Name: derivation of cardiac progenitor cells from embryonic stem cells
Uploaded: 2015-01-12T13:00:00.000+00:00
Duration: 8 min
Description: Watch this Scientific Journal Video about Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells at JoVE.com

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.

Fare EKH adlı Fare Embriyonik TBMlerin 1. türetilmesi <ol><li> Fare embriyonik fibroblastlar (MEFS) besleyici tabakası hazırlayın. <ol><li> Sıcak MEF ortam 37 ° C&#39;ye kadar (DMEM içinde% 10 FBS). </li><li> Jelatin kaplı tabak hazırlayın. <ol><li> 10 cm&#39;lik bir tabak içine bir 6-yuvalı plaka arasında bir çukur veya 5 ml su içinde,% 0.1 Jelatin 1 ml ilave edilir. </li><li> En az 30 dakika boyunca plakaları ya da yemekler, 37 ° C&#39;de ya da oda sıcaklığı bırakın. Kullanmadan önce jelatin talip. </li></ol></li><li> Çözülme hafifçe çalkalanarak, 37 ° C su banyosu içinde hızla MEFS maruz bırakıldı. </li><li> 15 ml veya 50 ml konik bir tüp yavaşça hücreler aktarın. Önceden ısıtılmış MEF ortam 5 ml. Yavaşça hücreler girdap ve 5 dakika için 200 x g&#39;de santrifüj. </li><li> MEF ortamda hücrelerin tekrar ve canlı hücreleri saymak. 10 cm&#39;lik bir tabak hücreleri 2 ml 6 yuvalı plakadan gelen başına hücre ve 10 ml: Aşağıdaki MEF ortam hacimleri kullanın. </li><li> 6-delikli plakalara Levha hücreleri veya 1-2 en az 10 cm yemeklerPlaka / tabak başına x 10 6. </li><li> 37 ° C&#39;de, MEF plakalar / tabaklar inkübe,% 5 CO2, en az 24 saat önce kullanımı. NOT: Bir haftadan daha eski MEF plakaları / yemekler atın. </li></ol></li><li> Fare EKH hazırlayın <ol><li> Kültür Isl1-CRE; Rosa26YFP / Mef2c-CRE; % 90 konfluent kadar MEF&#39;ler üzerinde Rosa26YFP fare EKH. Kullanım ES hücre ortamı 410 ml DMEM, 75 ml FBS, 0.1 mM MEM NEAA, 2 mM L-glutamin, 0.1 mM piruvat, 100 E Pen / Strep, ve 0.1 mM β-merkaptoetanol içermektedir. </li><li> 1.1.2 tarif edildiği gibi, jelatin kaplı 6 oyuklu plakalar hazırlamak. </li><li> Plakalardan aspire orta ve DPBS hücreleri durulayın. <ol><li> Aspire DPBS ve 6 oyuklu plakanın bir kuyuya 0.4 mi% 0.25 tripsin ekleyin. 5 dakika için 37 ° C,% 5 CO2 seviyesinde hücreleri inkübe edin. Tripsin reaksiyonu durdurmak için 1 ml önceden ısıtılmış ES ortamı ekleyin. </li></ol></li><li> Hasat hücreleri ve santrifüj hücreleri 200 xg ve aspire orta az. Süspanse 1 ml ES hücre ortamında hücre ve hücreleri saymak. &lt;/ Li&gt; <li> 3 x 10 4 / cm2 yoğunlukta, jelatin-kaplanmış plakalar üzerinde plaka EKH. EKH yaklaşık% 90 konfluent ulaştığında yaklaşık 2 gün boyunca 37 ° C&#39;de,% 5 CO2 inkübe edin. Orta gün değiştirin. </li><li> EKH% 90 konfluent olduğunda, tekrar tamamen MEF besleyiciler kaldırmak için 1.2.2-1.2.5 adımları. </li></ol></li><li> EB formasyonu TBM farklılaşmasını teşvik <ol><li> Ardından farklılaştırma ortamı hazırlayın: 36 ml IMDM, 12 mi Ham F12, 2 mM L-glutamin, 0.34 ml BSA (% 7.5), 0.25 mi, N2, 0.5 mi B27, 50 ug / ml AA, ve 0.45 mM 1-tiogliserol. </li><li> Aspire hücrelerinden orta ve DPBS ile durulayın. Aspire DPBS ve 6 oyuklu plakanın bir kuyuya 0.4 mi% 0.25 tripsin ekleyin. 