Name: assessing cardiomyocyte subtypes following transcription factor-mediated reprogramming of mouse embryonic fibroblasts
Uploaded: 2017-03-22T14:00:00
Description: Watch this Scientific Journal Video about Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts at JoVE.com

A.F.-P. was supported by the National Science Foundation Graduate Research Fellowship under Grant No.2015165336. N.V.M was supported by grants from the NIH (HL094699), Burroughs Wellcome Fund (1009838), and the March of Dimes (#5-FY14-203). We acknowledge Young-Jae Nam, Christina Lubczyk, and Minoti Bhakta for their important contributions to protocol development and data analysis. We also thank John Shelton for valuable technical input and members of the Munshi lab for scientific discussion.

涉及动物的做法，所有的实验程序进行的机构动物护理和使用委员会在UT西南医疗中心的批准。 1.隔离HCN4-GFP E12.5小鼠胚胎成纤维细胞（MEF中） <ol><li>建立纯合子HCN4-GFP男性和CD-1雌性间定时交配。 </li><li>在二氧化碳安乐死和随后的颈椎脱位E12.5牺牲怀孕的女性。 <ol><li>与解剖钳除去子宫角，如前所述22，23，并将其放置在不含Ca ...

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	<li>Sissman, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+landmarks+in+cardiac+morphogenesis:+comparative+chronology.">Developmental landmarks in cardiac morphogenesis: comparative chronology.</a> Am J Cardiol. 25 (2), 141-148 (1970).</li><li>Buckingham, M., Meilhac, S., Zaffran, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+the+mammalian+heart+from+two+sources+of+myocardial+cells.">Building the mammalian heart from two sources of myocardial cells.</a> Nat Rev Genet. 6 (11), 826-835 (2005).</li><li>Ali, S. R., 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=Existing+cardiomyocytes+generate+cardiomyocytes+at+a+low+rate+after+birth+in+mice.">Existing cardiomyocytes generate cardiomyocytes at a low rate after birth in mice.</a> Proc Natl Acad Sci U S A. 111 (24), 8850-8855 (2014).</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=Evidence+for+cardiomyocyte+renewal+in+humans.">Evidence for cardiomyocyte renewal in humans.</a> Science. 324 (5923), 98-102 (2009).</li><li>Senyo, S. E., 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=Mammalian+heart+renewal+by+pre-existing+cardiomyocytes.">Mammalian heart renewal by pre-existing cardiomyocytes.</a> Nature. 493 (7432), 433-436 (2013).</li><li>Lin, Z., Pu, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+cardiac+regeneration+and+repair.">Strategies for cardiac regeneration and repair.</a> Sci Transl Med. 6 (239), 239rv231 (2014).</li><li>Writing Group, 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=Heart+Disease+and+Stroke+Statistics-2016+Update:+A+Report+From+the+American+Heart+Association.">Heart Disease and Stroke Statistics-2016 Update: A Report From the American Heart Association.</a> Circulation. 133 (4), e38-e360 (2016).</li><li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126 (4), 663-676 (2006).</li><li>Song, K., 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=Heart+repair+by+reprogramming+non-myocytes+with+cardiac+transcription+factors.">Heart repair by reprogramming non-myocytes with cardiac transcription factors.</a> Nature. 485 (7400), 599-604 (2012).</li><li>Ieda, 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+reprogramming+of+fibroblasts+into+functional+cardiomyocytes+by+defined+factors.">Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors.</a> Cell. 142 (3), 375-386 (2010).</li><li>Qian, 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=In+vivo+reprogramming+of+murine+cardiac+fibroblasts+into+induced+cardiomyocytes.">In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes.</a> Nature. 485 (7400), 593-598 (2012).</li><li>Fu, J. D., 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+reprogramming+of+human+fibroblasts+toward+a+cardiomyocyte-like+state.">Direct reprogramming of human fibroblasts toward a cardiomyocyte-like state.</a> Stem Cell Reports. 1 (3), 235-247 (2013).</li><li>Zhou, H., Dickson, M. E., Kim, M. S., Bassel-Duby, R., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Akt1/protein+kinase+B+enhances+transcriptional+reprogramming+of+fibroblasts+to+functional+cardiomyocytes.">Akt1/protein kinase B enhances transcriptional reprogramming of fibroblasts to functional cardiomyocytes.</a> Proc Natl Acad Sci U S A. 112 (38), 11864-11869 (2015).</li><li>Zhou, Y., 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=Bmi1+Is+a+Key+Epigenetic+Barrier+to+Direct+Cardiac+Reprogramming.">Bmi1 Is a Key Epigenetic Barrier to Direct Cardiac Reprogramming.</a> Cell Stem Cell. 18 (3), 382-395 (2016).