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

1. Ableitung von embryonalen Maus-CPCs aus Maus WSR <ol><li> Bereiten embryonalen Maus-Fibroblasten (MEFs) Feeder-Schicht. <ol><li> Warm MEF Medium (10% FBS in DMEM) auf 37 ° C. </li><li> Bereiten Gelatine beschichteten Platten. <ol><li> 1 ml der 0,1% Gelatine in Wasser in eine Vertiefung Platte mit 6 Vertiefungen oder 5 ml in eine 10 cm-Schale. </li><li> Lassen Platten oder Gerichte bei 37 ° C oder bei Raumtemperatur für mindestens 30 min. Streben die Gelatine vor der Verwendung. </li></ol></li><li> Thaw bestrahlt MEFs schnell in einem 37 ° C Wasserbad, unter leichtem Schütteln. </li><li> Übertragen die Zellen vorsichtig in ein 15 ml oder 50 ml konischen Röhrchen. 5 ml vorgewärmten MEF Medium. Schwenken Sie die Zellen langsam und Zentrifuge bei 200 xg für 5 min. </li><li> Zellen in MEF Medium und zählen lebensfähigen Zellen. Verwenden Sie die folgenden Mengen von MEF Medium: 2 ml für Zellen pro Vertiefung einer 6-Well-Platte und 10 ml für Zellen aus einer 10 cm Schüssel. </li><li> Platte Zellen in 6-Well-Platten oder 10 cm-Schalen bei 1-2x 10 6 pro Platte / Schale. </li><li> Die MEF Platten / Geschirr bei 37 ° C, 5% CO 2 mindestens 24 Stunden vor der Verwendung. HINWEIS: Entsorgen Sie MEF Platten / Geschirr älter als eine Woche. </li></ol></li><li> Bereiten Maus WSR <ol><li> Kultur der Isl1-cre; Rosa26YFP / Mef2c-cre; Rosa26YFP Maus WSR auf MEFs bis 90% konfluent. Verwendung ES-Zellmedium bestehend aus 410 ml DMEM, 75 ml FBS, 0,1 mM MEM NEAA, 2 mM L-Glutamin, 0,1 mM Pyruvat, 100 U Pen / Strep und 0,1 mM β-Mercaptoethanol. </li><li> Bereiten Gelatine beschichtete 6-Well-Platten, wie in 1.1.2 beschrieben. </li><li> Absaugen Medium von Platten und spülen Zellen mit DPBS. <ol><li> Saugen Sie DPBS und fügen Sie 0,4 ml 0,25% Trypsin in einem gut 6-Well-Platte. Zellen bei 37 ° C, 5% CO 2 für 5 min. 1 ml vorgewärmten ES-Medium zu Trypsin Reaktion zu beenden. </li></ol></li><li> Ernten Sie die Zellen und Zentrifuge Zellen bei 200 xg und saugen Medium. Zellen in 1 ml ES-Zellmedium und zähle die Zellen. &lt;/ Li&gt; <li> Platte WSR auf Gelatine-beschichteten Platten bei einer Dichte von 3 x 10 4 / cm 2. Bei 37 ° C, 5% CO 2 für ca. 2 Tage bei WSR erreichen ca. 90% konfluent. Ändern Sie das Medium jeden Tag. </li><li> Wenn WSR sind 90% konfluent, wiederholen Sie die Schritte 1.2.2-1.2.5 die MEF Feeder vollständig zu entfernen. </li></ol></li><li> Induzieren CPC Differenzierung von EB Bildung <ol><li> Bereiten Differenzierungsmedium wie folgt: 36 ml IMDM, 12 ml Ham-F12, 2 mM L-Glutamin, 0,34 ml BSA (7,5%), 0,25 ml N 2, 0,5 ml B27, 50 ug / ml AA und 0,45 mM 1-Thioglycerin. </li><li> Absaugen Medium von Zellen, und spülen Sie mit DPBS. Saugen Sie DPBS und fügen Sie 0,4 ml 0,25% Trypsin in einem gut 6-Well-Platte. Bei 37 ° C für 5 min. </li><li> 1 ml Differenzierungsmedium, um die Reaktion und die Pipette bis zu beenden und auf Zellclustern in einzelne Zellen zu verteilen. </li><li> Centrifuge WSR bei 200 xg für 5 min und entsorgen Sie das Medium. <ol><li> Resuspendieren 1 x 10 6 &lt;/ Sup&gt; WSR in 1 ml Differenzierungsmedium. Zählen Sie die Zellen, und die Kultur der Zellen