Name: visualization of cell cycle variations and determination of nucleation in postnatal cardiomyocytes
Uploaded: 2017-02-24T14:00:00
Description: Watch this Scientific Journal Video about Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes at JoVE.com

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

All procedures of this protocol involving animals were in accordance with the ethical standards of the University of Bonn and complied with the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes.
1. In Vitro Visualization of Cell Cycle Activity in Postnatal Cardiomyocytes
<ol>
	<li>Postnatal cardiomyocyte dissociation
	<ol>
		<li>Pre-experimental preparations
		<ol>
			<li>Prepare culture media (IMDM, 1% Penicill...

<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>

There is a controversy over whether cardiomyocytes are able to reenter the cell cycle and divide after injury and during tissue homeostasis. Values for the basic turnover of cardiomyocytes have been given in the range between 1%1 and 80%7. Also after a cardiac lesion, the induction of cell cycle activity and the generation of new cardiomyocytes has been reported in the border zone, with values between 0.0083%8 and 25 - 40%7. T...

The correct identification of cardiomyocyte nuclei and the cell cycle status is of crucial importance for the determination of cardiac muscle turnover and regeneration. This is especially true for the use of nuclear markers, such as pHH3, Ki-67, or Thymidine analogs, for identifying cell cycle activity. As the proliferative capacity of adult mammalian cardiomyocytes is very small1, a false identification of a nucleus positive for a proliferation marker of a cardiomyocyte nucleus could make a crucial difference in the outcome of a proliferation assay. Moreover, cardiomyocytes are prone to variations in the cell cycle, such as endoreduplication and acytokinetic mitosis, which result in polyploid and multinucleated cells rather than in cell division. To this end, the interpretation of antibody staining against common cell cycle markers is not conclusive in all cases.
Here, we present methods for the straight-forward recognition of mouse cardiomyocytes and their nuclearity in native isolated cells and thick tissue sections at postnatal and adult stages by the unequivocal identification of their nuclei. For that purpose, a transgenic mouse line with cardiomyocyte-specific expression of a fusion protein consisting of human histone H2B and mCherry under the control of the Myh6 promoter (Myh6-H2B-mCh) was used2. Cross-breeding this mouse line with a transgenic proliferation indicator mouse line, in which the expression of an eGFP-anillin fusion protein is under the control of the ubiquitous chicken actin promoter with a CMV enhancer (CAG-eGFP-anillin), allows for the determination of the cell cycle status. The scaffolding protein anillin is specifically expressed in cell-cycle active cells3, and its differential subcellular localization during the cell cycle allows for live-tracking cell-cycle progression with a high resolution of M-Phase4. Therefore, the double transgenic mice can be used to discriminate between proliferating cardiomyocytes and those that undergo cell-cycle variations. This proves especially useful in screening for proliferation-inducing substances in vitro.