</li><li>Zhao, Y., 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=High-efficiency+reprogramming+of+fibroblasts+into+cardiomyocytes+requires+suppression+of+pro-fibrotic+signalling.">High-efficiency reprogramming of fibroblasts into cardiomyocytes requires suppression of pro-fibrotic signalling.</a> Nat Commun. 6, 8243 (2015).</li><li>Miki, K., Yoshida, Y., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+steady+progress+on+direct+cardiac+reprogramming+toward+clinical+application.">Making steady progress on direct cardiac reprogramming toward clinical application.</a> Circ Res. 113 (1), 13-15 (2013).</li><li>Atkinson, 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=Anatomical+and+molecular+mapping+of+the+left+and+right+ventricular+His-Purkinje+conduction+networks.">Anatomical and molecular mapping of the left and right ventricular His-Purkinje conduction networks.</a> J Mol Cell Cardiol. 51 (5), 689-701 (2011).</li><li>Bootman, M. D., Smyrnias, I., Thul, R., Coombes, S., Roderick, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atrial+cardiomyocyte+calcium+signalling.">Atrial cardiomyocyte calcium signalling.</a> Biochim Biophys Acta. 1813 (5), 922-934 (2011).</li><li>Miquerol, L., Beyer, S., Kelly, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+the+mouse+ventricular+conduction+system.">Establishment of the mouse ventricular conduction system.</a> Cardiovasc Res. 91 (2), 232-242 (2011).</li><li>Sp&#228;ter, D., Hansson, E. M., Zangi, L., Chien, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+make+a+cardiomyocyte.">How to make a cardiomyocyte.</a> Development. 141 (23), 4418-4431 (2014).</li><li>Nam, Y. J., 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=Induction+of+diverse+cardiac+cell+types+by+reprogramming+fibroblasts+with+cardiac+transcription+factors.">Induction of diverse cardiac cell types by reprogramming fibroblasts with cardiac transcription factors.</a> Development. 141 (22), 4267-4278 (2014).</li><li>Conner, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current Protocols in Molecular Biology. , (2001).</li><li>Jozefczuk, J., Drews, K., Adjaye, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+mouse+embryonic+fibroblast+cells+suitable+for+culturing+human+embryonic+and+induced+pluripotent+stem+cells.">Preparation of mouse embryonic fibroblast cells suitable for culturing human embryonic and induced pluripotent stem cells.</a> J Vis Exp. (64), (2012).</li><li>Burns, J. C., Friedmann, T., Driever, W., Burrascano, M., Yee, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+stomatitis+virus+G+glycoprotein+pseudotyped+retroviral+vectors:+concentration+to+very+high+titer+and+efficient+gene+transfer+into+mammalian+and+nonmammalian+cells.">Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells.</a> Proc Natl Acad Sci U S A. 90 (17), 8033-8037 (1993).</li><li>Ichim, C. V., Wells, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+high-titer+viral+preparations+by+concentration+using+successive+rounds+of+ultracentrifugation.">Generation of high-titer viral preparations by concentration using successive rounds of ultracentrifugation.</a> J Transl Med. 9, 137 (2011).</li><li>Qian, L., Berry, E. C., Fu, J. D., Ieda, M., Srivastava, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+mouse+fibroblasts+into+cardiomyocyte-like+cells+in+vitro.">Reprogramming of mouse fibroblasts into cardiomyocyte-like cells in vitro.</a> Nat Protoc. 8 (6), 1204-1215 (2013).</li><li>Yang, R., 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+conversion+of+mouse+and+human+fibroblasts+to+functional+melanocytes+by+defined+factors.">Direct conversion of mouse and human fibroblasts to functional melanocytes by defined factors.</a> Nat Commun. 5, 5807 (2014).</li><li>Muraoka, N., Ieda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+reprogramming+of+fibroblasts+into+myocytes+to+reverse+fibrosis.">Direct reprogramming of fibroblasts into myocytes to reverse fibrosis.</a> Annu Rev Physiol. 76, 21-37 (2014).</li><li>Coffin, J. M., Hughes, S. H., Varmus, H. E., Coffin, J. M., Hughes, S. H., Varmus, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Retroviruses. , (1997).</li><li>Bass, G. 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=Automated+image+analysis+identifies+signaling+pathways+regulating+distinct+signatures+of+cardiac+myocyte+hypertrophy.">Automated image analysis identifies signaling pathways regulating distinct signatures of cardiac myocyte hypertrophy.</a> J Mol Cell Cardiol. 52 (5), 923-930 (2012).</li></ol>

