 6個の細胞を希釈する。それぞれ5 / mlの、0.8 ng / mlであり、5 / mlの、の最終濃度で培地にVEGF、BMP4とアクチビンAを追加する。 Cultu37℃での第EB形成のためのペトリ皿で​​細胞を再。 </li><li>第二EB形成後に40時間、50ミリリットルチューブにEBを転送します。 5分間37℃で1ミリリットル0.25％トリプシンでのEBを解離、DPBSで一度すすぎます。上下にピペットで単一細胞にしたEBを解離する。 </li><li> 5 / mlのVEGF、10ng / mlのbFGFおよび12.5 / mlのFGF10とStempro-34培地中の細胞を懸濁します。ゼラチンコートプレートに2×10 5 / cm 2の細胞をプレート。 CPCの分離前に約32時間、37℃のインキュベーターで培養。 </li></ol></li><li> mCPCsを分離<ol><li>培地を吸引し、DPBSですすぐ。 5分間37℃で培養プレートに0.5ミリリットルの0.25％トリプシンを加える。反応を停止する4.5ミリリットル分化培地を加える。上下にピペットで単一細胞にしたEBを解離する。 FACS-精製のために、2％FBS / PBSで遠心分離し、再懸濁細胞によって細胞を回収します。 </li><li>陰性対照として選別機に未分化のESCを実行します。スキャットを楽しみに調整する変更電圧TER（FSC）および側方散乱（SSC）は、所望の人口を選択します。破片とダブレットを排除するために側方散乱と幅対前方散乱の高さ対飛散楽しみにしてください。選択されたサブ集団において、ESCコントロールの分布に応じて、細胞の自家蛍光を排除するためにヒストグラムに正のYFPのゲートを描く。 YFP +ゲーティングからYFP +のCPCを収集します。 </li><li>分化培地（1.3.1）を6ウェルプレート中で精製したクリック単価（YFP +細胞）プレート。心筋細胞および平滑筋細胞へのCPCの分化のために、37℃で培養追加の3〜5日。 </li></ol></li></ol>ヒトESCからのヒト胚性CPCの2導出<ol><li>フィーダを準備<ol><li> 2×10 4個 / cm 2の密度で、上記のように、マウス胚性線維芽細胞（MEF）フィーダー層を準備します。 </li></ol></li><li>ヒトESCを維持<ol><li> CRE; M上の日常のpCAG-FLOX-STOP-FLOX-GFPまたはRFPのESC：人間ISL1を維持正規のhESC培地を用いてEF（ノックアウトDMEM / F-12 385ミリリットル、ノックアウトSR 100mlの2mMのL-グルタミン、0.1mMのNEAA、0.1mMのβメルカプトエタノール、10ng / mlの塩基性FGF）。心臓分化する前に、少なくとも2継代のフィーダーフリーの状態でヒトESCを転送する。 </li></ol></li><li>フィーダーフリーの状態でヒトESCを準備<ol><li>ヒトESC培養のためのコーティングされたプレートを準備します。 <ol><li> 4℃で一晩マトリゲルを解凍する。氷上で50ミリリットルチューブを入れ、チューブに30ミリリットルの冷DMEM / F12培地を追加。 </li><li>冷たい培地に1ミリリットルマトリゲルを加え、よく混ぜる。 10cmディッシュに6ウェルプレートのウェルまたは5ミリリットルに1ミリリットルの冷マトリゲル-DMEM / 12培地を追加します。 </li><li> 2時間4℃で一晩または室温でプレート/皿のままにしておきます。使用前に培地を吸引。 </li></ol></li><li> MEFプレートから培地を吸引し、DPBSでヒトESCをすすぎ：人間のプレート上のESCを分割。その後、6ウェルプレートのウェルに1ミリリットルのディスパーゼ（1mg / ml）を加える。 37℃で細胞をインキュベートインキュベータは、コロニーの縁が剥離し始めるまで。 </li><li>ディスパーゼ溶液を除去。 DPBSで2回すすぎ、その後プレートによくmTeSR培地またはMEF馴化培地あたり2.5ミリリットルを追加/。 </li><li>セルスクレーパーを用いて細胞をこすり、慎重にピペッティング上下小さな塊に人間のESCコロニーを破る。 </li><li> ESC塊を移し、1を希釈：3をコーティングしたプレートに。 </li><li> 2ミリリットル/ウェルmTeSR培地またはMEF馴化培地を追加し、毎日培地交換。 </li><li> mTeSR媒体または少なくとも2継代のためにコーティングされたプレート上のMEF馴化培地中での培養ヒトESC。 </li></ol></li><li>ヒトESCからのCPCの分化を誘導<ol><li>細胞が約90％コンフルエントである場合には、培地を吸引し、DPBSですすぐ。その後、20分間、37℃で0.5 mg / mlのディスパーゼでのhESCを培養する。 </li><li>セルリフターでプレートから細胞を除去し、DPBSで2回のhESCをすすぎ、その後DMEM / F12は、18％のFBS、0.1 mMのNEAA、2 mMのを含む（分化培地中の細胞クランプを再懸濁L-グルタミン、0.1mMのβメルカプトエタノールおよび50μg/ mlのアスコルビン酸）。 </li><li> EB形成のために、6ウェルの超低付着性プレートに細胞を移す。 </li><li>翌日分化培地を変更します。 </li><li> 3日ごとに培地を交換し、懸濁培養でのEBを維持する。 </li></ol></li><li> HCPCSを分離<ol><li>遅くとも人間CPC分離のための分化の9日目よりのEBを収集しません。 </li><li>培地を吸引し、DPBSで2回のEBをすすぐ。 5分間37℃で6ウェル培養プレートの各ウェルに0.5ミリリットルの0.25％トリプシンを加える。 </li><li>反応を停止し、分化培地の等量を加える。上下にピペットで単一細胞にしたEBを解離する。 2％FBS / DPBS中で5分間再懸濁細胞を200×gで遠心分離して細胞を回収します。 40μmのセルストレーナーおよび1.4.3に記載のようにFACS精製RFP + CPC細胞とフィルタセル。 </li><li>プレートはgeltin被覆プレート上にHCPCSを選別した。 7のための分化培地で培養HCPCS-9心筋細胞および平滑筋細胞に分化する日数。 7日めっき後、培養物は、DMEM / F12培地中で5％のノックアウトSRで細胞を分化した。 </li></ol></li></ol>