in geringer Distanz Petrischale mit einer Konzentration von 1 x 10 5 Zellen / ml in Differenzierungsmedium bei 37 ° C für die ersten EB-Aggregation. </li></ol></li><li> Zwei Tage nach der EB Bildung, übertragen EVG vom Geschirr bis zu 50 ml Tuben. Spin bei 200 · g für 1 min. Entfernen Sie Medium und spülen EVG mit 10 ml DPBS ohne Ca 2+ und Mg 2+. </li><li> Saugen Sie DPBS und 1 ml 0,25% Trypsin. Bei 37 ° C für 5 min. Hinzufügen 4,5 ml Differenzierungsmedium, um die Reaktion zu stoppen. Pipette auf und ab, um EBs in einzelne Zellen zu trennen. </li><li> Zentrifugiere die Zellen bei 200 g für 5 min. Absaugen Medium resuspendieren Zellen in 1 ml Differenzierungsmedium. </li><li> Zählen Sie die Zellen und verdünnte 2 x 10 6 Zellen in 1 x 10 5 Zellen / ml in Differenzierungsmedium. Hinzufügen VEGF, Bmp4 und Activin A zu dem Medium in einer Endkonzentration von 5 ng / ml, 0,8 ng / ml und 5 ng / ml. Culture Zellen in Petrischale für die zweite EB Bildung bei 37 ° C. </li><li> 40 Stunden nach der zweiten EB Bildung, übertragen EVG auf 50-ml-Röhrchen. Spülen mit DPBS einmal dissoziieren EBs mit 1 ml 0,25% Trypsin bei 37 ° C für 5 min. Pipette auf und ab, um EBs in einzelne Zellen zu trennen. </li><li> Die Zellen in Stempro-34 Medium mit 5 ng / ml VEGF, 10 ng / ml bFGF und 12,5 ng / ml FGF10. Platte der Zellen bei 2 × 10 5 / cm 2 in Gelatine-beschichteten Platten. Kultur in 37 ° C Inkubator für etwa 32 Stunden vor dem CPC Isolation. </li></ol></li><li> Isolieren mCPCs <ol><li> Absaugen Medium und spülen Sie mit DPBS. 0,5 ml 0,25% Trypsin zu Kulturplatten bei 37 ° C für 5 min. Hinzufügen 4,5 ml Differenzierungsmedium, um die Reaktion zu stoppen. Pipette auf und ab, um EBs in einzelne Zellen zu trennen. Sammeln Sie die Zellen durch Zentrifugieren und resuspendieren Zellen in 2% FBS / PBS für FACS-Reinigung. </li><li> Führen undifferenzierten WSR am Sortierer als negative Kontrolle. Ändern Spannung nach vorne Scat einstellenter (FSC) und Seitwärtsstreuung (SSC), um die gewünschte Population auszuwählen. Verwenden Vorwärtsstreuung gegenüber Seitenstreuung und Vorwärtsstreuhöhe gegen die Breite, um Schmutz und Wams aus. In den ausgewählten Subpopulation, nach der Verteilung der ESC steuert, ziehe eine positive Gate-YFP auf Histogramm, um die Zellautofluoreszenz zu beseitigen. Sammeln YFP + CPCs aus YFP + Gating. </li><li> Platte der gereinigten CPCs (YFP + Zellen) in 6-Well-Platten mit Differenzierungsmedium (1.3.1). Kultur weitere 3-5 Tage bei 37 ° C für die Differenzierung von CPC in Kardiomyozyten und SMCs. </li></ol></li></ol> 2. Gewinnung menschlicher embryonaler CPCs von Menschen WSR <ol><li> Bereiten Feeders <ol><li> Bereiten embryonalen Maus-Fibroblasten (MEF) Nährschicht wie oben bei einer Dichte von 2 x 10 4 / cm 2 beschrieben ist. </li></ol></li><li> Pflegen menschlichen WSR <ol><li> Pflegen menschlichen ISL1: cre; pCAG-flox-AUS-flox-GFP oder RFP WSR routinemäßig auf MEF mit regulären hES Medium (Knockout DMEM / F-12 385 ml, Knockout SR 100 ml, 2 mM L-Glutamin, 0,1 mM NEAA, 0,1 mM β-Mercaptoethanol, 10 ng / ml basischen FGF). Vor der Herzdifferenzierung, übertragen menschliche WSR in Feeder-freien Zustand für mindestens 2 Passagen. </li></ol></li><li> Bereiten menschlichen WSR in Einzug freien Zustand <ol><li> Bereiten beschichteten Platten für den menschlichen WSR Kultur. <ol><li> Auftauen Matrigel bei 4 ° C über Nacht. Setzen Sie einen 50-ml-Röhrchen auf Eis und 30 ml kaltem DMEM / F12-Medium in den Schlauch. </li><li> 1 ml Matrigel in das kalte Medium und gut mischen. 