We thank S. Gr&#252;nberg (Bonn, Germany) and P. Freitag (Bonn, Germany) for their technical assistance.

<ol>
	<li>Bergmann, O., 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=Evidence+for+cardiomyocyte+renewal+in+humans.">Evidence for cardiomyocyte renewal in humans.</a> Science. 324 (5923), 98-102 (2009).</li><li>Raulf, 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=Transgenic+systems+for+unequivocal+identification+of+cardiac+myocyte+nuclei+and+analysis+of+cardiomyocyte+cell+cycle+status.">Transgenic systems for unequivocal identification of cardiac myocyte nuclei and analysis of cardiomyocyte cell cycle status.</a> Basic Res.Cardiol. 110 (3), 33 (2015).</li><li>Field, C. M., Alberts, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anillin,+a+contractile+ring+protein+that+cycles+from+the+nucleus+to+the+cell+cortex.">Anillin, a contractile ring protein that cycles from the nucleus to the cell cortex.</a> J.Cell Biol. 131 (1), 165-178 (1995).</li><li>Hesse, M., 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=Direct+visualization+of+cell+division+using+high-resolution+imaging+of+M-phase+of+the+cell+cycle.">Direct visualization of cell division using high-resolution imaging of M-phase of the cell cycle.</a> Nat.Commun. 3, 1076 (2012).</li><li>Eulalio, 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=Functional+screening+identifies+miRNAs+inducing+cardiac+regeneration.">Functional screening identifies miRNAs inducing cardiac regeneration.</a> Nature. 492 (7429), 376-381 (2012).</li><li>Di, S. V., Giacca, M., Capogrossi, M. C., Crescenzi, M., Martelli, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knockdown+of+cyclin-dependent+kinase+inhibitors+induces+cardiomyocyte+re-entry+in+the+cell+cycle.">Knockdown of cyclin-dependent kinase inhibitors induces cardiomyocyte re-entry in the cell cycle.</a> J.Biol.Chem. 286 (10), 8644-8654 (2011).</li><li>Hosoda, T., 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=Clonality+of+mouse+and+human+cardiomyogenesis+in+vivo.">Clonality of mouse and human cardiomyogenesis in vivo.</a> Proc.Natl.Acad.Sci.U.S.A. 106 (40), 17169-17174 (2009).</li><li>Soonpaa, M. H., Field, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+cardiomyocyte+DNA+synthesis+in+normal+and+injured+adult+mouse+hearts.">Assessment of cardiomyocyte DNA synthesis in normal and injured adult mouse hearts.</a> Am.J.Physiol. 272 (1 Pt 2), H220-H226 (1997).</li><li>Ang, K. L., 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=Limitations+of+conventional+approaches+to+identify+myocyte+nuclei+in+histologic+sections+of+the+heart.">Limitations of conventional approaches to identify myocyte nuclei in histologic sections of the heart.</a> Am.J.Physiol Cell Physiol. 298 (6), C1603-C1609 (2010).</li><li>Engel, F. B., Schebesta, M., Keating, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anillin+localization+defect+in+cardiomyocyte+binucleation.">Anillin localization defect in cardiomyocyte binucleation.</a> J Mol Cell Cardiol. 41 (4), 601-612 (2006).</li><li>Bergmann, O., 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=Dynamics+of+Cell+Generation+and+Turnover+in+the+Human+Heart.">Dynamics of Cell Generation and Turnover in the Human Heart.</a> Cell. 161 (7), 1566-1575 (2015).</li></ol>

The authors declare that they have no competing financial interests.