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			<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>
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			<td style="width:119px;">352360</td>
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			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">B0753</td>
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			<td height="21" style="height:21px;">G20x1 &#189; injection cannula, Sterican</td>
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			<td>4657519</td>
			<td></td>
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			<td height="21" style="height:21px;width:383px;">20 gauge needle</td>
			<td>Becton Dickinson GmbH</td>
			<td style="width:119px;">301300</td>
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			<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>
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			<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>
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		<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>
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			<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>
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			<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>
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			<td height="21" style="height:21px;width:383px;">70% ethanol</td>
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			<td style="width:119px;">27669</td>
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			<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>
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			<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>
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			<td height="21" style="height:21px;width:383px;">CaCl</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">C4901</td>
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			<td style="width:119px;">675986</td>
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			<td height="21" style="height:21px;width:383px;">Collagenase B</td>
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			<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>
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			<td style="width:119px;"></td>
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			<td height="21" style="height:21px;">donkey serum</td>
			<td>Jackson Immuno Research, Suffolk, GB</td>
			<td style="width:119px;">017-000-121</td>
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			<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>
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			<td height="30" style="height:30px;width:383px;">EDTA</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">E4884</td>
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			<td height="21" style="height:21px;">fetal calf serum</td>
			<td>PromoCell, Heidelberg</td>
			<td style="width:119px;"></td>
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			<td height="21" style="height:21px;">Formaldehyde solution (4%)</td>
			<td>PanReac AppliChem</td>
			<td style="width:119px;">A3697</td>
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			<td height="25" style="height:25px;">Gelatine from porcine skin, Type A</td>
			<td>Sigma-Aldrich, Steinheim</td>
			<td style="width:119px;">G2500</td>
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			<td height="21" style="height:21px;width:383px;">glass coverslips</td>
			<td style="width:331px;">VWR</td>
			<td style="width:119px;">631-0146</td>
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			<td height="21" style="height:21px;width:383px;">Glucose</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">G7021</td>
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			<td height="21" style="height:21px;width:383px;">Heidelberger extension tube</td>
			<td>IMPROMEDIFORM GmbH</td>
			<td style="width:119px;">MF 1833</td>
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			<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>
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			<td height="21" style="height:21px;width:383px;">HEPES</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3375</td>
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			<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>
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		<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>
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			<td height="21" style="height:21px;width:383px;">Insulin syringe</td>
			<td>Becton Dickinson GmbH</td>
			<td style="width:119px;">300334</td>
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			<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>
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			<td height="21" style="height:21px;width:383px;">KCl</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">P9333</td>
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			<td height="21" style="height:21px;width:383px;">Laminin</td>
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			<td height="21" style="height:21px;">Laser Scanning Mikroskop Eclipse Ti</td>
			<td>Nikoninstruments, D&#252;sseldorf</td>
			<td style="width:119px;"></td>
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			<td height="21" style="height:21px;">Lipofectamine RNAiMAX</td>
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			<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>
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		<tr height="21">
			<td height="21" style="height:21px;width:383px;">MGCl</td>
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			<td height="21" style="height:21px;width:383px;">microcentrifuge tube</td>
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			<td style="width:119px;">72690</td>
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			<td height="21" style="height:21px;width:383px;">Mini shaker</td>
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			<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>
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			<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>
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		<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>
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			<td height="21" style="height:21px;width:383px;">Opti-MEM, Reduced Serum Medium</td>
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			<td style="width:331px;">Ambion/Thermo Fischer Scientific</td>
			<td>4390771</td>
			<td></td>
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			<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>
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		<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>
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		<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>
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		<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>
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		<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 order to analyze the effects of siRNAs/miRNAs on the cell cycle activity of postnatal cardiomyocytes in vitro, cardiomyocytes of double-transgenic Myh6-H2B-mCh/CAG-eGFP-anillin mice were isolated on postnatal day 3 (P3) and transfected with cell cycle activity-inducing miR1995, siRNA p27, and siRNA Fzr1. Compared to the negative control (Figure 1A), the pictures of miR199- (Figure 1B) and siRNA p27- (Figure 1C</...

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.