1 ml kaltem Matrigel-DMEM / 12-Medium in eine Vertiefung Platte mit 6 Vertiefungen oder 5 ml in eine 10 cm-Schale. </li><li> Lassen Platten / Geschirr bei 4 ° C über Nacht oder bei Raumtemperatur für 2 Std. Absaugen Medium vor der Verwendung. </li></ol></li><li> Split menschlichen WSR auf die Teller: absaugen Medien aus MEF Platte und spülen menschlichen WSR mit DPBS. Dann 1 ml Dispase (1 mg / ml) in eine Vertiefung einer Platte mit 6 Vertiefungen. Inkubieren Zellen in 37 ° CInkubator, bis Ränder Kolonien beginnen zu lösen. </li><li> Entfernen Dispase-Lösung. Spülen Sie mit DPBS zweimal, dann fügen Sie 2,5 ml / pro Vertiefung mTeSR Medium oder MEF-konditionierten Mediums auf die Platte. </li><li> Kratzen Sie Zellen mit einem Zellschaber und sorgfältig pipettieren nach oben und unten, um die menschliche ESC Kolonien in kleine Klumpen zu brechen. </li><li> Übertragen Sie die ESC-Klumpen und verdünnt 1: 3 in die beschichteten Platten. </li><li> 2 ml / Vertiefung mTeSR Medium oder MEF-konditionierten Mediums und ändern Medium täglich. </li><li> Kultur menschlichen WSR in mTeSR Medium oder MEF-konditionierten Mediums auf beschichteten Platten für mindestens 2 Passagen. </li></ol></li><li> Induzieren CPC Differenzierung von humanen WSR <ol><li> Wenn Zellen sind ~ 90% konfluent, saugen das Medium und spülen Sie mit DPBS. Inkubieren der HES in 0,5 mg / ml Dispase bei 37 ° C für 20 min. </li><li> Spülen Sie die hES mit DPBS zweimal Entfernen Zellen aus Platten mit einer Zellheber, dann resuspendieren Zellschellen in Differenzierungsmedium (DMEM / F12 mit 18% FBS, 0,1 mM NEAA, 2 mML-Glutamin, 0,1 mM β-Mercaptoethanol und 50 ug / ml Ascorbinsäure). </li><li> Übertragen Sie Zellen in 6-Well-Ultra-Low-Befestigungsplatten für EB-Bildung. </li><li> Ändern Sie am nächsten Tag Differenzierungsmedium. </li><li> Ersetzen Medium alle 3 Tage und pflegen EVG in Suspensionskultur. </li></ol></li><li> Isolieren HCPCS <ol><li> Sammeln EVG spätestens 9. Tag der Differenzierung der Menschen CPC Isolation. </li><li> Absaugen Medium und spülen EVG mit DPBS zweimal. 0,5 ml 0,25% Trypsin in jede Vertiefung einer 6-Well-Kulturplatten bei 37 ° C für 5 min. </li><li> Hinzufügen gleichen Volumen Differenzierungsmedium, um die Reaktion zu stoppen. Pipette auf und ab, um EBs in einzelne Zellen zu trennen. Sammle die Zellen durch Zentrifugieren bei 200 xg für 5 Minuten und resuspendiere Zellen in 2% FBS / DPBS. Filterzellen mit 40 um Zellsieb und FACS-gereinigten RFP + CPC Zellen, wie in 1.4.3 beschrieben. </li><li> Platte nach HCPCS auf geltin beschichteten Platten. Kultur HCPCS in Differenzierungsmedium für 7-9 Tage in Kardiomyozyten und glatten Muskelzellen zu differenzieren. 7 Tage nach der Plattierung, die Kultur differenzierter Zellen mit 5% Knockout SR in DMEM / F12-Medium. </li></ol></li></ol>