心筋細胞は、細胞周期を再入力し、損傷後や組織の恒常性の間に分割することができるかどうかをめぐる論争があります。心筋細胞の基本的な売上高の値は1％1と80％7の範囲で与えられています。 40％7 - 、心臓病変後、細胞周期活性の誘導および新しい心筋細胞の生成は、0.0083％〜8と25の間の値と、境界域で報告されています。これらの不一致は、部分的に心臓の核を同定するための異なる実験的アプローチ、9組織切片の細胞分裂の間に非常に困難なプロセスによって説明することができます。心筋細胞は、細胞周期の変動を受けやすいように、倍数体および多核細胞をもたらす核内倍加およびacytokinetic有糸分裂、より本格的な細胞分裂を区別するために決定的に重要です。ために細胞分裂の同定は、そのような収縮リングとmidbodiesなどの細胞分裂の特徴を視覚化するだけでなく、二核心筋細胞のパーセンテージを決定する必要があります。我々は、詳細に心筋細胞における細胞周期活性を分析し、単離された細胞または厚い部分でnuclearityの程度を決定するための技術を開発しました。 eGFPの-anillinシステムは対称的収縮環（ 図1F）と中心体（ 図1G）、および核内倍加およびacytokinetic有糸分裂のための間接的な証拠の可視化を通じて、細胞分裂の直接の証拠を提供することに留意することが重要です。前述のように、収縮環の一方的な侵入の発生は二核性10の指標であり、これはのeGFP-anillinシステムで観察することができます。 核内倍加は無収縮環またはMI限りとして示唆されていますdbodyは必須これらのローカライズを検出する確率の計算を行いいる、観察されます。 25時間の細胞周期の継続時間、20分の収縮環の可視性の平均期間、および1時間の中心体の永続性を仮定すると、100分周のeGFP-anillin陽性細胞と4 midbodiesで1収縮環があるはずです。統計的に言えば、25のeGFP-anillin陽性細胞の最小値は、細胞周期の変動から細胞分裂を識別するために分析される必要があります。 （多くの場合不明である）は、細胞周期の持続時間が増加または減少すると、この数はそれに応じて変化します。これはまた、（1E図 ）のeGFP-anillin信号の大部分が核になることを意味し、M-相として、非核のローカライズを持つ唯一の相は、わずか約1時間持続します。 生後発育の間に、二核性は、マウス心臓で起こると心室の心筋細胞に90％まで（〜ヒトでは25％）11を上昇させます。フォー心臓における再生の分析をR、二核性の程度を決定することが重要です。すなわち、ランゲンドルフ解離および心臓組織の厚い部分の作成：私たちは、この重要な形態学的特徴に対処するための2つの方法を説明します。ランゲンドルフ分離は、簡単かつ迅速ですが、厚い部分の形態は、より多くの時間がかかるだけでなく、より正確です。興味深いことに、我々はランゲンドルフ分離は、二核心筋細胞の割合を過大評価することを見出しました。これは、このむしろ剛性の手順中の単核及び二核細胞の異なる生存率が原因である可能性があります。 出生後の心筋細胞における増殖の誘導は、心臓の再生に新興のアプローチであるように、このプロトコルは、2つのトランスジェニックマウス系統、MYH6-H2BmChとCAG-EGFP-anillinを組み合わせることにより、スクリーニングシステムを説明しています。生後日のP1上の心臓から単離された心筋細胞 - P6は簡単に培養し、miRNAをトランスフェクトすることができますかsiRNAまたは小分子のライブラリーで処理しました。心筋細胞の核を手動または自動ソフトウェアアルゴリズムによって検出することができ、潜在的な「ヒット」は、MCH +核数の増加によって決定することができます。核内倍加およびacytokinetic有糸分裂の細胞分裂の識別は、eGFPを-anillin信号の異なる局在の定量化によって行うことができます。このシステムは、出生後の心筋細胞の増殖を根底にある調節機構の上にいくつかの新しい光を当てるべきであり、心臓疾患の治療のための新たな治療薬の発見につながる可能性があります。

心筋細胞の核および細胞周期の状態の正確な同定は、心筋の代謝回転および再生の決意するための極めて重要です。これは、細胞周期活性を同定するための、そのようなpHH3、のKi-67、またはチミジン類似体のような核マーカーの使用のために特に当てはまります。哺乳類成体心筋細胞の増殖能が1非常に小さいので、心筋細胞核の増殖マーカーについて陽性の核の偽の身分証明書は、増殖アッセイの結果で決定的な違いを作ることができます。また、心筋細胞は倍数体および多核細胞ではなく、細胞分裂につながるような核内倍加およびacytokinetic有糸分裂などの細胞周期の変化、になりやすいです。この目的のために、一般的な細胞周期マーカーに対する抗体染色の解釈は、全ての場合において決定的ではありません。 ここで、我々は、直forwaのための方法を提示します<html> RDマウス心筋細胞の認識とそれらの核の明確な同定により生後および成体の段階でネイティブ単離された細胞と厚い組織切片でのnuclearity。そのために、MYH6プロモーターの制御下にヒトヒストンH2BおよびmCherryをからなる融合タンパク質（MYH6-H2B-MCH）の心筋細胞特異的発現を有するトランスジェニックマウス系統は、2を使用しました。 、のeGFP-anillin融合タンパク質の発現は、CMVエンハンサー（CAG-EGFP-anillin）を有するユビキタスニワトリアクチンプロモーターの制御下にあるトランスジェニック増殖インジケータマウス株、このマウス系統を交配することが可能細胞周期状態の決意。足場タンパク質のanillinは、具体的には、アクティブセル3細胞周期で発現され、細胞周期の間に、その差動細胞内局在は、M相の高解像度でライブトラッキング細胞周期の進行を可能にEF &quot;&gt; 4になるので、二重トランスジェニックマウスは、増殖性心筋細胞および細胞周期変動を受けるものとを区別するために使用することができる。これは、in vitroでの増殖を誘導する物質のスクリーニングに特に有用であることを証明。