5 dakika boyunca 37 ° C&#39;de inkübe edilir. </li><li> Tek hücre içine hücre kümeleri dağıtmak için aşağı reaksiyon ve pipet yukarı durdurma ve 1 ml farklılaşma orta ekleyin. </li><li> Santrifüj EKH 5 dakika için 200 xg&#39;de ve orta atın. <ol><li> Süspanse 1 x 10 6 &lt;/ Sup&gt; 1 mi farklılaşma ortamında EKH. İlk EB toplama için 37 ° C &#39;de ayırt ortamında 1 x 10 5 hücre / ml&#39; lik bir konsantrasyonda, düşük sıyrılma Petri kabındaki hücrelere hücre sayımı ve kültür. </li></ol></li><li> İki gün EB oluşumundan sonra, 50 ml&#39;lik tüplere yemekleri EBS aktarmak. 1 dakika için 200 xg&#39;de Spin. Orta çıkarın ve Ca 2+ ve Mg 2+ olmadan 10 ml DPBS ile EBS durulayın. </li><li> Aspire DPBS ve 1 mL% 0.25 tripsin ekleyin. 5 dakika boyunca 37 ° C&#39;de inkübe edilir. Reaksiyonu durdurmak için 4.5 mi farklılaşma orta ekleyin. Pipet yukarı ve aşağı tek hücre içine EBS ayırmak. </li><li> 5 dakika süreyle 200 x g&#39;de hücreleri santrifüjleyin. 1 ml farklılaşma ortamda aspire orta ve tekrar süspansiyon hücreleri. </li><li> Hücre sayımı ve farklılaşma ortamında 1 x 10 5 hücre / ml, 2 x 10 6 hücre seyreltin. 5 ng / ml, 0.8 ng / ml&#39;lik nihai konsantrasyonda ortama VEGF BMP4 ve aktivin A ilave edin ve 5 ng / ml idi. Cultu37 ° C &#39;de, ikinci EB formasyonu için Petri kabındaki hücrelere yeniden. </li><li> İkinci EB oluşumundan sonra 40 saat, 50 ml&#39;lik tüplere EBS aktarmak. 5 dakika için 37 ° C &#39;de, 1 ml% 0.25 tripsin ile EBS, ayırma DPBS ile bir kez yıkayın. Pipet yukarı ve aşağı tek hücre içine EBS ayırmak. </li><li> 5 ng / ml VEGF, 10 ng / ml bFGF ve 12.5 ng / ml FGF10 ile Stempro-34 ortamı içinde tekrar süspansiyon hücreleri. Jelatin kaplı plakalara, 2 x 10 5 / cm2&#39;de, hücreler plaka. TBM izole edilmesinden önce 32 saat süre ile 37 ° C inkübatör içinde kültürü. </li></ol></li><li> MCPCs yalıtmak <ol><li> Aspire orta ve DPBS ile durulayın. 5 dakika için 37 ° C &#39;de kültür plakalarına 0.5 mi% 0.25 tripsin ekleyin. Reaksiyonu durdurmak için 4.5 mi farklılaşma orta ekleyin. Pipet yukarı ve aşağı tek hücre içine EBS ayırmak. FACS-saflaştırılması için% 2 FBS / PBS santrifüj ve tekrar süspansiyon hücreleri hücreleri toplayın. </li><li> Negatif kontrol olarak sıralayıcı farklılaşmamış EKH çalıştırın. Değişim gerilimi ileri dışkılarını ayarlamak içinter (FSC) ve yan dağılım (SSC) istenen nüfus seçmek için. Enkaz ve dublet dışlamak için yan dağılım ve genişliği karşı ileri dağılım yüksekliği karşı dağılım ileri kullanın. Seçilen alt-popülasyonda, ESC kontrol dağılımına göre, hücre otofloresans ortadan kaldırmak için histogram üzerinde olumlu YFP bir kapı çizin. YFP + yolluk gelen YFP + TBM&#39;leri toplayın. </li><li> Farklılaştırma ortamının (1.3.1) &#39;e sahip 6-çukurlu plakalarda saflaştırılmış TBM (YFP + hücreleri) plaka. Kardiyomiyositler ve SMC&#39;lerin içine TBMlerin farklılaşması için 37 ° C&#39;de Kültür ek 3-5 gün. </li></ol></li></ol> İnsan EKH İnsan Embriyonik TBMlerin 2. türetilmesi <ol><li> Yemlikler hazırlayın <ol><li> 2 x 10 4 / cm2 yoğunluğunda, yukarıda tarif edildiği gibi, fare embriyonik fibroblastlar (MEF&#39;ler) besleyici tabaka hazırlanır. </li></ol></li><li> İnsan EKH koruyun <ol><li> CRE, M rutin pCAG-flox-STOP-flox-GFP veya RFP EKH: İnsan Isl1 koruyunNormal HESC ortamı (Nakavt DMEM / F-12 385 ml, Nakavt SR 100 mi, 2 mM L-glutamin, 0.1 mM NEAA, 0.1 mM β-merkaptoetanol, 10 ng / ml temel FGF) kullanılarak EF. Kalp farklılaşma önce, en az 2 pasajlar için besleyici serbest durumda insan EKH aktarmak. </li></ol></li><li> Besleyici serbest durumda insan EKH hazırlayın <ol><li> İnsan EKH kültür için kaplanmış tabak hazırlayın. <ol><li> Gece boyunca 4 ° C &#39;de Matrigel çözülme. Buz üzerinde 50 mi tüp koyun ve bir tüp içine, 30 ml soğuk DMEM / F12 ortamı ilave edin. </li><li> Soğuk ortama 1 ml Matrigel ekleyin ve iyice karıştırın. 