本研究通过心脏转录的逆转录病毒介导的表达提供的MEF转换的直接重新编程战略转化为一组不同的亚型心脏因素GATA4，MEF2C，Tbx5中，和HAND2（GHMT）。在使用具有特定的PM记者鼠标组合多重染色的方法，我们能够确定（IAM），网络视频监控软件，并在IPMS单细胞分辨率。这种试验可以在隔离能力个别转录因子对亚型多样性和肌节发展的贡献的体外系统的实验研究。与此同时，这可能会带?...

心脏是在胚胎1，2，开发的第一个功能器官。与循环系统相结合，其提供氧，营养物，和开发过程中的废物处理机制。受精后三个星期，人的心脏跳动，第一次和适当的调整则是由心肌细胞（CMS）维护。因此，这些特殊细胞的不可逆损失是潜在的进行性心脏衰竭的根本问题。而一些生物如斑马鱼和爪蟾具有用于心脏再生的潜力，成年哺乳动物心脏较为有限3，5，6。因此，由于心脏的重要功能，是不令人吃惊的是心脏疾病是全世界死亡的首要原因，占仅7美国60万人死亡。该refore，基于细胞的疗法有效地修理或更换损伤心肌是伟大的具有临床意义。 山中和同事8的开创性的研究表明，四种转录因子的强制表达足以完全分化的成纤维细胞转化为多能干细胞。不过，所有的多能干细胞的策略的致瘤能力已在其用于治疗目的使用的一个关键问题。这促使科学领域寻找替代方法转分化的细胞，同时避免多能的阶段。最近，一些团体已经通过与转录异位表达显示小鼠成纤维细胞直接转化为诱导的心肌样细胞（iCLMs）显示了这一战略的可行性因素GATA4，MEF2C，Tbx5中，后来，HAND2（GMT和GHMT分别）9，10。 Furthermore，同样的策略可以在体内和人衍生的组织9，11，12来进行。最近的研究已强调了额外的因素或可被调制以进一步改善心脏重编程效率13，14，15的信号通路。总之，这些研究表明定向转分化为再生疗法的潜力。然而，CM重新编程，未知的分子机制，重复性不一致由于方法上的差异16低效率，iCLMs的异质性仍未得到解决。 为了直接评价iCLM异质性，我们设计了一个离散的和强大的单细胞测定为肌节发展和心脏谱系specificatio的识别正两个功能的心肌细胞的必要特征。存在由它们的位置和独特的电特性定义在心脏的至少三个主要类型的CM：心房（AM），心室（VM）和起搏器（PM）17，18，19，20。在一个精心策划的组合，他们让血液正常抽水。在心脏损伤，一个或所有亚型可能会受到影响，细胞治疗的类型，需要根据具体情况逐案予以解决。目前，大多数战略，注重整体代心肌细胞，而少量的工作正在做研究，调节亚型规范的分子机制。 下面详细研究如何正确量化井井有条肌节，并确定一组不同的亚型心肌的。使用心脏起搏器（PM）专用鼠标的记者，我们可以申请一个我mmunocytochemical方法来区分诱发房状细胞（IAM），诱发室状细胞（IVM），并诱导般的PM肌细胞（IPMS）21。根据我们的观察，只表现出肌组织细胞能够自发跳动。这种独特的重新编程平台可以评估的作用 在肌组织，子规范，CM重编程效率的某些参数在单细胞分辨率。