<ol>
	<li>Garbern, J. C., Lee, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+stem+cell+therapy+and+the+promise+of+heart+regeneration.">Cardiac stem cell therapy and the promise of heart regeneration.</a> Cell Stem Cell. 12 (6), 689-698 (2013).</li><li>Passier, R., van Laake, L. W., Mummery, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem-cell-based+therapy+and+lessons+from+the+heart.">Stem-cell-based therapy and lessons from the heart.</a> Nature. 453 (7193), 322-329 (2008).</li><li>Bondue, A., Blanpain, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesp1:+a+key+regulator+of+cardiovascular+lineage+commitment.">Mesp1: a key regulator of cardiovascular lineage commitment.</a> Circulation Research. 107 (12), 1414-1427 (2010).</li><li>Bondue, 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=Mesp1+acts+as+a+master+regulator+of+multipotent+cardiovascular+progenitor+specification.">Mesp1 acts as a master regulator of multipotent cardiovascular progenitor specification.</a> Cell Stem Cell. 3 (1), 69-84 (2008).</li><li>Bu, 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=Human+ISL1+heart+progenitors+generate+diverse+multipotent+cardiovascular+cell+lineages.">Human ISL1 heart progenitors generate diverse multipotent cardiovascular cell lineages.</a> Nature. 460 (7251), 113-117 (2009).</li><li>Lei, I., Gao, X., Sham, M. H., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SWI/SNF+protein+component+BAF250a+regulates+cardiac+progenitor+cell+differentiation+by+modulating+chromatin+accessibility+during+second+heart+field+development.">SWI/SNF protein component BAF250a regulates cardiac progenitor cell differentiation by modulating chromatin accessibility during second heart field development.</a> The Journal of Biological Chemistry. 287 (29), 24255-24262 (2012).</li><li>Lei, I., Liu, L., Sham, M. H., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SWI/SNF+in+cardiac+progenitor+cell+differentiation.">SWI/SNF in cardiac progenitor cell differentiation.</a> Journal of Cellular Biochemistry. 114 (11), 2437-2445 (2013).</li><li>Wu, S. 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=Developmental+origin+of+a+bipotential+myocardial+and+smooth+muscle+cell+precursor+in+the+mammalian+heart.">Developmental origin of a bipotential myocardial and smooth muscle cell precursor in the mammalian heart.</a> Cell. 127 (6), 1137-1150 (2006).</li><li>Spater, 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=A+HCN4++cardiomyogenic+progenitor+derived+from+the+first+heart+field+and+human+pluripotent+stem+cells.">A HCN4+ cardiomyogenic progenitor derived from the first heart field and human pluripotent stem cells.</a> Nature Cell Biology. 15 (9), 1098-1106 (2013).</li><li>Verzi, M. P., McCulley, D. J., De Val, S., Dodou, E., Black, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+right+ventricle,+outflow+tract,+and+ventricular+septum+comprise+a+restricted+expression+domain+within+the+secondary/anterior+heart+field.">The right ventricle, outflow tract, and ventricular septum comprise a restricted expression domain within the secondary/anterior heart field.</a> Developmental Biology. 287 (1), 134-145 (2005).</li><li>Boheler, K. 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=Differentiation+of+pluripotent+embryonic+stem+cells+into+cardiomyocytes.">Differentiation of pluripotent embryonic stem cells into cardiomyocytes.</a> Circulation Research. 91 (3), 189-201 (2002).</li><li>Wada, 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=Induction+of+human+cardiomyocyte-like+cells+from+fibroblasts+by+defined+factors.">Induction of human cardiomyocyte-like cells from fibroblasts by defined factors.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (31), 12667-12672 (2013).</li><li>Kattman, S. 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=Stage-specific+optimization+of+activin/nodal+and+BMP+signaling+promotes+cardiac+differentiation+of+mouse+and+human+pluripotent+stem+cell+lines.">Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines.</a> Cell Stem Cell. 8 (2), 228-240 (2011).