10 cm&#39;lik bir tabak içine bir 6-yuvalı plaka oyuğuna ya da 5 ml &#39;si içerisine 1 ml soğuk Matrigel, DMEM / 12 orta ekleyin. </li><li> 2 saat boyunca levhalar / gece boyunca 4 ° C&#39;de yemekleri veya oda sıcaklığını bırakın. Orta aspire kullanmadan önce. </li></ol></li><li> MEF plaka aspire medya ve DPBS insan EKH durulayın: İnsan plakalar üzerine EKH bölün. Daha sonra 6-çukurlu plaka içinde bir kuyu 1 ml dispaz (1 mg / ml) ilave edilmektedir. 37 ° C de kuluçkaya bırakılırinkübatör kolonilerinin kenarları ayırmak başlayana kadar. </li><li> Dispase çözüm çıkarın. Daha sonra 2,5 ml / add iyi mTeSR orta veya MEF-klimalı ortamda başına plaka, iki kez DPBS ile durulayın. </li><li> Küçük öbekler halinde insan ESC kolonileri yıkmak için bir hücre kazıyıcı kullanarak hücreleri kazıyın ve dikkatlice pipetlemeyin ve. </li><li> ESC kümeleri aktarın ve 1 sulandırmak: 3 kaplanmış plakalar halinde. </li><li> 2 ml / kuyu mTeSR orta veya MEF-klimalı orta ekleyin ve günlük orta değiştirin. </li><li> MTeSR ortam veya en az 2 pasaj ile kaplanmış plakalar üzerine, MEF-şartlandırılmış ortam içinde kültür insan EKH. </li></ol></li><li> İnsan EKH TBM farklılaşmasını teşvik <ol><li> Hücreler ~% 90 konfluent olduğunda, orta aspire ve DPBS ile durulayın. Daha sonra 20 dakika boyunca 37 ° C&#39;de 0.5 mg / ml dispaz içinde HESC inkübe edin. </li><li> Hücre kaldırıcı plakalardan hücreleri çıkarmak iki kez DPBS HESC durulayın, sonra, DMEM / F12% 18 FBS, 0.1 mM NEAA, 2 mM ihtiva eden (farklılaştırma ortamının hücre kelepçeler tekrar süspansiyonL-glutamin, 0.1 mM β-merkaptoetanol ve 50 ug / ml askorbik asit). </li><li> EB oluşumu için 6-kuyu, ultra-düşük bağlantı plakaları içine hücreleri aktarın. </li><li> Ertesi gün farklılaşma ortamı değiştirin. </li><li> Her 3 günde bir orta takın ve süspansiyon kültürü içinde EBS korumak. </li></ol></li><li> HCPCS yalıtmak <ol><li> Geç insan TBM izolasyonu için farklılaşma 9 gün daha EBS toplayın. </li><li> Aspire orta ve iki kez DPBS ile EBS durulayın. 5 dakika için 37 ° C &#39;de 6-çukurlu kültür plakalarının her bir oyuğuna 0.5 mi% 0.25 tripsin ekleyin. </li><li> Reaksiyonu durdurmak için farklılaştırma ortamının eşit hacim. Pipet yukarı ve aşağı tek hücre içine EBS ayırmak. % 2 FBS / DPBS&#39;de 5 dk ve tekrar süspansiyon hücreleri için 200 xg&#39;de santrifüj hücreleri toplayın. 40 mikron hücre süzgeç ve FACS-saflaştırılmış RFP + TBM hücreleri ile Filtre hücreleri olarak 1.4.3 de anlatıldığı. </li><li> Plaka geltin kaplı plakalar üzerine HCPCS sıralanır. 7 için farklılaşma ortam içinde kültür HCPCS-9 Gün kardiyomiyosit ve düz kas hücrelerine ayırt etmek. 7 gün kuluçkadan sonra kültür, DMEM / F12 ortamı içinde% 5 Nakavt SR farklılaşmış hücreleri. </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

