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?...

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 border="0" cellpadding="0" cellspacing="0" width="999">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">10 cm petri dish</td>
			<td style="width:331px;">Sarstedt</td>
			<td style="width:119px;">821472</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">100 &#181;m cell strainer</td>
			<td style="width:331px;">Becton Dickinson GmbH/Falcon</td>
			<td style="width:119px;">352360</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">2,3-Butanedione monoxime (BDM)</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">B0753</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">G20x1 &#189; injection cannula, Sterican</td>
			<td style="width:331px;">Braun, Melsungen</td>
			<td>4657519</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">20 gauge needle</td>
			<td>Becton Dickinson GmbH</td>
			<td style="width:119px;">301300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">24-well plates</td>
			<td style="width:331px;">Becton Dickinson GmbH/Falcon</td>
			<td style="width:119px;">353047</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">2-Methyl-butane</td>
			<td style="width:331px;">Carl Roth GmbH + Co. KG</td>
			<td style="width:119px;">3927.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">37% formaldehyde solution</td>
			<td style="width:331px;">AppliChem GmbH&#160;</td>
			<td>A0936,1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">3-way stopcock</td>
			<td style="width:331px;">B. Braun Medical Inc.</td>
			<td style="width:119px;">16494C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">50 ml syringe</td>
			<td style="width:331px;">B. Braun Medical Inc.</td>
			<td style="width:119px;">8728810F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">70% ethanol</td>
			<td style="width:331px;">Otto Fischar GmbH</td>
			<td style="width:119px;">27669</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Alexa-Fluor-conjugated secondary antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td style="width:119px;">115-605-205</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Alpha-Aktinin EA-53, Mouse IgG</td>
			<td style="width:331px;">Sigma-Aldrich, Steinheim</td>
			<td style="width:119px;">A7811</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">CaCl</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">C4901</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Cell Culture Microplate, 96 Well, Half Area</td>
			<td style="width:331px;">Greiner bio-one</td>
			<td style="width:119px;">675986</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Collagenase B</td>
			<td style="width:331px;">Roche</td>
			<td style="width:119px;">11088815001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">confocal microscope Eclipse Ti-E</td>
			<td style="width:331px;">Nikon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">cryostat CM 3050S</td>
			<td style="width:331px;">Leica</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">donkey serum</td>
			<td>Jackson Immuno Research, Suffolk, GB</td>
			<td style="width:119px;">017-000-121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:383px;">EDTA</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">E4884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fetal calf serum</td>
			<td>PromoCell, Heidelberg</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formaldehyde solution (4%)</td>
			<td>PanReac AppliChem</td>
			<td style="width:119px;">A3697</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Gelatine from porcine skin, Type A</td>
			<td>Sigma-Aldrich, Steinheim</td>
			<td style="width:119px;">G2500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">glass coverslips</td>
			<td style="width:331px;">VWR</td>
			<td style="width:119px;">631-0146</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Glucose</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">G7021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Heidelberger extension tube</td>
			<td>IMPROMEDIFORM GmbH</td>
			<td style="width:119px;">MF 1833</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Heparin-Natrium</td>
			<td style="width:331px;">Ratiopharm</td>
			<td style="width:119px;">5394.02.00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">HEPES</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">HistoBond microscope slides</td>
			<td style="width:331px;">Marienfeld</td>
			<td style="width:119px;">0810000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33342 (1mg/ml)</td>
			<td>Sigma Aldrich, Taufkirchen</td>
			<td style="width:119px;">B2261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Insulin syringe</td>
			<td>Becton Dickinson GmbH</td>
			<td style="width:119px;">300334</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iscove&#8217;s ModifiedDulbecco&#8217;s Medium (IMDM)</td>
			<td>Gibco/Life Technologies, Darmstadt</td>
			<td>21980-032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">KCl</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">P9333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Laminin</td>
			<td style="width:331px;">Corning</td>