</li><li>Takahashi, 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=Ascorbic+acid+enhances+differentiation+of+embryonic+stem+cells+into+cardiac+myocytes.">Ascorbic acid enhances differentiation of embryonic stem cells into cardiac myocytes.</a> Circulation. 107 (14), 1912-1916 (2003).</li><li>Wamstad, J. 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=Dynamic+and+coordinated+epigenetic+regulation+of+developmental+transitions+in+the+cardiac+lineage.">Dynamic and coordinated epigenetic regulation of developmental transitions in the cardiac lineage.</a> Cell. 151 (1), 206-220 (2012).</li><li>Cong, 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=Multiplex+genome+engineering+using+CRISPR/Cas+systems.">Multiplex genome engineering using CRISPR/Cas systems.</a> Science. 339 (6121), 819-823 (2013).</li><li>Ding, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+efficiency+of+human+pluripotent+stem+cell+genome+editing+through+replacing+TALENs+with+CRISPRs.">Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs.</a> Cell Stem Cell. 12 (4), 393-394 (2013).</li><li>Gaj, T., Gersbach, C. S., Barbas, T. A. L. 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=ZFN,+TALEN,+CRISPR/Cas-based+methods+for+genome+engineering.">ZFN, TALEN, CRISPR/Cas-based methods for genome engineering.</a> Trends in Biotechnology. 31 (7), 397-405 (2013).</li><li>Joung, J. K., Sander, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TALENs:+a+widely+applicable+technology+for+targeted+genome+editing.">TALENs: a widely applicable technology for targeted genome editing.</a> Nature Reviews. Molecular Cell Biology. 14 (1), 49-55 (2013).</li><li>Yang, H., 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=One-step+generation+of+mice+carrying+reporter+and+conditional+alleles+by+CRISPR/Cas-mediated+genome+engineering.">One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering.</a> Cell. 154 (6), 1370-1379 (2013).</li><li>Mali, P., 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=RNA-guided+human+genome+engineering+via+Cas9.">RNA-guided human genome engineering via Cas9.</a> Science. 339 (6121), 823-826 (2013).</li><li>Laflamme, M. 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=Cardiomyocytes+derived+from+human+embryonic+stem+cells+in+pro-survival+factors+enhance+function+of+infarcted+rat+hearts.">Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts.</a> Nature. biotechnology. 25 (9), 1015-1024 (2007).</li><li>Burridge, P. W., 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=A+universal+system+for+highly+efficient+cardiac+differentiation+of+human+induced+pluripotent+stem+cells+that+eliminates+interline+variability.">A universal system for highly efficient cardiac differentiation of human induced pluripotent stem cells that eliminates interline variability.</a> PloS One. 6 (4), e18293 (2011).</li><li>Kouskoff, V., Lacaud, G., Schwantz, S., Fehling, H. J., Keller, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+development+of+hematopoietic+and+cardiac+mesoderm+during+embryonic+stem+cell+differentiation.">Sequential development of hematopoietic and cardiac mesoderm during embryonic stem cell differentiation.</a> Proceedings of the National Academy of Sciences of the United States of America. 102 (37), 13170-13175 (2005).</li><li>Xu, X. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemically+defined+medium+supporting+cardiomyocyte+differentiation+of+human+embryonic+stem+cells.">Chemically defined medium supporting cardiomyocyte differentiation of human embryonic stem cells.</a> Differentiation: Research in Biological Diversity. 76 (9), 958-970 (2008).</li><li>Lian, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+cardiomyocyte+differentiation+from+human+pluripotent+stem+cells+via+temporal+modulation+of+canonical+Wnt+signaling.">Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (27), E1848-1857 (2012).</li><li>Bizy, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+light+chain+2-based+selection+of+human+iPSC-derived+early+ventricular+cardiac+myocytes.">Myosin light chain 2-based selection of human iPSC-derived early ventricular cardiac myocytes.</a> Stem Cell Research. 11 (3), 1335-1347 (2013).</li></ol>

The authors declare no competing financial interests.

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

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

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

Erratum: Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Herzkrankheit bleibt die führende Ursache in der heutigen Welt und die Todesraten haben in den vergangenen zwei Jahrzehnten (American Heart Association) nahezu unverändert geblieben. Es gibt einen dringenden Bedarf für die Entwicklung neuer therapeutischer Strategien effektiv verhindern oder rückgängig Herzinsuffizienz. Ein vielversprechender Ansatz ist zellbasierte Therapie nach der raschen Entwicklung von Stammzellen biology 1. In dieser Hinsicht könnte multi CPCs eine ausgezeichnete Zellquelle für die Therapie aufgrund ihrer Fähigkeit, sich zu vermehren, aber nur, um die Herzlinie Differenzierung gebunden werden. Daher effiziente und robuste Methode zur Erzeugung und Isolierung CPCs ist von großer Bedeutung für die Herzzelltherapie-Studien. Dieses Protokoll konzentriert sich auf embryonale CPCs der frühen Embryogenese und wie ihre Generation von WSR identifiziert. Verschiedene CPCs wurden aus embryonalen und adulten Herzen isoliert worden, auch aus Knochenmark 2. Während der Embryonalentwicklung, Knochen morphogetische Proteine ​​(BMPs), flügellosen Typ MMTV Integrationsstelle Familienmitglieder (Wnts) und Nodal-Signale induzieren das Engagement der Mesp1 + multi Mesoderm 3. Mesp1 + Zellen in die embryonale CPCs 4 unterscheiden dann. Diese CPCs werden typischerweise durch HCN4 markiert, NK2 homeobox 5 (NKX2-5) Isl LIM homeobox 1 (Isl1), T-box 5 (Tbx5) und Myozyten-Enhancer Faktor 2C (Mef2c), bilden primären und zweiten Herzfelder, und während Kardiogenese 5-10 tragen zu der Hauptteile des Herzens. Sowohl NKX2-5 + und Isl1 + / + Mef2c CPCs Lage sind, in Kardiomyozyten differenzieren, glatte Muskelzellen (SMCs) und Endothelzellen 5-8. So sind diese CPCs werden die zu Herzgefäßsystem sowie Herzgewebe zu geben und sind eine ideale Zellquelle für zellbasierte Herztherapie. Folglich erzeugen CPCs in vitro war ein wichtiger Forschungsschwerpunkt in der Herz-Kreislauf-Studien. Seit WSR haben unbegrenzte Erweiterungsmöglichkeiten einnd stellen die ICM-Zellen im Blastozystenstadium, ist die Differenzierung von WSR in embryonale CPCs nach der Naturembryogenese als eine logische und effektiven Ansatz zur CPCs zu erhalten. Eine weit angelegte Herangehensweise an CPCs von WSR erhalten, ist die Wirtschafts- und Sozialräte in EBs 11 aggregieren. Die Differenzierung Effizienz zu verbessern, sind definierte chemische und Wachstumsfaktoren auf Basis der Kenntnis der Herzentwicklung verwendet worden, 12-14. Es gibt jedoch keine definitive CPC Marker, insbesondere keine Zelloberflächenmarkern, die allgemein auf dem Gebiet akzeptiert. Um dieses Problem anzugehen, WSR sind so konstruiert, Isl1 + oder Mef2c + CPCs und ihre Derivate mit fluoreszierenden Reporter mit Cre / loxP-System markieren. Die cre-Rekombinase in unter der Kontrolle Isl1 / Mef2c Promotor / Enhancer geklopft. Das modifizierte fluoreszierende Protein RFP oder YFP-Gen von einem konstitutiven Promotor angetrieben wird durch Exzision flox Stoppcodon mit cre-Rekombinase aktiviert werden(ISL1: cre; pCAG-flox-AUS-flox-GFP oder RFP / Isl1-cre; Rosa26YFP / Mef2c-cre; Rosa26YFP) 5,6. Sobald die Wirtschafts- und Sozialräte in zweiten Herzfeldes CPCs unterschieden werden Isl1 / Mef2c Promotor / Enhancer angetrieben cre die fluoreszierenden Reportern zu aktivieren und CPCs können durch FACS-Aufreinigung angereichert werden. Kurz gesagt, wird EB Aggregationsmethode verwendet werden, um ESC Differenzierung einzuleiten. Differenzierung Effizienz zu verbessern, werden die differenzierten Zellen mit Ascorbinsäure (AA) und Wachstumsfaktoren wie Bmp4, Activin A und VEGF 13,15 behandelt. Dieses Protokoll ermöglicht robuste und effiziente CPC Differenzierung sowohl mit Maus und Mensch WSR.