<table><tbody><tr><td>10 cm Petri dish</td><td>Sarstedt</td><td>821472</td><td></td></tr><tr><td>100 &amp;micro;m cell strainer</td><td>Becton Dickinson GmbH/Falcon</td><td>352360</td><td></td></tr><tr><td>2,3-Butanedione monoxime (BDM)</td><td>Sigma-Aldrich</td><td>B0753</td><td></td></tr><tr><td>G20x1 &amp;frac12; injection cannula, Sterican</td><td>Braun, Melsungen</td><td>4657519</td><td></td></tr><tr><td>20 gauge needle</td><td>Becton Dickinson GmbH</td><td>301300</td><td></td></tr><tr><td>24-well plates</td><td>Becton Dickinson GmbH/Falcon</td><td>353047</td><td></td></tr><tr><td>2-Methyl-butane</td><td>Carl Roth GmbH + Co. KG</td><td>3927.1</td><td></td></tr><tr><td>37% formaldehyde solution</td><td>AppliChem GmbH&amp;nbsp;</td><td>A0936,1000</td><td></td></tr><tr><td>3-way stopcock</td><td>B. Braun Medical Inc.</td><td>16494C</td><td></td></tr><tr><td>50 mL syringe</td><td>B. Braun Medical Inc.</td><td>8728810F</td><td></td></tr><tr><td>70% ethanol</td><td>Otto Fischar GmbH</td><td>27669</td><td></td></tr><tr><td>Alexa-Fluor-conjugated secondary antibody</td><td>Jackson ImmunoResearch</td><td>115-605-205</td><td></td></tr><tr><td>Alpha-Aktinin EA-53, Mouse IgG</td><td>Sigma-Aldrich, Steinheim</td><td>A7811</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C4901</td><td></td></tr><tr><td>Cell Culture Microplate, 96 Well, Half Area</td><td>Greiner bio-one</td><td>675986</td><td></td></tr><tr><td>Collagenase B</td><td>Roche</td><td>11088815001</td><td></td></tr><tr><td>confocal microscope Eclipse Ti-E</td><td>Nikon</td><td></td><td></td></tr><tr><td>cryostat CM 3050S</td><td>Leica</td><td></td><td></td></tr><tr><td>donkey serum</td><td>Jackson Immuno Research, Suffolk, GB</td><td>017-000-121</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E4884</td><td></td></tr><tr><td>fetal calf serum</td><td>PromoCell, Heidelberg</td><td></td><td></td></tr><tr><td>Formaldehyde solution (4%)</td><td>PanReac AppliChem</td><td>A3697</td><td></td></tr><tr><td>Gelatine from porcine skin, Type A</td><td>Sigma-Aldrich, Steinheim</td><td>G2500</td><td></td></tr><tr><td>glass coverslips</td><td>VWR</td><td>631-0146</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G7021</td><td></td></tr><tr><td>Heidelberger extension tube</td><td>IMPROMEDIFORM GmbH</td><td>MF 1833</td><td></td></tr><tr><td>Heparin-Natrium</td><td>Ratiopharm</td><td>5394.02.00</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>HistoBond microscope slides</td><td>Marienfeld</td><td>0810000</td><td></td></tr><tr><td>Hoechst 33342 (1&amp;nbsp;mg/mL)</td><td>Sigma Aldrich, Taufkirchen</td><td>B2261</td><td></td></tr><tr><td>Insulin syringe</td><td>Becton Dickinson GmbH</td><td>300334</td><td></td></tr><tr><td>Iscove&amp;rsquo;s ModifiedDulbecco&amp;rsquo;s Medium (IMDM)</td><td>Gibco/Life Technologies, Darmstadt</td><td>21980-032</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P9333</td><td></td></tr><tr><td>Laminin</td><td>Corning</td><td>354221</td><td></td></tr><tr><td>Laser Scanning Mikroskop Eclipse Ti</td><td>Nikoninstruments, D&amp;uuml;sseldorf</td><td></td><td></td></tr><tr><td>Lipofectamine