Kalp hastalığı dünyanın önde gelen nedeni, bugün kalır ve ölüm oranları son iki yılda (Amerikan Kalp Derneği) hemen hemen değişmeden kalmıştır. Etkili bir şekilde engellemek veya kalp yetmezliği ters yeni tedavi stratejilerinin geliştirilmesi için önemli bir ihtiyaç vardır. Bir umut verici bir strateji kök hücre biyolojisi 1 hızlı gelişimi aşağıdaki hücre tabanlı tedavi yöntemidir. Bu bağlamda, multipotent TBM bağlı olarak çoğalır, ancak sadece kalp dizi farklılaşma kararlı etme kabiliyetleri tedavisi için mükemmel bir hücre kaynağı olabilir. Bu nedenle, verimli ve sağlam bir yöntem oluşturmak ve kalp hücre tedavisi çalışmaları için büyük önem taşımaktadır TBM&#39;lerini izole etmek. Bu protokol, erken embriyogenezin ve nasıl EKH kendi üretim sırasında belirlenen embriyonik TBM&#39;lerle üzerinde duruluyor. Çeşitli TBM da kemik iliği 2, embriyonik ve yetişkin kalplerinden izole edilmiştir. Embriyo gelişimi sırasında, kemik morphogemanyetik proteinleri (BMP), kanatsız-tipi MMTV entegrasyon sitesi aile üyeleri (Wnts) ve Düğüm sinyaller Mesp1 + Multipotent mezoderm 3 bağlılığını neden. Mesp1 + hücreleri, daha sonra embriyonik TBM 4 içinde farklılık gösterir. Bu TBMler genellikle NK2 homeobox 5 (Nkx2-5), Isl LIM homeobox 1 (Isl1), T-box 5 (Tbx5), ve miyosit arttırıcı faktör 2C (Mef2c), birincil ve ikinci kalp alanları oluşturmak, HCN4 tarafından işaretlendiği ve cardiogenesis 5-10 sırasında kalbin ana bölümden katkıda bulunur. Nkx2-5 + ve Isl1 + / Mef2c + Her iki TBM kardiyomiyositlere ayırt edebiliyoruz, düz kas hücreleri (SMC&#39;lere) ve endotel hücreleri 5-8. Bu nedenle, bu TBM kalp damar yanı sıra kardiyak dokulara doğuran ve hücre bazlı kalp tedavisi için ideal bir hücre kaynağıdır olacaktır. Sonuç olarak, in vitro TBM&#39;leri üreten kardiyovasküler çalışmalarda önemli bir araştırma odağı olmuştur. EKH sınırsız genişleme kapasitesi a sahip olduğundannd blastosist aşamasında ICM hücreleri temsil doğal embriyogenez aşağıdaki embriyonik TBM&#39;lerle içine EKH farklılaşması TBM&#39;lerini elde etmek mantıklı ve etkili bir yaklaşım olarak kabul edilir. EKH gelen TBM&#39;ler elde etmek için bir çok uygulamalı bir yaklaşım EBS 11 içine EKH bir araya etmektir. Farklılaşma verimliliğini artırmak için, kardiyak gelişme bilgiye dayalı tanımlanmış kimyasal ve büyüme faktörleri 12-14 kullanılmıştır. Ancak, yaygın olarak alanda kabul edilen kesin TBM belirteçler, özellikle de herhangi bir hücre yüzeyi işaretleri vardır. Bu sorunu gidermek için, EKH Isl1 + veya Mef2c + TBM ve Cre / loxP sistemi kullanılarak floresan gazetecilere türevleri işaretlemek için tasarlanmıştır. CRE rekombinaz Isl1 / Mef2c promotör / güçlendirici kontrolü altında çaldı. Bir kurucu promoteri tarafından tahrik edilen modifiye floresan protein RFP veya YFP gen CRE rekombinaz ile flox durdurma kodonu çıkarılması ile aktive edilebilir(Isl1: CRE;-pCAG-flox-STOP-flox GFP veya RFP / Isl1-CRE; Rosa26YFP / Mef2c-CRE; Rosa26YFP) 5,6. EKH ikinci kalp alan TBM&#39;ler ayrılırlar sonra, Isl1 / Mef2c promotör / güçlendirici tahrik CRE floresan gazetecilere aktive edecek ve TBM FACS-saflaştırma ile zenginleştirilmiş olabilir. Kısaca, EB toplama yöntemi ESC farklılaşma başlatmak için kullanılır. Farklılaşma verimliliğini artırmak için, farklılaşmış hücreler, askorbik asit (AA) ve BMP4, aktivin A ve VEGF 13,15 gibi büyüme faktörleri ile muamele edilir. Bu protokol fare ve insan EKH her ikisini de kullanarak sağlam ve verimli TBM farklılaşmasını sağlar.

<table><tbody><tr><td>FBS</td><td>Thermo scentific</td><td>SH30070.03E</td><td></td></tr><tr><td>Knockout SR</td><td>Life technology</td><td>10828028</td><td></td></tr><tr><td>Knockout DMEM</td><td>Life technology</td><td>10829018</td><td></td></tr><tr><td>DMEM</td><td>Life technology</td><td>11965118</td><td></td></tr><tr><td>NEAA</td><td>Life technology</td><td>11140050</td><td></td></tr><tr><td>GlutaMAX</td><td>Life technology</td><td>35050061</td><td></td></tr><tr><td>N2</td><td>Life technology</td><td>17502048</td><td></td></tr><tr><td>B27</td><td>Life technology</td><td>12587010</td><td></td></tr><tr><td>Ham&amp;rsquo;s F12</td><td>Life technology</td><td>11765062</td><td></td></tr><tr><td>IMDM</td><td>Life technology</td><td>12440061</td><td></td></tr><tr><td>Pen/Strep</td><td>Life technology</td><td>15140122</td><td></td></tr><tr><td>Pyruvate</td><td>Life technology</td><td>11360070</td><td></td></tr><tr><td>Dispase</td><td>Life technology</td><td>17105041</td><td></td></tr><tr><td>Stempro-34</td><td>Life technology</td><td>10639011</td><td></td></tr><tr><td>DMEM/F12</td><td>Life technology</td><td>11330032</td><td></td></tr><tr><td>BSA&amp;nbsp;</td><td>Life technology</td><td>15260037</td><td></td></tr><tr><td>Trypsin&amp;nbsp;</td><td>Life technology</td><td>25200056</td><td></td></tr><tr><td>Ascobic Acid&amp;nbsp;</td><td>Sigma</td><td>A5960</td><td></td></tr><tr><td>1-Thioglycerol</td><td>Sigma</td><td>M1753</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma</td><td>M3148</td><td></td></tr><tr><td>VEGF</td><td>R&amp;amp;D</td><td>293-VE</td><td></td></tr><tr><td>Bmp4</td><td>R&amp;amp;D</td><td>314-BP</td><td></td></tr><tr><td>ActivinA</td><td>R&amp;amp;D</td><td>338-AC</td><td></td></tr><tr><td>bFgf</td><td>R&amp;amp;D</td><td>233-FB</td><td></td></tr><tr><td>Fgf10</td><td>R&amp;amp;D</td><td>345-FG</td><td></td></tr><tr><td>mTeSR</td><td>Stemcell technologies</td><td>5850</td><td></td></tr><tr><td>Matrigel</td><td>BD Biosciences</td><td>354277</td><td></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.

Protokol pek çok ES hücre hatları TBM türetilmesini göstermektedir. EKH TBM&#39;leri içine ayırt etmek için EBS oluşturmak için toplanır. EKH rutin MEF besleyiciler (Şekil 1A, F) tutulur ve besleyiciler farklılaşma önce kaldırılır. Farklılaşma ortamında EBS (Şekil 1B, G) içine EKH agregasyonu üzerine, ikinci oluşan EBS fare hücre hatları (Şekil 1C) içinde mezoderm farklılaşmasını geliştirmek için BMP4 ve aktivin A ile işleme tabi tutu...

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.

Embriyonik Kök Hücreleri Kardiyak Progenitör Hücrelerinin türetilmesi

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 Görüntüleme.  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

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Murin Embriyonik Kök Hücre Hematopoetik Kök Hücre türetilmesi

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Biyoloji

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

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

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

BMP rakip GREM2 kullanma Pluripotent Kök Hücreleri Atriyal kardiyomiyositlerde Farklılaşma

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

Gelişim Biyolojisi

Assessing Stem Cell DNA Integrity for Cardiac Cell Therapy

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data in all cells, including stem cells, and its integrity is fundamental to its regenerative ability. Stem cells undergo rapid propagation in labs to achieve the necessary numbers for transplantation. Accelerated cell growth leads to the loss of DNA integrity by accumulated metabolites, such as reactive oxygen, carbonyl, and alkylating agents. Transplanting these cells would result in poor engraftment and regeneration of the deteriorating organ. Moreover, transplanting DNA-damaged cells leads to mutations, DNA instability, cellular senescence, and possibly, life-threatening diseases such as cancer. Therefore, there is an immediate need for a quality control method to evaluate the cell's suitability for transplantation. Here, we provide step-by-step protocols for the assessment of the DNA integrity of stem cells prior to cell transplantation.

Here, we provide a detailed description of an experimental setup for an analysis of the assessment of DNA integrity in stem cells prior to cell transplantation.

Kardiyak hücre tedavisi için kök hücre DNA bütünlük değerlendirilmesi

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data ...

Genetik

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

Vitro İnsan Mezenkimal Kök hücre farklılaşma fonksiyonel Cardiomyocyte benzeri hücre içine

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is a pre-requisite for cell-based cardiac therapies and disease modeling. We have recently shown that Id genes are both necessary and sufficient to specify first heart field progenitors during vertebrate development. This differentiation protocol leverages these findings and uses Id1 overexpression in combination with Activin A as potent specifying cues to produce first heart field-like (FHF-L) progenitors. Importantly, resulting progenitors efficiently differentiate (~70&#8211;90%) into ventricular-like cardiomyocytes. Here we describe a detailed method to 1) generate Id1-overexpressing hPSCs and 2) differentiate scalable quantities of cryopreservable FHF-L progenitors and ventricular-like cardiomyocytes.