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derivation of cardiac progenitor cells from embryonic stem cells

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

Das Protokoll zeigt Ableitung CPCs aus mehreren ES-Zelllinien. WSR werden aggregiert, um EBs bilden in CPCs zu unterscheiden. WSR werden routinemäßig auf MEF Feeder (1A, F) gehalten und Zuführungen vor Differenzierung entfernt. Nach Aggregation der WSR in EBs in Differenzierungsmedium (1B, G), werden die zweiten geformten EVGs Bmp4 und Activin A behandelt, um das Mesoderm Differenzierung in Zelllinien der Maus (1C) zu verbessern. WSR in Herzlinie differenziert wie fl...

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

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

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

Ableitung von Herzvorläuferzellen aus embryonalen Stammzellen

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

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

Research

JoVE Journal

Developmental Biology

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

Serie: Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

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

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

Effiziente Ableitung von Retinapigmentepithel-Zellen aus Stammzellen

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

Forschung

Entwicklungsbiologie

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

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

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

Direkte Induktion von humanen neuralen Stammzellen aus peripherem Blut hämatopoetischen Vorläuferzellen

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

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

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

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

Feeder freie Ableitung von Melanozyten aus humanen pluripotenten Stammzellen

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

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

Epigenetische Regulation der Herz Differenzierung von embryonalen Stammzellen und Gewebe

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

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

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

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

Horizontale Ganze Mount: Ein neuartiges Verarbeitungs- und Imaging-Protokoll für dickes, dreidimensionales Gewebequerschnitt der Haut

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

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

Schnelle, gezielte Differenzierung der retinalen Pigment epithelialen Zellen aus menschlichen embryonalen oder induzierte pluripotente Stammzellen

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Single-cell Photoconversion in Living Intact Zebrafish

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

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

Einzellige Ladungszustand im lebendigen intakten Zebrafisch

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

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

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

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

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

Computergestützte Analyse der Keimbahn Caenorhabditis Elegans , die Verteilung der Kerne, Proteine und das Zytoskelett zu studieren

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

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

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

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

Ableitung und Differenzierung von Eckzahn Eierstock mesenchymalen Stammzellen

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

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

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

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

In vitro-Generation der Somiten Derivate aus menschlichen induzierten Zellen pluripotenten Stammzellen

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