RNAiMAX</td><td>Invitrogen/Life Technologies, Darmstadt</td><td>13778075</td><td></td></tr><tr><td>Mouse IgG Cy5 (donkey)</td><td>Jackson ImmunoResearch</td><td>715-175-151</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M8266</td><td></td></tr><tr><td>microcentrifuge tube</td><td>Sarstedt</td><td>72690</td><td></td></tr><tr><td>Mini shaker</td><td>VWR</td><td>12620-940</td><td></td></tr><tr><td>mirVana miRNA mimic, hsa-miR199a-3p</td><td>Ambion/Thermo Fischer Scientific</td><td>4464066</td><td></td></tr><tr><td>Biopsy Mold</td><td>Sakura Finetek/ VWR</td><td>4565</td><td></td></tr><tr><td>M-slide 8-well ibiTreat</td><td>ibidi</td><td>80826</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S9888</td><td></td></tr><tr><td>NaOH</td><td>Merck Millipore</td><td>567530</td><td></td></tr><tr><td>negative control(scrambled RNA)</td><td>Ambion/Thermo Fischer Scientific</td><td>AM4611</td><td></td></tr><tr><td>Neonatal Heart Dissociation Kit</td><td>Miltenyi Biotech, Bergisch Gladbach</td><td>130-098-373</td><td></td></tr><tr><td>NIS Elements AR 4.12.01-4.30.02-64bit</td><td>Nikoninstruments, D&amp;uuml;sseldorf</td><td></td><td></td></tr><tr><td>Non essential amino acids, NEAA</td><td>Gibco/Life Technologies, Darmstad</td><td>11140-035</td><td></td></tr><tr><td>Opti-MEM, Reduced Serum Medium</td><td>Gibco</td><td>51985-026</td><td></td></tr><tr><td>P21-siRNA</td><td>Ambion/Thermo Fischer Scientific</td><td>4390771</td><td></td></tr><tr><td>P27-siRNA</td><td>Ambion/Thermo Fischer Scientific</td><td>4390771</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco/Life Technologies, Darmstadt</td><td>15140-122</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Sigma-Aldrich, Steinheim</td><td>14190-094</td><td></td></tr><tr><td>Polyvinyl alcohol mounting medium with DABCO&amp;reg;, antifading</td><td>Sigma-Aldrich</td><td>10981</td><td></td></tr><tr><td>RNase A</td><td>Qiagen</td><td>1007885</td><td></td></tr><tr><td>RNaseZap</td><td>Invitrogen/Life Technologies, Darmstadt</td><td>AM9780</td><td></td></tr><tr><td>sample containers</td><td>Vitlab</td><td>80731</td><td></td></tr><tr><td>Serological pipette</td><td>Greiner</td><td>607180</td><td></td></tr><tr><td>software NIS Elements</td><td>Nikon</td><td></td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S0389</td><td></td></tr><tr><td>Tissue-Tek O.C.T. Compound</td><td>Sakura Finetek/ VWR</td><td>25608-930</td><td></td></tr><tr><td>ToPro3 iodide (642/661)</td><td>Molecular probes/ThermoFisher Scientific</td><td>T3605</td><td></td></tr><tr><td>Tris</td><td>Sigma-Aldrich</td><td>T1503</td><td></td></tr><tr><td>Triton X</td><td>Fluka</td><td>93418</td><td></td></tr><tr><td>Triton X-100</td><td>Fluka</td><td>93418</td><td></td></tr><tr><td>Trypsin</td><td>Sigma-Aldrich</td><td>T1426</td><td></td></tr><tr><td>Wheat germ agglutinin (WGA) Fluorescein labeled</td><td>Vector Laboratories</td><td>VEC-FL-1021-5</td><td></td></tr><tr><td>&amp;alpha;-actinin antibody</td><td>Sigma-Aldrich</td><td>A7811</td><td></td></tr><tr><td>&amp;beta;-Mercaptoethanol</td><td>Sigma-Aldrich, Steinheim</td><td>M3148</td><td></td></tr></tbody></table>