<table border="0" cellpadding="0" cellspacing="0" width="873">
	<tbody>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">DMEM</td>
			<td style="width:91px;">Sigma</td>
			<td style="width:119px;">D5796</td>
			<td style="width:347px;">Component of iCLM media, Plat-E media, fibroblast, and Transfection media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Medium 199</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11150059</td>
			<td style="width:347px;">Component of iCLM media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Fetal bovine serrum (FBS)</td>
			<td style="width:91px;">Sigma</td>
			<td style="width:119px;">F2442</td>
			<td style="width:347px;">Component of iCLM media, Plat-E media, fibroblast, and Transfection media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Insulin-Transferrin-Selenium G</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">41400-045</td>
			<td style="width:347px;">Component of iCLM media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">MEM vitamin solution</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11120-052</td>
			<td style="width:347px;">Component of iCLM media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">MEM amino acids</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">1601149</td>
			<td style="width:347px;">Component of iCLM media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Non-Essential amino acids</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11140-050</td>
			<td style="width:347px;">Component of iCLM media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Antibiotic-Antimycotics</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15240062</td>
			<td style="width:347px;">Component of iCLM media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">B-27 supplement</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">17504044</td>
			<td style="width:347px;">Component of iCLM media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Heat-Inactivated Horse Serum</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">26050-088</td>
			<td style="width:347px;">Component of iCLM media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">NaPyruvate</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11360-70</td>
			<td style="width:347px;">Component of iCLM media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Penicillin/Streptomycin</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">1514022</td>
			<td style="width:347px;">Component of Plat-E media and fibroblast media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Puromycin&#160;</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td>A11139-03</td>
			<td>Component of Plat-E media</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Blasticidin&#160;&#160;</td>
			<td style="width:91px;">Gemini Bio-Products</td>
			<td>400-128P</td>
			<td>Component of Plat-E media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Glutamax</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td>35050-061</td>
			<td>Component of Fibroblast media</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Confocal laser scanning LSM700</td>
			<td style="width:91px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td>For confocal analysis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">FuGENE 6 transfection Reagent</td>
			<td style="width:91px;">Promega</td>
			<td style="width:119px;">E2692</td>
			<td>Transfection reagent&#160;</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Opti-MEM Reduced Serum Medium</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">31985-070</td>
			<td>Transfection reagent&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Polybrene</td>
			<td style="width:91px;">Millipore</td>
			<td style="width:119px;">TR-1003-G</td>
			<td>Induction reagent. Use at a final concentration of 8um/mL</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">Platinium-E (PE) Retroviral Packagin Cell Line, Ecotropic</td>
			<td style="width:91px;">CellBiolabs</td>
			<td style="width:119px;">RV-101</td>
			<td>Retroviral pacaking cell line</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Trypsin 0.25% EDTA</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td>For MEFs and Plat-E dissociation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti &#945;-Actinin (Clone EA-53)</td>
			<td style="width:91px;">Sigma</td>
			<td>A7811</td>
			<td>Antibody for confocal analysis. Use at 1:200</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Chicken anti-GFP IgY</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td>A10262</td>
			<td>Antibody for confocal analysis. Use at 1:200</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rabbit Pab anti-NPPA&#160;</td>
			<td style="width:91px;">Abgent</td>
			<td>AP8534A</td>
			<td>Antibody for confocal analysis. Use at 1:400</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rabbit Pab anti Myl2 IgG&#160;</td>
			<td style="width:91px;">ProteinTech</td>
			<td>10906-1-AP</td>
			<td>Antibody for confocal analysis. Use at 1:200</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">Vectashield solution with DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td style="width:91px;">Vector Labs</td>
			<td style="width:119px;">H-1500</td>
			<td>Dye for confocal analysis</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Superfrost Plus Microscope slides</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">12-550-15</td>
			<td>25 x 75 x 1.0 mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">BioCoat Fibronectin 12mm coverslips</td>
			<td style="width:91px;">NeuVitro Corp</td>
			<td>GG-12-1.5</td>
			<td>Coverslips for confocal analysis</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">100um cell strainer</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td>08-771-19</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">0.45um Syringes filters SFCA 25MM</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">09-740-106</td>
			<td>For virus filtration</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">6ml Syringes</td>
			<td style="width:91px;">Covidien</td>
			<td>8881516937</td>
			<td>For virus filtration</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Goat anti-Chicken IgY (H&#38;L) A488</td>
			<td style="width:91px;">Abcam</td>
			<td>AB150169</td>
			<td style="width:347px;">Secondary antibody for confocal analysis. Use at 1:400</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Donkey anti-rabbit A647 IgG(H+L)&#160;</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td>A31573</td>
			<td style="width:347px;">Secondary antibody for confocal analysis. Use at 1:400</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Goat anti-mouse IgG(H+L) A555</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td>A21422</td>
			<td style="width:347px;">Secondary antibody for confocal analysis. Use at 1:400</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Triton X-100</td>
			<td style="width:91px;">Sigma</td>
			<td style="width:119px;">93443-100ml</td>
			<td>For cell permeabilization&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Dulbecco&#39;s PBS without CaCl2 and MgCl2 (D-PBS)</td>
			<td style="width:91px;">Sigma</td>
			<td style="width:119px;">D8537</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">Power Block 10X Universal Blocking reagent</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">NC9495720</td>
			<td>Dilute to 1X in H20</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:319px;">16% Paraformaldehyde aqueous solution (PFA)</td>
			<td style="width:91px;">Electro Microscopy Sciences&#160;</td>
			<td style="width:119px;">15710</td>
			<td>Use at 4% diluted in dH20</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">6 cm&#160; plates</td>
			<td style="width:91px;">Olympus</td>
			<td style="width:119px;">25-260</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">6-well plates</td>
			<td style="width:91px;">Genesee Scientific</td>
			<td style="width:119px;">25-105</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">24-well plates</td>
			<td style="width:91px;">Genesee Scientific</td>
			<td style="width:119px;">25-107</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">10 cm Tissue culture dishes</td>
			<td style="width:91px;">Corning</td>
			<td>4239</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">15 cm&#160; Tissue culture dishes</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td>5442</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">15 ml Conical tubes</td>
			<td style="width:91px;">Corning</td>
			<td style="width:119px;">4308</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">50 ml Conical tubes</td>
			<td style="width:91px;">Corning</td>
			<td style="width:119px;">4249</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">0.4% Trypan blue solution</td>
			<td style="width:91px;">Sigma</td>
			<td style="width:119px;">T8154</td>
			<td>For viability</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">Ethyl Alcohol 200 proof</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">7005</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">Bleach</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">6009</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing cardiomyocyte subtypes following transcription factor-mediated reprogramming of mouse embryonic fibroblasts

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

服用特定的PM记者鼠标的优势，我们开发了多重免疫策略，确定为如图1所示多样化的内源性肌细胞。以下在图2所示的重新编程的步骤，亚型特异性的CM的诱导可以早在4第21天虽然是在低速率检测。在第14天，在实验可以停止并评估肌组织（ 图3）和亚型规范（ 图4）。 图5总结玻片制备的用于I...

Watch this Scientific Journal Video about Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts at JoVE.com

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

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

评估心肌亚型以下鼠标胚胎成纤维细胞的转录因子介导的重编程

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

Research

JoVE Journal

Developmental Biology

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

小鼠胚胎植耳蜗的文化和基因转移通过电穿孔

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

科研

发育生物学

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

在胚胎小鼠心脏采用免疫荧光心肌发展分析

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

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

在成年小鼠V-SVZ细胞的神经分析的稳定，高效的基因修饰干细胞自主和非自主的影响

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

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

心肌细胞DNA的合成和倍性的同时评价：一个方法来帮助心肌细胞再生和营业额的量化

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

Kinetic Measurement and Real Time Visualization of Somatic Reprogramming

体细胞重编程的动力学测量和实时可视化

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease ...

Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells

协议小鼠胚胎成纤维细胞直接转化为滋养层干细胞

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining ...

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

重新编程的小鼠胚胎成纤维细胞转录因子诱导Hemogenic计划

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

Dissection and Culture of Mouse Embryonic Kidney

小鼠胚胎肾的解剖与培养

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes

促纤维化信号增强因子介导的小鼠胚胎成纤维细胞再编程诱导心肌细胞的抑制

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that ...

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

成人小鼠成纤维细胞对红细胞生成子细胞的直接线重编程

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...