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

The overall goal of this procedure is to visualize cell cycle activity in postnatal cardiomyocytes and to determine their nuclearity. This method can help answer key questions in the cardiac regeneration field, such as does a cardiomyocyte really divide or does it just increase its ploidy or become multi-nucleated? The main advantages of this technique are that the M phase of the cell cycle can be visualized in high spatiotemporal resolution and that cardiomyocyte nuclei can be identified by fluorescence.Demonstrating the procedure will be Patricia Freitag, a technician from our laboratory. If using non-homozygous breeding pairs, first identify the transgenic hearts via fluorescent microscopy to check for their EGFP analin and H2B-mCherry expression. EGFP analyin nuclear signals can be most easily detected at the edges of the atria by focusing through its tissue layers.To isolate the cardiomyocytes, place the appropriate number of hearts in 1.5 milliliter reaction tubes containing one milliliter of enzyme mix from a neonatal heart dissociation kit and cut the hearts into small pieces with scissors. Then, transfer the solution into 15 milliliter reaction tubes. Place the tube in an almost horizontal position at 37 degrees celsius for 15 minutes, mixing the tissue five to 10 times with a five milliliter pipette at the end of the incubation.At the end of the third digestion, stop the reaction with 7.5 milliliters of medium two and filter the cell suspension through a 70 micron cell strainer. Rinse the cell strainer with three milliliters of medium two, pooling the wash with the collected cells and collect the cells by centrifugation. Re-suspend the pellet in 500 microliters of medium one for counting.Then, seed one times ten to the fourth cells per well in 120 microliters of medium one in a 96-well flat-bottom plate and centrifuge the cells before their overnight culture in a cell culture incubator. The next day, most of the cardiomyocytes should be beating. Before beginning the transfection, wipe the bench and all of the transfection materials with RNAse decontamination solution to remove any RNAse.When all of the materials are ready, add 20 microliters of freshly prepared siRNA mix to each well and return the cells to the incubator for 48 hours. After two days, replace the supernatant in each well with 120 microliters of medium one and return the cells to the incubator for at least another 24 hours. At the end of the incubation, wash the cells one time with PBS, followed by fixation in 4%formaldehyde solution for 20 minutes.To analyze the cells by confocal video microscopy, transfer the plate to the confocal microscope stage and start the imaging software. Open the A1plus settings and check the channel one, two, three and four boxes. In the pull-down menu, set channel one to DAPI, channel two to eGFP, channel three to RFP, and channel four to Cy5.Click the channel voltage and use the slide bars to set the voltage to 80 for each channel. Use the slide bars to set the offset to zero and click on the home button to set the pinhole to the home position. Set the scan size to 1024 by 1024 pixels in the pull-down menu.Click optimize to open the XYZ size setup window and check the perfect voxel box under the suggested step Z.Select the 20x objective and adjust the laser intensities until the picture is neither under-nor overexposed. When all of the parameters have been set, open the acquire menu. Select scan large image then choose current position is at top left corner under area.Then set the number of fields in X and Y to three by three and click scan. To define the number of cardiomyocyte nuclei, click measure, manual measurement, and counts. Select the nuclei with H2B-mCherry signals, thereby marking and counting them.Then, select the alpha-actinin positive cells to determine the number of cardiomyocytes. To count the G1, S, G2 phase cardiomyocytes with nuclear EGFP analin signals, select measure, manual measurement, and counts. Mark the EGFP analin and H2B-mCherry expressing nuclei with clicking and manually count the cardiomyocytes with the mitosis-specific EGFP analin signals such as contractile rings and mid-bodies.Then, click measure, manual measurement, and counts and click the cardiomyocytes with mitosis-specific EGFP analin signals, cytoplasmatic signals, contractile rings, and mid-bodies. Compared to the negative controls, the miRNA and siRNA transfected cardiomyocytes demonstrate an induction of cell cycle activity. Cardiomyocytes that perform endoreduplication exhibit an exclusively nuclear EGFP analin expression, whereas cardiomyocytes undergoing cell division display EGFP analin expression in M phase typical localizations.After enzymatic digestion of the heart tissue at the Langendorff apparatus, the atria and ventricles can be mechanically separated and analyzed independently, allowing the visualization of H2B-mCherry transgenic ventricular cardiomyocytes with a high number of H2B-mCherry positive bi-nucleated cardiomyocytes. By contrast, the majority of atrial cardiomyocytes are mono-nucleated. Enzymatic digestion does not result in a 100%single-cell population revealing a pattern of cross-striation by alpha-actinin staining, further facilitating the discrimination between bi-nucleated cardiomyocytes as a continuous pattern of cross-striation and cell doublets.To analyze the bi-nucleation index of cardiomyocytes in thick slices, it is necessary to manually scroll through the stack as the nuclei do not necessarily lay within one Z-plane, with wheat germ agglutinin staining permitting the detection of the cell borders. 3D reconstructions of fixed slices of adult transgenic mouse hearts allow the detection and automated counting of hooks, stained, and H2B-mCherry positive nuclei to determine the proportion of cardiomyocyte nuclei under physiological conditions within the tissue. Once mastered, the heart dissociation can be completed in two to three hours if it is performed properly.While attempting this procedure, it is important to remember to work continuously to maintain the viability of the cells throughout the experiment. Following this procedure, it is possible to screen microRNAs or other small substances for their proliferation-inducing effects in postnatal cardiomyocytes.

visualization-cell-cycle-variations-determination-nucleation

Research

JoVE Journal

Developmental Biology

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases of the cell cycle, or the incorporation of nucleoside analogs. During S-phase, DNA polymerases incorporate thymidine analogs such as EdU or BrdU into chromosomal DNA, marking the cells for analysis. For C. elegans, the nucleoside analog EdU is fed to the worms during regular culture and is compatible with immunofluorescent techniques. The germline of C. elegans is a powerful model system for the studies of signaling pathways, stem cells, meiosis, and cell cycle because it is transparent, genetically facile, and meiotic prophase and cellular differentiation/gametogenesis occur in a linear assembly-like fashion. These features make EdU a great tool to study dynamic aspects of mitotically cycling cells and germline development. This protocol describes how to successfully prepare EdU bacteria, feed them to wild-type C. elegans hermaphrodites, dissect the hermaphrodite gonad, stain for EdU incorporation into DNA, stain with antibodies to detect various cell cycle and developmental markers, image the gonad and analyze the results. The protocol describes the variations in the method and analysis for the measurement of S-phase index, M-phase index, G2 duration, cell cycle duration, rate of meiotic entry, and rate of meiotic prophase progression. This method can be adapted to study the cell cycle or cell history in other tissues, stages, genetic backgrounds, and physiological conditions.

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

An imaging-based method is described that can be used to identify S-phase and analyze cell cycle dynamics in the C. elegans hermaphrodite germline using the thymidine analog EdU. This method requires no transgenes and is compatible with immunofluorescent staining.

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases ...