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. Derivazione embrionali di topo CPC da mouse CES <ol><li> Preparare il mouse fibroblasti embrionali (MEF) feeder layer. <ol><li> Medio MEF caldo (10% FBS in DMEM) a 37 ° C. </li><li> Preparare piatti gelatina rivestito. <ol><li> Aggiungere 1 ml di 0,1% di gelatina in acqua in un pozzetto della piastra da 6 pozzetti o 5 ml in un piatto 10 cm. </li><li> Lasciare piatti o piatti a 37 ° C oa temperatura ambiente per almeno 30 min. Aspirare la gelatina prima dell&#39;uso. </li></ol></li><li> Scongelare irradiato MEF rapidamente in un bagno d&#39;acqua a 37 ° C, agitando delicatamente. </li><li> Trasferire le cellule delicatamente per un tubo da 15 ml o 50 ml. Aggiungere 5 ml di terreno MEF pre-riscaldato. Agitare le cellule lentamente e centrifugare a 200 xg per 5 min. </li><li> Risospendere le cellule in terreno MEF e contare le cellule vitali. Utilizzare i seguenti volumi di MEF medio: 2 ml per cellule per bene da un 6-pozzetti e 10 ml per le cellule da un piatto di 10 cm. </li><li> Cellule piastra in 6 pozzetti o 10 piatti cm a 1-2x 10 6 per piatto / piatto. </li><li> Incubare le piastre MEF / stoviglie a 37 ° C, 5% CO 2 almeno 24 ore prima dell&#39;uso. NOTA: Eliminare MEF piatti / stoviglie più vecchi di una settimana. </li></ol></li><li> Preparare CES del mouse <ol><li> Culture del Isl1-cre; Rosa26YFP / MEF2C-cre; Rosa26YFP del mouse CES sul MEF fino al 90% confluenti. Media cell Uso ES comprende 410 ml DMEM, 75 ml FBS, 0,1 mm MEM NEAA, 2 mM L-glutammina, 0.1 mM piruvato, 100 U Pen / Strep, e 0,1 mm β-mercaptoetanolo. </li><li> Preparare 6 pozzetti gelatina rivestito come descritto al punto 1.1.2. </li><li> Aspirare media da piatti e lavare le cellule con DPBS. <ol><li> Aspirare DPBS e aggiungere 0,4 ml 0,25% tripsina in un pozzetto di 6-pozzetti. Incubare le cellule a 37 ° C, 5% CO 2 per 5 min. Aggiungere 1 ml di pre-riscaldato medie ES cessare reazione tripsina. </li></ol></li><li> Celle di raccolta e le cellule centrifugare a 200 xg e aspirare media. Risospendere le cellule in 1 ml di mezzo ES cellulare e contare le cellule. &lt;/ Li&gt; <li> CES piastra su piastre di gelatina rivestite a densità di 3 x 10 4 / cm 2. Incubare a 37 ° C, 5% di CO 2 per circa 2 giorni in cui i CES raggiungono circa il 90% confluenti. Cambiare la media di tutti i giorni. </li><li> Quando CES sono 90% confluenti, ripetere i punti 1.2.2-1.2.5 per rimuovere completamente gli alimentatori MEF. </li></ol></li><li> Indurre CPC differenziazione dalla formazione EB <ol><li> Preparare medio differenziazione seguito: 36 ml IMDM, 12 ml F12 di Ham, 2 mM L-glutammina, 0,34 ml BSA (7,5%), 0,25 ml N2, 0,5 ml B27, 50 mg / ml AA, e 0,45 mm 1-thioglycerol. </li><li> Medio Aspirare dalle cellule e risciacquare con DPBS. Aspirare DPBS e aggiungere 0,4 ml 0,25% tripsina in un pozzetto di 6-pozzetti. Incubare a 37 ° C per 5 min. </li><li> Aggiungere 1 ml di mezzo di differenziazione di cessare la reazione e la pipetta su e giù per disperdere gruppi di cellule in cellule singole. </li><li> Centrifugare CES a 200 xg per 5 minuti e scartare il medium. <ol><li> Risospendere 1 x 10 6 &lt;/ Sup&gt; CES in 1 ml di mezzo di differenziazione. Contare le cellule, e coltura le cellule a partire distacco piatto Petri a una concentrazione di 1 x 10 5 cellule / ml in terreno di differenziamento a 37 ° C per prima aggregazione EB. </li></ol></li><li> Due giorni dopo la formazione EB, trasferire EBs dai piatti a 50 ml provette. Spin a 200 g per 1 min. Rimuovere medie e risciacquare EBs con 10 ml DPBS senza Ca 2+ e Mg 2+. </li><li> Aspirare DPBS e aggiungere 1 ml 0,25% tripsina. Incubare a 37 ° C per 5 min. Aggiungere 4,5 ml di mezzo di differenziazione per arrestare la reazione. Pipettare su e giù per dissociarsi EB in cellule singole. </li><li> Centrifugare le cellule a 200 xg per 5 min. Aspirare media e risospendere le cellule in 1 ml di mezzo di differenziazione. </li><li> Contare le cellule, e diluire 2 x 10 6 cellule per 1 x 10 5 cellule / ml in terreno di differenziamento. Aggiungere VEGF Bmp4 e Activin A al mezzo a una concentrazione finale di 5 ng / ml, 0,8 ng / ml, e 5 ng / ml, rispettivamente. Culture le cellule in capsula di Petri per la seconda formazione EB a 37 ° C. </li><li> 40 ore dopo la seconda formazione EB, trasferimento EBs a 50 ml provette. Risciacquare con DPBS una volta, dissociare EBs con 1 ml 0,25% tripsina a 37 ° C per 5 minuti. Pipettare su e giù per dissociarsi EB in cellule singole. </li><li> Risospendere le cellule in Stempro-34 media con 5 ng / ml VEGF, 10 ng / ml bFGF e 12,5 ng FGF10 / ml. Piastra le cellule a 2 x 10 5 / cm 2 in piastre rivestite di gelatina. Cultura in 37 ° C incubatore per circa 32 ore prima di isolamento CPC. </li></ol></li><li> Isolare mCPCs <ol><li> Aspirare il terreno e risciacquare con DPBS. Aggiungere 0,5 ml di 0,25% tripsina a piastre di coltura a 37 ° C per 5 min. Aggiungere 4,5 ml di mezzo di differenziazione per arrestare la reazione. Pipettare su e giù per dissociarsi EB in cellule singole. Raccogliere le cellule cellule centrifuga e risospendere in 2% FBS / PBS per FACS-purificazione. </li><li> Eseguire CES indifferenziate sul sorter come controllo negativo. Modifica tensione per regolare scat avantiter (FSC) e side scatter (SSC) per selezionare la popolazione desiderata. Utilizzare avanti spargere contro scatter lato e altezza scatter in avanti rispetto a larghezza di escludere detriti e farsetto. Nel sub-popolazione selezionata, in base alla distribuzione dei controlli ESC, elaborare un cancello di YFP positivo sulla istogramma per eliminare autofluorescenza cellule. Raccogliere YFP + CPC da YFP + gating. </li><li> Piastra i CPC purificati (cellule YFP +) a 6 pozzetti con terreno di differenziamento (1.3.1). Cultura ulteriori 3-5 giorni a 37 ° C per la differenziazione del CPC in cardiomiociti e SMC. </li></ol></li></ol> 2. Determinazione dei embrionale umano CPC dal CES umani <ol><li> Preparare Alimentatori <ol><li> Preparare topo fibroblasti embrionali (MEF) strato alimentatore come descritto sopra ad una densità di 2 x 10 4 / cm 2. </li></ol></li><li> Mantenere CES umani <ol><li> Mantenere Isl1 umana: cre; pCAG-flox-STOP-flox-GFP o RFP CES di routine su MEF utilizzando media hESC regolare (Knockout DMEM / F-12 385 ml, Knockout SR 100 ml, 2 mM L-glutammina, 0.1 NEAA mM, 0,1 mm β-mercaptoetanolo, 10 ng / ml FGF base). Prima di differenziazione cardiaca, trasferire CES umani in alimentatore condizione libera per almeno 2 passaggi. </li></ol></li><li> Preparare CES umani in condizioni di alimentazione gratuito <ol><li> Preparare piastre rivestite per la cultura CES umani. <ol><li> Scongelare matrigel a 4 ° C durante la notte. Mettere un tubo da 50 ml su ghiaccio e aggiungere 30 ml fredda terreno DMEM / F12 nel tubo. </li><li> Aggiungere 1 ml matrigel nel mezzo freddo e mescolare bene. Aggiungere 1 ml di freddo Matrigel-DMEM / 12 di media in un pozzo di 6-pozzetti o 5 ml in un piatto di 10 cm. </li><li> Lascia piatti / stoviglie a 4 ° C durante la notte o temperatura ambiente per 2 ore. Aspirare il terreno prima dell&#39;uso. </li></ol></li><li> Split CES umane sulle piastre: Aspirare il terreno dalla piastra MEF e sciacquare CES umani con DPBS. Poi aggiungere 1 ml dispasi (1 mg / ml) in un pozzetto di piastra da 6 pozzetti. Incubare le cellule in 37 ° Cincubatrice fino bordi delle colonie cominciano a staccarsi. </li><li> Rimuovere la soluzione dispasi. Risciacquare con DPBS due volte, quindi aggiungere 2,5 ml / per medie dimensioni, ben mTeSR o medio MEF condizionata alla piastra. </li><li> Raschiare le cellule con un raschietto cellulare e con attenzione pipettare su e giù per rompere le colonie CES umani in piccoli grumi. </li><li> Trasferire i ciuffi ESC e diluire 1: 3 nelle piastre rivestite. </li><li> Aggiungere 2 ml / e medio mTeSR o medio MEF condizionata e cambiare media giornaliera. </li><li> Cultura CES umani in mTeSR medio o medio MEF condizionata su piastre rivestite per almeno 2 passaggi. </li></ol></li><li> Indurre CPC differenziazione dalla CES umani <ol><li> Quando le cellule sono ~ 90% confluenti, aspirare il terreno e risciacquare con DPBS. Poi incubare le hESC a 0,5 mg / ml dispasi a 37 ° C per 20 min. </li><li> Sciacquare le hESC con DPBS due volte, rimuovere le cellule dai piatti con sollevatore cellulare, quindi risospendere morsetti cellulari in terreno di differenziamento (DMEM / F12 contenente 18% FBS, 0.1 NEAA mm, 2 mmL-glutammina, 0,1 mM β-mercaptoetanolo e 50 ug / ml acido ascorbico). </li><li> Trasferire le cellule in piastre di fissaggio ultra-bassi 6 pozzetti per la formazione EB. </li><li> Cambiare medio differenziazione giorno successivo. </li><li> Sostituire media ogni 3 giorni e mantenere EBs in coltura in sospensione. </li></ol></li><li> Isolare HCPCS <ol><li> Raccogliere EBs entro e non oltre il giorno 9 di differenziazione per l&#39;isolamento CPC umana. </li><li> Aspirare il terreno e lavare EBs con DPBS due volte. Aggiungere 0,5 ml di 0,25% tripsina in ciascun pozzetto di piastre di coltura 6 pozzetti a 37 ° C per 5 min. </li><li> Aggiungere uguale volume di terreno di differenziamento per arrestare la reazione. Pipettare su e giù per dissociarsi EB in cellule singole. Raccogliere le cellule centrifugare a 200 xg per 5 minuti e risospendere le cellule in 2% FBS / DPBS. Celle filtranti con 40 micron filtro cellulare e FACS purificato la RFP + cellule CPC come descritto in 1.4.3. </li><li> Piatto allineati HCPCS su piastre geltin rivestite. HCPCS culturali in media di differenziazione per 7Giorni -9 a differenziarsi in cellule muscolari lisce e cardiomiociti. 7 giorni dopo la placcatura, cultura cellule con 5% Knockout SR in terreno DMEM / F12 differenziato. </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

Le malattie cardiache rimane la causa nel mondo leader di oggi e il tasso di mortalità sono rimasti pressoché invariati negli ultimi due decenni (American Heart Association). Vi è una necessità critica per lo sviluppo di nuove strategie terapeutiche per prevenire o invertire l&#39;insufficienza cardiaca in modo efficace. Una strategia promettente è terapia cellulare a seguito del rapido sviluppo della biologia delle cellule staminali 1. A questo proposito, CPC multipotenti potrebbero essere una fonte eccellente cellule per la terapia a causa della loro capacità di proliferare ma impegnata solo differenziazione lineage cardiaca. Pertanto, il metodo efficiente e robusto per generare ed isolare CPC è di grande importanza per gli studi di terapia cellulare cardiaca. Questo protocollo si concentra su CPC embrionali individuati durante l&#39;embriogenesi precoce e di come la loro generazione dal CES. Vari CPC sono stati isolati dai cuori embrionali e adulte, anche da midollo osseo 2. Durante lo sviluppo embrionale, morphoge ossoproteine ​​tiche (BMP), senza ali di tipo familiari sito di integrazione MMTV (Wnts) e segnali nodali inducono l&#39;impegno della Mesp1 + mesoderma multipotenti 3. + Cellule Mesp1 poi differenziano nelle CPC embrionali 4. Questi CPC sono tipicamente caratterizzati da HCN4, NK2 homeobox 5 (NKX2-5), Isl LIM homeobox 1 (Isl1), T-box 5 (Tbx5), e dei miociti fattore enhancer 2C (MEF2C), formano campi cuore primarie e seconde, e contribuire alle principali parti del cuore durante cardiogenesis 5-10. CPC Sia NKX2-5 + e Isl1 + / MEF2C + sono in grado di differenziarsi in cardiomiociti, cellule muscolari lisce (SMC), e le cellule endoteliali 5-8. Così questi CPC daranno luogo a vascolare cardiaca così come i tessuti cardiaci e sono una fonte di cellule ideale per la terapia cardiaca cell-based. Di conseguenza, generando CPC in vitro è stato un obiettivo importante di ricerca in studi cardiovascolari. Dal CES hanno la capacità di espansione illimitata and rappresentano le cellule ICM allo stadio di blastocisti, la differenziazione dei CES in CPC embrionali seguito della embriogenesi naturale è considerato un approccio logico ed efficace per ottenere CPC. Un approccio ampiamente applicata per ottenere CPC dal CES è quello di aggregare CES in EBs 11. Per migliorare l&#39;efficienza differenziazione, fattori chimici e crescita definiti sulla base della conoscenza di sviluppo cardiaco sono stati utilizzati 12-14. Tuttavia, non ci sono marcatori CPC definitive, in particolare marcatori della superficie cellulare, che sono ampiamente accettate nel settore. Per risolvere questo problema, i CES sono progettati per contrassegnare Isl1 + o MEF2C CPC + e loro derivati, con i giornalisti fluorescenti che utilizzano il sistema / loxP Cre. Il ricombinasi cre è buttato in sotto il controllo di Isl1 / MEF2C promoter / enhancer. Modificata la proteina fluorescente o RFP gene YFP guidata da un promotore costitutivo possono essere attivate da escissione di arresto flox codone con cre ricombinasi(Isl1: cre; pCAG-flox-STOP-flox-GFP o RFP / Isl1-cre; Rosa26YFP / MEF2C-cre; Rosa26YFP) 5,6. Una volta che i CES sono differenziati in CPC secondo campo cuore, Isl1 / MEF2C promoter / enhancer cre guidato attiverà i reporter fluorescenti e CPC può essere arricchita da FACS-purificazione. In breve, il metodo di aggregazione EB viene utilizzato per avviare ESC differenziazione. Per migliorare l&#39;efficienza differenziazione, le cellule differenziate sono trattati con acido ascorbico (AA) e fattori di crescita come Bmp4, Activin A e VEGF 13,15. Questo protocollo permette robusto ed efficiente differenziamento CPC utilizzando sia il mouse e CES umani.

<table border="0" cellpadding="0" cellspacing="0" width="415">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Name</td>
			<td style="width:156px;">Company</td>
			<td style="width:131px;">Catalog Number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>Thermo scentific</td>
			<td>SH30070.03E</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Knockout SR</td>
			<td>Life technology</td>
			<td align="right">10828028</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Knockout DMEM</td>
			<td>Life technology</td>
			<td align="right">10829018</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td>Life technology</td>
			<td align="right">11965118</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NEAA</td>
			<td>Life technology</td>
			<td align="right">11140050</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GlutaMAX</td>
			<td>Life technology</td>
			<td align="right">35050061</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N2</td>
			<td>Life technology</td>
			<td align="right">17502048</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B27</td>
			<td>Life technology</td>
			<td align="right">12587010</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ham&#8217;s F12</td>
			<td>Life technology</td>
			<td align="right">11765062</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IMDM</td>
			<td>Life technology</td>
			<td align="right">12440061</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pen/Strep</td>
			<td>Life technology</td>
			<td align="right">15140122</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pyruvate</td>
			<td>Life technology</td>
			<td align="right">11360070</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dispase</td>
			<td>Life technology</td>
			<td align="right">17105041</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stempro-34</td>
			<td>Life technology</td>
			<td align="right">10639011</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F12</td>
			<td>Life technology</td>
			<td align="right">11330032</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA&#160;</td>
			<td>Life technology</td>
			<td align="right">15260037</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin&#160;</td>
			<td>Life technology</td>
			<td align="right">25200056</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ascobic Acid&#160;</td>
			<td>Sigma</td>
			<td>A5960</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1-Thioglycerol</td>
			<td>Sigma</td>
			<td>M1753</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M3148</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VEGF</td>
			<td>R&#38;D</td>
			<td>293-VE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bmp4</td>
			<td>R&#38;D</td>
			<td>314-BP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ActivinA</td>
			<td>R&#38;D</td>
			<td>338-AC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">bFgf</td>
			<td>R&#38;D</td>
			<td>233-FB</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fgf10</td>
			<td>R&#38;D</td>
			<td>345-FG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mTeSR</td>
			<td>Stemcell technologies</td>
			<td align="right">5850</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel</td>
			<td>BD Biosciences</td>
			<td align="right">354277</td>
		</tr>
	</tbody>
</table>

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.

Il protocollo mostra derivazione del CPC da diverse linee di cellule ES. CES sono aggregati per formare EB di differenziarsi in CPC. CES sono regolarmente mantenuti su alimentatori MEF (Figura 1A, F) e alimentatori vengono rimossi prima di differenziazione. Su aggregazione dei CES in EBs in terreno di differenziamento (Figura 1B, G), il secondo EBs formati vengono trattati con Bmp4 e Activin A per migliorare la differenziazione mesoderma in linee cellulari di topo (Figura 1C).</...

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.

Derivazione di cellule cardiache progenitrici di cellule staminali embrionali

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 Views.  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.

Video: 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.

Derivazione efficiente di cellule retiniche epitelio pigmentato da cellule staminali

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 ...

Ricerca

Biologia dello sviluppo

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.

Induzione diretta di cellule staminali neurali umane da cellule periferiche del sangue emopoietiche progenitrici

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.

Derivazione senza alimentatore di melanociti da cellule staminali umane pluripotenti

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.

Regolazione epigenetica del Cardiac differenziazione delle cellule staminali embrionali e Tessuti

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.

Supporto orizzontale intero: un protocollo di elaborazione e imaging novello per le sezioni trasversali tessute spesse e tridimensionali della pelle

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.

Rapida, regia di differenziazione delle cellule epiteliali del pigmento retinico da cellule staminali pluripotenti umane embrionali o indotto

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.

Cella singola fotoconversione in Zebrafish intatto vivente

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.

Analisi computazionale della linea germinale Caenorhabditis elegans di studiare la distribuzione dei Nuclei, le proteine ed il citoscheletro

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.

Derivazione e differenziazione delle cellule mesenchimali ovarico canini

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.

Generazione in Vitro di Somite derivati da umani indotta da cellule staminali pluripotenti indotte

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