Here we describe a scalable method, using a simple combination of Activin A and lentivirus-mediated Id1-overexpression, to generate first heart field-like cardiac progenitors and ventricular-like cardiomyocytes from human pluripotent stem cells.

İlk kalp alan benzeri kardiyak ataları ve ventrikül benzeri Cardiomyocytes insan Pluripotent kök hücrelerden nesil

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is ...

Induction of Endothelial Differentiation in Cardiac Progenitor Cells Under Low Serum Conditions

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are extremely scarce, but expanded CPCs could be useful for cell therapy. A prerequisite for their use is their ability to differentiate in a controlled manner into the various cardiac lineages using defined and efficient protocols. In addition, upon in vitro expansion, CPCs isolated from patients or preclinical disease models may offer fruitful research tools for the investigation of disease mechanisms.
Current studies use different markers to identify CPCs. However, not all of them are expressed in humans, which limits the translational impact of some preclinical studies. Differentiation protocols that are applicable irrespective of the isolation technique and marker expression will allow for the standardized expansion and priming of CPCs for cell therapy purpose. Here we describe that the priming of CPCs under a low fetal bovine serum (FBS) concentration and low cell density conditions facilitates the endothelial differentiation of CPCs. Using two different subpopulations of mouse and rat CPCs, we show that laminin is a more suitable substrate than fibronectin for this purpose under the following protocol: after culturing for 2 - 3 days in medium including supplements that maintain multipotency and with 3.5% FBS, CPCs are seeded on laminin at &#60;60% confluence and cultured in supplement-free medium with low concentrations of FBS (0.1%) for 20 - 24 hours before differentiation in endothelial differentiation medium. Because CPCs are a heterogeneous population, serum concentrations and incubation times may need to be adjusted depending on the properties of the respective CPC subpopulation. Considering this, the technique can be applied to other types of CPCs as well and provides a useful method to investigate the potential and mechanisms of differentiation and how they are affected by disease when using CPCs isolated from respective disease models.

This protocol describes an endothelial differentiation technique for cardiac progenitor cells. It particularly focuses on how serum concentration and cell-seeding density affect the endothelial differentiation potential.

İndüksiyon düşük Serum koşullar altında kalp Progenitor hücreler endotel farklılaşma

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are ...

In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in developmental cardiology have led to generating cardiac cells from pluripotent stem cells, it is unclear if the two cardiac fields - the first and second heart fields (FHF and SHF) &#8212; are induced in pluripotent stem cells systems. To address this, we generated a protocol for in vitro specification and isolation of heart field-specific cardiac progenitor cells. We used embryonic stem cells lines carrying Hcn4-GFP and Tbx1-Cre; Rosa-RFP reporters of the FHF and the SHF, respectively, and live cell immunostaining of the cell membrane protein Cxcr4, a SHF marker. With this approach, we generated progenitor cells which recapitulate the functional properties and transcriptome of their in vivo counterparts. Our protocol can be utilized to study early specification and segregation of the two heart fields and to generate chamber-specific cardiac cells for heart disease modelling. Since this is an in vitro organoid system, it may not provide precise anatomical information. However, this system overcomes the poor accessibility of gastrulation-stage embryos and can be upscaled for high-throughput screens.

The purpose of this method is to generate heart field-specific cardiac progenitor cells in vitro in order to study the progenitor cell specification and functional properties, and to generate chamber specific cardiac cells for heart disease modelling.

In vitro Generation fare kalp alanı-spesifik kardiyak progenitor hücreler

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Küçük Molekül İndüksiyon Pluripotent İnsan Embriyonik Kök Hücreleri İnsan Kardiyak Öncüleri ve kardiyomiyositlerde Verimli türetilmesi

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

İnsan kardiyomiyositleri Üretimi: Besleyici-ücretsiz İnsan Bağlı Pluripotent Kök Hücre A Farklılaşma Protokolü

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

Sitometrisi ile Yüksek Verim kardiyomiyositlerde İnsan pluripotent kök hücrelerin Farklılaşma ve Karakterizasyonu

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...