visualization of cell cycle variations and determination of nucleation in postnatal cardiomyocytes

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and cytokinesis) and acytokinetic mitosis (karyokinesis but no cytokinesis). Such atypical cell cycle variations result in polyploid and multinucleated cells rather than in cell division. Therefore, to determine cardiac turnover and regeneration, it is of crucial importance to correctly identify cardiomyocyte nuclei, the number of nuclei per cell, and their cell cycle status. This is especially true for the use of nuclear markers for identifying cell cycle activity, such as thymidine analogues Ki-67, PCNA, or pHH3. Here, we present methods for recognizing cardiomyocytes and their nuclearity and for determining their cell cycle activity. We use two published transgenic systems: the Myh6-H2B-mCh transgenic mouse line, for the unequivocal identification of cardiomyocyte nuclei, and the CAG-eGFP-anillin mouse line, for distinguishing cell division from cell cycle variations. Combined together, these two systems ease the study of cardiac regeneration and plasticity.

in vitroでの出生後の心筋細胞の細胞周期活性に対するsiRNAを/ miRNAの効果を分析するために、二重トランスジェニックMYH6-H2B-MCH / CAG-EGFP-anillinマウスの心筋細胞は、生後3日目（P3）で単離し、でトランスフェクトしました細胞周期活性誘導miR199 5は、siRNA P27、およびsiRNA Fzr1。陰性対照（ 図1A）と比較して、miR199-（ 図1B）およびsiRNA p27-（ 図1C）の写真は、心筋細胞が細胞周期活性の誘導を示すトランスフェクトしました。 Fzr1の阻害がトランスフェクトされた細胞の核内でのeGFP-anillin融合タンパク質の蓄積をもたらし、Fzr1の損失がAPCの阻害をもたらすようにeGFP-anillinマウスモデルにおいて、Fzr1に対するsiRNAは、トランスフェクションコントロールとして使用することができますFzr1。 Fzr1はプロテアのためanillinをターゲットと後期促進複合体のE3リガーゼの補因子であり、OMAL劣化。 図1Dは、〜45％のトランスフェクション効率を示す、トランスフェクションの3日後にsiRNAをFzr1トランスフェクト心筋細胞の共焦点概観写真を示します。 （P27 6のノックダウンした後に、例えば ）核内倍加を行う心筋細胞はもっぱら核のeGFP-anillin発現（ 図1E）を示すかのeGFP-anillin陰性（説明を参照）です。核内倍加のみエンドS期（EGFP-anillin陽性）およびエンド- G相（EGFP-anillin陰性で構成として彼らは、典型的なM相のローカライズ（ 図1FおよびG）におけるEGFP-anillinを発現しません）。エンド-Gの段階では、APCは、プロテアソームにおけるEGFP-anillinのユビキチン化および分解の結果、アクティブです。 成人期におけるモノおよび二核心筋部の定量化は、ラン後の単一細胞レベルでのいずれかで行うことができますMYH6-H2B-MCHトランスジェニックの心臓のまたはトランスジェニック心の厚いcryoslicesでgendorff解離。ランゲンドルフ装置、心房及び心室における心臓組織の酵素消化後に機械的に分離され、互いに独立して分析することができます。核H2B-MCHの発現によって示されるように、 図2Aは 、二核心筋度の高いランゲンドルフ分離後の非固定H2B-MCHトランスジェニック心室の心筋細胞の代表的な画像を示しています。対照的に、心房心筋細胞の大部分が（ 図2B）単核されています。酵素消化は100％単セルにならないように、αアクチニン染色によって明らかにされたクロスストライエーションのパターンは、二核心筋細胞（クロスストライエーションの連続パターン、 図2C）と細胞ダブレット間の識別を容易にします。 図2Dは 、中二核心筋細胞の識別の例を示しています田舎のスライス。 成人MYH6-H2B-MCHトランスジェニックの心臓の厚いスライスの3D再構成は、組織内の生理学的条件下で心筋細胞核の割合を決定することができます。 図2Eに示すように、イメージングソフトウェアの3Dモジュールを使用して、ヘキスト染色した核およびH2B-MCH陽性核は、自動的に検出し、計数することができます。最終的な結果は、この場合には、互いに直接手を触れ核を意味し、二重のために手動で修正する必要があります。厚いスライスにおける心筋細胞の核の指標を分析するために、核が1 z平面内にない、必要な嘘がそうであるように、手動で、スタックをスクロールすることが必要です。 WGA染色は、セルの枠線の検出を可能にします。 <img alt="図1" src="/files/ftp_upload/55204/55204fig1.jpg" /> 図1：In の例siRNAによるトランスフェクション後の生後心筋細胞における細胞周期活性の in vitro 可視化。 （AD）αアクチニン（白）について染色のeGFP-anillin / MYH6-H2B-MCHの心からP3心筋細胞。心筋細胞核はヘキスト核色素（青色）によってのeGFP-anillin信号（緑色）、及び核によってH2B-MCH信号（赤）、細胞周期活性によって同定されます。スクランブルsiRNAをトランスフェクト（A）P3の心筋細胞は、陰性対照としての役割を果たす。バーは100μmです。 （B）P3の心筋細胞は、コントロール（A）よりのmiRNA-199ディスプレイかなり多くのeGFP-anillin信号をトランスフェクトしました。バーは100μmです。 （C）のp27のsiRNAでトランスフェクションした後に、もっぱら核のeGFP-anillin信号の例、核内倍加を示します。バーは80μmです。 （D）トランスフェクション効率の決意ためFzr1に対するsiRNAのトランスフェクション。 eGFP-anillin交流として、eGFP-anillin +心筋細胞の数は、トランスフェクション効率を示し、累積。バーは80μmです。 （EG）細胞周期の間の心筋細胞におけるEGFP-anillin（緑）の異なるローカライズ：核局在化（Eの矢印）、収縮環（F中の矢印）、および中心体の局在化（G中の矢印）。バーは、（E）中の20ミクロンと（FとG）で10μmです。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55204/55204fig1large.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/55204/55204fig2.jpg" /> 図2：H2B-MCHマウスからの心筋細胞のランゲンドルフ分離により多核化の評価のため、厚いスライスの3D解析による例。 （A、B）心室（A）及び心房（B）成体H2から心臓から心筋細胞ランゲンドルフ解離によって分離した後、B-MCHトランスジェニックマウス。バーは50μmです。 αアクチニン（緑色）について染色MYH6-H2B-MCHの心から（C）心筋細胞。心筋細胞核はH2B-MCH信号（赤）によって識別されます。バーは10μmです。厚いスライス（矢印）中の（D）二核の心筋細胞。心筋細胞核はH2B-MCH信号（赤）、TOPRO3による細胞WGA染色によって境界（緑）および核（白）によって識別されます。バーは50μmです。 （E）ワークフローの厚さにスライスにおける二核性の3D分析のため。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55204/55204fig2large.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 Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes at JoVE.com

Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes

To distinguish cell division from cell cycle variations in cardiomyocytes, we present protocols using two transgenic mouse lines: Myh6-H2B-mCh transgenic mice, for the unequivocal identification of cardiomyocyte nuclei, and CAG-eGFP-anillin mice, for distinguishing cell division from cell cycle variations.

生後心筋細胞における細胞周期変化の可視化と核の決意

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and ...

visualization-cell-cycle-variations-determination-nucleation

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

8.6K ビュー.  University of Bonn. To distinguish cell division from cell cycle variations in cardiomyocytes, we present protocols using two transgenic mouse lines: Myh6-H2B-mCh transgenic mice, for the unequivocal identification of cardiomyocyte nuclei, and CAG-eGFP-anillin mice, for distinguishing cell division from cell cycle variations.