			<td style="width:119px;">354221</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laser Scanning Mikroskop Eclipse Ti</td>
			<td>Nikoninstruments, D&#252;sseldorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lipofectamine RNAiMAX</td>
			<td>Invitrogen/Life Technologies, Darmstadt</td>
			<td>13778075</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:383px;">Mouse IgG Cy5 (donkey)</td>
			<td style="width:331px;">Jackson ImmunoResearch</td>
			<td>715-175-151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">MGCl</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">M8266</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">microcentrifuge tube</td>
			<td style="width:331px;">Sarstedt</td>
			<td style="width:119px;">72690</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Mini shaker</td>
			<td style="width:331px;">VWR</td>
			<td>12620-940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">mirVana miRNA mimic, hsa-miR199a-3p</td>
			<td style="width:331px;">Ambion/Thermo Fischer Scientific</td>
			<td>4464066</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Biopsy Mold</td>
			<td style="width:331px;">Sakura Finetek/ VWR</td>
			<td>4565</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">M-slide 8-well ibiTreat</td>
			<td style="width:331px;">ibidi</td>
			<td style="width:119px;">80826</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">NaCl</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">S9888</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">NaOH</td>
			<td style="width:331px;">Merck Millipore</td>
			<td style="width:119px;">567530</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">negative control(scrambled RNA)</td>
			<td style="width:331px;">Ambion/Thermo Fischer Scientific</td>
			<td>AM4611</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neonatal Heart Dissociation Kit</td>
			<td>Miltenyi Biotech, Bergisch Gladbach</td>
			<td style="width:119px;">130-098-373</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NIS Elements AR 4.12.01-4.30.02-64bit</td>
			<td>Nikoninstruments, D&#252;sseldorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non essential amino acids, NEAA</td>
			<td>Gibco/Life Technologies, Darmstad</td>
			<td>11140-035</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Opti-MEM, Reduced Serum Medium</td>
			<td style="width:331px;">Gibco</td>
			<td style="width:119px;">51985-026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">P21-siRNA</td>
			<td style="width:331px;">Ambion/Thermo Fischer Scientific</td>
			<td>4390771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">P27-siRNA</td>
			<td style="width:331px;">Ambion/Thermo Fischer Scientific</td>
			<td>4390771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptomycin</td>
			<td>Gibco/Life Technologies, Darmstadt</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich, Steinheim</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:383px;">Polyvinyl alcohol mounting medium with DABCO&#174;, antifading</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">10981</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">RNase A</td>
			<td style="width:331px;">Qiagen</td>
			<td>1007885</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNaseZap</td>
			<td>Invitrogen/Life Technologies, Darmstadt</td>
			<td style="width:119px;">AM9780</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">sample containers</td>
			<td style="width:331px;">Vitlab</td>
			<td style="width:119px;">80731</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Serological pipette</td>
			<td style="width:331px;">Greiner</td>
			<td style="width:119px;">607180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">software NIS Elements</td>
			<td style="width:331px;">Nikon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Sucrose</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">S0389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Tissue-Tek O.C.T. Compound</td>
			<td style="width:331px;">Sakura Finetek/ VWR</td>
			<td>25608-930</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">ToPro3 iodide (642/661)</td>
			<td style="width:331px;">Molecular probes/ThermoFisher Scientific</td>
			<td style="width:119px;">T3605</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Tris</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">T1503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Triton X</td>
			<td style="width:331px;">Fluka</td>
			<td style="width:119px;">93418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Triton X-100</td>
			<td style="width:331px;">Fluka</td>
			<td style="width:119px;">93418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Trypsin</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">T1426</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Wheat germ agglutinine (WGA) Fluorescein labeled</td>
			<td>Vector Laboratories</td>
			<td>VEC-FL-1021-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">&#945;-actinin antibody</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7811</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#946;-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）?...