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Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemically+defined+medium+supporting+cardiomyocyte+differentiation+of+human+embryonic+stem+cells.">Chemically defined medium supporting cardiomyocyte differentiation of human embryonic stem cells.</a> Differentiation: Research in Biological Diversity. 76 (9), 958-970 (2008).</li><li>Lian, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+cardiomyocyte+differentiation+from+human+pluripotent+stem+cells+via+temporal+modulation+of+canonical+Wnt+signaling.">Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (27), E1848-1857 (2012).</li><li>Bizy, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+light+chain+2-based+selection+of+human+iPSC-derived+early+ventricular+cardiac+myocytes.">Myosin light chain 2-based selection of human iPSC-derived early ventricular cardiac myocytes.</a> Stem Cell Research. 11 (3), 1335-1347 (2013).</li></ol>

The authors declare no competing financial interests.

This protocol combines a method using growth factors to guide mESCs differentiation and spontaneous differentiation of human ESCs into CPCs. CPC lineage marked with fluorescent reporter is used to efficiently identify and isolate CPCs by FACS. The FACS-purified CPCs retain the capacity to differentiate into cardiomyocytes, smooth muscle, and endothelial cells and have a comparable expression profile to the in vivo cells. Thus, these CPCs can serve as a great resource for cell based heart therapy because of their ability ...

A correction to the author list was made to: <a href="http://www.jove.com/video/52047/derivation-of-cardiac-progenitor-cells-from-embryonic-stem-cells">Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells</a>.
The following author is now listed as a co-corresponding author:
Lei Bu

A correction to the author list was made to: Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells.