ビデオ: Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated discs, and costameres requires the coordinated assembly of multiple components in time and space. Disruption in assembly of these components leads to developmental heart defects. Immunofluorescent staining techniques are used commonly in cultured cardiomyocytes to probe myofibril maturation, but this ex vivo approach is limited by the extent to which myocytes will fully differentiate in culture, lack of normal in vivo mechanical inputs, and absence of endocardial cues. Application of immunofluorescence techniques to the study of developing mouse heart is desirable but more technically challenging, and methods often lack sufficient sensitivity and resolution to visualize sarcomeres in the early stages of heart development. Here, we describe a robust and reproducible method to co-immunostain multiple proteins or to co-visualize a fluorescent protein with immunofluorescent staining in the embryonic mouse heart and use this method to analyze developing myofibrils, intercalated discs, and costameres. This method can be further applied to assess cardiomyocyte structural changes caused by mutations that lead to developmental heart defects.

Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

胚性マウスの心臓に免疫蛍光を用いて、心筋細胞の開発の分析

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

発生生物学

Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover

Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover occur during normal ageing, quantification of these events remains challenging. This is in part due to the rarity of the process and the fact that multiple cellular sources contribute to myocardial maintenance. Furthermore, DNA duplication within cardiomyocytes often leads to a polyploid cardiomyocyte and only rarely leads to new cardiomyocytes by cellular division. In order to accurately quantify cardiomyocyte turnover discrimination between these processes is essential. The protocol described here employs long term nucleoside labeling in order to label all nuclei which have arisen as a result of DNA replication and cardiomyocyte nuclei identified by utilizing nuclei isolation and subsequent PCM1 immunolabeling. Together this allows the accurate and sensitive identification of the nucleoside labeling of the cardiomyocyte nuclei population. Furthermore, 4&#8242;,6-diamidino-2-phenylindole labeling and analysis of nuclei ploidy, enables the discrimination of neo-cardiomyocyte nuclei from nuclei which have incorporated nucleoside during polyploidization. Although this method cannot control for cardiomyocyte binucleation, it allows a rapid and robust quantification of neo-cardiomyocyte nuclei while accounting for polyploidization. This method has a number of downstream applications including assessing the potential therapeutics to enhance cardiomyocyte regeneration or investigating the effects of cardiac disease on cardiomyocyte turnover and ploidy. This technique is also compatible with additional downstream immunohistological techniques, allowing quantification of nucleoside incorporation in all cardiac cell types.

Quantification of cardiomyocyte turnover is challenging. The protocol described here makes an important contribution to this challenge by enabling accurate and sensitive quantification of neo-cardiomyocyte nuclei generation and nuclei ploidy.

心筋細胞のDNA合成と倍数性の同時評価：心筋細胞再生の定量化を支援する方法および売上高

Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover ...

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, and allow for the study of the cellular and molecular basis of normal and abnormal cardiac morphogenesis. Recent emerging technologies of retrospective single clonal analyses make the study of cardiac morphogenesis at single cell resolution feasible. However, tissue opacity and light scattering of the heart as imaging depth is increased hinder whole-heart imaging at single cell resolution. To overcome these obstacles, a whole-embryo clearing system that can render the heart highly transparent for both illumination and detection must be developed. Fortunately, in the last several years, many methodologies for whole-organism clearing systems such as CLARITY, Scale, SeeDB, ClearT, 3DISCO, CUBIC, DBE, BABB and PACT have been reported. This lab is interested in the cellular and molecular mechanisms of cardiac morphogenesis. Recently, we established single cell lineage tracing via the ROSA26-CreERT2; ROSA26-Confetti system to sparsely label cells during cardiac development. We adapted several whole embryo-clearing methodologies including Scale and CUBIC (clear, unobstructed brain imaging cocktails and computational analysis) to clear the embryo in combination with whole mount staining to image single clones inside the heart. The heart was successfully imaged at single cell resolution. We found that Scale can clear the embryonic heart, but cannot effectively clear the postnatal heart, while CUBIC can clear the postnatal heart, but damages the embryonic heart by dissolving the tissue. The methods described here will permit the study of gene function at a single clone resolution during cardiac morphogenesis, which, in turn, can reveal the cellular and molecular basis of congenital heart defects.

We describe a protocol to volumetrically image fluorescent protein labeled cells deep inside intact embryonic and postnatal hearts. Utilizing tissue-clearing methods in combination with whole mount staining, single fluorescent protein-labeled cells inside an embryonic or postnatal heart can be imaged clearly and accurately.

イメージングは​​、単一細胞の解像度で胚および出生後の心をクリア

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, ...

Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates. Conventional aggregate formation of hPSCs involves dissociating cell colonies into smaller clumps, with size control of the clumps crudely controlled by pipetting the cell suspension until the desired clump size is achieved. One of the main challenges of conventional aggregate-based cardiac differentiation of hPSCs is that culture heterogeneity and spatial disorganization lead to variable and inefficient cardiomyocyte yield. We and others have previously reported that human embryonic stem cell (hESC) aggregate size can be modulated to optimize cardiac induction efficiency. We have addressed this challenge by employing a scalable, microwell-based approach to control physical parameters of aggregate formation, specifically aggregate size and shape. The method we describe here consists of forced aggregation of defined hPSC numbers in microwells, and the subsequent culture of these aggregates in conditions that direct cardiac induction. This protocol can be readily scaled depending on the size and number of wells used. Using this method, we can consistently achieve culture outputs with cardiomyocyte frequencies greater than 70%.

Conventional methods to initiate suspension aggregate based cardiac differentiation of human pluripotent stems cells (hPSCs) are plagued with culture heterogeneity with respect to aggregate size and shape. Here, we describe a robust method for cardiac differentiation employing microwells to generate size-controlled hPSC aggregates cultured under cardiac-promoting conditions.

ヒト多能性幹細胞の懸濁液ベースの心臓分化のためのマイクロウェル中の凝集体サイズの最適化

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates.  Conventional aggregate formation ...

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events in cardiac myocytes such as proliferation and apoptosis require an accurate identification of cardiac myocyte nuclei to analyze cellular renewal in homeostasis and in pathological conditions2. Here, we provide a method to isolate cardiomyocyte nuclei from post mortem tissue by density sedimentation and immunolabeling with antibodies against pericentriolar material 1 (PCM-1) and subsequent flow cytometry sorting. This strategy allows a high throughput analysis and isolation with the advantage of working equally well on fresh tissue and frozen archival material. This makes it possible to study material already collected in biobanks. This technique is applicable and tested in a wide range of species and suitable for multiple downstream applications such as carbon-14 dating3, cell-cycle analysis4, visualization of thymidine analogues (e.g. BrdU and IdU)4, transcriptome and epigenetic analysis.

Cardiac nuclei are isolated via density sedimentation and immunolabeled with antibodies against pericentriolar material 1 (PCM-1) to identify and sort cardiomyocyte nuclei by flow cytometry.

事後組織から心筋細胞核の分離

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

医学

Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage specification. In particular, the conversion of fibroblasts into induced cardiomyocyte-like myocytes (iCLMs) by Gata4, Hand2, Mef2c, and Tbx5 (GHMT) represents an important avenue for generating de novo cardiac myocytes in vitro and in vivo. Recent evidence suggests that GHMT generates a greater diversity of cardiac subtypes than previously appreciated, thus underscoring the need for a systematic approach to conducting additional studies. Before direct reprogramming can be used as a therapeutic strategy, however, the mechanistic underpinnings of lineage conversion must be understood in detail to generate specific cardiac subtypes. Here we present a detailed protocol for generating iCLMs by GHMT-mediated reprogramming of mouse embryonic fibroblasts (MEFs). 
We outline methods for MEF isolation, retroviral production, and MEF infection to accomplish efficient reprogramming. To determine the subtype identity of reprogrammed cells, we detail a step-by-step approach for performing immunocytochemistry on iCLMs using a defined set of compatible antibodies. Methods for confocal microscopy, identification, and quantification of iCLMs and individual atrial (iAM), ventricular (iVM), and pacemaker (iPM) subtypes are also presented. Finally, we discuss representative results of prototypical direct reprogramming experiments and highlight important technical aspects of our protocol to ensure efficient lineage conversion. Taken together, our optimized protocol should provide a stepwise approach for investigators to conduct meaningful cardiac reprogramming experiments that require identification of individual CM subtypes.

This manuscript describes a step-by-step protocol for the generation and quantification of diverse reprogrammed cardiac subtypes using a retrovirus-mediated delivery of Gata4, Hand2, Mef2c, and Tbx5.

マウス胚線維芽細胞の転写因子媒介リプログラミングに続いて心筋細胞のサブタイプを評価します

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

小分子の誘導と多能性ヒト胚性幹細胞からヒト心筋前駆体と心筋細胞の効率的な導出

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

生物学

High-Throughput Cardiotoxicity Screening Using Mature Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Monolayers

Human induced stem cell-derived cardiomyocytes (hiPSC-CMs) are used to replace and reduce the dependence on animals and animal cells for preclinical cardiotoxicity testing. In two-dimensional monolayer formats, hiPSC-CMs recapitulate the structure and function of the adult human heart muscle cells when cultured on an optimal extracellular matrix (ECM). A human perinatal stem cell-derived ECM (maturation-inducing extracellular matrix-MECM) matures the hiPSC-CM structure, function, and metabolic state in 7 days after plating. 
Mature hiPSC-CM monolayers also respond as expected to clinically relevant medications, with a known risk of causing arrhythmias and cardiotoxicity. The maturation of hiPSC-CM monolayers was an obstacle to the widespread adoption of these valuable cells for regulatory science and safety screening, until now. This article presents validated methods for the plating, maturation, and high-throughput, functional phenotyping of hiPSC-CM electrophysiological and contractile function. These methods apply to commercially available purified cardiomyocytes, as well as stem cell-derived cardiomyocytes generated in-house using highly efficient, chamber-specific differentiation protocols. 
High-throughput electrophysiological function is measured using either voltage-sensitive dyes (VSDs; emission: 488 nm), calcium-sensitive fluorophores (CSFs), or genetically encoded calcium sensors (GCaMP6). A high-throughput optical mapping device is used for optical recordings of each functional parameter, and custom dedicated software is used for electrophysiological data analysis. MECM protocols are applied for medication screening using a positive inotrope (isoprenaline) and human Ether-a-go-go-related gene (hERG) channel-specific blockers. These resources will enable other investigators to successfully utilize mature hiPSC-CMs for high-throughput, preclinical cardiotoxicity screening, cardiac medication efficacy testing, and cardiovascular research.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer an alternative to using animals for preclinical cardiotoxicity screening. A limitation to the widespread adoption of hiPSC-CMs in preclinical toxicity screening is the immature, fetal-like phenotype of the cells. Presented here are protocols for robust and rapid maturation of hiPSC-CMs.