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

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

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

生後神経発生の研究のための器官型スライス培養

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

発生生物学

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 ...

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for &#945;-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.

The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

新生児ラット心筋細胞の単離と凍結保存

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is ...

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel&#8217;s Cartilage

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. Patterning of the Meckel&#39;s cartilage, a first pharyngeal arch derivative, involves the migration of cranial neural crest (CNC) cells and the progressive partitioning, proliferation and organization of differentiated chondrocytes. Several studies have described CNC migration during lower jaw&#160;morphogenesis, but the details of how the chondrocytes achieve organization in the growth and extension of Meckel&#8217;s cartilage remains unclear. The sox10 restricted and chemically induced Cre recombinase-mediated recombination generates permutations of distinct fluorescent proteins (RFP, YFP and CFP), thereby creating a multi-spectral labeling of progenitor cells and their progeny, reflecting distinct clonal populations. Using confocal time-lapse photography, it is possible to observe the chondrocytes behavior during the development of the zebrafish Meckel&#8217;s cartilage.
Multispectral cell labeling enables scientists to demonstrate extension of the Meckel&#8217;s chondrocytes.&#160;During extension phase of the Meckel&#8217;s cartilage, which prefigures the mandible, chondrocytes intercalate to effect extension as they stack in an organized single-cell layered row. Failure of this organized intercalating process to mediate cell extension provides the cellular mechanistic explanation for hypoplastic mandible that we observe in mandibular malformations.

Cell organization of craniofacial bones has long been hypothesized but never directly visualized. Multi-spectral cell labeling and in vivo live imaging allows visualization of dynamic cell behavior in zebrafish lower jaw. Here, we detail the protocol to manipulate Zebrabow transgenic fish and directly observe cell intercalation and morphological changes of chondrocytes in the Meckel&#8217;s cartilage.

胚性メッケル軟骨でZebrabowクローン細胞分析によって軟骨細胞のインターカレーションと方向性増殖の可視化

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. ...

Differentiation of Atrial Cardiomyocytes from Pluripotent Stem Cells Using the BMP Antagonist Grem2

Protocols for generating populations of cardiomyocytes from pluripotent stem cells have been developed, but these generally yield cells of mixed phenotypes. Researchers interested in pursuing studies involving specific myocyte subtypes require a more directed differentiation approach. By treating mouse embryonic stem (ES) cells with Grem2, a secreted BMP antagonist that is necessary for atrial chamber formation in vivo, a large number of cardiac cells with an atrial phenotype can be generated. Use of the engineered Myh6-DSRed-Nuc pluripotent stem cell line allows for identification, selection, and purification of cardiomyocytes. In this protocol embryoid bodies are generated from Myh6-DSRed-Nuc cells using the hanging drop method and kept in suspension until differentiation day 4 (d4). At d4 cells are treated with Grem2 and plated onto gelatin coated plates. Between d8-d10 large contracting areas are observed in the cultures and continue to expand and mature through d14. Molecular, histological and electrophysiogical analyses indicate cells in Grem2-treated cells acquire atrial-like characteristics providing an in vitro model to study the biology of atrial cardiomyocytes and their response to various pharmacological agents.

Generating cardiomyocytes from pluripotent stem cells in vitro allows access to large amounts of cardiac tissue in vitro for basic science and clinical applications. This protocol uses the atrializing factor Grem2 to both increase the numbers of cardiomyocytes obtained and to generate cardiomyocytes with an atrial phenotype.

BMPアンタゴニストGrem2を使用した多能性幹細胞から心房心筋細胞の分化

Protocols for generating populations of cardiomyocytes from pluripotent stem cells have been developed, but these generally yield cells of mixed ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

再現性の高い小分子ベースの分化プロトコルを使用したヒト多能性幹細胞から心筋細胞の大規模生産

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

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, ...

Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their potential applications in cardiac research, including drug discovery, disease modeling, tissue engineering, and regenerative medicine. However, cells produced by existing protocols display a range of immaturity compared with native adult ventricular cardiomyocytes. Many efforts have been made to mature hPSC-CMs, with only moderate maturation attained thus far. Therefore, an engineered system, called biowire, has been devised by providing both physical and electrical cues to lead hPSC-CMs to a more mature state in vitro. The system uses a microfabricated platform to seed hPSC-CMs in collagen type I gel along a rigid template suture to assemble into aligned cardiac tissue (biowire), which is subjected to electrical field stimulation with a progressively increasing frequency. Compared to nonstimulated controls, stimulated biowired cardiomyocytes exhibit an enhanced degree of structural and electrophysiological maturation. Such changes are dependent upon the stimulation rate. This manuscript describes in detail the design and creation of biowires.

The cardiac biowire platform is an in vitro method used to mature human embryonic and induced pluripotent stem cell-derived cardiomyocytes (hPSC-CM) by combining three-dimensional cell cultivation with electrical stimulation. This manuscript presents the detailed setup of the cardiac biowire platform.

電気刺激を使用してBiowires中のヒト幹細胞由来の心筋細胞の成熟

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their ...

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes (hPSC-CMs) Using Multi-electrode Arrays (MEAs)

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived cardiomyocytes (hPSC-CMs) are increasingly recognized as having great value for modeling cardiovascular diseases in humans, especially arrhythmia syndromes. They have also demonstrated relevance as in vitro systems for predicting drug responses, which makes them potentially useful for drug-screening and discovery, safety pharmacology and perhaps eventually for personalized medicine. This would be facilitated by deriving hPSC-CMs from patients or susceptible individuals as hiPSCs. For all applications, however, precise measurement and analysis of hPSC-CM electrical properties are essential for identifying changes due to cardiac ion channel mutations and/or drugs that target ion channels and can cause sudden cardiac death. Compared with manual patch-clamp, multi-electrode array (MEA) devices offer the advantage of allowing medium- to high-throughput recordings. This protocol describes how to dissociate 2D cell cultures of hPSC-CMs to small aggregates and single cells and plate them on MEAs to record their spontaneous electrical activity as field potential. Methods for analyzing the recorded data to extract specific parameters, such as the QT and the RR intervals, are also described here. Changes in these parameters would be expected in hPSC-CMs carrying mutations responsible for cardiac arrhythmias and following addition of specific drugs, allowing detection of those that carry a cardiotoxic risk.

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes hPSC-CMs Using Multi-electrode Arrays MEAs

Electrophysiological characterization of cardiomyocytes derived from human Pluripotent Stem Cells (hPSC-CMs) is crucial for cardiac disease modeling and for determining drug responses. This protocol provides the necessary information to dissociate and plate hPSC-CMs on multi-electrode arrays, measure their field potential, and a method for analyzing QT and RR intervals.

多電極アレイ（MEA）を用いたヒト多能性幹細胞由来心筋細胞（hPSC-CM）の電気生理学的解析

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

マウスにおけるヒト化骨髄微小環境のバイオエンジニアリングとライブイメージングによるそれらの可視化

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

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

要約

さらに動画を探す

この動画の章

生後神経発生の研究のための器官型スライス培養

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

新生児ラット心筋細胞の単離と凍結保存

胚性メッケル軟骨でZebrabowクローン細胞分析によって軟骨細胞のインターカレーションと方向性増殖の可視化

BMPアンタゴニストGrem2を使用した多能性幹細胞から心房心筋細胞の分化

再現性の高い小分子ベースの分化プロトコルを使用したヒト多能性幹細胞から心筋細胞の大規模生産

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

電気刺激を使用してBiowires中のヒト幹細胞由来の心筋細胞の成熟

多電極アレイ（MEA）を用いたヒト多能性幹細胞由来心筋細胞（hPSC-CM）の電気生理学的解析

マウスにおけるヒト化骨髄微小環境のバイオエンジニアリングとライブイメージングによるそれらの可視化

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

要約

さらに動画を探す

この動画の章

生後神経発生の研究のための器官型スライス培養

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

新生児ラット心筋細胞の単離と凍結保存

胚性メッケル軟骨でZebrabowクローン細胞分析によって軟骨細胞のインターカレーションと方向性増殖の可視化

BMPアンタゴニストGrem2を使用した多能性幹細胞から心房心筋細胞の分化

再現性の高い小分子ベースの分化プロトコルを使用したヒト多能性幹細胞から心筋細胞の大規模生産

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

電気刺激を使用してBiowires中のヒト幹細胞由来の心筋細胞の成熟

多電極アレイ（MEA）を用いたヒト多能性幹細胞由来心筋細胞（hPSC-CM）の電気生理学的解析

マウスにおけるヒト化骨髄微小環境のバイオエンジニアリングとライブイメージングによるそれらの可視化

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