Erratum: Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

心臓疾患は、今日の世界で主要な原因のままであり、死亡率は、過去20年間（米国心臓協会）に事実上変わっていない。効果的に、心不全を予防または逆転するための新規な治療戦略を開発するための重要な必要性がある。一つの有望な戦略は、幹細胞生物学1の急速な発展以下の細胞ベースの治療である。この点では、多能性のCPCは増殖だけ心臓系統への分化にコミットする能力のために、治療のための優れた細胞源である可能性があります。したがって、クリック単価を生成し、単離するための効率的かつロバストな方法は、心臓の細胞療法の研究のために非常に重要である。 このプロトコルは、初期胚発生とどのように彼らの世代のESCからの間に識別胚のCPCに焦点を当てています。各種のCPCにも骨髄2から、胚および成体の心臓から単離されている。胚発生時には、骨のmorphoge電磁タンパク質（BMP）、ウィングレス型MMTV組込み部位ファミリーメンバー（のWnt）と節点の信号がMesp1 +多分化中胚葉3のコミットメントを誘導する。 Mesp1 +細胞はその後、胚のCPC 4に分化する。これらのCPCは、一般的にHCN4でマーク、NK2ホメオボックス5（Nkx2-5）、カルチのLIMホメオボックス1（ISL1）、T-ボックス5（TBX5）、及び筋細胞エンハンサー因子2C（MEF2C）、一次および第二の心臓フィールドを形成されており、心臓発生5-10の間に心臓の主要な部分に貢献しています。 Nkx2-5 +及びISL1 + / MEF2C +両方のCPCは、心筋細胞に分化することができる、平滑筋細胞（SMC）、および内皮細胞5-8。したがって、これらのCPCは、心臓血管系、ならびに心臓組織を生じさせると細胞ベースの心臓療法のための理想的な細胞源であるであろう。その結果、in vitroでのCPCを生成する心血管研究における主要な研究の焦点となっている。 ESCは、無制限の拡張容量aを有しているのでND胚盤胞段階でICM細胞を表し、自然の胚発生以下の胚のCPCへのESCの分化は、クリック単価を取得するために論理的かつ効果的なアプローチと考えられている。 ESCのからのCPCを得るための1つの広く適用のアプローチは、EBを11にESCのを集約することです。分化効率を向上させるために、心臓の開発の知識に基づいて定義された化学的および成長因子は12-14使用されてきた。しかし、広く分野で受け入れられている明確なCPCマーカー、特に無細胞表面マーカーは、存在しない。この問題に対処するために、ESCのはISL1 +またはMEF2C +クリック単価とのCre / loxPシステムを用いた蛍光レポーターとその誘導体をマークするために設計されている。 CreリコンビナーゼはISL1 / MEF2Cプロモーター/エンハンサーの制御下で落札された。構成的プロモーターによって駆動される修正された蛍光タンパク質RFPまたはYFP遺伝子のcreリコンビナーゼでFLOX停止コドンの切除により活性化され得る（ISL1：CRE;-のpCAG-FLOX-STOP-FLOX GFPまたはRFP / ISL1-CRE。Rosa26YFP / MEF2C-CRE。Rosa26YFP）5,6。 ESCは第二の心臓フィールドのCPCに分化されると、ISL1 / MEF2Cプロモーター/エンハンサー駆動CREは蛍光レポーターを活性化するとのCPCをFACS-精製によって濃縮することができる。簡単に説明すると、EB凝集法は、ESCの分化を開始するために使用される。分化効率を高めるために、分化した細胞は、アスコルビン酸（AA）およびBMP4、アクチビンAとのVegf 13,15などの増殖因子で処理される。このプロトコルは、マウスおよびヒトESCの両方を使用して、堅牢で効率的なCPCの分化を可能にする。

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derivation of cardiac progenitor cells from embryonic stem cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

プロトコルは、いくつかのES細胞株からのCPCの導出を示す。 ESCのは、CPCのに分化するのEBを形成するために集約されます。 ESCは、日常的MEFフィーダー（ 図1A、F）に維持され、フィーダは、分化前に削除されます。分化培地中のEB（ 図1B、G）へのESCの凝集の際に、第二形成EBをマウス細胞株（ 図1C）における中胚葉分化を増強するために、BMP4とアクチビン...

Watch this Scientific Journal Video about Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells at JoVE.com

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

胚性幹細胞から心臓前駆細胞の誘導

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

derivation-of-cardiac-progenitor-cells-from-embryonic-stem-cells

Research

JoVE Journal

Developmental Biology

8.1K ビュー.  University of Michigan. In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

ビデオ: Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

幹細胞から網膜色素上皮細胞の効率的な導出

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

発生生物学

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

末梢血造血前駆細胞からヒト神経幹細胞の直接誘導

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

ヒト多能性幹細胞からメラノサイトのフィーダーフリーの導出

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

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 ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

水平全マウント：皮膚の厚い3次元組織断面のための新規処理および画像化プロトコール

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

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 ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

生活そのままゼブラフィッシュの単一セルの光電

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

核の分布、蛋白質および細胞骨格を研究する線虫生殖細胞の解析

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies have demonstrated the wide differentiation capacity that these cells possess. The ovary represents a promising candidate for cell-based therapies due to the fact that it is rich in MSCs and that it is frequently discarded after ovariectomy surgeries as biological waste. This article describes procedures for the isolation, expansion, and differentiation of MSCs derived from the canine ovary, without the necessity of cell-sorting techniques. This protocol represents an important tool for regenerative medicine because of the broad applicability of these highly differentiable cells in clinical trials and therapeutic uses.

Herein, we describe a method for the isolation, expansion, and differentiation of mesenchymal stem cells from canine ovarian tissue.

導出とイヌの卵巣間葉系幹細胞の分化

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

体節誘導体誘導される人間からの体外世代多能性幹細胞します。

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...