成熟ヒト人工多能性幹細胞由来心筋細胞単分子膜を用いたハイスループット心毒性スクリーニング

Human induced stem cell-derived cardiomyocytes (hiPSC-CMs) are used to replace and reduce the dependence on animals and animal cells for preclinical ...

生物工学

Model of Ischemic Heart Disease and Video-Based Comparison of Cardiomyocyte Contraction Using hiPSC-Derived Cardiomyocytes

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with small-animal models such as rodents. However, the physiology of the human heart differs significantly from that of the rodent heart, underscoring the need for clinically relevant models to study heart disease. Here, we present a protocol to model ischemic heart disease using cardiomyocytes differentiated from human induced pluripotent stem cells (hiPS-CMs) and to quantify the damage and functional impairment of the ischemic cardiomyocytes. Exposure to 2% oxygen without glucose and serum increases the percentage of injured cells, which is indicated by staining of the nucleus with propidium iodide, and decreases cellular viability. These conditions also decrease the contractility of hiPS-CMs as confirmed by displacement vector field analysis of microscopic video images. This protocol may furthermore provide a convenient method for personalized drug screening by facilitating the use of hiPS cells from individual patients. Therefore, this model of ischemic heart disease, based on iPS-CMs of human origin, can provide a useful platform for drug screening and further research on ischemic heart disease.

We present a model of ischemic heart disease using cardiomyocytes derived from human induced pluripotent stem cells, together with a method for quantitative evaluation of tissue damage caused by ischemia. This model can provide a useful platform for drug screening and further research on ischemic heart disease.

hiPSC由来心筋細胞を用いた虚血性心疾患のモデルと心筋細胞収縮の映像的比較

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

フローサイトメトリーによる高効率心筋細胞へのヒト多能性幹細胞の分化と特性

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

生後心筋細胞における細胞周期変化の可視化と核の決意

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胚性マウスの心臓に免疫蛍光を用いて、心筋細胞の開発の分析

心筋細胞のDNA合成と倍数性の同時評価：心筋細胞再生の定量化を支援する方法および売上高

イメージングは、単一細胞の解像度で胚および出生後の心をクリア

ヒト多能性幹細胞の懸濁液ベースの心臓分化のためのマイクロウェル中の凝集体サイズの最適化

事後組織から心筋細胞核の分離

マウス胚線維芽細胞の転写因子媒介リプログラミングに続いて心筋細胞のサブタイプを評価します

小分子の誘導と多能性ヒト胚性幹細胞からヒト心筋前駆体と心筋細胞の効率的な導出

成熟ヒト人工多能性幹細胞由来心筋細胞単分子膜を用いたハイスループット心毒性スクリーニング

hiPSC由来心筋細胞を用いた虚血性心疾患のモデルと心筋細胞収縮の映像的比較

フローサイトメトリーによる高効率心筋細胞へのヒト多能性幹細胞の分化と特性

生後心筋細胞における細胞周期変化の可視化と核の決意

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この動画の章

胚性マウスの心臓に免疫蛍光を用いて、心筋細胞の開発の分析

心筋細胞のDNA合成と倍数性の同時評価：心筋細胞再生の定量化を支援する方法および売上高

イメージングは​​、単一細胞の解像度で胚および出生後の心をクリア

ヒト多能性幹細胞の懸濁液ベースの心臓分化のためのマイクロウェル中の凝集体サイズの最適化

事後組織から心筋細胞核の分離

マウス胚線維芽細胞の転写因子媒介リプログラミングに続いて心筋細胞のサブタイプを評価します

小分子の誘導と多能性ヒト胚性幹細胞からヒト心筋前駆体と心筋細胞の効率的な導出

成熟ヒト人工多能性幹細胞由来心筋細胞単分子膜を用いたハイスループット心毒性スクリーニング

hiPSC由来心筋細胞を用いた虚血性心疾患のモデルと心筋細胞収縮の映像的比較

フローサイトメトリーによる高効率心筋細胞へのヒト多能性幹細胞の分化と特性

イメージングは、単一細胞の解像度で胚および出生後の心をクリア